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

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1989, p /89/ $02.00/0 Copyright ) 1989, American Society for Microbiology Vol. 55, No. 6 Protection of Rainbow Trout against Vibriosis and Furunculosis by the Use of Attenuated Strains of Vibrio anguillarum ANDERS NORQVIST,* AKE HAGSTROM, AND HANS WOLF-WATZ Department of Microbiology, University of Umea, Umea, Sweden Received 28 November 1988/Accepted 3 March 1989 The fish pathogen Vibrio anguillarum causes a lethal infection in rainbow trout (Salmo gairdneri). Three different avirulent mutants, constructed by transposon insertion mutagenesis (VAN20 and VAN70) or as antibiotic-resistant mutants (VAN00), were isolated by screening 200 individual isolated mutants for avirulence. When used as live vaccines, all three avirulent mutants were able to induce protective immunity against the homologous as well as a heterologous strain of V. anguillarum. When VAN00 was used, protective immunity could be recorded 1 week after bath vaccination with 7 bacteria per ml of water for 30 min. A single-dose immunization was effective for at least 12 weeks. Western immunoblotting showed that strains of V. anguillarum have antigenic determinants in common with Aeromonas strains. Therefore, we tested and confirmed that VAN00 also was able to induce protective immunity against challenge with Aeromonas salmonicida. Vibrio anguillarum is an important infectious agent, causing vibriosis in farmed fish. Vaccines against bacterial diseases are playing an increasingly important role in the farming of fish, and vaccines against vibriosis and enteric red mouth have been developed. However, improved vaccines against furunculosis are still needed. So far, vaccines against vibriosis have been Formalin-killed bacteria or membrane constituents (2). The efficacy of these vaccines has been tested, and the route of administration of the vaccine plays an important role in the result of the vaccination. Injection of the bacterin gives the best result with the longest duration of protection, whereas immersion and oral administration give less protection (12, 20). During recent years, interest has increased in the use of live, attenuated bacterial strains as vaccines in human diseases, especially enteric diseases (9, 18). To test the possibility of constructing and using a live antivibriosis vaccine consisting of attenuated bacteria, we mutated a strain of V. anguillarium and isolated avirulent offspring by a screening procedure. Three avirulent mutants were obtained and tested as live, attenuated vaccines in terms of the degree and duration of protection and the presence of cross-protection against 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 their sources are listed in Table 1. All of the fish-pathogenic bacteria used in this study had been passaged once in fish before being used in infections of fish or in genetic experiments. Conjugation and transposition experiments. Plasmid prk2013 was transferred from Escherichia coli K-12 C600(pRK2013::Tn5-132) to V. anguillarum by conjugation on filters (19). The donor and recipient were cultured overnight in Luria broth and brain heart infusion broth supplemented with 2% NaCl, respectively, inoculated into fresh * Corresponding author media, and grown to 1 x 8 bacteria per ml. A mixture containing 1.5 ml each of donor and recipient was passed through a zm-pore-size membrane filter. The filter was placed for 3 h at 18 C on Trypticase soy agar (TSA; BBL Microbiology Systems) plates supplemented with 2% NaCl. After incubation, the filter was transferred to TSA plates supplemented with 2% NaCl plus tetracycline (15 Kg/ml), rifampin (200,ug/ml), and streptomycin (0 pkg/ml) to select for V. anguillarum exconjugants. Colonies appearing on the filter were transferred to new TSA plates supplemented with 2% NaCl plus tetracycline, rifampin, and streptomycin. After incubation, the exconjugants were frozen at -70 C before being used in experimental infections of fish. Construction of antibiotic-resistant mutants. Approximately V. anguillarum 775 organisms were spread on TSA-2% NaCl plates containing rifampin (200 plg/ml). Colonies growing on the plates were picked and restreaked on TSA-2% NaCl-rifampin plates. A number of these rifampinresistant colonies were spread on TSA-2% NaCl-rifampinstreptomycin (0,ug/ml) plates. Colonies appearing on these plates were restreaked and then frozen at -70 C before being used in experimental infections of fish. Experimental infections of fish. Experimental infections were performed by using -liter plastic bags assembled in a plastic rack (Fig. 1). The water was added and removed by a simple pumping system. The bags were oxygenated by assembled aeration stones. The bacterial challenges were carried out with rainbow trout (Salmo gairdneri) weighing 5 to 15 g each. The fish were tempered up from the holding tank temperature, which was 0 to 3 C, to 18 C during the 12 h before infection. When immersion infections were performed, the bags were filled with 2 liters of water, five fish were put into each bag, and the bacteria were added. After an incubation time of 30 min, the water containing the bacterial suspension was removed and 5 liters of fresh water was added. For intraperitoneal infections, fish were anesthetized with tricaine methane sulfonate (0 mg/liter), and 0.1 ml of bacteria was injected into the peritoneal cavity. The fish were then placed into 5 liters of fresh water. Experiments were normally run for 7

