MFR PAPER 1332. 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, Rockville,MD 20852. Copies of Marine Fisheries Review are available Irom theSuperintendent of Documents, U.S. Government Printing Office, Washington,DC 20402 for $1.10 each.

inlraperiloneal injection and hyperosmotic in­filtration of Vibrio angaillamm andAeromonas salmonicida bactcrins. J. Fish.Res. Board Can. 34:203-208.

Evelyn. T. P. T. 1971. First records of vibriosisin Pacific salmon cultured in Canada. andtaxonomic status of the responsible bacterium,Vibrio angail/aram. J. Fish. Res. Board Can.28:517-525.

Harrell, L. W., A. J Novotny, M. H. Schiewe,and H. O. Hodgins. 1976. Isolation and de­scription of two vibrios pathogenic to Pacificsalmon in Puget Sound, Washington. Fish.Bull., C.S. 74:447-449.

Sawyer. E. S, and R. G. Strout. 1977. Survivaland growth of vaccinated. medicated and un­treated coho salmon (Oncorhvnchas kisalch)expos~d to Vibrio angail/aru'm. Aquaculture10:311-315.

Strout. R. G., 1:::. S. Sawyer, and B. A.Coulermarsh. 1978. Pathogenic vibrios inconfincment-reared and feral fishes of theMaine-New Hampshire coast. 1. Fish. Res.Board Can. 35:403-408.

MFR PAPER 1333 Lanny R. Udey is with the De­partment of Microbiology, Universityof Miami School of Medicine, Miami,FL 33152.

Anaerobic Bacteria as PossibleDisease Agents in Fish

LANNY R. UDEY

There are currently 23 recognizedgenera of heterotrophic, obligatelyanaerobic bacteria. Within this groupare gram-positive rods and cocci andgram-negative rods and cocci. Many ofthe genera have representative specieswhich are clinically importantpathogens of man and animals. Themost familiar genus, Clostridium, con­tains the species C. botulinum, C.tetani, and C. perjringens, causingbotulism toxicity, tetanus, and gasgangrene, respectively. Other genera ofanaerobes such as Propionibacterium,Fusobacterium, Bacteroides, andEubacterium are less familiar, but alsosignificantly involved in human dis­ease.

Although anaerobic bacteria havebeen studied since the beginnings ofmicrobiology, it has been only recentlythat they have been identified as a majorgroup of pathogens in animals and man.A number of reasons can be cited forthis lag. Probably the most importantwas that many people did not believe

10

that strict anaerobes could survive andproliferate in "oxygenated" tissues ofthe body. Lungs, brain, liver, and othervital organs were good candidates forfacultative bacteria, but not foranaerobes. Frequent coinfections withfacultative organisms further com­plicated the picture.

The development of standardizedprocedures for working with anaerobicbacteria has helped to dispell some ofthese beliefs. The popularization of theroll-tube method (Hungates' method)by workers at the Virginia PolytechnicInstitute's Anaerobe Laboratory andcommercialization of the Gas-Pak l

(BBL) anaerobic jar have both greatlyaided in standardizing and facilitatingprocedures for cultivating anaerobicbacteria. Recent advances in the pro­cedures used to classify anaerobes havemade the task of identifying these

I Mention of trade names or commercial productsdoes not imply endorsement by Ihe NationalMarine Fisheries Service, NOAA.

organisms somewhat less tedious. Inparticular, the use of gas-liquidchromatography to identify the majormetabolic by-products from ferment­able substrates allows rapid identi­fication to genus and sometimes tospecies. Although other confirmatorytests are required, much time can besaved by knowing which tests to run.Many of these advances have comeabout within the past 3-5 years, andtheir adoption by clinical microbiologylaboratories is still underway. It seemsto me that in the not too distant future anumber of fish disease laboratories willalso adopt these procedures and de­velop new ones particularly suited tofish disease studies.

