Vol. 55, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1989, p. 1400-14050099-2240/89/061400-06$02.00/0Copyright ©) 1989, American Society for Microbiology

Protection of Rainbow Trout against Vibriosis and Furunculosis bythe Use of Attenuated Strains of Vibrio anguillarum

ANDERS NORQVIST,* AKE HAGSTROM, AND HANS WOLF-WATZ

Department of Microbiology, University of Umea, 5-901 87 Umea, Sweden

Received 28 November 1988/Accepted 3 March 1989

The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Threedifferent avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or asantibiotic-resistant mutants (VAN1000), were isolated by screening 200 individual isolated mutants foravirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunityagainst the homologous as well as a heterologous strain of V. anguillarum. When VAN1000 was used, protectiveimmunity could be recorded 1 week after bath vaccination with 107 bacteria per ml of water for 30 min. Asingle-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains ofV. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested andconfirmed that VAN1000 also was able to induce protective immunity against challenge with Aeromonassalmonicida.

Vibrio anguillarum is an important infectious agent, caus-ing vibriosis in farmed fish. Vaccines against bacterial dis-eases are playing an increasingly important role in thefarming of fish, and vaccines against vibriosis and enteric redmouth have been developed. However, improved vaccinesagainst furunculosis are still needed. So far, vaccines againstvibriosis have been Formalin-killed bacteria or membraneconstituents (2). The efficacy of these vaccines has beentested, and the route of administration of the vaccine playsan important role in the result of the vaccination. Injection ofthe bacterin gives the best result with the longest duration ofprotection, whereas immersion and oral administration giveless protection (12, 20).During recent years, interest has increased in the use of

live, attenuated bacterial strains as vaccines in human dis-eases, especially enteric diseases (9, 18). To test the possi-bility of constructing and using a live antivibriosis vaccineconsisting of attenuated bacteria, we mutated a strain of V.anguillarium and isolated avirulent offspring by a screeningprocedure. Three avirulent mutants were obtained andtested as live, attenuated vaccines in terms of the degree andduration of protection and the presence of cross-protectionagainst homologous as well as heterologous strains of V.anguillarum and Aeromonas salmonicida.An experimental setup for screening a substantial number

of different fish-pathogenic strains was also developed.

MATERIALS AND METHODS

Bacterial strains. The strains used in this study and theirsources are listed in Table 1. All of the fish-pathogenicbacteria used in this study had been passaged once in fishbefore being used in infections of fish or in genetic experi-ments.

Conjugation and transposition experiments. PlasmidpRK2013 was transferred from Escherichia coli K-12C600(pRK2013::Tn5-132) to V. anguillarum by conjugationon filters (19). The donor and recipient were cultured over-night in Luria broth and brain heart infusion broth supple-mented with 2% NaCl, respectively, inoculated into fresh

* Corresponding author.

media, and grown to 1 x 108 bacteria per ml. A mixturecontaining 1.5 ml each of donor and recipient was passedthrough a 0.45-.zm-pore-size membrane filter. The filter wasplaced for 3 h at 18°C on Trypticase soy agar (TSA; BBLMicrobiology Systems) plates supplemented with 2% NaCl.After incubation, the filter was transferred to TSA platessupplemented with 2% NaCl plus tetracycline (15 Kg/ml),rifampin (200 ,ug/ml), and streptomycin (100 pKg/ml) to selectfor V. anguillarum exconjugants. Colonies appearing on thefilter were transferred to new TSA plates supplemented with2% NaCl plus tetracycline, rifampin, and streptomycin.After incubation, the exconjugants were frozen at -70°Cbefore being used in experimental infections of fish.

Construction of antibiotic-resistant mutants. Approxi-mately 1010 V. anguillarum 775 organisms were spread onTSA-2% NaCl plates containing rifampin (200 pLg/ml). Col-onies growing on the plates were picked and restreaked onTSA-2% NaCl-rifampin plates. A number of these rifampin-resistant colonies were spread on TSA-2% NaCl-rifampin-streptomycin (100 ,ug/ml) plates. Colonies appearing onthese plates were restreaked and then frozen at -70°Cbefore being used in experimental infections of fish.

