APPUED MICROBIOLOGY, June 1971, p. 1024-1031.Copyright @ 1971 American Society for Microbiology

Accumulation and Elimination of Coliphage S-13by the Hard Clam, Mercenaria mercenaria

WALTER J. CANZONIEROyster Research Laboratory, New Jersey Agricultural Experiment Station,

Rutgers-The State University, New Brunswick, New Jersey 08903

Received for publication 1 February 1971

Accumulation and elimination of viral particles by hard clams, Mercenariamercenaria, were studied with the coliphage S-13 as a working model. Escherichiacoli uptake and elimination were simultaneously monitored. Clams were exposed tolow levels of S-13 (7 particles/ml) in running seawater for several days, achievingtiters in tissues from 2 to more than 1,000 times the levels to which they had beenexposed. Bacterial accumulation (previously established by other workers) was com-parable. Upon exposure to virus-free running water, clams polluted to relativelylow levels (100 plaque-forming units/ml) eliminated most of their bacterial con-taminants in 24 to 48 hr. Viral contaminants, however, persisted for several days toweeks even under ideal conditions for clam activity, provided that the temperatureremained below the inactivation threshold for the virus. Most of the accumulatedvirus appeared to be sequestered in the digestive gland. These sequestered particlesare refractory to those mechanisms responsible for elimination of bacterial con-taminants. This discrepancy points out the need for caution in evaluating theefficiency of shellfish depuration processes, especially if only a bacterial criterion isused as a monitoring system.

The indictment of shellfish as probable carriersof enteric viruses (3, 12, 15, 16, 18-20; 0. C. Liuet al., Bacteriol. Proc., p. 151, 1968) has generatedconsiderable interest in development of methodsof cleansing oysters and clams of viral contami-nants (depuration). Several laboratory studieshave demonstrated the accumulation by clamsand oysters of various viruses and their subse-quent reduction upon the return of the shellfishto virus-free seawater (5, 10, 11, 13, 14; Liu et al.,Bacteriol. Proc., p. 151, 1968). Some workers havealso compared Escherichia coli with viruses intheir accumulation and elimination by shellfish(6, 14).The virus of principal public health significance

is the etiological agent of infectious hepatitis. Theisolation of this agent, however, is yet to be per-fected and all work with shellfish has been con-fined to the use of model systems employing otherviral entities. In these model systems shellfish areexposed from a few hours to several days toknown concentrations of virus, in aquaria withstanding, recirculated, or flow-through watersupplies. Poliovirus and coxsackievirus (13),poliovirus (5, 10, 14), and bacteriophage (7) havebeen used in other laboratories. S. Y. Feng (Proc.Nat. Shellfish Ass. 57:2, 1967), working in thislaboratory with the hard clam, Mercenaria mer-

cenaria, initiated studies with a phage ofStaphylo-coccus aureus 80 and later introduced a smallerphage of E. coli C designated S-13. This workwas continued by C. Fenton (S. Y. Feng andC. Fenton, unpublished data).Feng and Fenton initially utilized high dosage

of particles during the pollution phase whichusually resulted in viral concentrations in excess of2 X 103 particles per ml of clam tissue extract.At these high levels, clams eliminated a large per-centage of the viral load within a short period ofexposure to clean water (24 to 48 hr). This cor-responded to an equivalent efficiency in simul-taneous elimination of E. coli. However, a lowtiter of phage often persisted for several days inmany of these experiments. The persistence ofviral particles even after 48 hr of depurationraises the question of infectious potential, espe-cially since the minimal infective dose for hepatitisand other enteric viruses is not well established.The exposure levels and subsequent levels of con-tamination in the experiments ofFeng and Fentonwere usually in excess of concentrations one mightencounter under natural conditions. In the workreported here, therefore, the final contaminationtiters in clam tissue were held below 100 particles/ml by reducing the levels in the seawater to lessthan 10/ml.

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S-13 COLIPHAGE IN THE HARD CLAM

A secondary objective of the present studies hasbeen to determine the capacity of hard clams inaccumulating and concentrating viruses when ex-

posed continuously to very low levels in a run-

ning-seawater system. The concentrating capacityof hard clams and oysters has been establishedfor bacteria (E. coli) and viruses when exposed tolevels in excess of 40 particles or more per ml ofseawater (2, 9, 11, 13, 14).

