APPuED MICROBIOLOGY, Mar., 1967, P. 307-315Copyright © 1967 American Society for Microbiology

Viral Depuration of the Northern QuahaugtOSCAR C. LIU, HELEN R. SERAICHEKAS, AND BERT L. MURPHY2

Northeast Shellfish Sanitation Research Center, U.S. Public Health Service Narragansett, Rhode Island

Received for publication 3 October 1966

A study was conducted to evaluate critically the feasibility of using the self-cleans-ing mechanism as a practical means to obtain virus-free shellfish. Two systemssupplied with fresh running seawater, three strains of human enterovirus and the North-ern quahaug, were used as working models. Preliminary experiments in the experi-mental system under arbitrarily selected conditions showed that depuration ofpoliovirus-polluted quahaugs could be achieved by the method used for the Easternoyster. The factors affecting viral depuration studied so far included: (i) initial con-

centration of shellfish pollution; (ii) temperature of seawater; and (iii) salinity ofseawater. It was shown that purification of the lightly polluted shellfish was achievedsooner than of the heavily polluted ones. The efficiency of viral depuration was

roughly a function of the water temperature within the range tested (5 to 20 C). Re-duction of salinity to 50 to 60% of the original level stopped this process completely,but 25% reduction in salinity did not affect significantly the rate of depuration. Pre-liminary study in the pilot system showed that viral depuration in the large tank ap-

peared to be equally as efficient as that in the small experimental tanks under theparticular conditions.

In the past decade, several outbreaks of in-fectious hepatitis (12, 15, 18, 19, 20, 21) have beentraced to the consumption of raw shellfish. Thetotal number of cases reported in these outbreaksamounted to approximately 1,700. Although it isdifficult to account for all sporadic cases as-

sociated with shellfish, there is indication thatsuch cases do occur from time to time (4). Fur-ther, secondary cases resulting from contact withshellfish-associated patients may be sizable on a

yearly basis. Clinically, this disease has a pro-tracted course, is extremely debilitating to thepatients, and may even result in occasional deaths.Thus, there is a definite need for research in thisarea with the aim of developing means of mini-mizing or eradicating this mode of transmission.Effort has been made in this area in our laboratorywith the following objectives: (i) to gain more

understanding on the basic mechanisms as to howshellfish are able to transmit the infectious hepa-titis virus to man; (ii) to evaluate whether or notthe depuration process may be used as a practicalmeans in obtaining clean shellfish. The presentcommunication describes the experimental re-

sults concerning the latter objective. The patternof viral accumulation and elimination by theEastern oyster, Crassostrea virginica, has been

1 Contribution no. 19 from this center.2Present address: Technical Branch, Communi-

cable Disease Center, Phoenix, Ariz.

reported previously (21). Thus, the present studyhas been devoted to the Northern quahaug,Mercenaria mercenaria. Since the human hepatitisvirus was not available for this study, three strainsof human enterovirus were used as the workingmodels.

MATERIALS AND METHODS

Viruses. The LSc 2ab strain of type 1 and the Leon12ab strain of type 3 of poliovirus, and the POWstrain of B-4 coxsackievirus, were used in the experi-ments. The stock polioviruses were grown in primaryAfrican green monkey kidney (MK) tissue culturesand were kindly supplied by the Lederle ResearchLaboratories, American Cyanamide Co., Inc. Thevirus pools contained approximately 10Y0 plaque-forming units (PFU)/ml and were stored at -20 Cbefore use. The coxsackievirus seed was obtainedfrom S. Kibrick, Boston University School of Medi-cine. The stock virus was propagated in our labora-tory in 3-oz (90-ml) prescription bottles containingmonolayers of African green MK cells. After thecells showed 4+ cytopathic effect (CPE), the bottleswere frozen and thawed three times. The fluids werepooled and centrifuged at 2,000 rev/min for 30 min,and the sedimented cell debris was discarded. Thevirus pool was stored at -20 C until it was used. Thevirus content was also approximately 108°0 PFU/mI.

Tissue culture. Primary African green MK cellswere used throughout the experiments. The monkeys,weighing 2 to 3 kg each, were purchased from theHurtlast and Thoresen Farm, Neptune, N.J. The pro-cedures used for preparation of monolayer tissue

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cultures in 3-oz prescription bottles were essentiallythose described by Hsiung (7).

