JOURNAL OF BACTERIOLOGY, OCt., 1965Copyright © 1965 American Society for Microbiology

Vol. 90, No. 4Printed in U.S.A.

Bacillary Necrosis, a Disease of Larval and JuvenileBivalve Mollusks

1. Etiology and EpizootiologyHASKELL S. TUBIASH,' PAUL E. CHANLEY,2 AND EINAR LEIFSON

Biological Laboratory, U.S. Bureau of Commercial Fisheries, Milford, Connecticut, and Stritch School ofMedicine and Graduate School, Loyola University, Chicago, Illinois

Received for publication 26 April 1965

ABSTRACTTUBIASH, HASKELL S. (U.S. Bureau of Commercial Fisheries, Milford, Conn.), PAUL

E. CHANLEY, AND EINAR LEIFSON. Bacillary necrosis, a disease of larval and juvenilebivalve mollusks. I. Etiology and epizootiology. J. Bacteriol. 90:1036-1044. 1965.-Lethal bacterial infections of a variety of hatchery-spawned bivalve mollusk larvaeand juveniles have been studied. The symptoms of the disease and the course of theinfection are described. Four biotypes and five antigenic types of bacteria, pathogenicfor the larvae of five species of bivalve mollusks, were isolated and described in somedetail. All are gram-negative motile rods. Comparative studies were made of a fairlylarge number of similar bacteria isolated from presumably normal marine fauna. Noneof these was pathogenic for the bivalve larvae nor did they have antigens in commoniwith the pathogenic group. The four biotypes had a number of characteristicsin common that rarely were present in other cultures from marine fauna. Several anti-biotic preparations proved to be of value in the treatment and control of the infection.

Marine pelecypods commonly reproduce by re-leasing gametes into the water, where externalfertilization occurs. Usually, shelled larvae de-velop within 24 hr (Fig. 1). The larvae are plank-tonic and swim by the ciliary action of the ex-tended velum. The larvae of the hard clam,Mercenaria (Venus) mercenaria, remain in thefree-swimming form for approximately 20 days.After their planktonic existence, they meta-morphose into relatively sedentary juvenileforms more closely resembling adults. At meta-morphosis, most of the more common marinebivalves average 200 to 400 ,u in length(J0rgensen, 1946).Advances in culture techniques have estab-

lished the feasibility of culturing desirable speciesof bivalves commercially (Loosanoff and Davis,1963; Davis and Ukeles, 1961). However, re-search groups and pilot-plant shellfish hatcheriesare hindered by a disturbingly high incidence offatal epizootics among larval and juvenile stocks.Sporadic fungal infections believed to be causedby members of the genus Sirolpidium were re-

1 Present address: Biological Laboratory, U.S.Bureau of Commercial Fisheries, Oxford, Md.

2 Present address: Eastern Shore Laboratory,Virginia Instittute of Marine Science, Wacha-preague, Va.

ported in early work on methodology of larvalculture of Lamellibranchia (Davis et al., 1954).Guillard (1959) isolated a number of bacterialstrains from a single moribund hard clam larva.Broth cultures of two of these strains, identifiedas Pseudomonas sp. and Vibrio sp., proved patho-genic to M. mercenaria larvae in small axenictest-tube cultures. Recurring losses of larval andjuvenile bivalve mollusks in the Milford Labora-tory prompted a search for causal microbialagents. The outbreaks were characterized bysudden onset and heavy mortality with charac-teristic "swarming" of bacteria around the deadand moribund larvae. Many ciliated protozoa werealso present.

