MFR PAPER 1297

ANTHONYJ,NOVOTNY

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Figure I.-Pugel Sound. WashinglOn.This inland arm oflhe northeaslem PacificOcean is Ihe site of major acti vilies for Ihemarine culture of Pacific salmon.

can also occur in marine fish other thansalmon (Evelyn, 1971a, b; Kennedy,1974; Egidius and Andersen, 1975;Novotny, 1975). The typical symptomsof both diseases are almost identical: ageneral hemorrhagic septicemia, exter­nal lesions, hemorrhaging of the fins,and bloody discharges from the vent.

Vibriosis in fish is an infectioncaused by the marine bacterium Vibrioanguillarum (Bergman, 1909), amotile, gram-negative rod which hasbeen thoroughly described by Evelyn(1971b). At least two pathogenicserotypes have been identified (Nov­otny, 1975; Harrell et aI., 1976).

Furunculosis in fish is an infectioncaused by the nonmotile, gram­negative rod, Aeromonas sa/monicida(Lehmann and Neumann, 1896). Thisdisease has been described (McCraw,1952; Scott, 1968), but is generallyconsidered to be a serious problem infreshwater environments only. Unlike

Anthony J. Novotny is with theNorthwest and Alaska FisheriesCenter, National Marine FisheriesService, NOAA, 2725 Mont/akeBlvd. East, Seattle, WA 98112.

Marine Fisheries Review

DESCRIPTION OF VIBRIOSISAND FURUNCULOSIS

The two most common diseases oc­curring in salmon cultured in seawaterin Puget Sound are vibriosis and furun­culosis. Both of these bacterial diseasescan be either epizootic or chronic; they

physiological stresses caused by directtransfer from fresh water to seawater,high population densities, repeatedhandling, and increasing water temper­atures during the summer are conduciveto diseases (Wedemeyer, 1970;Wedemeyer and Wood, 1974).

During an experiment to determinethe effects of rearing densities on thegrowth and survival of chinook salmonin seawater pens, repeated epizootics ofvibriosis and furunculosis were encoun­tered. The objectives of this paper areto: I) present a history of these diseasesduring the course of the experiment; 2)demonstrate drug resistance in furun­culosis; 3) demonstrate the infectiousnature of furunculosis in seawater; and4) show that the presence of multipleinfectious agents can be a serious threatto marine cultured salmonids.

Since 1969, the Northwest andAlaska Fisheries Center of the NationalMarine Fisheries Service has been con­ducting research on the marine cultureof salmonids (Oncorhynchus sp. andSa/mo sp.) at its Aquaculture Experi­ment Station near Manchester, Wash.(Fig. I). The major research effort atthis station in central Puget Sound isfocused on the culture of coho, O.kisutch, and chinook, O. tshawytscha,salmon in floating net pens.

The principle of this type of culture isthe same as for an agriculture feedlot:the fish are concentrated to minimizespace, materials, and labor, and are fedcommercial pelleted rations. Tidal cur­rents provide an almost continuous ex­change of water through the knotlessnylon net pens, insuring adequate sup­ply of dissolved oxygen to the fish andthe dilution and removal of excretorywaste products.

The normal procedure is to transportunacclimated juvenile chinook or cohosalmon from a freshwater hatchery andplace them directly into seawater pens.The fish are then cultured for 6 monthsto a year prior to harvesting. The

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ABSTRACT-Infections in marine cultured Pacific salmon (genus Oncorhyn­chus) and trout (genus Salmo) can include those caused by two bacterial patho­gens, Vibrio anguillarum (vibriosis) and Aeromonas salmonicida (furunculosis), Inthe Puget Sound area, two distinct serotypes of V, anguillarum have causedextensive mortalities in net-pen culture, Although furunculosis is probably carriedby fish from fresh water, it can be transmitted in seawater, and in the close confinesof net-pen culture can reach epizootic proportions. In large-scale experiments,epizootics of vibriosis and furunculosis reduced the population of two sea cages(300,000) of chinook salmon (0. tshawytscha) by 80 percent during approximately5 months ofmarine culture. Laboratory tests of the bacterial pathogen isolated frommoribund fish indicated that the furunculosis organism was resistant to oxytetracy­cline and sulfa drugs, but was sensitive to furazolidone. Multiple infections of bothdiseases proved difficult to treat. The results of these experiments indicate theneed for better management of furunculosis during the freshwater culture stages ofsalmon.