Shellfish. The Northern quahaug was used through-out the study. The shellfish were purchased from localfishermen, and were of the size served in restaurantsas "cherrystones"; each weighed 100 d 10 g withshell, and measured 2.5 by 3.0 inches (6.4 by 7.6 cm).Upon arrival in the laboratory, they were stored inlarge tanks in the wet laboratory, supplied with con-stant incoming fresh seawater from Narragansett Bay,Rhode Island. The period of storage was generallymore than 1 week, offering ample time for the shell-fish to be adapted to the conditions of this laboratory.This also served as a process of purification ofcontaminants they had acquired in nature. Thevolume of shell liquor from each shellfish of this sizeamounted to 11 to 22 ml. Each digestive diverticulumwith stomach weighed from 0.7 to 2.0 g. The combina-tion of the gill and mantle membranes from eachweighed 4 to 6 g, and the remaining body, 4.5 to 10.0g. The shellfish harvested during experiments, if notimmediately dissected or homogenized, or both, werestored at -20 C until time for processing.

Preparation of homogenates. In all experiments,each sample contained 5 or 10 quahaugs unless other-wise indicated. First, the shells were scrubbed withsoap and water, and were rinsed thoroughly with tapwater. The valves were opened by using a sterile clamknife to sever the adductor muscles. The mantle cavityfluids from quahaugs in each group were collectedinto a sterile beaker. Approximately 30 sec wereallowed for draining the fluids from each shellfish,and this process was facilitated by lifting the mantlecavity membranes from the shell surface. Both thevolume and weight of the fluid samples were meas-ured. The meat was then shucked, and the shuckedmeat from the shellfish of each group was pooled andweighed. The meat was homogenized at the medianspeed of a Waring Blendor for 3 min after adding anequal weight of phosphate-buffered water at pH 7.2or Nutrient Broth. The 50% (w/w) homogenates andthe fluid samples were then centrifuged at 2,000rev/min for 20 min. The supernatant fractions werestored at -20 C. On the day of assay, the sampleswere quickly thawed in a water bath at 37 C andclarified again at 2,000 rev/min for 20 min. Thesupernatant fluids were assayed for viral content byplaquing.

Plaque Assay. The procedures were essentiallythose described by Hsiung and Melnick (8). Through-out the text, the total number of PFU contained ineach milliliter or gram of various specimens is pre-sented.

Depuration system and seawater supply. The contactsurfaces of the entire system were made of eitherpolyvinyl chloride material or epoxy-based com-pounds. The seawater was continuously pumped infrom the deep part of Narragansett Bay, RhodeIsland. Duplicate intakes and pumps were providedin case of mechanical difficulties. The water waspumped into an 800-gal balance tank in the wetlaboratory from which the water was distributed tovarious experimental systems by gravity. The flowrate to each system was controlled by polyvinyl

valves. The entire system from the balance tank downto disposal of the waste water is depicted in Fig. 1.The water first passed through a heat exchanger, bywhich its temperature was maintained at a desiredlevel, 15 to 20 C. After passing through a Purdy typeof ultraviolet treatment box (9) for sterilization, thewater flowed into a constant head box. From thisbox, the water was distributed into four mixingboxes, 6 by 9 by 10 inches (15.2 by 22.8 by 25.4 cm)each. In general, virus at appropriate dilutions waspumped into these boxes by a diaphragm pump andmixed thoroughly with the water by flowing throughseveral baffles in the boxes. From each mixing box,the water flowed through a plastic tubing into a tankof 1 by 1 by 2 ft (30 by 30 by 60 cm), where shellfishwere kept. The flow rate from the constant head boxto the mixing box was usually adjusted to 2 liters permin. With the total capacity of each shellfish tankbeing 54 liters, the average retaining time of water ineach tank was approximately 27 min.The number of shellfish in each tank in each experi-

ment has never exceeded 120 quahaugs for accumula-tion phase, whereas the number has not exceeded 50for depuration phase. During the course of depurationafter each sample of 5 or 10 quahaugs was removed,an equivalent number of clean shellfish was addedinto the tank, thereby keeping the number of shellfishin the tank constant for the entire experiment.