Previous workers at this laboratory and mostat other laboratories had exposed clams toviruses by immersion in standing water with highviral titers. In the present work, to simulate moreprobable natural pollution situations and toachieve lower but more uniform contaminationlevels in the clam at the initiation of depuration,the presentation of phage (dosing) was at lowlevels for prolonged periods.Feng and Fenton used pools of whole clams in

their assay procedure, and no effort was made toconcentrate or refine the homogenized tissue. Inthis present work, sensitivity at lower phage titerswas enhanced by using only an extract of thedigestive glands of individual clams.

MATERIALS AND METHODSClams. Northem hard clams (Mercenaria merce-

naria; 100 to 150 g) were obtained from local com-mercial sources and conditioned in the aquariumsystem prior to initiation of the experiment. In anyexperiment, clams were selected to be approximatelythe same size.

Phage model. The bacteriophage used was desig-nated S-13 (1), the host cell being E. coli C. This isone of the smaller viruses, approximately 20 nm indiameter, about the size of the poliovirus. Phage andhost cells were obtained from E. Simon and E. S.Tessman, Purdue Univ. High phage titers (107 to109/ml) were produced in 250-ml nutrient broth(Difco) cultures of E. coli, the cells were removed bycentrifugation, and the supernatant fluid was storedover chloroform at 4 C until dispensed.

Assay. Extracts of clam tissue were prepared byhomogenizing in a blendor the digestive gland ofindividual clams in an equal weight of 0.8% nutrientbroth (Difco) for 1 min at high speed. Ten millilitersof the brei was placed in 13-ml tubes to which 2 ml ofchloroform was added; the tubes were capped andshaken and then centrifuged at 25,000 X g at 4 C for1 hr. A 1-ml sample of supernatant fluid (includinggelatinous layer), or an appropriate dilution innutrient broth, was spread uniformly over a base layerof plate count agar (Difco) with 0.0025% CaCl2added. After 10 min, this was overlaid with about 5ml of 1.5% agar containing 107 to 108 host cells per mland 0.05 M tris(hydroxymethyl)aminomethane bufferadjusted to pH 7.6. Plates were incubated 4 to 6 hr at37 C.

Each sample consisted of 10 to 20 clams, and 5 or10 ml of extract was plated for each clam. It wasestimated that the lower limit of recoverability was

accordingly 0.2 or 0.1 plaque-forming unit (PFU) perml of clam tissue extract.

Bacterial system. A strain of fecal E. coli B wasgrown in nutrient broth at 45 C and assayed by a pourplate technique, developed by V. Cabelli (2), em-ploying a modified MacConkey agar. The clam diges-tive glands were ground in a blendor as for phage, butwhole brei or dilutions thereof were plated. Six to 10clams, processed individually, were used for eachsample.

Experimental aquaria and seawater system. Sea-water was pumped continuously from the Shrews-bury River, Monmouth County, N.J. Salinities rangein this river from 17 to 26%. During all experimentsreported, they were 20% or greater.The piping and tank system was constructed of

polyvinylchloride, polyethylene, and expoxy-coatedwood. The clam treatment tanks measured 61 by 244cm with a flow of 150 to 200 liters per hr per tank.

Water temperature was controlled when necessaryby passage through a Teflon heat exchanger. All waterwas sterilized by passage through a thin layer (1 to2 mm) beneath 15 G. E. Germicidal Lamps (30-w;UV). This UV-treatment system is essentially asdescribed for the unit used in the Public HealthService Laboratory at Purdy, Wash. (8), with theexception that a smooth rather than riffled surfacewas used.

During the pollution phase, phage and bacterialsuspensions were refrigerated at 4 C and dispensed bya peristaltic pump into a small mixing chamber re-ceiving the inflowing water before its entry into theaquarium. This chamber also counterbalanced aspring-loaded switch controlling the peristaltic pump,thus preventing excessive dosing in the event of atemporary reduction in water flow.

During the course of the experiment, aquaria weredrained and flushed daily to remove accumulateddetritus, mucus, and feces.