2 VOL. 55, 1989 TABLE 1. Bacterial strains used in this work Strain Source' Markers V. anguillarum 775 J. L. Larsen, Copenhagen B This study Rif' Strr derivative of 775" VAN00 This study Rif' Strr derivative of 775" VAN20 This study Exconjugant (see text) VAN70 This study Exconjugant (see text) NB This study J. L. Larsen ATCC ATCC A. salmonicida NCMB 12 NCMB 11 NB8601 NCMB NCMB This study E. coli K12 C600 B.-E. Uhlin, Umea prk2013::tn5-132 Tetr Amprc "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 bags without 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 was collected each day, and kidney samples were tested for the presence of bacteria. For determination of the 50% lethal dose (LD50), three different dilutions of bacteria were used to infect five fish at each dilution. LD50 calculations were made by the method of Reed and Muench (14). Vaccination experiments and PI. Fish were immersed for 30 min at 18 C in water containing 7 avirulent mutants of FIG. 1. Experimental setup for fish infections. A system for screening large numbers of mutated bacteria for their virulence in rainbow trout was developed. The principle for the system was to keep the water volumes during and after infection as small as possible throughout the experiment. The handling of the infections was rationally designed, so that the same plastic bag was used both during and after infection. This was made possible by using a simple pumping system. The stress factors for the fish caused by the suction off of the infected water and addition of fresh water were not severe enough to cause death in any uninfected fish treated in the same way as the infected fish. VACCINATION OF RAINBOW TROUT 1401 V. anguillarum (VAN20, VAN70, or VAN 00) per ml. Thereafter, the fish were transferred to tanks and held at 0 to 30C. Vaccinated and nonvaccinated fish were challenged by immersion 1, 2, 4,, 12, 17, and 24 weeks after vaccination. The bacterial strains used for challenge were V. anguillarum B and NB1O and A. salmonicida NB8601. The results of the immunization are given as a protection index (PI = LD50 for vaccinated fish/ld50 for nonvaccinated fish) instead of the real LD50s. The reason for this is that the LD50 differed during the experiments, leading to higher LD50s for both the vaccinated and the control fish during the warmer season. However the LD50 differences between vaccinated and nonvaccinated fish were of the same magnitude during the experiments. Isolation of outer membrane proteins. Outer membrane proteins were prepared essentially by the method of Filip et al. (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) with 12% acrylamide and 0.3% bisacrylamide. The gels were run at 7 ma for 20 h. Proteins were fixed in 40% methanol-% acetic acid, stained with 0.1% Coomassie brilliant blue in 40% methanol-% acetic acid, and destained in 40% methanol-% acetic acid. Preparation of antisera. The antigens that were used were whole Formalin-killed cells of V. anguillarum B grown in TSA-2% NaCl to the stationary phase. Before immunization, a serum pool was taken from the rabbit. Antigens were administered with or without complete Freund adjuvant at a final concentration of 8 bacteria per ml by using the following schedule: day 1, 0.5 ml of antigen in complete adjuvant in each hind footpad; day 3, 5, 7, two 0.5-ml doses of antigen in complete adjuvant subcutaneously; days 14 and 16, 0.5 ml of antigen intramuscularly with no adjuvant; and day 18, two 0.5-ml doses of antigen intramuscularly with no adjuvant. Further intramuscular injections of 0.5 ml of antigen were given weekly up to week 6. The rabbit was test bled on day 14 and, starting on day 21, was bled every to 12 days from the ear veins. Boosters (1.0 ml of antigen, no adjuvant) were given intramuscularly at monthly intervals. Western blotting. Western immunoblotting of outer membrane proteins after SDS-PAGE was performed as described by Swanson et al. (16). Rabbit antiserum diluted 1:500 was used. RESULTS Construction of transposon mutants. V. anguiillarum B 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 a broad-host-range transfer system. It can therefore be transferred to many nonenteric gram-negative bacteria. In strains in which the vector is unable to replicate, selection for resistance against tetracycline results in transposition (6). Conjugations were performed on filters, which gave a frequency of transposition of about -7. The donor and recipient were mixed on a filter and placed on nonselective media for 3 h. The filters were transferred to a selective plate and incubated at 18 C. They were incubated on selective media, and each colony that appeared was certain to be a unique exconjugant. The exconjugants were picked and

