disease of lobsters (genus HomanIS). J. Fish.Res. Board Can. 31: 1873-1879.

____, and H. Rabin. 1970. Gaffkemia. abacterial disease of lobsters (genus Homarus).

/n S. F. Snieszko (editor). A symposium ondiseases of fishes and shellfishes. p. 431-439.Am. Fish. Soc., Spec. Publ. 5.

____, and B. M. Zwicker. 1972. Natural

and induced bactericidal activities in thehemol ymph of the lobster. Homarusamerican tiS: products of hemocyte-plasma in­teraction. Can. J. Microbiol. 18: 1499-1509.

MFR PAPER 1330

MFR Paper 1329. From Marine Fisheries Review. Vol. 40, No. 10, October1978. Copies of this paper, in limited numbers, are available from 0822, UserServices Branch, Environmental Science Information Center, NOAA, Rockvilfe,MD 20852. Copies of Marine Fisheries Review are available from the Superin­tendent of Documents, U.S. Government Printing Office, Washington, DC20402 for $1.10 each.

R. A. Robohm and C. Brown are withthe Milford Laboratory, NortheastFisheries Center, National MarineFisheries Service. NOAA, Milford,CN 06460.

A New Bacterium (Presumptive VibrioSpecies) Causing Ulcers in Flatfish

R. A. ROBOHM and C. BROWN

week and progressively for 4 weeks byulcers in other summer flounder in thesame tank, as well as in winter floun­der, Pseudopleuronectes americanus,and hogchokers, Trinectes maculatus,in an adjacent tank. Although the le­sions resemble those caused by Vibrio

Summer flounder, Paralichthys den­tatus, captured in the wild and held forlO days in a tank supplied with flowingseawater (about 25 ppt salinity) fromMilford harbor developed ulcerative le­sions of the tail and dorsal muscle. Le­sions began as a white patch at the flex­ure point of the tail (where the caudalfin meets the body) and sometimes wereaccompanied by hemorrhagic necrosisat the tip of the caudal fin. Frank ulcera­tion extending into the skeletal musclecould be seen within a few days whenthe epithelium sloughed off under thepatches (Fig. I). Frequently, bony raysof the entire caudal fin were exposedby progression of the ulcerati ve processeither anteriorly from the tip of the finor from the initial lesion caudally (Fig.2).

First appearance of the ulcers in twosummer flounder was followed within I

OCfober /978

Figure I.-Posterior portion of a summer flounder infected with thenew Vibrio species. Ulcers are seen to extend deep within the dorsalmuscle.

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Figure 2.-Caudal fin of a summer flounder infected wilh the newVibrio species. Epithelium is sloughed off 10 expose the underlyingbony rays.

anguillarum infections (Haastein andHolt, 1972; Levin et aI., 1972; ZoBelland Wells, 1934), other bacteria areknown to cause similar ulcers in fish.These include lesions due toAeromonas hydrophila (Amlacher,1961), A. salmonicida (Herman,1968), Hemophilus piscium (Snieszkoet aJ., 1950), and Cytophaga psychro­phila (Borg, 1960; Rucker et aI.,1954). The disease in summer fl ounderwas arrested by daily treatment withoxytetracycline (13 mg/liter of water)for I week, thus confirming the sus­pected bacterial etiology of the lesions.

Material swabbed from ulcers insummer flounder prior to antibiotictreatment was streaked onto Difco 1

TCBS agar, a medium containing 0.05percent yeast extract, a.05 percent pep­tone, 1.5 percent agar and seawater,and modified Twedt agar (Vanderzantand Nickelson, 1972). Four distinctbacterial types were isolated. Prelimi­nary biochemical testing indicated thattwo of the isolates were Vibrio sp., onewas vibriolike, and one a Pseudomonas

'Mention of (rade names or commercial produclsdoes nol imply endorsement by the NationalMarine Fisheries Service, NOAA.