The pilot depuration tank was made of wood, andits interior was painted with epoxy material. Theinternal dimension was 2.9 by 4 by 6.6 ft (88.4 by121.9 by 201.2 cm). After passing through a Purdy-type ultraviolet box, the water flowed into this tankfrom numerous side holes on two polyvinyl pipes onthe bottom extending from the front to the middle ofthe tank. The flow rate into the tank ranged from 32to 64 liters per min. The total capacity of the tank wasapproximately 1,700 liters. The tank could hold twolayers of eight baskets of shellfish. Each basket held1.5 bushels of shellfish. During the depuration experi-ment, these baskets were filled with normal quahaugsof various sizes. Study was carried out on the shellfishwhich were previously polluted in the experimentaltanks and planted among the "dummy" shellfish. Ingeneral, the experimental shellfish were planted in un-favorable positions among the dummies, e.g., in themiddle of basket, in the lowest level of sbellfish in abasket, etc.

RESULTSPreliminary experiments on viral depuration.

Mitchell et al. (17) showed that the Eastern oys-ters eliminated the poliovirus and Escherichia colivery rapidly when the depuration process wascarried out in a flow-through system. As a pre-liminary test of whether this finding holds truewith quahaugs, the following experiment wasconducted. One hundred quahuags were pollutedby appropriate concentrations of the type 1 polio-virus and a strain of E. coli in a running watersystem for 48 hr. The shellfish were then rinsedthoroughly with tap water and transferred into a

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SOURCE OFSEA WATER

THIS DISTANCECONTROLS FLOW

5-BULB U.V.TREATMENT BOX

CON STA NTH EAD BOX J

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DETENTION TROUGH TEMP AT THIS POINT

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FIG. 1. Diagram ofexperimental depuration system and seawater supply.

clean tank for depuration. At varying times, asample of 10 shellfish and a sample of seawater inthe tank were obtained. The shellfish were pro-cessed and the shell liquor and 50% meat homoge-nates were prepared, as described. All preparationsand the seawater samples were assayed for bothviral and bacterial contents. The results of viralassays are illustrated in Fig. 2.As shown, the seawaters contained very little

or no virus when the shellfish were depurating ina clean tank. The virus was eliminated rapidlyfrom both the shell liquor and meat of the shell-fish under the particular experimental conditions.After 72 to 96 hr of treatment, no virus wasdetectable by the technique used, whereas smallamounts of bacteria persisted in the shellfish formore than 96 hr. These data in general confirmedthose observed for the oysters by Mitchell et al.(17).

Factors affecting viral depuration. From thepreliminary experiments described above andthose reported by Mitchell et al. (17), it appearsclear that depuration of virus-polluted shellfishwas feasible under proper conditions. The majorway in which our experiments and those ofMitchell differed from those described previously(5, 16; S.Y. Feng, personal communication) was inthe use of running-water system. In view of theencouraging results obtained in arbitrarily chosen

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HOURS OF DEPURAT ION

FiG. 2. Preliminary experimental results of depura-tion ofquahaugs polluted with poliovirus type 1.

VOL. 15, 1967

EXPERIMENTALSET -UP

VIRUSFEEDER

H EATEXCHAN GER

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TEMP- 20-31.2"CSALINIIV= 31.2 -31.5%.POLSOVIRUS IAUGUSt 1965

E

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ALIQUOR

ond MEAT

LD POELUTION \

.SEAWATER

LEI I

24 48 72 96

HOURS OF DEPURATION

FIG. 3. Effect of initial viral concentration in shell-fish on viral depuration (poliovirus type 1).

conditions, further effort was made to evaluatethoroughly various factors which may possiblyaffect the efficiency of depuration. From theseexperiments, it is hoped, first, that the factorswhich can facilitate this process may be singledout, and, second, that by using a combination ofthe favorable factors the depuration process maybe developed into a practical means for com-mercial use. The factors studied thus far are dis-cussed separately below.