At the Fish and Shellfish PathologyLaboratory at the University of Miami,we have been involved with the study ofanaerobes from marine fish for the past2 years. Our studies began followingthe occurrence of several large fish killsin Biscayne Bay. In addition to thekills, moribund fish were present whichexhibited a "twirling" symptom not un­like that observed in trout with whirlingdisease. Analysis of toxicological andparasitological as well as aerobicbacteriological data revealed no con­sistent cause of this symptom. Webegan a search for anaerobic bacteria inthe brains (because of the likelihood ofneurologic involvement) of fish

Marine Fisheries Review

Figure I.-Long filaments and shorter rods of Eubacterium tarantellus seen in initialcultures using Brewer's lhioglycollate medium. Phase contrast, 400 x.

samples contained E. tarantellus. In nocase was the anaerobe isolated from anorgan other than the brain if the brainwas not also infected. This clearly indi­cates that this particular anaerobe has adefinite tissue specificity. Of the 12species sampled in this study, thebluefish, seatrout, menhaden, andsnook all had a 100 percent incidence.In the past, the striped mullet alsoshowed a 100 percent incidence. Allstrains which we have isolated to dateappear to be closely related serolog­ically (Fig. 2). Infections do not seemto be correlated with the type of feedinghabits; menhaden and mullet are filterfeeders, while bluefish and seatrout arecarnivores. It seems likely that the fishare either all equally exposed to thesource of infection or that it is beingtransmitted up the food chain. Morework will be needed in this area in thefuture.

The question which arises most oftenin discussions of E. tarantellus in­fections is "What, if anything, is theorganism doing to the large number offish which seem to be infected?" This isa particularly germane question and onewhich is extremely difficult to answerfor native fish populations. We areconfident that, in the past, the anaerobehas been responsible for fish kills in

Figure 2.-Double immunodiffusionplate demonstrating the serological re­·Iatedness of Eubacterium taranrellusisolates. Outer wells: suspensions of son­ically disrupted E. rarantel/us cells of sixstrains isolaled from d ifferenl fishspecies. Center well: rabbit anli-E.rarantel/us (strain 128) serum.

Florida and in Texas (Henley andLewis, 1976). If we can use twoexamples from the field of cultured fishdiseases, the importance becomes moreevident. The best example, perhaps,would be that of bacterial kidney dis­ease (BKD). Fish can be infected withthe BKD bacterium for several yearsand not show any outward symptoms,

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/showing these symptoms. At the time,only thioglycollate cultures were taken,but we were fortunate in isolating ananaerobe, in pure culture, from thebrains of all the mullet, MugUcephalus, sampled.

This bacterium has since been namedby us as Eubacterium tarantellus sp.nov. (Udey et aI., 1977). It is a gram­positive rod measuring 1.3-1.6 x10.0-/7.0 [Jill which upon initial isola­tion can occur as long filaments manytimes that length (Fig. I). The organismdoes not form spores and is nonmotile.Colonies on BHI blood agar are trans­parent, filamentous, and often form a"pinwheel" design. Colonies are al­ways surrounded by a large zone of betahemolysis. The organism also produceslecithinase upon initial isolation, butmay lose this characteristic upon re­peated transfer. The minimum growthtemperature of E. tarantellus is 15°C,which is probably a reflection of the factthat it was isolated in the tropics. Un­less the organism can adapt to coldertemperatures, its growth in many areas(outside the tropics) would be limited tosummer months. It is interesting to notethat E. tarantellus will not grow atsal inities of greater than 20 ppt or abovepH 8.0 (at least in laboratory media).This strongly suggests that the or­ganism is an obligate fish pathogen, islimited to growth in estuaries, or isfrom exogenous sources such as landrunoff or sewage effluents. Fortu­nately, E. tarantellus presents littlethreat to humans since it does not pro­duce toxins; it is not pathogenic forguinea pigs. This is a particularly im­portant consideration when workingwith anaerobes because numerousspecies of Clostridium produce potenttoxins, making them a hazard in thelaboratory.

To determine if E. tarantellus causesinfection primarily of the central ner­vous system or a more generalized in­fection, an organ distribution study wasundertaken. Organ samples from 38fish belonging to 12 species werecultured on BHI blood agar. Sixty-sixpercent ofthe fish yielded positive braincultures. Only 5 percent of the liver andkidney samples were positive, while 7percent of the intestinal content

October /978 II

MFR Paper 1333. 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,Rockville, MD 20852. Copies of Marine Fisheries Review are available from theSuperintendent of Documents, U.S. Government Printing Office, Washington,DC 20402 for $1.10 each.

but, if they are subjected to an appro­priate environmental stress, they mayquickly succumb to the infection. Sec­ondly, Aeromonas salmonicida is oftenpresent in a large percentage of fish in acarrier state which can again betriggered into an active infection by astressful environment. It is our feeling,therefore, that any significant deeporgan infection (particularly the brain)is of major consequence to the fishpopulation.