Vibriosis and Furunculosis inMarine Cultured Salmon inPuget Sound, Washington

the vibrios, A. salmonicida is not con­sidered to be a marine organism. Insalmonids, the disease is undoubtedlyfirst contracted during the freshwaterculture stages. At low temperatures, thedisease is latent and in a carrier state ininfected fish (Snieszko, 1969). Whenthe fish are transferred to seawater, thecombined effect of osmotic stress andother environmental factors weakensthe hosts, and the disease become infec­tious. Transmission in seawater canundoubtedly occur by direct contact(Scott, 1968) and probably by contactwith fecal casts.

DISEASE OBSERVATIONSDURING 1972-73

Severe outbreaks of vibriosis andfurunculosis among chinook salmonduring an experiment to determine theeffect of different fish densities on fishgrowth and survival in pens allowedextensive observation of the diseases.Observations on diagnosis, drug sen­sitivity tests, treatment, and mortalityassessment are mentioned below.

In June 1972, ju venile chinooksalmon were transferred from trucks tofloating net pens by gravity flowthrough a large pipe. The fish went di­rectly from the fresh water in the trans­port trucks to 30%0 seawater. The float­ing hexagon-shaped pens, made fromknotless nylon webbing, measured 4.9m on a side, were 3.8 m deep, and hadan approximate volume of 220 m3 .

One hundred thousand salmon,weighing an average of 5.4 g each wereplaced in Pen I and 200,00 in Pen U.The temperature of the water in thetransport truck was 13.9°C for Pen Ifish and 17. 9°C for Pen II fish. Thetemperature of the seawater wasII. 20c. The fish. were to be reared inthe pens for approximately I year, dur­ing which time they were to be fed adaily ration of Oregon Moist Pellets(aMP), based on a percentage of bodyweight.

Diagnostic procedures

Sick or freshly dead fish wereroutinely examined for disease. Sam­ples of kidney tissue were asepticallystreaked onto Petri dishes containing

March /978

Trypticase Soy Agar (TSA)l with 1.5percent NaCI or Tryptose Blood Agar(TBA). Sterile discs impregnated withthe vibriostatic agent 2,4-diamino­6,7-di-iso-propyl pteridine phosphate(0/129) were placed on freshly streakedTSA plates. All plates were incubatedat 24°C for 24 to 72 hours. A diagnosisof vibriosis was based on: I) growth ofsmooth, opaque colonies in 24 to 48hours on TSA, 2) motility, and 3) azone of inhibition to the 0/ 129 disc of 8mm or more. Furunculosis was consi­dered to be the causative agent of dis­ease when: I) Pin-point colonies ap­peared within 72 hours on TBA, 2) theorganisms were nonmotile, and 3) theTBA plates would be pigmented a lightto chocolate brown.

Because of the large quantities ofdiseased fish that were processed, it wasimpossible to confirm all cultures bybiochemical reactions. However, cul­ture samples were periodicallyanalyzed by the Department of Micro­biology, University of Washington, forconfirmation. In general, the accuracyof the field diagnosis was high.

Drug Sensitivity Tests

Drug sensitivity tests were con­ducted by using discs containing 30 p.gof oxytetracycline or 100 p.g offurazolidone placed on freshly streakedTSA or TBA plates. Triple sulfa (SSS)was tested by placing discs containing250 p.g SSS on freshly streaked platescontaining Mueller-Hinton's agar. Allplates were incubated at 24°C untilsufficient growth occurred to indicatezones of inhibition. A zone of inhibi­tion of less than 10 mm indicated aquestionable effective value.

Therapeutic Treatment Procedures

All of the fish were placed on OMP­TM-50D (OMP-TM-50D is a commer­cial pellet containing 4 percent TM­50D; eleven percent of TM-50D is ac­tive oxytetracycline) medicated dietsfor the first 5 days after transfer as aprecautionary measure to assist in com­bating any stress infection induced bytransfer shock. Routinely thereafter,

I Reference to trade names does not imply en­dorsement by the National Marine Fisheries Ser­vice, NOAA.

whenever the mortalities reached O. Ipercent/day and vibriosis or furun­culosis was determined to be the cause,OMP-TM-50D diets were fed the fishfor at least 5 days, or until the mor­talities subsided to less than O. Ipercent/day. When it became evidentlater in the study that the furunculosisorganism was resistant to oxytetracyc­line, standard aMP diets were prepareddaily with commercial agriculturegrade Furox 50. The Furox 50 (which is50 percent active furazolidone) wasmixed with herring oil and sprayed overthe pellets at a rate of 2 percent oil byweight. The amount of Furox 50 addedto the oil was adjusted to provide 0.8 gof Furox 50/kg fish per day.