3 1402 NORQVIST ET AL. restreaked on a selective plate. In parallel, the recipient strain, B, was also passaged for the same number of times on plates, to ensure that an eventual change in pathogenicity of the exconjugants was not a result of too many passages. Plasmid preparation of the exconjugants showed that the delivery plasmid vector prk2013 did not replicate in strain B (data not shown). Isolation of antibiotic-resistant mutants. It has previously been shown that isolation of spontaneous rifampin- or streptomycin-resistant mutants of pathogenic bacteria may lead to avirulence (3, 4). To test whether this strategy for isolating avirulent mutants also could be applied to strains of V. anguiillariin, we used two steps to isolate mutants of strain 775 resistant to rifampin (200 p.g/ml) and streptomycin (0 p.g/ml). Spontaneous rifampin-resistant mutants were first isolated, and then rifampin-streptomycin-resistant double mutants were isolated. In each step, the frequency of mutation to resistance was -8. Screening for avirulent exconjugants. We screened 200 transposon and antibiotic-resistant mutants for their virulence properties by infecting rainbow trout. The challenge was carried out at 18 C by immersing fish for 30 min in water containing 5 x 6 bacteria per ml. The fish were monitored daily for mortality, and the experiments were run for 7 days. Two of the tested transposon mutants and one of the antibiotic-resistant mutants were found to have reduced virulence. We subjected these three mutants, VAN20 and VAN70 (transposon mutants) and VAN00 (antibiotic-resistant mutant), to further tests of their virulence properties by infecting fish both intraperitoneally and by immersion. The LD50 for intraperitoneal infection and immersion exceeded 1 x 6 and 1 x 8 bacteria per ml, respectively, for all strains. The corresponding LD50s for the parental strain were <1 x 2 bacteria and 2 x 6 bacteria per ml for intraperitoneal infection and immersion, respectively. Thus, strains VAN20, VAN70, and VAN00 were all avirulent. Use of avirulent strains of V. anguillarum as live, attenuated vaccines. VAN20, VAN70, and VAN00 were tested for their potential ability as live vaccines against vibriosis. Fish were immersed at 18 C for 30 min in a bacterial suspension containing VAN20, VAN70, or VAN 00 at 7 bacteria per ml. Challenge was performed 1, 2, 4,, 17, and 24 weeks after vaccination. Two strains were used as challenge organisms, the homologous strain B and the heterologous strain NB. The latter strain is an isolate from the Sea of Bothnia and exhibits LD50s of about 1 x 2 bacteria per ml and < 1 x 2 bacteria for infection by immersion and intraperitoneal injection, respectively. The challenge was carried out by bathing the fish in water containing three different concentrations of the respective test strain. This made it possible to determine the LD50s for the vaccinated fish compared with the nonvaccinated fish. For strains VAN20 and VAN70, the vaccinated fish showed an increased resistance against the homologous strain 2 weeks after immunization. This was observed as a - to 30-fold increase in the LD50 (Fig. 2A and B). After 4 and weeks, protection by VAN20 was still significant, while protection by VAN70 was intermediate. At 12 weeks after immunization, the fish were boostered with new doses of VAN20 and VAN70. This gave protection upon challenge 17 and 24 weeks after the initial immunization. When the immunized fish were challenged with the heterologous strain, NB1O, no significant protective effect was observed after the first immunization (Fig. 3A and B). After the booster dose had been given, protection was observed, "0 0 *-4 U *- 2 A 01 C ND ND IND APPL. ENVIRON. MICROBIOL Weeks after immunization FIG. 2. Immunization with V. anguillarum VAN20 (A), VAN70 (B), and VAN00 (C). Protective immunity of fish after challenge with V. angiiillarurn B is shown. The effects of the vaccination are given in terms of the P1. Thus, the PI for nonvaccinated control fish is always 1. Booster vaccination with VAN20 and VAN70 was performed 12 weeks after the first immunization. The P1 after the booster is shown (ED). LD50s in nonvaccinated fish after challenge with B were as follows: 3 x 6 cells per ml (week 1), 2 x 6 cells per ml (week 2), 5 x ' cells per ml (week 4), 5 x 6 