Effect of initial viral concentration in shellfish.At the outset, one may suspect that depurationfor the lightly polluted shellfish may require lesstime than for the heavily polluted ones. To docu-ment this assumption, the following experimentwas conducted. Two groups of 50 quahaugs eachwere polluted with a high and a low level of thetype 1 poliovirus in seawater. After 48 hr, thepolluted shellfish were rinsed and transferred intoa clean tank for each group. Groups of five qua-haugs were harvested from the two depurationtanks periodically up to 96 hr. The shell liquor andmeat from the shellfish of each group were pooledand homogenized. All homogenates and seawaterwere assayed for viral content, with the resultsshown in Fig. 3. As shown, at the time of startingdepuration, the lightly polluted shellfish con-tained 50 PFU/ml of the homogenate, and the

heavily polluted ones contained approximately1,000 PFU/ml. The time required to depurate theformer group to nondetectable level was 24 hrand that for the latter group was 72 hr. This find-ing definitely bears out the original suspicion thatthe time of depuration is proportional to the de-gree of pollution of the shellfish.The same experimental design was repeated

with the type 3 poliovirus. Both the shell liquorand meat samples from each group were collectedand prepared separately. The assay results aresummarized in Fig. 4. As shown, the lightly pol-luted shellfish were cleansed at 24 hr, whereasthe heavily polluted ones were cleansed at 72 hr.These data confirmed those observed in the pre-vious experiment with the type 1 poliovirus.

Effect of seawater temperature. The effect ofwater temperature on viral depuration was studiedin a series of experiments during the winter of1965-66. The range of temperatures tested wasfrom 5 to 20 C. The viruses used in these experi-ments were poliovirus types 1 and 3 and thecoxsackievirus type B-4. The general proceduresfor each of these experiments were as follows.One hundred quahaugs were polluted for 24 hrin running seawater of 15 C, containing approxi-mately 50 to 100 PFU per ml. At the end of pol-

TEMP 13-leCSALINITY 30.31%.POLIOVIEUS III

MEAT (NIGH POLLUTION)

tIQUOR (HIGH POLLUTION)

48 72

HOURS OF DEPURATION

96

FIG. 4. Effect of initial viral concentration in shell-fish on viral depuration (poliovirus type 3).

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POLIOVIRUS TYPESALINITY 30-31 '/ooEXP DONE DEC 1965

A MEAT

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FIG. 5. Effect ofseawater temperature on depurationofpoliovirus type 1-polluted quahaugs.

lution, all shellfish were scrubbed and rinsed withtap water. Two groups of five shellfish were har-vested at this time and considered as the zero-hoursample of depuration. The remaining quahaugswere divided equally into three groups, each ofwhich was transferred into a clean tank suppliedwith seawater at different temperatures. Duringthe next 72 to 96 hr, samples of shellfish and sea-water were removed periodically. The shellliquors and meat from the shellfish in each groupwere pooled and processed in the usual manner.The viral assay for all samples in each experimentwas generally done at one time. The results fromthree experiments are illustrated in Fig. 5-7. Ex-cept for the zero-hour sample, all seawater sam-ples were found to contain no detectable virus;thus, these values are not included in the figures.

In Fig. 5 are summarized the results of depura-tion of the type 1 poliovirus-polluted quahaugs.The three groups of shellfish were treated in thetanks containing seawater at 7 to 8 C, 13 to 14C, and 19 C. The solid lines represent the results ofmeat samples, whereas the broken lines representthose of liquor samples. At 19 C, the virus in shellliquors was reduced to nondetectable level after24 hr of treatment, whereas the virus in meat sam-

ples was reduced to the same level at 72 hr. In13 to 14 C water, the viral content of liquorreached nondetectable level by 72 hr, and that of

311

meat was reduced considerably but has never

reached the nondetectable level. In 7 to 8 Cwater, there was little depuration occurring ineither liquor or meat for the entire experimentalperiod.

In Fig. 6 are summarized the depuration re-sults of the type 3 polluted shellfish. The threegroups of shellfish were treated with seawater at5 to 6 C, 14 C, and 18 to 20 C. It may be notedthat at 18 to 20 C the viral content of both shellliquors and meat reached nondetectable levelwithin 24 hr. The same level was also reached atlower temperatures, but at a slower pace. Thedifference in time of reaching the nondetectablelevel, especially in meat samples, between thesetwo experiments may be attributable in part tothe difference in initial level of pollution of theseshellfish.