Environmental data. Various physical parameters in-cluding temperature, salinity, turbidity, and chloro-phyll levels of incoming water were determined dailyor at more frequent intervals. As a direct index ofsuitability of the conditions for clam activity, shellmovements of 12 to 24 clams were recorded continu-ously throughout these studies. In addition, a popula-tion of 100 clams was held under observation in theaquarium system, and the number with valves openand siphons extended was recorded three times daily.All experiments were conducted during periods ofrelatively high activity.

RESULTSAccumulation of phage. Two hundred clams

were placed in running water and dosed withapproximately five phage particles/ml. Samplesof 20 clams were removed at 16, 40, 160, and 184hr. Samples of seawater were removed from theaquarium at frequent intervals (24 hr or less)throughout the experiment to monitor phagelevels. Exposure levels never exceeded seven perml and had a mean of five per ml. At 212 hr, the

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TABLE 1. Uptake of bacteriophage S-13 byhard clams"

Date

20 January28 January16 April29 April7 May27 May19 June26 June3 August9 September18 September23 September8 October

27 October

14 May5 June

Expo-suretime(days)

10151828971677777

1

1

Exposureleveib

0.054-73-5

19

4-63-7706

0.4-2.24

0.4-1.20.8-8.0

2

3,000475

Meantiterc

577590549210770

1,754147

245

83395

1,816151

Accumula-tion factord

1,140142254102314252.33.265

10220

0.70.3

a All in flow-through seawater except last twoin aerated standing water.

b Concentration of S-13 in seawater in PFU/ml.c Concentration of S-13 in digestive glands of

10 or 20 clams in PFU/ml.d Concentration in clams/concentration in sea-

water.

dosing was increased by a factor of 14 (70 per ml),and the clams were again sampled at 231 hr.The means for 20 clams in each sample through

184 hr ranged between 52 and 86 per ml. Themean for all samples was 70 per ml. This titer isapproximately 10 times the level to which theywere exposed.

After 19 hr at the higher dosage, the samplemean was 1,754 per ml, somewhat more than 25times the dosage level.

Capacity of clams to accumulate phage in theirtissues considerably in excess ofthe levels to whichthey were exposed was demonstrated in the pollu-tion phase of 14 additional experiments (Table1). In these experiments, the accumulation factor(ratio of clam titer to seawater titer) for themeans ofeach sample ranged between 2 and 1,100.Individuals often had titers in excess of 100 andoccasionally as high as 1,500 times the dosagelevels.

In a few cases, negligible accumulation wasattributable to generally low activity as indicatedby other observations. For example, in the lasttwo experiments listed in Table 1 the clams wereexposed in standing water. Under such conditions,activity is severely inhibited and many clams failto open for long periods.

5_

4

v ECOLI IN CLAMS

\ S-13 IN CLAMS

3 OS-13 IN SEAWATER

-J

o.iI- i _ _ __

LL20,

0

I LI I24 48 72 96 120 1

T IME (HR S)

FIG. 1. Elimination of phage S-13 and E. coli inclams and inactivation of S-13 in seawater at 16 C(October 1968). Confidence intervals of 95% indicatedfor samples representing 20 individual clams or 30 mlof seawater.

Depuration experiments. These experimentswere conducted throughout the year and underbroadly varying conditions. Four experimentsare considered representative and illustrate fea-tures of retention and elimination of viral par-ticles in hard clams.

(i) This experiment (October 1968), conductedat 16 C, illustrates clearly that bacteria and phageare handled quite differently by the hard clam(Fig. 1). The mean viral titer for 10 clams in theinitial sample was 33 PFU per ml. The initialE. coli level was 105 colony-forming units (CFU)per ml. At the time of initial sampling, threeflasks, each containing seawater with phage at6 PFU/ml, were immersed in the aquanum as acontrol for phage inactivation at the temperatureof the depuration phase. E. coli elimination pro-ceeded rather rapidly; within 24 hr the level haddropped to 10 CFU/ml and by 48 hr to less than1 CFU/ml, approaching the lower limit of reliableassay by the method used.

In contrast, reduction in phage proceeded veryslowly and in 144 hr (6 days) reached a level lessthan one log below the initial titer. Phage in sea-water exhibited an identical pattern of reduction.