In the future, I believe that additionalanaerobic bacterial fish pathogens willbe discovered. Much of the ground-

work is set, and, as more people beginto look for this group of organisms,more will be found.

LITERATURE CITEDHenley, M. w., and D. H. Lewis. 1976.

Anaerobic bacteria associated wilh epizootics

in grey mullet (Mugil cephalus) and redfish(Sciaellops ocel/ata) along lhe Texas Gulfcoast. J. Wild!. Dis. 12:448-453.

Udey. L. R.. E. Young. and B. Sallman. 1977.. Isolation and characterization of an anaerobic

bacterium, Eubacrerium raramel/us sp.nov.,associated wilh striped mullet (Mugil cepha­Ius) mortalily in Biscayne Bay, Florida. J.Fish. Res. Board Can. 34:402-409.

MFR PAPER 1334 J. A. Plumb and P. R. Bowser arewith the Department of Fisheries andAllied Aquacultures, Auburn Univer­sity, Auburn, AL 36830

Recent Developments in ChannelCatfish Virus Research

J. A. PLUMB and P. R. BOWSER

Channel catfish virus (CCV) is wellestablished as a highly virulentpathogen in some cultured channelcatfish fingerling populations. Thishighly contagious disease of juvenilechannel catfish occurs during the warmsummer months. Although mostepizootics have occurred in the south­ern states, the virus has been isolatedfrom channel catfish fingerlings inseveral northern and western states.Mortality rates of CCV -infectedpopulations may approach 100 percent.CCV is caused by herpesvi'rus ofictalurids (Wolf and Darlington, 1971).The virus has been isolated only fromfish when fry or fingerlings are actuallydying. Epizootics appear to beassociated with some form of stress.

The brown bullhead (BB) cell line isnormally used for isolation of CCV. Acell line is being developed fromchannel catfish ovaries (CCO) which

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is now in the 75th passage andapproximately 20 months old. CCOcells appear to be more sensitive toCCV than the BB cells; CCO cells de­velop initial microscopic cytopathiceffect (CPE) 12-24 hours before the 88cells, and total CPE occurs 24-48 hoursin CCO cells before it does in 8B cells.Also, the CCV titers in CCO cells are0.5-1.0 log (base 10) higher than in theB8 cells.

A fluorescent antibody (FA)technique has been developed for de­tecting CCV in vitro. Rabbit anti-CCVserum was conjugated with fluoresceinisothiocyanate (FITC) using themethod of Cherry (1974). CCOcultures in Leighton tubes were in­fected with CCV, and, at 2-hour in­tervals after infection, coverslipcultures were removed, fixed inacetone, and incubated with FITC con­jugate. Infected CCO cells fluoresced

at 4 hours postinfection (PI) with focalcytoplasmic fluorescence. Fluores­cence increased with larger focal areasthrough 6 and 8 hours PI, until at 10hours the entire cell sheet was fluor­escing. The FA technique has not beenapplied to in vivo infections.

Confirmed CCV epizootics havebeen found only in farm-raised channelcatfish populations. Different strains ofchannel catfish vary in theirsusceptibility to CCV (Plumb et at.,1975); however, data on susceptibilityof other species of ictalurids to CCV arelacking. Young brown, black, andyellow bullhead catfish were exposed toCCV by incorporating the virus into thefeed, intraperitoneal OP) injection, andby cohabitation with CCV-infectedchannel catfish. Although somemortality occurred in all of thesebullheads in each method of exposure,CCV was not isolated from any of theexposed fish. It is interesting to notethat the BB cells support CCV re­plication but that the fish itselfapparently does not.

In another study, blue and channelcatfish fingerlings and their reciprocalcrosses were compared for CCV

Marine Fisheries Review