In Fig. 7, the results of depuration of thecoxsackievirus-polluted shellfish are summarized.Only the assay results of the meat samples fromtwo groups of shellfish are shown, because a

mechanical failure of the third tank occurred inthis experiment. The effect of temperature on theefficiency of depuration is clearly shown, con-firming, in general, the observations with thepolioviruses.By using only the approximate time required

to depurate the polluted quahaugs to contain the

3-POLIOVIRUS TYPE 3

SALINITY 30-331°ooEXP DONE JUNE 1966

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FIG. 6. Effect ofseawater temperature on depurationofpoliovirus type 3 polluted quahaugs.

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E

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FIG. 7. Effect ofseawater temperature on depurationof coxsackievirus B4 polluted quahaugs (meat speci-mens).

nondetectable level of virus, the data from foursuch experiments are combined and tabulated inTable 1. It is clear from these results that theefficiency of viral depuration is roughly a functionof the seawater temperature within the rangetested. The highest temperature used, 20 C, hasgiven the best results. Whether or not this willhold true for quahaugs harvested in other localesor for other species of shellfish remains to bedetermined. Although temperatures above 20 Chave not been tested, on the basis of currentknowledge of shellfish physiology one suspectsthat the efficiency of depuration may be reducedat a temperature not too much above 20 C.

Effect ofsalinity. The effect of varying salinitiesof the seawater on viral depuration was studiedin several experiments while the water tempera-ture was kept at a constant range. In one experi-ment, 100 quahaugs were polluted with the type1 poliovirus in the usual manner for 24 hr. Aftersampling the duplicate samples of shellfish at theend of pollution, the shellfish were divided equallyinto three groups and were transferred into cleantanks. The seawater flowing through these tankswas at 18 to 20 C and with the following salinities:17 to 21, 23 to 28, and 31%, The quahaugs andseawater were sampled periodically up to 96 hr.

The specimens were processed and assayed in theusual manner (Fig. 8 and 9).

In Fig. 8 the data of shell liquor assays areshown, and in Fig. 9 those of meat are shown.Depuration proceeded rapidly in the water withsalinities of 31%o and 23 to 28%0, whereas littledepuration was obtained with those treated inseawater with salinities of 17 to 21 %00. Although aslightly better depuration was shown for the shell-fish depurated in 31 %O salinity than in 23 to 28%,in this particular experiment, this difference wasnot observed in other experiments when thesalinity of seawater only reduced approximately25% of the original. It has been repeatedly ob-served, however, that a reduction of salinity to 50to 60% of the original seawater completelystopped the process.

Depuration in pilot system. A preliminary studyof depuration in the pilot tank was initiated tosee what difficulty may be encountered during theprocess of scaling up. A total of 120 quahaugswere polluted with the type 1 poliovirus and astrain of group A E. coli in the experimental sys-tem. At the end of 24 hr, three groups of eightquahaugs were harvested and considered as zero-hour samples. The remaining 96 quahaugs wereplanted among the dummy quahaugs in eightbaskets. At varying intervals, triplicate samples ofeight quahaugs each were harvested. In general, asample consisted of one shellfish from each of thebaskets. Both liquor and meat from shellfish ofeach group were pooled and homogenized. Thehomogenates were assayed for bacterial contentimmediately and for viral content later. Duringeach sampling, a seawater sample was also ob-tained and titrated. The results of viral assaysfrom two such experiments are summarized inFig. 10. The bacterial results will be described byCabelli et al. elsewhere. As shown, the great ma-jority of samples were cleansed to contain a non-detectable level of virus at 48 hr. There was nodetectable virus in all samples at 72 hr. In general,the trend of viral depuration in the large tank ap-

TABLE 1. Effect of seawater temperature ondepuration of enteric viruses (four

experiments)

Time required to depurate atPrepn Values

20C i5c lOc 5C

hr hr hr hr

Liquor Average 48 60 60 >72Range 24-72 48-72 48-72 >72

Meat Average 48 78 84 >96Range 24-72 48-96 72-96 >96

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-.'~~~~~~~~

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ti X /o'-.-

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FIG. 8. Effect of seawater salinition ofquahaugs (liquor specimens).

types of enteric virus may be needed in the future,in addition to that for infectious hepatitis virus.(ii) Studies with the model viruses would offerinformation as guidelines for improvement of