Experimental infections of fish. Experimental infectionswere performed by using 10-liter plastic bags assembled in aplastic rack (Fig. 1). The water was added and removed by asimple pumping system. The bags were oxygenated byassembled aeration stones.The bacterial challenges were carried out with rainbow

trout (Salmo gairdneri) weighing 5 to 15 g each. The fishwere tempered up from the holding tank temperature, whichwas 0 to 3°C, to 18°C during the 12 h before infection. Whenimmersion infections were performed, the bags were filledwith 2 liters of water, five fish were put into each bag, andthe bacteria were added. After an incubation time of 30 min,the water containing the bacterial suspension was removedand 5 liters of fresh water was added. For intraperitonealinfections, fish were anesthetized with tricaine methanesulfonate (100 mg/liter), and 0.1 ml of bacteria was injectedinto the peritoneal cavity. The fish were then placed into 5liters of fresh water. Experiments were normally run for 7

1400

on March 28, 2021 by guest

http://aem.asm

.org/D

ownloaded from

VACCINATION OF RAINBOW TROUT 1401

TABLE 1. Bacterial strains used in this work

Strain Source' Markers

V. anguillarum775 J. L. Larsen,

Copenhagen775.17B This study Rif' Strr derivative of 775"VAN1000 This study Rif' Strr derivative of 775"VAN20 This study Exconjugant (see text)VAN70 This study Exconjugant (see text)NB10 This study53-507 J. L. LarsenATCC 19264 ATCC

A. salmonicidaNCMB 1102 NCMBNCMB 1110 NCMBNB8601 This study

E. coli K12 C600 B.-E. Uhlin, Umea pRK2013::Tn5-132 TetrAmprc

"ATCC, American Type Culture Collection, Rockville, Md.; NCMB,National Collection of Marine Bacteria, Torry Research Station, Aberdeen,Scotland.

b Chromosomal resistance to rifampin and streptomycin.' Plasmid resistance to tetracycline and ampicillin.

days, and during that time, no fish died in control bagswithout bacteria.The bacteria were 24-h cultures grown in Trypticase soy

broth (BBL) supplemented with 2% NaCl (V. anguillarum)or Luria broth (A. salmonicida) at 18°C. Dead fish wascollected each day, and kidney samples were tested for thepresence of bacteria. For determination of the 50% lethaldose (LD50), three different dilutions of bacteria were usedto infect five fish at each dilution. LD50 calculations weremade by the method of Reed and Muench (14).

Vaccination experiments and PI. Fish were immersed for30 min at 18°C in water containing 107 avirulent mutants of

FIG. 1. Experimental setup for fish infections. A system forscreening large numbers of mutated bacteria for their virulence inrainbow trout was developed. The principle for the system was tokeep the water volumes during and after infection as small as

possible throughout the experiment. The handling of the infectionswas rationally designed, so that the same plastic bag was used bothduring and after infection. This was made possible by using a simplepumping system. The stress factors for the fish caused by thesuction off of the infected water and addition of fresh water were notsevere enough to cause death in any uninfected fish treated in thesame way as the infected fish.

V. anguillarum (VAN20, VAN70, or VAN1000) per ml.Thereafter, the fish were transferred to tanks and held at 0 to30C.

Vaccinated and nonvaccinated fish were challenged byimmersion 1, 2, 4, 10, 12, 17, and 24 weeks after vaccination.The bacterial strains used for challenge were V. anguillarum775.17B and NB1O and A. salmonicida NB8601.The results of the immunization are given as a protection

index (PI = LD50 for vaccinated fish/LD50 for nonvaccinatedfish) instead of the real LD50s. The reason for this is that theLD50 differed during the experiments, leading to higherLD50s for both the vaccinated and the control fish during thewarmer season. However the LD50 differences betweenvaccinated and nonvaccinated fish were of the same magni-tude during the experiments.