MATERIALS AND METHODSIsolation of pathogens. Moribund larvae of the

hard clam, M. mercenaria, sturrounded by swarm-ing bacteria were collected individually by capil-lary pipette and washed by threefold centrifu-gation in sterile seawater. The final washing wasdecanted, and 1 ml of seawater-Trypticase-glucose-yeast (TGY) broth was added. (TGYbroth was composed of 0.4% Trypticase, 0.1%glucose, and 0.1% yeast extract. The mediumwas adjusted to a final pH of 7.4 and autoclavedat 15 psi for 10 min. For solid medium, 1.25%agar was added.) After incubation at 28 C for 4

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BACILLARY NECROSIS OF BIVALVE MOLLUSKS

FIG. 1. Anatomy of a typical bivalve mollusklarva. The velum extends beyond the shell when theanimal is swimming. After Jorgensen (1946).

hr, streak and pour plates were made with TGYagar. The plates were incubated at 28 C for 48 to72 hr, and colonies were subcultured to agarslants for pathogenicity screening. Tisstue wasalso teased from the gaping shells of moributidjuvenile oysters, Crassostrea virginica, and ju-venile hard clams, M. mercenaria, and was cul-tured in TGY broth as with the larvae. A lesstedious technique, adopted stubsequently, calledfor washing dozens to hundreds of affected ju-veniles or larvae in sterile, round-bottorned,screw-capped centrifuge tubes by fivefold stis-pension and decantationi in sterile seawater. Thespecimens were grounid to a slurry by shakingwith sterile glass beads. This homogenate wasthen plated omit in serial dilutions for isolation ofpredominianit bacteria. On occasion, portions ofthe homogenate were also seeded into healthylarval cultures for rapid determinations of patho-genicity. Isolates which proved pathogenic werepreserved by lyophilization in equal volumes ofsterile skim milk and seawater.

Pathogenicity screening and range of host sus-ceptibility. A standard test for pathogenicity wasperformed by inoculating 24-hr suspensions of thetest organism into 400-ml cultures of 2- to 7-day-old larvae. Clean, but not sterile, 1-pint (0.47-liter) polyethylene frozen-food containers wereemployed as convenient cultture vessels. Eachcontained approximately 10,000 larvae in seawater,which had been filtered to remove silt and detritusand ultraviolet-treated to kill protozoa and otherplankton. Candidate pathogens were spread onTGY agar slants, incubated for 24 hr at 28 C,washed off with 10 ml of sterile seawater, washedby centrifuigation to remove traces of TGY andbacterial metabolites, and diluted to approxi-mately 5 X 108 organisms per milliliter. Larvalcultures were seeded with 1- and 2-ml inocula ofthese bacterial suspensions, incubated at 28 C,and examined for mortality of the larvae after24 and 28 hr. Stuitable pathogenic and saprophyticcontrols were included, as well as 2-ml seedings ofidentical bacterial suspensions which had beenheated for 30 min at 65 C.

Host susceptibility ranges were determined in a

similar manner, except that in addition to M.mercenaria and C. virginica, larvae of the Euro-pean oyster (Ostrea edulis), bay scallop (Aequi-pecten irradians), and shipworm (Teredo navalis)served as hosts for challenge by strain M 17, apathogen of high virulence. Larvae of T. navaliswere obtained as the result of a fortuitous singlespawning, and incubation was at room temper-ature (abouit 20 C) rather than 28 C. Mortalitycurves were constructed by setting up multiplelarval cultures, seeding all simultaneously, andcounting viable larvae hourly, one culture perhour, for up to 24 hr. In this manner, an entirelarval culture could be sampled and discardedwithout disturbing the remaining cultures. Differ-ential counts of morbidity and mortality weremade by filtering the test larval cultures througha small-mesh (60-,u) stainless-steel wire screen anddistributing a representative portion of the larvaethus removed on a Sedgewick-Rafter countingcell. With practice, the dead and moribund larvae,as well as those showing early signis of infection,could be readily differentiated from the normalanimals under 100 times magnification. Differ-ential counts were made on 100 evenly distributedlarvae. Moribund and dead larvae were countedas "dead"; the rest of the sturvivors were cotunitedas "alive." Moribund larvae always died within afew hours.

Twenty-seven marine bacterial cultures withmorphological and biochemical properties similarto those of the Milford pathogenis were screenedfor pathogenicity and antigenicity. These organ-isms had been isolated from a variety of normalmarine fauna in Long Island Sound and had beenstudied at Loyola University, Chicago, Ill.