P, i8-2@Cf depuration conditions, both in terms of biologyloviRus and of engineering, since the success of viral de-IL. 1966 puration is probably hinged on shellfish biology.OUOR) (iii) Biologically, the human enteroviruses simu-

late in many aspects the hepatitis virus. It is hopedthat the information obtained from our study onthese viruses is applicable to handling the hepatitisvirus-poluted shelfish. We, however, are keenlyaware of the possibility, remote as it may be, thatthe hepatitis virus may behave differently fromother types of virus. This problem can only beresolved by testing shellfish which actually arepolluted with the hepatitis virus per se.Not long before the current study was initiated,

several laboratories yielded experimental datawhich had cast some shadow on this approach.From their data, these investigators seemed toimply that, although depuration of bacteriallycontaminated shellfish had been successful (1, 22),depuration of virus-polluted ones was not likelybecause of the nature of the contaminants. C. B.

2 ! --- Kelly (personal communication) first pointed outthat the failure of these investigators to depurateshellfish was chiefly due to the experimental sys-

ty on viral depura- tems used. The previous studies were uniformly

peared similar to those observed in the experi-mental system. One of the six samples did notdepurate well at 48 hr, indicating variation amongsamples which might occur from time to time.

DIscUSSION

The present study attempted a critical evalua-tion of whether or not viral depuration of shellfishmay be attained experimentally. If so, what arethe best conditions under which this process can

be conducted? For studies carried out to date, theworking models selected included three strains ofthe human enterovirus and the Northern qua-haug. The problem virus, infectious hepatitisvirus, however, was unfortunately not availablefor this study. It is felt that information obtainedwith the viruses studied may not be completelywithout merit for the following reasons. (i) Asreported by Metcalf and Stiles (16), strains ofcoxsackievirus and echovirus were recovered fromEastern oysters from the New Hampshire area.

Although only hepatitis outbreaks have been as-sociated with consumption of raw shellfish, thepotential hazard of the transmission by shellfishof other types of virus cannot be overlooked.Therefore, cleansing of shellfish of numerous

HOURS OF DEPURATION

FIG. 9. Effect ofseawater salinity on viral depurationof quahaugs (meat specimens).

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3.0

2.0

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FIG. 10. Depuration of poliovirus type I pollutedquahaugs in pilot size tanks (results of two experi-ments).

done in systems which contained only stationarywater. Although the water was exchanged peri-odically, in retrospect such a measure was ob-viously unable to provide the shellfish withadequate condition to function. This reasoningwas substantiated first by the work of Mitchellet al. (17), and later by ourselves (13) when run-

ning seawater was supplied to the depurationtanks. The two major species of shellfish for raw

consumption in this country, the Eastern oysterand the Northern quahaug, were shown to be ableto unload their viral contaminants rapidly when

they were treated in these systems. The success ofthese experiments was also partially attributed tothe use of the Purdy type of ultraviolet system (9),by which the seawater was completely sterilizedbefore being used to treat the shellfish.

Although under arbitrary conditions, the pre-liminary results of viral depuration offered suffi-cient encouragement for the study to continue.Several factors which may affect viral depurationhave been evaluated. As shown in Fig. 5-7, thewater temperature seems to play a major role inaffecting the process of purification. In manyexperiments, purification of the shellfish frompoliovirus contamination was achieved within24 to 48 hr at 19 to 20 C. This type of result cer-

tainly sheds more light on the practicability of theprocess. Whether or not this observation will holdtrue for other species of shellfish and the samespecies of shellfish harvested at other geographicallocations remains to be determined.Although salinity experiments did not reveal a

particular range in which shellfish will depuratemore efficiently, they provide a definite guidelinein that the salinity of seawater used for depurationshould not be lower than 50 to 60% of the originallevels to which the shellfish were conditioned.Excessive reduction of the salinity is definitelydetrimental to the process. As shown recentlyby C. N. Shuster, Jr., and F. C. Garb (personalcommunication), reduction of salinity to such anextent would also stop the shellfish from func-tioning.The first factor studied was the degree of shell-