Mortality Assessments

Dead fish were removed from thepens each day and counted, exceptwhen mortalities were excessivelyhigh. In this case, total weights weretaken, and an estimate of mortality wasobtained from subsample weights andcounts. Subsamples of the live fishpopulation were obtained periodicallyto measure growth rates and to adjustfeeding levels.

RESULTS AND DISCUSSION

The initial 10-day mortality in thehigh density pen (Pen II) was 0.5 per­cent compared to 0.1 percent in the lowdensity pen (Pen I). This was believedto be due to the greater temperatureshock during the transfer of Pen II fish.

Within 3 weeks of transfer, the netsbecame heavily fouled with sessilefilamentous algae, reducing water flowthrough the pens. Dissolved oxygenconcentrations in the center of the pensoccasionaJly dropped to 3.5 ppm, creat­ing an oxygen stress condition. Thiscondition was somewhat alleviated byforcing large volumes of compressedair through perforated pipes beneath thepens. The rising air bubbles brought innew water, creating a partial exchange.This problem was solved in late July,when larger mesh net pens were instal­led.

Water temperatures reached 12°Cearly in July (Fig. 2). At this time, thefirst serious epizootic of vibriosis oc­curred. At times, the mortalities were

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" Of O[AO OR OYIN<O fiSH WITH POSITIVE: viOftlOSIS

• 1'4;911 d.1Ki'yptll

• Low dtflS,I)~

CULTURE. P(RIOD

nolnw,"10fY

40

20

60

80

Figure 3.-Estimated cumulative losses ofchinook salmon in the high and low densitypens. The large disparity between the twopens in November is an estimating error. Thehigh density pen was physically inventoried,whereas the low density pen was not inven­toried until the following March.

'00

fish in the pen through a photocell fishcounter. In spite of Furox 50 feedingsbefore and after inventory, over 7,000mortalities were recorded within 10days. The inventory indicated acumulative loss of 80 percent of the fishin Pen II (Fig. 3). Although the data inFigure 3 indicate a better survival inPen I in November, this is an unreliableestimate, and the cumulative mortalitywas more likely between 70 and 80percent. We continued the Furox 50treatment in both pens periodicallythrough the winter.

By February 1973, the surviving fishin both pens appeared to be in goodenough condition to inventory. The fishwere weighed and samples collected todetermine average weights. We esti­mated only 6,000 fish surviving in Pen Iand 14,000 in Pen II, for a total survivalof 6 and 7 percent, respectively. At notime during the course of the experi­ment did we ever reach a planned highloading density of 32 kg/m3 (Fig. 4).

This total mortality of 93.5 percent iscontrasted with a loss attributed to vib­riosis of 3.2 percent during the 1971-72rearing period at Manchester and a1972-73 loss of 15.7 percent (directlyattributable to vibriosis) of a differentstock of chinook salmon (Moring,1973). These lots represented stocks ofchinook salmon from other hatcheries,no incidence of furunculosis wasfound, and all epizootics of vibriosiswere easily controlled with OMP-TM­SOD.

When the growth of the fish in the

Table 1.-Sallnlty tolerance and anllblotic sen-sillvlty 01 Aeromonas ••/mon/c/da '.

Sampleno. III T", SSS F,oo

1 +0 +M -N 0 11 252 +0 +M +N 0 8 123 +0 +L -N 0 7 154 + 0 + 0 -N Not conducted5 +0 +M - N 0 11 226 +0 + L -N Not conducted7 +0 +M -N 0 5 128 +0 +M -N 0 a 109 +0 TM ·N 0 5 20

10 +0 +M + N 0 a 911 +0 + D -N 0 0 1512 +0 +M + N a a 1513 + D +M + L a a 1514 +0 +M -N 0 a 12

the vibrio was not) and triple sulfacompounds, but sensitive to fura­zolidone, and 2) it would grow onmedia containing between 2 and 3 per­cent NaCI (Table I). This secondfinding indicated that this strain offurunculosis might be transmittable inseawater. Later, we isolated this or­ganism from adult coho salmon cul­tured in pens within a IS-mile radius ofPens I and II. In addition, we isolatedthe organism from marine fish that ac­cidentally entered the pens with the in­fected chinook salmon (Novotny,1975).

