8 Laboratory and Field Observations

8/13/2019 8 Laboratory and Field Observations

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Laboratory and Field Observationson ntivibriosis VaccinesT.P.T. EVELYN and J.E. KETCHESON

IntroductionAttempts in British Columbia at rearing Pacific salmon in floating netpens in sea waterwere first undertaken on an experimental (pilot-scale) basis at the Pacific BiologicalStation, Nanaimo, in 1974. Objectives of the study were to determine just how rapidlymarket-size salmon could be produced and to determine whether all of the fiveindigenous species of Pacific salmon were suitable mariculture candidates. After onlyone year of operation, it appeared obvious that mariculture ventures would likely onlybe successful if effective control measures for infectious diseases such as furunculosis,bacterial kidney disease, and vibriosis could be developed. Consequently, studies toexamine the feasibility of using vaccination to control these diseases were initiated.

The purpose of this paper is to report our findings with respect to antivibriosisvaccines. Our experience had shown vibriosis to be a particularly acute problem, and,as with the US findings in Puget Sound, Washington (Harrell et aI., 1976; Harrell,1978), to be due to two serologically and biochemically distinct vibrios. In this paper,we refer to these vibrios as Vibrio anguillarum and Vibrio sp.

Materials and MethodsBacteriaTwo vibrios were used for vaccinating and challenging fish: V anguillarum, strain VI(Evelyn 1971), and Vibrio sp., strain 74/48. The latter was isolated in 1974 from a sea-water-reared sockeye salmon Oncorhynchus nerka) that had died of vibriosis. The vibrios were the biochemical and serological equivalents of the Puget Sound isolates 775and 1669, respectively (see Harrell et al., 1976), and their virulence was maintainedby passage in fish and storage at 90C.

Fish and Fish HandlingTest fish were reared from eggs obtained from stocks of British Columbia salmon. Thesockeye were derived from the Fulton River stock and the coho 0. kisutch) andchinook 0. tshawytscha)from ig Qualicum River stocks. During handling forI Department of Fisheries and Oceans, Pacific Biological Station, Nanaimo, B.C., Canada

W. Ahne (ed.),Fish Diseases

Springer-Verlag Berlin Heidelberg 1980


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46 T.P.T. Evelyn and J.E. Ketchesontagging, vaccination, and challenge, the fish were anesthetized with 2-phenoxyethanol(1/7,000). Tagging to denote the variously vaccinated and control fish was necessarybecause following (or during) challenge, fish in any given experiment were held in acommon tank or netpen to ensure that all treatments experienced the same environmental conditions. Fish in field experiments received coded wire nose tags; fish inshort-term laboratory experiments were appropriately fin-clipped.

Vaccines and Vaccination MethodsCultures serving as the source of vaccines were grown at 21 24 c in aerated brainheart infusion (BHI) broth (Difco) for 30 to 64 h. The cultures were then all killed bythe addition of formalin (0.3 V V .

Vaccines took several forms and were administered by injection, feeding, orimmersion_ Injected vaccines (intact cells, spent culture fluids) were given in a volumeof 0.1 ml and as a single intraperitoneal (IP) injection; in some cases, the vaccines alsocontained a Freund's-type adjuvant. Fed vaccines consisted of intact cells; the cells insaline were sprayed onto the partially dried fish diet (Oregon Moist Pellets), and thelatter was then fed to the fish for 14 or 15 days. Immersion-administered vaccines contained intact cells, disrupted cells, whole broth cultures, or spent culture medium(broth). They were administered to fish by a two-step hyperosmotic infiltration procedure very similar to that described by Croy and Amend (1977). The procedure involved a 2-min dip in oxygenated brine (7.1 NaCI and 0.9 Hanks' balanced salt mixture; pH 7.0) followed by a 3-min dip in oxygenated vaccine. The brine and vaccinewere used at or below ambient water temperature, and Antifoam B (Dow CorningCorp.) was added, as necessary, to suppress foaming.