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sp. Sera drawn 3 weeks after the start ofsymptoms, from five antibiotic-treatedsummer flounder, contained antibodiesagainst one of the Vibrio isolates atreciprocal agglutination titers of 64 to256. Since the five sera had low anti­body titers (less than 16) against thethree other bactelial isolates, the fi rstisolate was strongly implicated as theulcer-disease bacteri urn. This organismwas a short, gram-negative rod with asingle polar flagellum when grown onliquid or solid medium.

Subcutaneous inoculation of varyingnumbers of the bactelium into 36 winterflounder produced small ulcers at theinoculation site in 31 fish; ulcers wereaccompanied by subcutaneous hemor­rhages along the base of nearby fins andpetechia on the nonpigmented side.Fish with these symptoms usually diedwithin 2 days of inoculation. The or­ganism was recovered repeatedly fromthe spleens of dead or dying fish­usually in pure culture. The subcutane­ous LDoo calculated by the Reed andMuench (1938) method was 15,985bacteria, but some fish died at a dose of7,000 bacteria. Fish inoculated with700 bactelia routinely survived. Be­cause subcutaneous inoculation pro-

duced a disease different in form fromthat seen naturally in the holding tanks,another method of disease inductionwas tlied. After first scraping awayscales from a small patch at the tail­flexure point with a scalpel blade,winter flounder were placed in 76-1 itertanks containing recirculating,charcoal-filtered seawater. Ulcerativelesions resembling those of the naturaldisease could be induced in the fishwithin 3-7 days by inoculating thewater on alternate days with 360bacteria/ml. Within 24 hours, bacterialcounts were 10/ml; within 48 hours,counts could no longer be made be­cause of competing microflora. Thediseased fish died within 10 days; theorganism was recovered from the le­sions and from the spleens of dead fishand confirmed biochemically. An in­oculum of 36 bacteria/ml into tankwater on alternate days failed to inducedisease.

Eight characteristics of the ulcer­disease organism are recognized by theSubcommittee on Vibrios of the Inter­national Committee on Systematic Bac­teriology as necessary to place an or­ganism in the genus Vibrio (Hugh andSakazaki, 1972; Subcommittee onTaxonomy of Vibrios, 1972). Theseare: gram reaction -, motility +,single polar flagellum +, oxidase +,catalase +, glucose fermentation +,gas from glucose -, and acid from glu­cose +. A ninth characteristic whichshould be included is a 40-50 percentguanine plus cytosine (G-C) content inthe bacterial DNA. Repeat analyses ofthe G-C content has given us conflictingresults. Final analysis is yet to be com­pleted. However, a total of53 biochem­ical and physical tests indicate that theulcer-disease organism is sufficientlydissimilar to currently known vibriosand closely related organisms to meritprovisional designation as a newspecies.

We examined the sera of a variety ofwild fish species for antibodies againstthe ulcer-disease organism. Sera ofhealthy blackfish, Tautoga onitis, andcunners, Tautogolabrus adspersus,from Long Island Sound near Milfordhad antibody levels to the ulcer-diseasebactelium which rivaled levels in in-

Marine Fisheries Review

fected summer flounder. The antibodyin cunners appeared to be protectiveagainst the disease; 50 percent of thefish had significant antibody levels and50 percent survived intraperitonealinjections of bacteria. Most of thesera from 13 windowpane flounder,Scophthalmus aquosus, 15 weakfish,Cynoscion regalis, 54 summer floun­der, and 101 winter flounder from LongIsland Sound and New York Bight hadlow antibody titers to the organism.These results suggest that the high sus­ceptibility of flounder species to thedisease may be due to low natural expo­sure to the organism; whereas, immu­nity in two fish species near Milfordmay be due to a high degree of exposureto the organism; this could arise from afood source or from proliferation of theorganism locally in nutrient-rich wa­ters.