Furox 50 was tried as a treatment forfurunculosis. The first Furox 50 treat­ment period was extended to approxi­mately 12 days in an attempt to reducethe numbers of low-level infectious orcarrier fish as much as possible. Weassumed that once the temperaturesstarted dropping in early fall, our prob­lems of recurring epizootics would beover. The rate of observed mortalitydeclined through August, and a secondtreatment in August reduced the mortal­ity rate to an even lower level in Sep­tember (Fig. 2). However, by mid­October, the rate of mortality againbegan to climb in spite of decreasingwater temperatures. Aeromonas sal­monicida was isolated from almost allsubsamples of dead or dying fish.

In late October, we inventoried thehigh density pen by passing all of the

'Tests were per10rmed with direct isolates from kid·ney smears on Tryptose Blood Agar and Mueller­Hinton's Agar. Samples collected 1·2 November1972. I = 1.5 percent NaCI; II = 2.0 percent NaCI; III=3.0 percent NaCL + = good colony growth; - =nogrowth. Agar pigmentation: D = dark; M =medium; L= lighl; N = none. Antibiotic sensitivity discs (distanceis the radius in mm): T", = 30 1"9 oxytetracycline; SSS= 250 1"9 triple sulla; F '00 = 100 1"9 furizolidone.

~ Of O[AO OR OYING fiSH WITH POSITlV[ fURUNCULOSIS

June Ju Iy Au<;!uSl September October NoyemberCULTURE PERIOD

I I,80

60

40

20

20 nof-----'L--~---J1'--~-~­

'00

Figure 2.-Incidence of vibriosis and furun­culosis in addition to estimated rates of mor­tality in the high and low density pens inrelation to season and seawater temperature.Note that the declining seawater temperaturein the fall did not inhibit an epizootic oi furun­culosis.

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60

so numerous that it was difficult to es­timate mortality rates or standing popu­lations. Deterioration of dead fish wasso rapid that many were never recov­ered. Therefore, lO-day mortality ratesin Figure 2 are based on estimates of thestanding population. Mortality rateswere generally in excess of 1 percent(~O.I percent/day), and the rate ofmortality in Pen II was always higherthan in Pen I, with the exception of aperiod in October (Fig. 2).

Our procedure for treatment, basedon past experience, was to use OMP­TM-50D exclusively to combat vib­riosis. Normally, an OMP-TM-50Ddiet was only necessary for a period of5-7 days to control the disease. Rarelyhad oral medication been used morethan three times during a growing sea­son (summer-fall). However in thisstudy, the mortalities were so numerousthat we doubted we could control thedisease with medicated feeds.

Figure 2 clearly indicates that thedominant problem was furunculosis.When the first epizootics of furuncu­losis appeared in July, we began test­ing cultures immediately. We foundthat the furunculosis organism was: I)Resistant to oxytetracycline (whereas

54 Marine Fisheries Review

200

June July AU9 Sept Oct Nov Dec Jon Feb MOt

1972 1973

CULlURE PERIOD

LITERATURE CITEDEgidius, E., and K. Andersen. 1975. Report on

an epizootic of vibriosis in the young saithepopulation along the Norwegian coast. Int.Counc. Explor. Sea. C. M. 1975/EE:43, p.1-7.

Evelyn. T. P. T. 1971a. An aberrant strain of thebacterial fish pathogen Aeromonas sal­monicida isolated from a marine host, the sa­blefish (Anoplopomo fimbria), and from twospecies of cultured Pacific salmon. J. Fish.Res. Board Can. 28: 1629-1634.

----. 1971b. First records of vibriosis inPacific salmon cultured in Canada, andtaxonomic status of the responsible bacterium,Vibrio anguillarum. 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., U.S. 74:447-449.

Kennedy, W. A. 1974. Sablefish culture-finalreport. Fish. Res. Board Can., Tech. Rep.452, 15 p.

McCraw, B. M. 1952. Furunculosis offish. U.S.Fish Wildl. Serv., Spec. Sci. Rep. Fish. 84,87 p.

Moring, J. R. 1973. Aspects of growth, and theeffects of some environmental factors on pen­reared chinook salmon, Oncorhynchustshawytscha (Walbaum), in Puget Sound,Washington. Ph.D. thesis, Univ. Wash., Seat­tle, 225 p.