Serological typing. Antigens consisted of liveorganisms grown for 24 hr on TGY slants, washed,suspended in seawater, and adjusted to a densityof approximately 1.5 X 109 organisms per milli-liter. Rabbits were immunized by four subcu-taneous doses, varying from 0.5 to 2 ml, adminis-tered weekly. At 1 week after the final injectioni,they were bled by cardiac puncture. The sera wereseparated, inactivated, and tube-tested for ag-glutinating titer against homologous and heterol-ogous strains of the pathogens. A 0.2-ml volumeof the serially diluted serum was mixed with a0.1-ml suspension of a 24-hr culture of the bacteria,incubated for 4 hr at 15 C, and examined foragglutination. All strains exhibiting pathogenicityfor larval bivalves, and the 27 Loyola strains, weretested in this manner.

Identification of pathogens. A typical strain ofeach of the five antigenic types of the larvalpathogens was studied in some detail. Tests forgelatin liquefaction, nitrate reduction, catalasedetermination, carbohydrate metabolism, andindole formation were performed as described byLeifson et al. (1964). Artificial seawater (one-halfstrength, pH 7.5 to 8.0) was uised for preparingthe liquid base medium. Starch-agar was pre-pared by adding 0.2% soluble starch and 1.5%agar to the base medium. Abouit 1 ml of a 1:10

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TUBIASH, CHANLEY, AND LEIFSON J. BACTERIOL.

dilution of Gram's iodine was added to a 2- to3-day-old slant culture to test for starch utili-zation. Flagella stains were made according tothe method of Leifson (1960), from both agar andslant cultures.

Application and screening of antibacterials.Three proprietary antibacterial formulations weretested, along with chloramphenicol, for thera-peutic use against experimental infection withstrain M 17: an aqueous preparation containing250 mg of streptomycin and streptomycin sulfateper ml (Combistrep; Chas. Pfizer & Co., Inc.,Brooklyn, N.Y.), sodium sulfamethazine solublepowder (Sulmet; American Cyanamid Co., Wayne,N.J.), and a complex of polyvinylpyrrolidonewith 10% free iodine (PVP-iodine).These formulations had previously been em-

ployed empirically to abate shellfish hatcheryepizootics. They were evaluated by adding gradedlevels of the drug, usually 5 to 200 ppm, to 400-mlcultures of clam larvae which had been seeded 4hr previously with the virulent pathogen. Larvalcultures were incubated with the drug for 48 hrat 28 C, and then for 24 hr in new, drug-freewater. After the total 72-hr incubation period,counts were made to determine percentage mor-tality. Controls for drug toxicity were included inall cases.As the number of pathogenic isolates increased,

they were screened for sensitivity to antibioticsby the use of sensitivity discs (Colab Laboratories,Inc., Chicago Heights, Ill.) on TGY agar platesspread with 0.2 ml of 24-hr broth cultures of thetest organisms. Antibiotics tested in this mannerincluded: chloramphenicol, kanamycin, neomycin,penicillin, polymyxin B, dihydrostreptomycin,and tetracycline.

RESULTS

Pathology and epizootiology. Two strains ofgram-negative bacilli from the isolated moribundclam larvae and three obtained by centrifugationof a group of dead and moribund larvae provedto be highly virulent for larval bivalves. Theseinitial bacterial strains proved to be the etiolog-ical agents of a disease we have termed bacillarynecrosis. They were typical of many subsequentpathogenic isolates.The course of this disease in experimentally

exposed larvae is swift and dramatic (Fig. 2a).Within 4 to 5 hr after seeding with a virulentpathogen, prodromal signs are a reduction ofmotility and a tendency for many larvae to liequiescent with either rudimentary foot or velumextended (Fig. 4b). "Swarms" of bacteria origi-nating from discrete foci on the margins ofscattered larvae appear simultaneously. Theseswarms are a pathognomonic sign of bacillarynecrosis, although at this point larvae may seemotherwise normal. The swarms become pro-gressively more dense and ubiquitous, in a manner

OSTREA EDULIS

60F

40

b0 2 4 6 8 10 12 14

TIME IN HOURS

100

AEOUIPECTEN IRRADIANS80 _

,40~

20 Czo . c

O 2 4 6 a 10 12 14TIME IN HOURS

TEREDO NAVALIS

0 2 4 6 a lo 14 Ie 20TIME IN HOURS

FIG. 2. Mortality of larvae after challenge withpathogen M 17. (a) Mercenaria mercenaria. (b)Ostrea edulis. (c) Aequipecten irradians. (d) Teredonavalis.