Isolation of outer membrane proteins. Outer membraneproteins were prepared essentially by the method of Filip etal. (7).SDS-PAGE. Outer membrane proteins were analyzed by

sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) by the method of Laemmli and Favre (13) with12% acrylamide and 0.3% bisacrylamide. The gels were runat 7 mA for 20 h. Proteins were fixed in 40% methanol-10%acetic acid, stained with 0.1% Coomassie brilliant blue in40% methanol-10% acetic acid, and destained in 40% meth-anol-10% acetic acid.

Preparation of antisera. The antigens that were used werewhole Formalin-killed cells of V. anguillarum 775.17Bgrown in TSA-2% NaCl to the stationary phase. Beforeimmunization, a serum pool was taken from the rabbit.Antigens were administered with or without complete Fre-und adjuvant at a final concentration of 108 bacteria per mlby using the following schedule: day 1, 0.5 ml of antigen incomplete adjuvant in each hind footpad; day 3, 5, 7, two0.5-ml doses of antigen in complete adjuvant subcutane-ously; days 14 and 16, 0.5 ml of antigen intramuscularly withno adjuvant; and day 18, two 0.5-ml doses of antigenintramuscularly with no adjuvant. Further intramuscularinjections of 0.5 ml of antigen were given weekly up to week6. The rabbit was test bled on day 14 and, starting on day 21,was bled every 10 to 12 days from the ear veins. Boosters(1.0 ml of antigen, no adjuvant) were given intramuscularlyat monthly intervals.Western blotting. Western immunoblotting of outer mem-

brane proteins after SDS-PAGE was performed as describedby Swanson et al. (16). Rabbit antiserum diluted 1:500 wasused.

RESULTS

Construction of transposon mutants. V. anguiillarum775.17B was mated with E. coli C600(pRK2013::Tn5-132)harboring the transposon TnS-132 (tetracycline resistance).TnS-132 is carried on a transposon delivery vector,pRK2013, which is based on the ColEl replicon and has abroad-host-range transfer system. It can therefore be trans-ferred to many nonenteric gram-negative bacteria. In strainsin which the vector is unable to replicate, selection forresistance against tetracycline results in transposition (6).Conjugations were performed on filters, which gave a fre-quency of transposition of about 10-7. The donor andrecipient were mixed on a filter and placed on nonselectivemedia for 3 h. The filters were transferred to a selective plateand incubated at 18°C. They were incubated on selectivemedia, and each colony that appeared was certain to be aunique exconjugant. The exconjugants were picked and

VOL. 55, 1989

on March 28, 2021 by guest

http://aem.asm

.org/D

ownloaded from

1402 NORQVIST ET AL.

restreaked on a selective plate. In parallel, the recipientstrain, 775.17B, was also passaged for the same number oftimes on plates, to ensure that an eventual change inpathogenicity of the exconjugants was not a result of toomany passages. Plasmid preparation of the exconjugantsshowed that the delivery plasmid vector pRK2013 did notreplicate in strain 775.17B (data not shown).

Isolation of antibiotic-resistant mutants. It has previouslybeen shown that isolation of spontaneous rifampin- or strep-tomycin-resistant mutants of pathogenic bacteria may leadto avirulence (3, 4). To test whether this strategy for isolatingavirulent mutants also could be applied to strains of V.anguiillariin, we used two steps to isolate mutants of strain775 resistant to rifampin (200 p.g/ml) and streptomycin (100p.g/ml). Spontaneous rifampin-resistant mutants were firstisolated, and then rifampin-streptomycin-resistant doublemutants were isolated. In each step, the frequency of muta-tion to resistance was 10-8.