(ii) A similar experiment (September 1968),

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S-13 COLIPHAGE IN THE HARD CLAM

ILll I

24 48 72TINE (HRS)

FIG. 2. Elimination of phage S-13 in clams and in-activation of S-13 in seawater at 24 C (September1968). Confidence intervals of95% for samples repre-senting 20 individual clams or 10 ml ofseawater.

conducted at 24 C, illustrates the effect of highertemperature on the rate of phage reduction.Again the rate of change in phage titer in clamsparalleled that in seawater although the slope forboth is considerably steeper than in the previousexperiment and the limits of reliable assay werereached in 72 hr (Fig. 2). Additional studies ofthe kinetics of inactivation of S-13 and P-80 phagehave indicated that temperature is indeed theprimary factor in phage inactivation in a sea-water medium and that there can be considerabledifferences between two viral types in response tothis factor.

(iii) Depuration at the Monmouth BeachLaboratory was compared with that at a tempo-rary facility located at Brigantine in southernNew Jersey. Water quality at that site, as judgedby clam activity, was superior to that obtainablein the Shrewsbury River. One experiment (Fig. 3)again illustrates the discrepency in elimination ofviruses and bacteria. Elimination of E. coli atBrigantine was rapid, dropping to less than 0.1CFU/ml in 24 hr, but was quite unsatisfactory atthe Monmouth Beach Laboratory. Reduction ofS-1 3 at both sites was better than that obtainedfor E. coli at Monmouth Beach, but even atBrigantine some phage were detectable after 168hr. This experiment illustrates the lack of correla-tion between elimination of phage and bacteria.The slightly better elimination of phage at Brigan-

t0

L

I0

D'L.a.

L ' l l l l20 40 60 sO .00

TIME (HRS)

FIG. 3. Comparison ofelimination ofphage S-13 andE. coli by clams depurated at Monmouth Beach andBrigantine. A reduction in seawater temperature oc-

curred at both sites during the period of exposure(September 1969).

tine was apparently a function of water tempera-ture, which attained a peak of 29 C at this siteduring the period of observation while remainingless than 25 C at Monmouth Beach.

Thirty-three experiments with varying condi-tions of temperature and clam activity have beenconducted in addition to those illustrated. Thedata strongly suggest that a portion of the phageis sequestered at low levels in the clam tissues andthis sequestered phage is not diminished appre-ciably by activity of the clam. The reduction inphage titer in every case could be accounted forby temperature-dependent inactivation in situ.

(iv) To test this hypothesis, an experiment wasdesigned to compare reduction in phage titer inactive and inactive clams at temperatures aboveand below 16 C. Above this temperature, rate ofS-13 inactivation in seawater is greatly accelerated(unpublished observation). A large group of clamswere exposed for 8 days to low levels of phage at

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CANZONIER A

VF,.QOLI -WET-14& 25Ca 14 A 25 S-13 - WETOI4*1425* S-13 - DRY0 14' *25* S-13 - IN SEAWATER

2 4 48TIME (HRS)

24 48 72TIME (HRS)

FIG. 4. Comparison of phage S-13 elimination byclams exposed to runningseawaterandconfinedin plasticbags at two temperatures. Elimination of E. coil inrunning seawater was essentially the same at both tem-peratures and is represented by a single series of ob-servations (November 1969).

14 C. All clams were also exposed to E. coli forthe last 24 hr of the phage pollution phase. Forthe depuration phase, four lots were treated asfollows: (i) running seawater at 14 C, (ii) run-ning seawater at 25 C, (iii) dry storage in plasticbags immersed in the 14 C aquarium, and (iv)dry storage in plastic bags immersed in the 25 Caquarium.Phage in flasks of seawater were also immersed

in the aquaria at 14 and 25 C, as an inactivationcontrol during the depuration phase.Clams in lots (i) and (ii) were sampled at 24, 48,

and 72 hr. There were insufficient clams to samplegroups (iii) and (iv) at 72 hr (Fig. 4). The E. colicells, initially 200 CFU/ml, were reduced to lessthan 0.1 CFU/ml at 24 hr and were undetectedat 48 hr. This indicated high clam activity. Nodifference in E. coli elimination was detectablebetween warm and cool treatment.Clams at 14 C (group i) retained essentially