cells per ml (week ), 2 x 6 cells per ml (week 17), and 2 x 6 cells per ml (week 24). ND, Not determined. first for VAN70-immunized fish but also later for the fish immunized with VAN20. VAN00 generated a good protection after 4 weeks against both the homologous and the heterologous strain (Fig. 2C and 3C). VAN00 also gave protection which lasted at least 17 weeks, although no booster dose was given. Immunoblotting experiments. Since we observed protective immunity against a homologous as well as a heterologous strain of V. anguillarum, we were interested in the immunological relatedness between some different isolates of V. angiiillarurn and also the possibility of cross-reaction with another member of the family Vibrionaceae which is a significant fish pathogen: A. salmonicida. Antiserum obtained after immunization of a rabbit with whole cells of V. anguillaruim B was used in immunoblotting experiments in which separated outer membrane proteins after SDS-PAGE analysis were used as the antigen source. Outer membrane preparations from four different isolates of V. anguillarurn, two strains of A. salmonicida subsp. salmonicida, and one strain of A. salmonicida subsp. achromogenes were studied. All isolates of V. anguiillarium exhibited a very ND 24

4 VOL. 55, 1989 VACCINATION OF RAINBOW TROUT 1403 X 4 '0 = ND ND Booster 1o6. C ND ND ND ND Weeks after immunization FIG. 3. Immunization with V. anguillarum VAN20 (A), VAN70 (B), and VAN00 (C). Protective immunity of fish after challenge with V. anguillarum NB1O is shown. The results are given as PI values. Booster vaccination with VAN20 and VAN70 was performed 12 weeks after the first immunization. The PI after the booster is shown (E). LD50s in nonvaccinated fish were as follows: < cells per ml (weeks 4,, and 17) and 2 x 2 cells per ml (week 24). ND, Not determined. similar antigenic response. Also, three strains of A. salmonicida gave detectable bands in the immunoblot, indicating that V. anguillarum and A. salmonicida had antigenic determinants in common (Fig. 4). Protection of Vibrio-vaccinated fish against furunculosis. The results of the immunoblotting experiments suggested that it might be possible to immunize fish against vibriosis by using a live vaccine and also to obtain protection against disease caused by A. salmonicida. Fish were therefore immunized with strain VAN00 by following the protocol outlined above. This strain was used because it gave the best protection against vibriosis (Fig. 2C and 3C). Protective immunity was observed against both the homologous and the heterologous strains of V. anguillarum, and protection against challenge with A. salmonicida could also be demonstrated (Fig. 5). An interesting observation was that significant protection against both V. anguillarum and A. salmonicida was already present 1 week after immunization. The duration 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-...,4"it t.. FIG. 4. Immunoblotting after SDS-PAGE of outer membrane proteins, using rabbit antisera directed against whole cells of V. anguillarum B. Lanes: A, V. anguillarum ATCC 19264; B, V. anguillarum NB; C, V. anguillarum B; D, V. anguillarum ; E, A. salmonicida NCMB 12; F, A. salmonicida NB8601; G, A. salmonicida NCMB 11. nity against different strains of V. anguillarum as well as against A. salmonicida subsp. salmonicida. All three avirulent mutants, VAN20, VAN70, and VAN00, were able to mediate protective immunity, although at different levels, after bath vaccination of S. gairdneri. The best results were obtained when the antibiotic-resistant strain VAN00 was used. A single dose of vaccine at a final concentration of 7 bacteria per ml of water and treatment for 30 min at 18 C was enough to protect the fish for at least 12 weeks against both vibriosis and furunculosis. In this study we chose to determine and calculate the LD50 of the challenge bacteria when infecting vaccinated and nonvaccinated fish. This allowed us to distinguish different levels of immunity of the fish after the use of different vaccine strains. Our results are presented as the PI rather than the relative percent protection (RPP) (1). The PI value is calculated as the fraction between the increased LD50 after vaccination over the LD50 for nonvaccinated fish after challenge. By using LD50 measurements and PI values, we can compensate for changes in experimental parameters such as water quality, temperature, fish size, exposure time, degree of virulence of the challenge strains, etc., during long-term vaccination studies, because the PI value gives a relative measure of protection at each challenge time. In addition, the PI value gives a quantitative assessment of the level of protective immunity, which is the most important parameter in vaccine trials. Since the LD50 determinations require more challenge facilities than the normal challenge.--,.'.