Screening for avirulent exconjugants. We screened 200transposon and antibiotic-resistant mutants for their viru-lence properties by infecting rainbow trout. The challengewas carried out at 18°C by immersing fish for 30 min in watercontaining 5 x 106 bacteria per ml. The fish were monitoreddaily for mortality, and the experiments were run for 7 days.Two of the tested transposon mutants and one of theantibiotic-resistant mutants were found to have reducedvirulence. We subjected these three mutants, VAN20 andVAN70 (transposon mutants) and VAN1000 (antibiotic-re-sistant mutant), to further tests of their virulence propertiesby infecting fish both intraperitoneally and by immersion.The LD50 for intraperitoneal infection and immersion ex-ceeded 1 x 106 and 1 x 108 bacteria per ml, respectively, forall strains. The corresponding LD50s for the parental strainwere <1 x 102 bacteria and 2 x 106 bacteria per ml forintraperitoneal infection and immersion, respectively. Thus,strains VAN20, VAN70, and VAN1000 were all avirulent.Use of avirulent strains of V. anguillarum as live, attenuated

vaccines. VAN20, VAN70, and VAN1000 were tested fortheir potential ability as live vaccines against vibriosis. Fishwere immersed at 18°C for 30 min in a bacterial suspensioncontaining VAN20, VAN70, or VAN 1000 at 107 bacteria perml. Challenge was performed 1, 2, 4, 10, 17, and 24 weeksafter vaccination. Two strains were used as challenge organ-isms, the homologous strain 775.17B and the heterologousstrain NB10. The latter strain is an isolate from the Sea ofBothnia and exhibits LD50s of about 1 x 102 bacteria per mland <1 x 102 bacteria for infection by immersion andintraperitoneal injection, respectively. The challenge wascarried out by bathing the fish in water containing threedifferent concentrations of the respective test strain. Thismade it possible to determine the LD50s for the vaccinatedfish compared with the nonvaccinated fish. For strainsVAN20 and VAN70, the vaccinated fish showed an in-creased resistance against the homologous strain 2 weeksafter immunization. This was observed as a 10- to 30-foldincrease in the LD50 (Fig. 2A and B).

After 4 and 10 weeks, protection by VAN20 was stillsignificant, while protection by VAN70 was intermediate. At12 weeks after immunization, the fish were boostered withnew doses of VAN20 and VAN70. This gave protectionupon challenge 17 and 24 weeks after the initial immuniza-tion.When the immunized fish were challenged with the heter-

ologous strain, NB1O, no significant protective effect wasobserved after the first immunization (Fig. 3A and B). Afterthe booster dose had been given, protection was observed,

A

"00

U10*-4

*-

2

1001 C

75.

50

25

ND ND IND

1 2 4 10 12 17

Weeks after immunization

ND

24

FIG. 2. Immunization with V. anguillarum VAN20 (A), VAN70(B), and VAN1000 (C). Protective immunity of fish after challengewith V. angiiillarurn 775.17B is shown. The effects of the vaccina-tion are given in terms of the P1. Thus, the PI for nonvaccinatedcontrol fish is always 1. Booster vaccination with VAN20 andVAN70 was performed 12 weeks after the first immunization. The P1after the booster is shown (ED). LD50s in nonvaccinated fish afterchallenge with 775.17B were as follows: 3 x 106 cells per ml (week1), 2 x 106 cells per ml (week 2), 5 x 10' cells per ml (week 4), 5 x106 cells per ml (week 10), 2 x 106 cells per ml (week 17), and 2 x106 cells per ml (week 24). ND, Not determined.

first for VAN70-immunized fish but also later for the fishimmunized with VAN20. VAN1000 generated a good pro-tection after 4 weeks against both the homologous and theheterologous strain (Fig. 2C and 3C). VAN1000 also gaveprotection which lasted at least 17 weeks, although nobooster dose was given.Immunoblotting experiments. Since we observed protec-

tive immunity against a homologous as well as a heterolo-gous strain of V. anguillarum, we were interested in theimmunological relatedness between some different isolatesof V. angiiillarurn and also the possibility of cross-reactionwith another member of the family Vibrionaceae which is asignificant fish pathogen: A. salmonicida. Antiserum ob-tained after immunization of a rabbit with whole cells of V.anguillaruim 775.17B was used in immunoblotting experi-ments in which separated outer membrane proteins afterSDS-PAGE analysis were used as the antigen source. Outermembrane preparations from four different isolates of V.anguillarurn, two strains of A. salmonicida subsp. salmoni-cida, and one strain of A. salmonicida subsp. achromogeneswere studied. All isolates of V. anguiillarium exhibited a very