constant phage titers for at least 72 hr. There wasno difference between wet and dry treatment inthe first 48 hr. Phage titers in seawater at 14 Calso remained constant.At 25 C, the titer in both clam groups (ii and

iv) and in seawater was reduced at a rate similarto that described in the 24 C experiment above(Fig. 2).Retention of S-13 has been observed in clams

in the laboratory for as long as 34 days duringperiods of good clam activity. Field experiments,in conjunction with relaying of clam from con-demned to approved shellfish waters, show evengreater periods of phage retention. Marked clamspolluted with S-13 at 76 PFU/ml were mixed withtransplants. After a period of 2 months (10October to 12 December) on the bottom in anarea considered good for clamming, 50 to 90% ofthe virus was retained. Water temperatures during50 days of this period ranged between 8 and 16 C,sufficient for clam activity, although always belowthe inactivation threshold level for S-13. During asubsequent period of 3 months at temperatureslower than 8 C, the phage persisted in both wetstored and refrigerated samples.

DISCUSSIONThe experimental evidence reported here illus-

trates several aspects of viral accumulation andelimination by hard clams. In comparison withearlier work, the use of low viral titers allows amore reasonable projection for the prediction ofthe kinetics of viral uptake and elimination innaturally polluted clams. Indeed, the pattern ofelimination when initial titers are less than 100PFU/ml is strikingly different from that in casesof clams polluted to levels in excess of 1,000PFU/ml. In the latter, elimination of a major

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portion of the viral contaminants is quite rapidand parallels the elimination of such bacterialcontaminants as E. coli.The accumulation of bacterial and viral par-

ticles by clams and other shellfish has been re-ported for several species. In some cases, viralaccumulation has not exceeded the exposure levelsand was often several orders of magnitude less.Mitchell et al. (14) have obtained concentrationsranging from 10 to 27 times the exposure levels inoysters with poliovirus, and accumulation factorsof 1 to 180 have been reported for poliovirus inOlympia oysters and Manila clams (6). In thecurrent studies, accumulation factors in excess of1,500 have been obtained for individuals; forpooled samples, however, the factor usually fellbetween 6 and 50 times the exposure levels, with amaximum level of 1,100. It should be noted thatresults of others are probably based on wholeclam samples, whereas the determinations re-ported here utilized digestive gland only. Correc-tion factors of 0.15 or 0.3 may be applied toaccumulation factors in Table 1 to render themcomparable with accumulations determined onthe basis of assays of total clam or drained meat,respectively. These correction factors are based ondeterminations of viral distribution in varioustissues (unpublished data).A significant feature in all accumulation studies

in which levels above environmental titers havebeen achieved is the use of a flow-through system.Accumulation of both bacteria and viral particlesis generally poor in standing water systems (5, 11,13; Feng and Fenton, unpublished data). Thisreflects unfavorable conditions for continuedphysiological activity (e.g., ventilation and feed-ing). Polluted clams may be those individuals thathave opened temporarily to sample the aquariumwater. The variation in accumulation in this series(Table 1) is perhaps due to clam activity and thequantity and quality of particulate material inthe seawater. The significance of this secondfactor was not investigated in this series but, asHoff and Becker (6) point out in their study ofpoliovirus uptake, the presence of particles towhich virus may adhere greatly enhances uptake.It should be noted that other workers used rela-tively high exposure levels, ranging from 40 to1,000 PFU/ml of seawater. The experiments re-ported here generally utilized pollution levels of1 to 8 PFU/ml of seawater in the pollution phase.This difference in the exposure levels plus thehigh stability and recoverability of S-13 must beconsidered in comparing accumulation reportedhere with that in studies with poliovirus. Exten-sion of accumulation studies to poliovirus andother enteroviruses requires development of im-proved recovery procedures at low titers and

determination of the inactivation kinetics of theviruses under the conditions of the experimentalsystem.The accumulation of viruses to high levels in

certain tissues emphasizes the potential healthhazard of shellfish exposed to even the lowestlevels ofviral pollutants.The pattern of viral elimination reported here is