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BACILLARY NECROSIS OF BIVALVE MOLLUSKS

FIG. 3. Swarms of motile bacteria emanatingfrom heavily parasitized presetting larvae ofCrassostrea virginica challenged with pathogenM 17. Swarming proceeds from discrete foci. Softtissues of most larvae have been or are being necro-tized. Viable larvae at top and right corner. X 140.

resembling the swarming of bees (Fig. 3). By 8hr after challenge, death with granular necrosis iswidespread. Under low power (100 times), thelarval tissues often appear to scintillate from theactivity of invading bacteria. In a heavily in-fected culture, mortality is often complete within18 hr. Loosely attached or detached portions ofthe velum frequently continue their ciliary actionafter all other soft tissues have been destroyed.The course of an infection induced in straight-

hinge larvae of the European oyster, 0. edulis, byexposure to pathogen M 17 is illustrated in Fig. 4.European oysters are larviparous; fertile ova de-velop in a brood chamber of the adult for 1 weekto 10 days before their release as pelagic larvae.The larvae illustrated were challenged 24 hr afterrelease and experienced an overwhelming, fulmi-nating infection. Ciliated protozoa appeared asscavengers after the height of the bacteria-in-duced epizootic, and apparently played no partin the primary infection (Fig. 4c). Larvae chal-lenged by pathogenic bacteria that had previously

been heated to 65 C for 30 min suffered no mor-tality and appeared normal in every respect (Fig.4d).

Histological sections of the larvae, sampledhourly for 24 hr after challenge with pathogenMI 17, confirmed a massive bacterial invasion andproliferation, with extensive cellular destruction.These findings support the designation of this dis-ease as bacillary necrosis. A detailed study of thehistopathology is in preparation.

Host range. Strain M 17, an early virulent iso-late from Milford clam larvae, was pathogenicfor all of the lamellibranch larvae challenged(Fig. 2). Subsequently, groups of pathogenic iso-lates were recovered from moribund juvenileoysters and clams at iMilford, as well as fromsamples of affected juvenile clams from Virginia.The pathogenic strains obtained from oysterswere neither as virulent nor as active as thosefrom clams, but each was interchangeably patho-genic for larvae of homologous or heterologousspecies of all of the lamellibranchs challenged.None of the 27 morphologically and biochemicallysimilar isolates from fauna of Long Island Soundcaused appreciable mortality in challenge againstlarvae of 1l. mercenaria.

Serology. All rabbit antisera agglutinatedhomologous antigens in dilutions from 1:128 to1:512 and were tested for cross-agglutinationagainst heterologous antigens. In this manner, fiveserological strains of pathogens were typed andlabeled after their geographical origins: MilfordA, J, and 0, and Virginia C and D (Table 1).Type A includes four Mlilford and two Virginiaisolates and types C and D each include twoVirginia isolates. The three strains comprisingtype 0 originated from outbreaks in C. virginicalarvae. Type J is the latest established, and con-sists entirely of pathogens isolated from diseasedjuvenile clams, lI. mercenaria, at the MilfordLaboratory hatchery.None of the 27 Loyola strains, originating from

normal fauna of Long Island Sound, was aggluti-nated to a significant titer by the antisera for theMilford and Virginia strains (Table 2), nor werethey pathogenic for any of the bivalve larvaetested.

Characteristics. The characteristics of the lar-val pathogens are listed in Table 3.

Morphology. All five antigenic types of larvalpathogens, Milford 17 (type A), Milford 27 (type0), Milford 74 (type J), Virginia 65 (type D), andVirginia 67 (type C), were composed of gram-negative small rods, measuring about 0.5 by 1.5A. Some individuals of strains AI 17 and V 65showed slight somatic curvature. Members of theother strains were quite straight. All strains

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TUBIASH, CHANLEY, AND LEIFSON

II

aI Ib

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FIG. 4

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J. BACTERIOL.