APPL. ENVIRON. MICROBIOL.

on March 28, 2021 by guest

http://aem.asm

.org/D

ownloaded from

VACCINATION OF RAINBOW TROUT 1403

X 4'0= 3

10100

7 ND ND Booster10

1o6. C

105

41.0

310

210

0 ND ND ND ND

1 2 4 10 12 17 24

Weeks after immunizationFIG. 3. Immunization with V. anguillarum VAN20 (A), VAN70

(B), and VAN1000 (C). Protective immunity of fish after challengewith V. anguillarum NB1O is shown. The results are given as PIvalues. Booster vaccination with VAN20 and VAN70 was per-formed 12 weeks after the first immunization. The PI after thebooster is shown (E). LD50s in nonvaccinated fish were as follows:<10 cells per ml (weeks 4, 10, and 17) and 2 x 102 cells per ml (week24). ND, Not determined.

similar antigenic response. Also, three strains of A. salmo-nicida gave detectable bands in the immunoblot, indicatingthat V. anguillarum and A. salmonicida had antigenic deter-minants in common (Fig. 4).

Protection of Vibrio-vaccinated fish against furunculosis.The results of the immunoblotting experiments suggestedthat it might be possible to immunize fish against vibriosis byusing a live vaccine and also to obtain protection againstdisease caused by A. salmonicida. Fish were thereforeimmunized with strain VAN1000 by following the protocoloutlined above. This strain was used because it gave the bestprotection against vibriosis (Fig. 2C and 3C). Protectiveimmunity was observed against both the homologous and theheterologous strains of V. anguillarum, and protectionagainst challenge with A. salmonicida could also be demon-strated (Fig. 5). An interesting observation was that signifi-cant protection against both V. anguillarum and A. salmo-nicida was already present 1 week after immunization. Theduration of the protection was at least 12 weeks.

DISCUSSION

We show in this report that attenuated strains of V.anguillarum 775, are capable of inducing protective immu-

FIG. 4. Immunoblotting after SDS-PAGE of outer membraneproteins, using rabbit antisera directed against whole cells of V.anguillarum 775.17B. Lanes: A, V. anguillarum ATCC 19264; B, V.anguillarum NB10; C, V. anguillarum 775.17B; D, V. anguillarum53-507; E, A. salmonicida NCMB 1102; F, A. salmonicida NB8601;G, A. salmonicida NCMB 1110.

nity against different strains of V. anguillarum as well asagainst A. salmonicida subsp. salmonicida. All three aviru-lent mutants, VAN20, VAN70, and VAN1000, were able tomediate protective immunity, although at different levels,after bath vaccination of S. gairdneri. The best results wereobtained when the antibiotic-resistant strain VAN1000 wasused. A single dose of vaccine at a final concentration of 107bacteria per ml of water and treatment for 30 min at 18°C wasenough to protect the fish for at least 12 weeks against bothvibriosis and furunculosis.

In this study we chose to determine and calculate the LD50of the challenge bacteria when infecting vaccinated andnonvaccinated fish. This allowed us to distinguish differentlevels of immunity of the fish after the use of differentvaccine strains. Our results are presented as the PI ratherthan the relative percent protection (RPP) (1). The PI valueis calculated as the fraction between the increased LD50 aftervaccination over the LD50 for nonvaccinated fish afterchallenge. By using LD50 measurements and PI values, wecan compensate for changes in experimental parameterssuch as water quality, temperature, fish size, exposure time,degree of virulence of the challenge strains, etc., duringlong-term vaccination studies, because the PI value gives arelative measure of protection at each challenge time. Inaddition, the PI value gives a quantitative assessment of thelevel of protective immunity, which is the most importantparameter in vaccine trials. Since the LD50 determinationsrequire more challenge facilities than the normal challenge

VOL. 55, 1989

...