somewhat different from that obtained by mostother workers. Their conclusions, at least implicit,have been that viral particles are readily purgedby the direct physiological activity of the con-taminated shellfish. The present study stronglysuggests that under certain conditions, i.e., lowtiters accumulated over an extended period, re-tention of some of the virus can be quite pro-longed and independent of clam activity as meas-ured by bacterial elimination. Indeed, reductionof viral titers in clams contaminated at low levelscan be accounted for solely as a result of inactiva-tion in situ under the influence of temperatureand other physical factors prevailing duringdepuration. The results of Seraichekas et al. (21),also with low initial titers, suggest a prolongedretention of small numbers of poliovirus in a fewof the shellfish sampled, even after 72 hr of de-puration. At higher initial titers, these workersfound poliovirus to be rapidly reduced andhandled essentially by the same mechanism as theE. coli contaminants. Simultaneous observationson viral inactivation under the condition of de-puration were not reported. It should be notedthat in the experiments of Seraichekas et al. thepollution phase was at 15 C and depuration at20 C. The significance of residual contaminationis difficult to interpret without some knowledgeof the persistence of the viral particle of interest.This is especially true in the case of prolongeddepuration and low initial titers. Considerablevariation in the uptake and elimination of twophages (S-13 and a large phage of Staphylococcusaureus) indicated the potential influence of viralstability in the kinetics of these two phenomena.The phage of S. aureus was not readily accumu-lated, and residual particles were rapidly elimi-nated from the clam. This phage was quicklyinactivated under the conditions prevailing duringthe experiments as determined by seawater con-trols (S. Y. Feng and W. J. Canzonier, unpub-lished data).

It has been assumed in the design of controlprocedures recommended for depuration proc-esses that elimination of particulate biologicalmaterial is a function of the physiological activityof the shellfish. This is certainly well documentedfor some bacterial contaminants and appears tohold true for a large portion of the viral particlesharbored by heavily polluted shellfish. The cur-

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rent work and some results of other workers indi-cate that accumulation of viral particles may involve two related but distinct processes. The firstprocess, and this may apply to all particles enter-ing the mantle cavity, is the collection and entrap-ment of particles (food, bacteria, viruses) and thesubsequent entry of many of these particles intothe digestive tract. Smaller particles may be ad-sorbed onto larger particles or entrapped inmucus. Most of these particles will be eliminatedin the feces and pseudofeces when a heavily pol-luted clam is placed in clean water under condi-tions suitable for ventilating and feeding. In thisphase, viral and bacterial elimination should pro-ceed at the same rate and this has indeed beenwell demonstrated (11, 14) by others and in thislaboratory. The conditions necessary for ade-quate clam activity to effect removal of bacteriaand a large portion of the viral contaminants canbe defined. The effectiveness of elimination ofthese contaminants can also be monitored byappropriate bacterial assays.

It appears, however, that small numbers ofviral particles can, during prolonged exposure, besequestered in clam tissues. This second processmay occur even with low environmental levels ofvirus. The majority of such sequestered particlesmay be recovered from the digestive gland andassociated tissues and a smaller fraction from thehemolymph (10, 11; unpublished data). Theseparticles apparently are not easily dislodged andtheir elimination is quite independent of clamactivity. In the case of heavily contaminatedclams, the majority of viral particles are rapidlyeliminated, creating the impression of extremelyeffective depuration. In the case of clams exposedto low levels for extended periods, the eliminationpattern is quite different since the majority ofparticles have been effectively sequestered and notlabile to rapid release. This has been confirmed byobservations of clams exposed at different levelsunder various conditions and subsequent studiesof elimination. All experiments listed in Table 1were followed by elimination studies. In caseswhere the titer was quite high, there was initially arapid decline to a lower level, but in all cases thephage persisted for periods commensurate withwith phage inactivation observed in seawater atthe temperature of the experi nents.The degree of coliform contamination is the

criterion in accepting shellfish for depuration andin evaluating the subsequent effectiveness of thedepuration process (4). This seems to assumethat bacterial elimination is a reliable index toviral elimination. The results described here showthat viruses can persist in shellfish depurated toacceptable coliform levels. The stability of par-

ticular viral pathogens under the conditions ofdepuration and handling is of considerable sig-nificance if the small numbers that remain se-questered in tissues are potentially an infectivedose. The stability of viruses under such condi-tions is yet to be established. On the basis of theepidemiological evidence of shellfish-associatedhepatitis (3, 12, 20) and of the persistence of theetiological agent of this disease in water (17), thisvirus could be expected to remain in clams in-definitely under conditions prevailing in depura-tion and storage.