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BACILLARY NECROSIS OF BIVALVE MOLLUSKS

TABLE 1. Serological typing of bivalve pathogens

Type Strain Isolate

Milford A 1720A20C63VA64VA70

J 7172737476

0 273032

Virginia C 61VA671-A

1) 65VA66VA

showed polar monotrichous flagellation whenstained from liquid cultures (Fig. 5a and b).The average wavelength of the polar flagella

was 1.8 to 2.0 ,u for all five cultures. When stainedfrom either broth or agar slant cultures, strainsM 27 and M 74 were polar monotrichousflagellate. Strain M 17 showed a few individualswith one or more lateral flagella of relatively shortwavelength (about 1.2 ,u). In addition to the polarflagellum of normal wavelength, strains V 65 andV 67 showed numerous loose flagella of shortwavelength. This phenomenon was described byLeifson (1964) and is characteristic of polarflagellate marine bacteria which "swarm" onagar media.

Cultural characteristics. Growth was uniformlyturbid, colorless, and dense in the liquid basemedium. On agar, growth was smooth and color-less; strains M 17 and V 65 were very mucoid, andV 67 swarmed on solid media. All strains werehalophilic and grew luxuriantly in simple peptone-yeast extract medium with the addition of 3%sodium chloride. All strains were more or lesspsychrophilic, and at 37 C either failed to grow orgrew very slowly as compared with growth at 20

TABLE 2. Agglutination titers of Loyola strains ofnmarine bacteria

Antigen

Loyola strains23 strains1........LA 7............LO 20...........LO 23..........LC 26............

ControlsA 17............J 71..............0 27............C 61.............D 65............

Antiserum

A J 0O

8* ~

8*4

128

8

128

4

8

256

C

256

D

128

* Aggltutiniation titer equals reciprocal of sertumdilution cauising aggluitinationi. Loyola seriesshowed no significaiit agglutinationi with Milfordantisera.

and 30 C. All grew well in media of pH 7 to 9. Acharacteristic of all five strains was their brieflongevity when cultures were stored at 4 C. Moststrains of fermentative marine bacteria in theLeifson collection could be maintained in a re-frigerator by transfer every 2 months, whereasthe larval pathogens invariably were dead afterthis period of storage. The same situation wasfound with cultures of Pseudomonas piscicida, afish pathogen, which also died after a few weeksof storage. However, larval pathogens proved tobe viable after more than 2 years of lyophilizedstorage.

Physiological characteristics. Strains 1\I 27 andMI 74 were morphologically and physiologicallyindistinguishable. The remaining strains hadmany characteristics in common but also ex-hibited differences (Table 3).

Perhaps the most definitive characteristics ofthe pathogenic group are: polar monotr ichousflagellation in liquid media, fermentative anaero-genic metabolism of carbohydrates, gelatinliquefaction, starch hydrolysis, and a negative orweak catalase reaction. Few cultures of bacteriaisolated from presumably normal Long IslandSound animals had all of the characteristics com-mon to the larval pathogens.

FIG. 4. Larvae of European oyster challenged with M 17. X 140. (a) At 1 hr after infection. Larvae appearnormal. (b) At 7 hr after infection. Several heavily infected larvae show necrosis of internal tissuies; arrowsindicate two exhibiting characteristic extension of the velum after infection. Larvae have beenfixed informalinand do not show swarming bacteria which were seen in the living preparation. (c) At 13 hr after infection.Almost all have been infected and necrotized by the pathogen. Arrows indicate secondary invasion by ciliatedprotozoa. (d) At 24 hr after seeding with a suspension of pathogen which had been heated at 65 C for 30 min.Larvae appear healthy and identical to normal controls.