,4"itt.. .--,

.'.

on March 28, 2021 by guest

http://aem.asm

.org/D

ownloaded from

1404 NORQVIST ET AL.

107

106

105xA.C 104.O

10

101

10°ND

2 12

Weeks after immunization

FIG. 5. Effect of immunization of fish with V. anguillarulmVAN1000 after challenge with V. angiuillacrum 775.17B ( S), V.angiillariin NB1O (E), and A. sahnonicida NB8601 (l). Thevalues are given as a PI after challenge. LD50s in nonvaccinated fishwere as follows: V. angiuillarum 775.17B: 2 x 106 cells per ml (week1), 5 x 105 cells per ml (week 2), 2 x 106 cells per ml (week 5), and2 x 106 cells per ml (week 12); V. anguillarunm NB10: <10 cells perml (weeks 1, 2, and 5) and 2 x 102 cells per ml (week 12); A.salmnonicida NB8601: 1 x 102 cells per ml (weeks 1 and 5) and3 x 102 cells per ml (week 12). ND, Not determined.

experiments with one fixed challenge dose, a novel experi-mental setup was designed to facilitate these experiments(see Fig. 1 for details).

If international challenge test strains for vaccine purposescould be agreed upon, the PI value could be useful whenvaccine trials in different laboratories are compared, sinceuse of the PI reduces the differences among different labo-ratories. Thus, comparison of our vaccine based on the live,attenuated vaccine strain VAN1000 with results of otherstudies would be facilitated.

If we give our protection calculated as RPP values (1), theRPP for challenge with V. anguillarlim would be 100, whilethe RPP for furunculosis would vary from 60 to 100 depend-ing on the challenge dose used. However, the high PI value(about 1,000) suggests that full protection would be obtainedagainst natural infections in fish farms. Thus, vaccinationwith VAN1000 gives a protection which is at the same levelas that for other vibriosis vaccines (10, 11), but the effectseems to be elevated with respect to furunculosis (1). The PIvalues can in some cases underestimate the protective effectof a vaccine if the challenge strain has a comparatively highLD50. This can be observed for PI values after challengeagainst strain 775.17B, for which the LD50 for infection byimmersion is about 2 x 106 bacteria per ml for nonvaccinatedfish and immersion challenge doses higher than 2 x 108/mlare difficult to use because of oxygen limitation and othertoxic effects of high bacterial density.

Fish vaccines can be delivered by different routes: orallyin food, by intraperitoneal injection, or by immersion. Usu-ally, intraperitoneal injection gives the best results withrespect to protection and duration, but for practical reasons

this route has its limitations. However, in contrast, bathvaccination is easy to carry out. The vaccine presented heregives a high degree of protection after bath vaccination,although a relatively small dose is used, probably becausethe bacteria multiply in the fish after infection and thus thevaccine signal is amplified in situ. However, the mutant doesnot cause any disease symptoms and is probably cleared by

the fish defense mechanisms after a short period, since wehave been unable to demonstrate the presence of bacteriaafter vaccination.The use of a live, attenuated vaccine strain can of course

be a matter of discussion. However, V. anguillarum 775 hasa considerably higher LD5( after bath infection than does afresh clinical isolate, strain NB10. Thus, strain 775 is alreadyattenuated after years under laboratory conditions, and it isa matter of definition whether this strain should be consid-ered virulent. Strain VAN1000 is less virulent than strain775. The mutation frequency to rifampin resistance andstreptomycin resistance is about 10-8 in each step, and thereversion frequency is of the same order. Furthermore, wehave been unable to isolate spontaneous revertants duringthese studies. Therefore, if the strain is cultivated underselective conditions, the probability of getting a revertantafter vaccination is extremely low. Consequently, it shouldbe possible to use strain VAN1000 as a safe live, attenuatedvaccine.There is a limited correlation between protective immu-