Dosages of bacterial cells used in the vaccines (see Tables 1, 2, 3, 6, 7) are expressedin terms of wet weight. When disrupted cells were administered, the vaccine containedboth the soluble and insoluble cell components. Cell disruption was accomplished withglass beads in a cell homogenizer (Bronwill Scientific, Inc.). When spent broth wastested as an immunogen, t was first freed of cells by centrifugation and filtration(0.45 / lm pore diameter filter, Millipore Corp.). Further fractionation of the broth wasachieved by ultrafiltration using an XMlOOA membrane (Am icon Corp.). The ultrafiltered fractions were kindly provided by Dr. R. Lallier, University of Montreal,Faculty of Veterinary Medicine.Following vaccination, fish in the first of two field experiments were held in freshwater at 6C for 8 weeks and at 16C for 2 weeks until challenged. Vaccinated fish inthe second field experiment were held in freshwater at 12 _13C for a month untilchallenged. In the laboratory experiments, vaccinated fish were, with one exception(Table 3), held in water at 12 15.5 c until challenged.

Challenge MethodsThe efficacy of vaccination was determined by natural and experimental challenge.Natural challenge was used in two field experiments (Tables 1 and 2); it occurred whenfish were transferred to sea water during warmwater periods.


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Laboratory and Field Observations on Antivibriosis Vaccines 47Experimental challenge was accomplished by IP injection (0.1 ml). t was usually

effected by injecting vaccinated and control fish with a dose of vibrio cells sufficientto kill 70 -85 of the controls. In a few cases, the efficacy of vaccination was assessedby determining and comparing the doses (LDso values) of injected vibrio cells requiredto kill 50 of the vaccinated and control fish. LDso values were calculated by themethod of Reed and Muench (1938).

All mortalities resulting from natural and experimental challenge were cultured todetermine the cause of death and examined to identify the treatment received. Inaddition, fish receiving experimental challenge were held in water at 13 0 15.5 toallow the challenge to express itself. Experimental challenge experiments were terminated only when mortalities had ceased for 3 or 4 days.

Results and DiscussionTables 1 and 2 give the results of two experiments initiated in 1975 and 1976, respectively, to investigate, in part, the efficacy of vaccination against vibriosis under conditions of natural challenge in sea water.

In the first experiment, sockeye fingerlings, vaccinated as outlined in Table 1, weretransferred to sea water on August 21. Losses due to vibriosis occurred for 14 weeks.The major challenge came from Vibrio sp., which killed four times as many controlfish as V anguillllrum (Challenge from Aeromonas salmonicida did not occur.)

The results (Table 1) clearly demonstrate the value of vaccination. All vaccinesprovided Significant protection (P=


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48 T.P.T. Evelyn and J.E. KetchesonTable 2. Mortalities due to vibriosis among variously vaccinated 12.5-g sockeye salmon during theirfust 12 weeks of natural challenge in sea watera

Percent fish dead of:Vaccine treatmentb No. fish treated Va VxNone (control) 383 42.0 0Immersion method: c

2 dips in vaccine, 14 days apart 384 0.8 01 dip in vaccine 367 1.9 01 dip in vaccine diluted 10-1 378 4.2 01 dip in vaccine diluted 10-2 383 5.0 0

Single IP injection: dVaccine in saline 415 0.7 0Vaccine in adjuvant 421 0 0Vaccine (diluted 10-1 ) in adjuvant 465 0.2 0Vaccine (diluted 10-2 ) in adjuvant 450 1 1 0

OraleVaccine fed for 14 days 393 17.6 0

a Water temperatures at 8 m were ll.5 -12 C during epizooticb All vaccines contained cells (interact or disrupted) of 4 fish pathogens: V anguil/arum Vibrio

sp., A. salmonicida and the kidney disease bacteriumc The undiluted immersion vaccine contained 3.5 g of disrupted cells of each pathogen/liter of