Novotny, A. J. 1975. Net-pen culture of Pacificsalmon in marine waters. Mar. Fish. Rev.37( 1):36-47.

Scott, M. 1968. The pathogenicity ofAeromonassalmonicida (Griffin) in sea and brackish wa­ters. J. Gen. Microbiol. 50:321-327.

Snieszko, S. F. 1969. Fish furunculosis. U.S.Fish Wildl. Serv., Fish. Disease LeaR. FDL­17,4 p.

Wedemeyer, G. A. 1970. The role of stress in thedisease resistance of fishes. In S. F. Snieszko(editor), A symposium on diseases of fishesand shellfishes, p. 30-35. Am. Fish. Soc.,Spec. Publ. 5.

Wedemeyer, G. A., and J. W. Wood. 1974.Stress as a predisposing factor in fish diseases.U.S. Fish Wildl. Serv., Fish. Disease LeaR.FDL-38, 8 p.

monicida isolated in this study is resis­tant to oxytetracycline and sulfa com­pounds but sensitive to furazolidone.Severe combined epizootics of vib­riosis and furunculosis could not be ef­fectively controlled by feeding medi­cated diets containing oxytetracycline.

4) These observations suggest that aserious effort must be put forth to con­trol furunculosis in fresh water.

MFR Paper 1297. From Marine Fisheries Review, Vol. 40, No.3, March 1978.Copies of this paper, in limited numbers, are available from 0822, User Ser­vices Branch, Environmental Science Information Center, NOAA, Rockville,MO 20852. Copies of Marine Fisheries Review are available from the Superin­tendent of Documents, U.S. Government Printing Office, Washington, DC20402 for $1. 10 each.

I) Juvenile chinook salmon arehighly susceptible to vibriosis andfurunculosis when subjected to exces­sive physical, environmental, and/orosmotic stress following transfer toseawater culture systems. Seawatertemperatures in excess of 12°C contri­bute to the severity of epizootics of vib­riosis and furunculosis. Epizootics offurunculosis recurred in the fall, in spiteof declining seawater temperatures.

2) Furunculosis can be carried by thehost fish in a latent state from fresh tosaltwater. Furthermore, the growth offurunculosis on culture media contain­ing 20-30%0 salt and other evidencesuggests that the organism may betransmitted in saltwater.

3) Drug sensitivity tests dem­onstrated that the strain of A. sal-

ses and the fact that the average size ofthe juvenile chinook salmon that weretransferred to the net pens was consi­dered to be too small for successfuladaptation to saltwater. (Juvenilechinook salmon should average at least8 g when transferred to seawater.) Os­motic stress is an important factor inpredisposing fish to disease (Wede­meyer, 1970; Wedemeyer and Wood,1974).

Approximately 20 percent of the fishwere tagged, and the entire populationwas released between May and July1973. The fish that were released wereapparently healthy survivors. On thebasis of actual tag recoveries, Moring(1973) estimated a O. I percent con­tribution to the sport fishery, and con­tributions from the tag recoveries of thereleased density-study fish were 0.3percent. However, neither of these con­tributions is high. Moring (1973) notesthat chinook salmon reared for 9months in pens at Manchester and re­leased in April 1971 had a 12 percentrecovery rate in the sport fishery-arecovery 40 times greater than that ofthe fish used in the density study.

CONCLUSIONS

Feb Mo.

1973CULTURING PERIOD

June July Aug S~ OCI Nov Dec

1972

• Hiott density pen

• Low density pen

A Averoge growth of severol stocks ofchinooks cultured In previous yeors

Figure 5.-Growth of chinook salmon in thedensity tests. A comparison is made with av­erage growth rates for previous years.

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Figure 4.-Estimated loading densities ofchinook salmon in the two pens during thespring-winter culturing periods. No physicalinventory of the low density pen was taken inNovember, and the fall estimate of this pen isprobably in error.

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loading density study was comparedwith the average growth of severalstocks of chinook salmon cultured inprevious years (Fig. 5), there is a de­pression in the rate of growth in thedensity of fish. Moring's (1973) aver­age fish weights were 145 g in February1972 and 92 g in February 1973. Bycontrast, the average weights of the PenI and Pen II chinook salmon in February1973 were 80 g and 59 g, respectively.

We felt that we had been unsuccess­ful in combating the infectious diseasesfor three primary reasons: I) Environ­mental stress caused by low dissolvedoxygen, 2) a carrier state of furun­culosis, and 3) high loading densities.Secondary factors were handling stres-

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