nity and the level of antibodies in fish after vaccination (5,15, 17), and it has been shown in several studies that the fishelicit a nonspecific immune response upon injection ofnon-antigen-containing agents such as mineral oil-based ad-juvants (1). However, stimulation of this nonspecific im-mune reaction gives a limited protection against furunculosis(1). We also observed a good protective immunity againstfurunculosis, suggesting that a specific immunity was ob-tained. This is supported by the fact that V. anguillaruim andA. salmonicida had surface-located antigenic determinantsin common. We suggest, on the basis of the high level ofprotection, that the observed protective immunity againstfurunculosis was caused at least in part by induction ofspecific immunity. Whether this is due to humoral or cellularactivity is at present not known, but the fact that a live strainof V. anguillarumn induces a high protective immunity, whichis even higher than when killed A. salmonicida bacteria areused, indicates the possibility that cellular immune defensemechanisms, which are stimulated by the replicating bacte-ria, are involved in protection. It is well known that cellularimmunity in mammals can be induced when live, attenuatedbacteria are used as vaccines (8). This assumption is alsosupported by the findings that a killed Vibrio vaccine medi-ates only a limited protection against furunculosis (1).The avirulent mutants used in this study were isolated by

a screening procedure in which individual isolates wereallowed to infect groups of five fish. Mutants showing areduced virulence were tested further, and 3 of the 200mutants were found to be avirulent. Two of these, VAN20and VAN70, were transposon generated. VAN1000 was aspontaneous rifampin-streptomycin-resistant double mutant,in which the mutation from virulence to avirulence appearedin the second step in the mutation to streptomycin resis-tance. In the latter group, avirulent offspring appeared at afrequency of 25%, whereas in the former group, about 1% ofthe transposon-generated mutants were found to be aviru-lent. The underlying molecular mechanisms leading to avir-ulence are at present unclear. It is likely that VAN1000 hasa defect in the regulation of virulence-associated moleculesrather than being a structural-gene mutant, since the mutantmost probably has a changed L12 ribosomal protein. Suchmutants have been observed before, e.g., in Yersiniapseiudotiuberelulosis and Y. pestis (3, 4), and in these twocases the mutations leading to antibiotic resistance had apleiotropic effect on the cells, resulting in avirulence.VAN20 and VAN70, on the other hand, are more likely to be

APPL. ENVIRON. MICROBIOL.

on March 28, 2021 by guest

http://aem.asm

.org/D

ownloaded from

VACCINATION OF RAINBOW TROUT 1405

structural-gene mutants of virulence-associated functions.The corresponding wild-type genes have been cloned, andthese genes and their products are presently being analyzedin our laboratory.

ACKNOWLEDGMENTS

We thank Stina Olofsson and Ulla Eriksson for their skillfultechnical assistance. We also thank Trevor Trust for critical readingof and comments on the manuscript.

This study was supported by The Swedish Council for Forestryand Agricultural Research and by The Swedish Board for TechnicalDevelopment.

LITERATURE CITED1. Adams, A., N. Auchinachie, A. Bundy, M. F. Tatner, and M. T.

Horne. 1988. The potency of adjuvanted injected vaccines inrainbow trout (Salmo gairdneri Richardson) and bath vaccinesin Atlantic salmon (Salmo salar L.) against furunculosis. Aqua-culture 69:15-26.

2. Agius, C., M. T. Horne, and P. D. Ward. 1983. Immunization ofrainbow trout, Salmo gairdneri Richardson, against vibriosis:comparison of an extract antigen with whole cell bacterins byoral and intraperitoneal routes. J. Fish Dis. 6:129-134.

3. Bolin, I., D. A. Portnoy, and H. Wolf-Watz. 1985. Expression ofthe temperature-inducible outer membrane proteins of yersin-iae. Infect. Immun. 48:234-240.