0.1 NaCI. When the vaccine was diluted, the diluent was 0.1 NaCId Each fish was injected with 2.0 mg (or less as indicated) of cells of each pathogen suspended insaline or a Freund's-type adjuvante Each fish received a total of 14.0 mg of cells of each pathogen

to Vibrio sp. were reduced by 50 ). Such cross-protection has recently been reportedby Schiewe and Hodgins (1977) and by Harrell (1978). More interesting were theresults obtained with the univalent Vibrio sp. vaccine. This vaccine gave essentiallycomplete protection against both homologous and heterologous challenge, thus raisingthe possibility that it could be used in place of a bivalent vibrio vaccine. Cross-protectionby Vibrio sp. against V anguillarum has been demonstrated by Schiewe and Hodgins(1977), but the protection was only partial. The different findings may be due to thefact that we used an unheated vaccine, or may simply reflect an inadequate challengeby V anguillarum in the present experiment. Until this matter can be resolved the re-placement of a bivalent vibrio vaccine with the univalent Vibrio sp. vaccine would beunjustified.

Table 2 summarizes the results obtained in a second field experiment in whichvariously vaccinated sockeye fingerlings were transferred to sea water in July. On thisoccasion, natural challenge came only from V anguillarum but the challenge was substantial. Once again, significant protection (P


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Laboratory and Field Observations on Antivibriosis VaccinesTable 3. Relative immunity at 7 months post vaccination of sockeye salmona vaccinated against

ibrio sp.

49

Vaccinationmethodb

Relative increase in immunity(LDso control-LDso treated)None (control)IP injectionImmersionOral

10-7 010 2 010-3 610 ...6

lO S o-fold103.4 -fold10 2 4 -folda Fish averaged 12.5 g at vaccination and were held, post vaccination, in freshwater averaging

10 C (temperatures gradually fell from 17 0 to 4 C)b Fish received 2 mg of cells by injection and 14 mg of cells by feeding; the vaccine used in the

immersion method contained 3.5 g of disrupted cells/liter of 0.1 NaClc LDso values represent the dilution of a ibrio sp. challenge cell suspension required to kill 50of the test fish. Challenged fish were held in freshwater at 14 0 C

suited in virtually full protection (mortalities of only 1.1 ). This high degree of im-munogenicity was also evident with the immersion vaccine, which, following only oneapplication, provided significant protection even when diluted lOO-fold. The ability toeffect immunity using dilute vaccines (see also Croy and Amend, 1977) could reducethe costs of vaccinating fish against vibriosis.

The strength and duration of protection elicited by the various vaccination methodswas briefly investigated using the LDso approach and ibrio sp. (Table 3). The datasupport our field results which suggested that injection and feeding, respectively, conferthe highest and lowest levels of immunity. They differ in this respect from the fieldfindings of Antipa and Amend (1977) which indicated immersion to be more protective than injection. The data also suggest that antivibriosis immunity is quite durable.The durability may have been enhanced somewhat by the low water temperatures(4 7 0c) prevailing during the last 8 weeks of holding, but it could not have beenaffected by fish-pathogen contact because contact was precluded by holding the fish infreshwater.

Concerns regarding the duration of antivibrio immunity in sea water are probablyunnecessary because natural challenge with vibrios probably serves to elicit or perpetuate immunity. t has been shown that contact with living vibrios stimulates immunity(Braaten and Hodgins, 1976; Egidius and Andersen, 1979), and this phenomenon isprobably the major reason for the increased immunity displayed by our unvaccinated(control) fish during their second summer in sea water (Table 4).