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TUBIASH, CHANLEY, AND LEIFSON

TABLE 3. Characteristics of larval bivalve pathogens*

Characteristic M27 M74 M17 V65 V67

NaCl-free base brothBase broth pltis 370 NaCl......... +++ +++ +++Growth at 37C...........+,-+W ++Gelatin liquefaction ....... ++ ++ ++ ++ ++Dextrose fermentationi ..... ++ + +++ + + +++Sucrose fermentatioii ...... +-+ +++ +±+ +++ +++Maltose fermentationi ...... ++- +++ +++ +++ +++Starch hydrolysis ......... +++ +++ +++ +++ +++Lactose fermentatioi-...__Xylose fermentatio .......Catalase reaction.......7...+/- +/ - +/- +/-Nitrate to nitrite....... ..... +++ +++++ ++Indole formation .......... +++ +++ _ ++++Mannitol fermentation ... +++ +++ +++Phenylalanine deaminase.. +++ +++ - ++++Swarming on agar ......... + +++Lateral flagella on agar .... - - + +++ +++Wavelength of polar

flagella.................. 1.9 1.9 2.0 1.7 1.9Somatic curvature e - - + + -

* Symbols: - = lack of growth, acid produiction, or aniy negative reaction; + =acid production, or any positive reaction; +, = slight or weak growth.

/

a

degree of growth,

/

b. dcFIG. 5. Flagellation of pathogenic strains. Leifson stain. (a) Strain V 65 showing polar monotrichous

flagellation and slight somatic curvature. Stainedfrom liquid culture. Strain M 17 had the same morphology.(b) Strain V 67 stained from liquid medium, showing polar monotrichous flagellation and straight soma.Strains M 27, M 74, and V 67 showed the same morphology when stained from liquid media. (c) Strain V 67stained from agar slant culture. The polar flagellum is present along with the lateral flagella of shorter wave-length. (d) Strain V 67 stained from agar slant culture. Only lateral flagella are seen. Strain V 65 showedthis same morphology when stained from slant culture. Strain M 17 also showed an occasional lateral fla-gellum.

Control of infection with antibacterials. In vitroassays with antibiotic sensitivity discs againsttype cultures of the pathogenic serotypes rie-vealed that all were in some degree sensitive tofour antibiotics: chloramphenicol, polymyxin 13,erythromycin, and neomycin (Table 4).

Combistrep added to the seawater in larvalcultures proved very effective as a therapeuticagent against challenge with organism M 17(Table 5). We found that 50 to 100 ppm of the

antibiotic (200 to 400 ppm of Combistrep) wereuseful, experimentally, for therapeutic applica-tion. This veterinary preparation, developed fortreatment of poultry, also enhanced larval growthin concentrations up to 2,000 ppm (Hidu andTubiash, Proc. Natl. Shellfisheries Assoc., inpress). Chloramphenicol was equally effectiveagainst pathogen M 17. Although Sulmet waswell tolerated by larvae at very high levels, itdemonstrated no protective properties. PVP-

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BACILLARY NECROSIS OF BIVALVE MOLLUSKS

TABLE 4. Antibiotic sensitivity of bi

Seroty]

AntibioticM17 (A) M27

Chloramphenicol ... +* +Erythromycin....... + +Kanamycin......... + 0Neomycin........... + +Penicillin ........... 0 +Polymyxin B........ + +Streptomycin ....... + 0Tetracycline ........ 0 0

* Plus indicates zone of inhibitconcentration Colab Multidisks.

TABLE 5. Therapeutic effect of achallenged with pathogen

Drug level inwater of larval

culture

PPM0

5102050

100

200

300

Drug

Unprotected (posi-tive) control

ChloramphenicolChloramphenicolChloramphenicolChloramphenicolCombistrepPVP-iodineChloramphenicolCombistrepPVP-iodineSulmetCombistrepSulmetSulmet

* All uninoculated drug controfactory except PVP-iodine, whichlarvae at 100 ppm.

iodine proved toxic to larvae at potconcentrations. Preliminary testsmyxin B sulfate, erythromycin, ,sulfate indicate that these drugs arlarvae at concentrations which mantically useful.