4. Brubaker, R. R., and M. J. Surgalla. 1962. Genotypic alterationassociated with avirulence in streptomycin-resistant Pasteurellapestis. J. Bacteriol. 84:615-624.

5. Croy, T. R., and D. F. Amend. 1977. Immunization of sockeyesalmon, Oncorhynchus nerka, against vibriosis using the hyper-osmotic infiltration technique. Aquaculture 12:317-325.

6. Ely, B. 1985. Vectors for transposon mutagenesis of nonentericbacteria. Mol. Gen. Genet. 200:302-304.

7. Filip, C., G. Fletcher, J. L. Wulif, and C. F. Earhart. 1973.Solubilization of the cytoplasmic membrane of Escherichia coliby the ionic detergent sodium lauryl sarcosinate. J. Bacteriol.115:717-722.

8. Hahn, H., and S. H. E. Kaufmann. 1981. The role of cell-mediated immunity in bacterial infections. Rev. Infect. Dis.3:1221-1250.

9. Hone, D., R. Morona, S. Attridge, and J. Hackett. 1987. Con-

struction of defined galE mutants of Salmonella for use asvaccines. J. Infect. Dis. 156:167-174.

10. Horne, M. T., M. Tatner, S. McDerment, C. Agius, and P.Ward. 1982. Vaccination of the rainbow trout, Salmo gairdneriRichardson, at low temperatures and the long-term persistenceof protection. J. Fish Dis. 5:343-345.

11. Johnson, K. A., J. K. Flynn, and D. F. Amend. 1982. Duration ofimmunity in salmonids vaccinated by direct immersion withYesinia ruckeri and Vibrio anguillarum bacterins. J. Fish Dis.5:207-213.

12. Kawano, K., T. Aoki, and T. Kitao. 1984. Duration of protectionagainst vibriosis in ayu, Plecoglossus altivelis, vaccinated byimmersion and oral administration with Vibrio anguillarum.Bull. Jpn. Soc. Sci. Fish. 50:771-774.

13. Laemmli, U. K., and M. Favre. 1973. Maturation of the head ofbacteriophage T4. J. Mol. Biol. 80:575-599.

14. Reed, L. J., and H. Muench. 1938. A simple method of estimat-ing fifty per cent endpoints. Am. J. Hyg. 27:493-497.

15. Sakai, M., T. Aoki, T. Kitao, J. S. Rohovec, and J. L. Fryer.1984. Comparison of the cellular immune response of fishvaccinated by immersion and injection of Vibrio anguillarum.Bull. Jpn. Soc. Sci. Fish. 50:1187-1192.

16. Swanson, J., L. W. Mayer, and M. R. Tam. 1982. Antigenicity ofNeisseria gonorrhoeae outer membrane protein(s) III detectedby immunoprecipitation and Western blot transfer with a mono-clonal antibody. Infect. Immun. 38:668-672.

17. Thuvander, A., T. Hongslo, E. Jansson, and B. Sundquist. 1987.Duration of protective immunity and antibody titres measuredby ELISA after vaccination of rainbow trout, Salmo gairdneriRichardson, against vibriosis. J. Fish Dis. 10:479-486.

18. Wahdan, M. H., C. Serie, Y. Cerisier, S. Sallam, and R.Germanier. 1982. A controlled field trial of live Salmonella typhistrain Ty 21a oral vaccine against typhoid: three-year results. J.Infect. Dis. 145:292-295.

19. Walter, M. A., S. A. Potter, and J. H. Crosa. 1983. Iron uptakesystem mediated by Vibrio anguillarum plasmid pJM1. J. Bac-teriol. 156:880-887.

20. Ward, P. D., M. F. Tatner, and M. T. Horne. 1985. Factorsinfluencing the efficacy of vaccines against vibriosis caused byVibrio anguillarum, p. 221-229. In M. J. Manning and M. F.Tatner (ed.), Fish immunology. Academic Press, Inc. (London),Ltd., London.

VOL. 55, 1989

on March 28, 2021 by guest

http://aem.asm

.org/D

ownloaded from