As a measure of the ability of theorganism to persist and be disseminatedin seawater, we inoculated a washedbroth culture of the bacterium intofiltered, sterilized seawater (25 ppt sa­linity) at a level of 106 cells/ml. Viablenumbers decreased over as-monthperiod at two temperatures; however,

by 155 days, 89 and 141 bacterial/mlremained at 25°C and 15°C, respec­tively. This appears to be a relativelylong survival when compared with thatof V. anguillarum; Evelyn (197 I) re­ported that 107 V. anguillarum cellswere reduced to 10 1 in 14 days at 0°_3 0c. These results suggest that a re­quirement for direct fish to fish contactmay be more important in maintaininginfection with V. anguillarum than withthe ulcer-disease organism.

From the results of this work, we findthat a previously undescribed bac­terium I) is pathogenic for flatfish, 2) isprobably a new Vibrio species whichsurvives for extended periods of time inseawater, and 3) has a capacity to causeulcers which may be modulated byroute of entry and/or the presence ofcompeting micrcftora.

LITERATURE CITEDAmlacher, E. 1961. Taschenbuch der Fischkran­

kheiten. Gustav Fischer Verlag, Jena, 286 p.Borg, A. F. 1960. Sludies on myxobacteria as­

socialed with diseases in salmonid fishes.Wildl. Dis. 8: 1-85.

Evelyn, T. P. T. 1971. First records of vibriosisin Pacific salmon cultured in Canada, andtaxonomic status of the responsible baclerium,

Vibrio angllillarum. J. Fish. Res. Board Can28:517-525.

Haastein, T., and G. Holl. 1972. The occurrenceof vibrio disease in wild Norwegian fish. 1.Fish BioI. 4:33-37.

Herman, R. L. 1968. Fish furunculosb 1952­1966. Trans. Am. Fish. Soc. 97:221-230.

Hugh, R., and R. Sakazaki. 1972. Minimalnumber of characters for the identification ofVibrio species, Vibrio cholera and VibrioparahaemolYlicus. J. Conl'. Public Health Lab.Dir. 30:133-137.

Levin, M. A., R. E. Wolke, and V. 1. Cabelli.1972. Vibrio angllillorum as a cause of diseasein winler flounder (Pselldopleuroneclesamericanlls). Can. 1. Microbiol. 18: 1585­1592.

Reed, L. J., and H. Muench. 1938. A simplemelhod of eSlimating fifty per cenl endpoints.Am. J. Hyg. 27:493-497.

Rucker, R. R., B. J. Earp, and E. J. Ordal. 1954.Infectious diseases of Pacific salmon. Trans.Am. Fish. Soc. 83:297-312.

Snieszko, S. F., P. 1. Griffin, and S. B. Friddle.1950. A new bacterium (Hemophilus pisciumn. sp.) from ulcer disease of trout. J. Bacleriol.59:699-710.

Subcommittee on Taxonomy of Vibrios, Inlerna­tional Committee on Systematic Bacteriology.1972. Minules of the meeting, 22 July 1971.Int. J. SYSI. Bacleriol. 22: 189-190.

Vanderzant, c., and R. Nickelson. 1972. Proce­dure for isolation and enumeration of Vibrioparahaemoly/iclls. Appl. Microbiol. 23:26­33.

ZoBell, C. E., and N. A. Wells. 1934. An infec­tious dermatilis of certain marine fishes. J.Infect. Dis. 55:299-305.

MFR Paper 1330. From Marine Fisheries Review, Vol. 40, No. 10, October1978. Copies of this paper, in limited numbers, are avaifable from 0822, UserServices Branch, Environmental Science fnformation Center, NOAA, Rockville,MD 20852. Copies of Marine Fisheries Review are avaifable from the Superin­tendent of Documents, U.S. Government Printing Office, Washington, DC20402 for $1.10 each.

The National Marine Fisheries Service (NMFS) does not approve, recommend orendorse any proprietary producl or proprietary material mentioned in Ihis publica­tion. No rderence shall be made 10 NMFS. or to this publication furnished by NM FS,in any advertising or sales promotion whil'h would indicate or imply lhat NMFSapproves, recommends or endorses any proprietary product or proprielary materialmentioned herein. or which has as ilS purpose an intent to cause directly or indirectlythe advertised product to be used or purchased because of lhis :"JMFS publication.

October 1978 7