DISCUSSIONThe bacilli found to be patho

various bivalve larvae were of the tnclassified as either Aeromonas sp. orslight somatic curvature of somestrains M 17 and V 65 probably knomic significance. The five larNhad many characteristics in comm

ivalve pathogens but could be classified into four biotypes, as wellas into the five antigenic types. A multiplicity of

pe cultures serological types is no surprise in view of similarV67 V65 M74 situations among bacterial and viral disease(C) (D) (J) agents of higher animals.

None of the cultures isolated from presumably+ + + normal marine fauna, and with generally similar+ + + morphological and physiological characteristics,0 + 0 had antigens in common with the larval patho-+ + + gens, and none was pathogenic for bivalve larvae.0 0 + Despite these results, it is felt that, in all prob-+ + + ability, the etiological agents of bacillary necrosis+ + + normally exist as widely distributed saprophytes___

+or commensals of marine forms, since typable

ion with high- pathogens were frequently isolated from culturesmaintained in unsanitary or otherwise unfavor-able physical environment.

ntibacterials The route of infection has not yet been definedM1 7 but, as a rule, the few malformed larvae found in

both experimentally and spontaneously infectedPer cent larval cultures were the last to show signs of infection

survival* (72 hr) and survived the longest. Since, unlike healthylarvae, these abnormal forms seldom feed, it is

0 probable that the initial invasion of the patho-gen is through the larval alimentary tract. The78 question of the route of entry should be resolved91 by current histopathological studies. On the91 other hand, adult hard clams, American oysters,98 blue mussels (Mytilus edulis), and soft clams99 (Mya arenaria), exposed to massive concentra-0 tions of all pathogenic serotypes for 24 hr in

100 standing seawater cultures, showed no ill effects,99 although these filter-feeders ingested vast num-0 bers of the test bacteria.

i0o Antibiotic preparations apparently can be0 used effectively in the prevention and treatment0 of larval hatchery infections and may offer a prac-

tical short-term means of control. Discovery ofIs were satis- the sources of the infectious agents, of conditionswas toxic to that encourage their proliferation, and of en-

vironmental factors which predispose bivalvelarvae to invasion by these bacteria might serve

sentally useful to clarify the epizootiology of bacillary necrosisawith poly- and establish a rational basis for hatchery sanita-and neomycin tion procedures.ueoernuareu Dy

y be therapeu-

)genic for theype commonlyVibrio sp. Theindividuals ofias little taxo-val pathogension (Table 3),

ACKNOWLEDGMENTSWe thank Manton Botsford for preparing the

figures and photomicrographs, and Rudolph Hughand Graham L. Bullock for examining the patho-gens. Chloramphenicol was supplied through thecourtesy of Parke, Davis & Co., Detroit, Mich.

LITERATURE CITEDDAVIS, H. C., V. L. LOOSANOFF, W. H. WESTON,AND C. MARTIN. 1954. A fungus disease in clamand oyster larvae. Science 120:36-38.

DAVIS, H. C., AND R. UKELES. 1961. Mass culture

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1044 TUBIASH, CHANLEY, AN) LEIFSON

of phytoplankton as foods for metazoans.Science 134:562-564.

GUILLARD, R. R. L. 1959. Fuirther evidence of thedestruction of bivalve larvae by bacteria. Biol.Bull. 117:258-266.

J0RGENSEN, C. B. 1946. Lamellibranchia, p.277-311. In Gunnar Thorson [ed.], Reproductionandlarval development of Danish marine bottominvertebrates. C. A. Reitzels Forlag, Copen-hagen.

J. BACTERIOL.

LEIFSON, E. 1960. Atlas of bacterial flagellation,p. 3-7. Academic Press, Inc., New York.

LEIFSON, E., B. J. COSENZA, R. MURCHELANO,AND R. C. CLEVERDON. 1964. Motile marinebacteria. I. Techniques, ecology, and genieralcharacteristics. J. Bacteriol. 87:652-666.

LOOSANOFF, V. L., AND H. C. DAVIS. 1963. Rearingof bivalve mollusks, p. 1-128. In F. S. RIussell[ed.], Advances in marine biology, vol. 1. Aca-demic Press, Inc., New York.

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