If depuration of hard clams is to be accepted asa commercial practice, additional controls will benecessary to assure a safe product. Such controlsmight include a monitoring system more criticalthan the coliform standard now recommended.Additional processing, such as inclusion of apostdepuration period of warm storage (25 C orhigher) to inactivate residual viruses, might beconsidered. The feasibility of such a procedurewould be limited by the inactivation thresholds ofthe viruses of concern and the tolerance of theclams to the temperatures necessary.

ACKNOWLEDGNMENTS

The author gratefully acknowledges the interest and guidanceof H. H. Haskin and L. A. Stauber during the course of this in-vestigation and their helpful criticism of the manuscript. Thetechnical assistance of the staff of this laboratory, especially thatof J. K. Tani and T. M. Menko, is deeply appreciated.

This investigation was supported by Public Health Servicecontract Ul-00155 from the National Center for Urban andIndustrial Health and FD00086, H. H. Haskin, principal investi-gator.

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2. Cabelli, V. J., and W. P. Heffernan. 1970. Accumulation ofEscherichia coli by the Northern quahaug. Appl. Microbiol.19:239-244.

3. Dougherty, W. J., and R. Altman. 1962. Viral hepatitis inNew Jersey. Amer. J. Med. 32:704-736.

4. Furfari, S. A. 1966. Depuration Plant Design, p. 19. U. S.Dept. Health, Educationi, and Welfare, Public HealthService, Div. Environ. Eng. Food Protection.

5. Hedstrom, D. C., and E. Lycke. 1964. An experimental studyon oysters as virus carriers. Amer. J. Hyg. 79:134-144.

6. Hoff, J. C., and R. C. Becker. 1968. The accumulation andelimination of crude and clarified polio virus suspensions byshellfish. Amer. J. Epidemiol. 90:53-61.

7. Hoff, J. C., W. Jakubowski, and W. J. Beck. 1966. Studies onbacteriophage accumulationi and elimination by the Pacificoyster (Crassostrea gigas), p. 74-90. Proc. 1965 NorthwestShellfish Sanit. Res. Planning Conf., Public Health ServicePubl. No. 999-FP-6.

8. Kelly, C. B. 1961. Disinfection of seawater by ultraviolet radia-tion. Amer. J. Public Health. 51:1670-1680.

9. Kelly, C. B., W. Arcisz, and M. W. Presnell. 1960. Bacterialaccumulation by the oyster, Crassostrea virginkca, on theGulf Coast. RATSEC, U. S. Public Health Service Tech.Rep. F60-4.

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11. Liu, 0. C., H. R. Seraichekas, and B. L. Murphy. 1966. Viralpollution in shellfish. 1. Some basic facts of uptake. Proc.Soc. Exp. Biol. Med. 123:481-487.

12. Mason, J. O., and W. R. McLean. 1962. Infectious hepatitistraced to consumption of raw oysters. Amer. J. Hyg. 75:90-111.

13. Metcalf, T. C., and W. C. Stiles. 1965. The accumulation ofenteric viruses by the oyster, Crassostrea virginica. J. Infec.

Dis. 115:68-76.14. Mitchell, J. R., M. W. Presnell, E. W. Akin, J. M. Cummins,

and 0. C. Liu. 1966. Accumulation and elimination of poliovirus by the Eastern oyster. Amer. J. Epidemiol. 84:1-10.

15. Mosley, J. W. 1964. Clam-associated epidemics of infectioushepatitis. Hepatitis Surveillance Rep. No. 18, p. 14-17.

16. Mosley, J. W. 1964. Shellfish-associated infectious hepatitis.Hepatitis Surveillance Rep. No. 19, p. 30-36.

17. Neefe, J. R., and J. Stokes. 1945. An epidemic of infectioushepatitis apparently due to a water borne agent. J. Amer.Med. Ass. 128:1063-1078.

18. Rindge, M. W. 1962. Infectious hepatitis in Connecticut.Conn. Health Bull. 76:125-130.

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