The value of immersion vaccination as a means of mass immunization against vib-riosis was considerably increased when it was found that very small fish could be effectively immunized. Also, vaccine preparation was greatly Simplified when testsshowed that vaccines consisting of disrupted cells could be replaced by killed brothcultures. Early tests on the foregoing in our laboratory utilized 2.2 g chinook salmonthat had been dip-vaccinated in a killed broth culture of ibrio sp. At 17 days postvaccination, these fish were found by the LDso challenge method to be over 100times as resistant to homologous challenge as their unvaccinated counterparts. Strong


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50 T.P.T. Evelyn and J.E. KetchesonTable 4. Mortalities due to vibriosis among unvaccinated (control) sockeye salmon during their firstand second summers in sea waterSockeyea No. of summers V - , - - , - , i b ~ r , " " i o : . . . . : c : : h : : : a l l : : : e = = - n " , g c : : . e Percent fish dead (no. dead/no.(Experiment No.) in sea water No. Date challenged) of vibriosis

1 Aug. 26-Dec. 9, 1975 56.5 (257/455)2 2 Sept. I-Oct. 14, 1976 1.20/84

2 1 1 Sept. I-Oct. 14,1976 40.0057/3742 2 July 6-Aug. 18,1977 1.5 0/66

3b 1 July 6-Aug. 18,1977 19.3 (29/150)2

a Sockeye in the various experiments were held in adjacent netpensb Observations on this group of fish had to be prematurely terminated during a period of vibrio

challenge because of a sudden bloom of the phytoplankton Chaetoceros convo[utus whichkilled virtually all of the fish

Table 5. The immune response following immersion (dip-)vaccination with ibrio sp. of 1.5 -2.0-gcoho salmon held in diluted (20 0) sea water and in freshwater at 15C

No. of fish Percent killed by ibrio sp.Holding water Treatmenta treated challengeb

20 0 sea water Dip-vaccinated 73 OcFreshwater Dip-vaccinated 83 OcFreshwater Unvaccinated 78 85.5

a The immersion vaccine consisted of a killed 48-h ibrio sp. BHI broth culture, diluted 1/2 with0.1 NaCIb At 21 days post vaccination, all fish were converted to 20 0 sea water at 15C and challenged

by an IP injection of ibrio sp. (1.25 X10 5 cells/fish)c Chi square test: P=


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Laboratory and Field Observations on Antivibriosis Vaccines 51

Table 6. Tests on the immunogenicity of intact cells and spent culture fluid (broth) derived from akilled 48-h BHI broth culture of V. anguillarum

Method of No. of fishb Percent killed byVaccinea administration treated V. anguillarum challengeCIntact cells IP injection 20 OdSpent broth IP inject ion 20 OdIntact cells Immersion 20 5dSpent broth Immersion 20 5dNone 20 70a Intact cells were 3x saline-washed; they were injected 2 mg/fish) or used at 4 g/liter of

0.85 NaCl as an immersion vaccine. Injected vaccines contained a Freund s-type adjuvantb Coho salmon averaging 10 g were used; they held in freshwater at 13 _14C throughout the

experimentc Fish were challenged at 35 days post vaccination by injection 1.5x106 V. anguillarum

cells/fish)d Chi square tes t: P=


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52 T.P.T. Evelyn and J.E. Ketcheson: Laboratory and Field ObservationsEgidius Ee Andersen K 1979) Bath immunization - a practical and non-stressing method of

vaccinating sea farmed rainbow trout Salrno gairdneri Richardson against vibriosis. J FishDis 2:405-410

Evelyn TPT 1971) First records of vibriosis in Pacific salmon cultured in Canada, and taxonomicstatus of the responsible bacterium, Vibrio anguillarurn. J Fish Res Board Can 28 :51 7-525

Harrell LW 1978) Vibriosis and current salmon vaccination procedures in Puget Sound, Washington. Mar Fish Rev 40: 24-25Harrell LW Novotny AJ, Schiewe MH Hodgins HO 1976) Isolation and description of two vibrios

pathogenic to Pacific salmon in Puget Sound, Washington. Fish Bull US 74 :447 -449Reed LJ, Muench H 1938) A simple method of estimating fifty percent end points. Am J Hyg 27:

493-497Schiewe MH Hodgins HO 1977) Specificity of protection induced in coho salmon Oncorhynchus

kisutch) by heat-treated components of two pathogenic vibrios. J Fish Res Board Can 34:1026-1028

8 Laboratory and Field Observations

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