fish pollution in relation to the time required fordepuration. Since there is little information avail-able concerning what amounts of virus werepresent in the shellfish which produced humandiseases, conclusion from these results would beonly conjectural. Metcalf and Stiles (16) haveisolated enteric viruses from oysters obtained inNew Hampshire areas which were known to bepolluted with raw sewage. The viruses present inthese specimens apparently were not numerous inview of the technique used to isolate the viruses.According to T. G. Metcalf (Symp. Natl. Conf.Depuration, lst), by plaquing out some of thesespecimens, the highest counts were not over 5PFU per 10 oysters. During the period whenMetcalf and Stiles did their sampling, no outbreakof any type of virus diseases was noted. Therefore,one still wonders how much virus would be foundin shellfish if they were examined during thehepatitis outbreaks. Since no simple technique isavailable for isolating the infectious hepatitisvirus at this time, to assay viral content of shell-fish for this virus is impossible. Although attemptswere made to determine what constitutes a mini-mal infectious dose for the infectious hepatitisvirus in human volunteers (2, 11), the data ob-tained so far appear unrealistic. From these data,one would be reluctant to guess how many viralparticles were in these shellfish which producedthe illness. Thus, in reality, we have no idea re-garding what levels of viral contaminants we aretrying to depurate. The problem of the virus con-centration in polluted shellfish in the field situa-tion remains to be solved by further investigation.It is felt, however, that the levels of pollution usedin the model systems in various laboratories areprobably much higher than those which areactually occurring in nature. At least, the experi-ments conducted in this study have demon-strated that shellfish polluted with low levels of

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virus could be purified to a certain level fasterthan those polluted with high levels. From thisguideline, one would be encouraged to deduce thatthe naturally polluted shellfish would be purifiedeven faster and easier than those containing thelowest concentration of virus tested in the labora-tory.

Lastly, the data obtained for depuration studiesin both experimental and pilot scales have beenencouraging. For the past four decades, the factthat no obvious hepatitis outbreaks were as-sociated with shellfish in some European coun-tries may be due to the practice of depuration (6).All of the evidence indicates that this process maybe useful commercially.At this time, it should be stressed, however, that

many problems remain to be solved before adop-tion of this process in our country. To achievethis step, the many conditions which may influ-ence this process should be explored and studied.Pitfalls in scaling up to a commercial size shouldbe uncovered, and definite measures should bedesigned to avoid these. After more is learnedfrom using the model systems, other species ofshellfish and the same species of shellfish grownin other locations should be thoroughly investi-gated. Only from such comprehensive studies cana set of generalized conditions and the manyexceptions to these be clearly defined.

ACKNOWLEDGMENTSOur appreciation is extended to Annette D. Harris

and Stanley F. Sekunda, Jr., for valuable technicalassistance in completing this work.

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10. KJELLANDER, J. 1956. Hygienic and microbiologicviewpoints on oysters as vectors of infection.Am. J. Public Health, 53:1009-1016.

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12. LiNDBERG-BROMAN, A. M. 1956. Clinical observa-tions in the so-called oyster hepatitis. Am. J.Public Health, 53:1003-1009.

13. LiU, 0. C., H. R. SERAICHEKAS, AND B. L.MURPHY. 1965. Viral pollution and self-cleans-ing mechanism of hard clams. Symp. VirusDiseases Transmitted by the Water Route.

14. Liu, 0. C., H. R. SERAICHEKAS, AND B. L.MURPHY. 1966. Fate of poliovirus in northernquahaugs. Proc. Soc. Exptl. Biol. Med. 121:601-607.

15. MASON, J. 0., AND W. R. MCLEAN. 1962. Infec-tious hepatitis traced to the consumption ofraw oysters. Am. J. Hyg. 75:90-111.

16. METCALF, T. G., AND W. C. SULES. 1965. Theaccumulation of the enteric viruses by theoysters, Crassostrea virginica. J. Infect. Dis-eases 115:68-76.

17. MITCHELL, J. R., M. W. PRESNELL, E. W. AKIN,J. M. CUMMINS, AND 0. C. Lw. 1966. Accumu-lation and elimination of poliovirus by theEastern oysters. Am. J. Epidemiol. 86:40-50.

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19. MOSLEY, J. W. 1964. Clams associated epidemicsof infectious hepatitis. Hepatitis SurveillanceRept. No. 18, p. 14-17; No. 19, 30-36. Com-municable Disease Center, Atlanta, Ga.

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