5 1404 NORQVIST ET AL O xạc 4 1 ND 2 12 Weeks after immunization FIG. 5. Effect of immunization of fish with V. anguillarulm VAN00 after challenge with V. angiuillacrum B ( S), V. angiillariin NB1O (E), and A. sahnonicida NB8601 (l). The values are given as a PI after challenge. LD50s in nonvaccinated fish were as follows: V. angiuillarum B: 2 x 6 cells per ml (week 1), 5 x 5 cells per ml (week 2), 2 x 6 cells per ml (week 5), and 2 x 6 cells per ml (week 12); V. anguillarunm NB: < cells per ml (weeks 1, 2, and 5) and 2 x 2 cells per ml (week 12); A. salmnonicida NB8601: 1 x 2 cells per ml (weeks 1 and 5) and 3 x 2 cells per ml (week 12). ND, Not determined. experiments with one fixed challenge dose, a novel experimental setup was designed to facilitate these experiments (see Fig. 1 for details). If international challenge test strains for vaccine purposes could be agreed upon, the PI value could be useful when vaccine trials in different laboratories are compared, since use of the PI reduces the differences among different laboratories. Thus, comparison of our vaccine based on the live, attenuated vaccine strain VAN00 with results of other studies would be facilitated. If we give our protection calculated as RPP values (1), the RPP for challenge with V. anguillarlim would be 0, while the RPP for furunculosis would vary from 60 to 0 depending on the challenge dose used. However, the high PI value (about 1,000) suggests that full protection would be obtained against natural infections in fish farms. Thus, vaccination with VAN00 gives a protection which is at the same level as that for other vibriosis vaccines (, 11), but the effect seems to be elevated with respect to furunculosis (1). The PI values can in some cases underestimate the protective effect of a vaccine if the challenge strain has a comparatively high LD50. This can be observed for PI values after challenge against strain B, for which the LD50 for infection by immersion is about 2 x 6 bacteria per ml for nonvaccinated fish and immersion challenge doses higher than 2 x 8/ml are difficult to use because of oxygen limitation and other toxic effects of high bacterial density. Fish vaccines can be delivered by different routes: orally in food, by intraperitoneal injection, or by immersion. Usually, intraperitoneal injection gives the best results with respect to protection and duration, but for practical reasons this route has its limitations. However, in contrast, bath vaccination is easy to carry out. The vaccine presented here gives a high degree of protection after bath vaccination, although a relatively small dose is used, probably because the bacteria multiply in the fish after infection and thus the vaccine signal is amplified in situ. However, the mutant does not cause any disease symptoms and is probably cleared by APPL. ENVIRON. MICROBIOL. the fish defense mechanisms after a short period, since we have been unable to demonstrate the presence of bacteria after vaccination. The use of a live, attenuated vaccine strain can of course be a matter of discussion. However, V. anguillarum 775 has a considerably higher LD5( after bath infection than does a fresh clinical isolate, strain NB. Thus, strain 775 is already attenuated after years under laboratory conditions, and it is a matter of definition whether this strain should be considered virulent. Strain VAN00 is less virulent than strain 775. The mutation frequency to rifampin resistance and streptomycin resistance is about -8 in each step, and the reversion frequency is of the same order. Furthermore, we have been unable to isolate spontaneous revertants during these studies. Therefore, if the strain is cultivated under selective conditions, the probability of getting a revertant after vaccination is extremely low. Consequently, it should be possible to use strain VAN00 as a safe live, attenuated vaccine. There is a limited correlation between protective immunity and the level of antibodies in fish after vaccination (5, 15, 17), and it has been shown in several studies that the fish elicit a nonspecific immune response upon injection of non-antigen-containing agents such as mineral oil-based adjuvants (1). However, stimulation of this nonspecific immune reaction gives a limited protection against furunculosis (1). We also observed a good protective immunity against furunculosis, suggesting that a specific immunity was obtained. This is supported by the fact that V. anguillaruim and A. salmonicida had surface-located antigenic determinants in common. We suggest, on the basis of the high level of protection, that the observed protective immunity against furunculosis was caused at least in part by induction of specific immunity. Whether this is due to humoral or cellular activity is at present not known, but the fact that a live strain of V. anguillarumn induces a high protective immunity, which is even higher than when killed A. salmonicida bacteria are used, indicates the possibility that cellular immune defense mechanisms, which are stimulated by the replicating bacteria, are involved in protection. It is well known that cellular immunity in mammals can be induced when live, attenuated bacteria are used as vaccines (8). This assumption is also supported by the findings that a killed Vibrio vaccine mediates only a limited protection against furunculosis (1). The avirulent mutants used in this study were isolated by a screening procedure in which individual isolates were allowed to infect groups of five fish. Mutants showing a reduced virulence were tested further, and 3 of the 200 mutants were found to be avirulent. Two of these, VAN20 and VAN70, were transposon generated. VAN00 was a spontaneous rifampin-streptomycin-resistant double mutant, in which the mutation from virulence to avirulence appeared in the second step in the mutation to streptomycin resistance. In the latter group, avirulent offspring appeared at a frequency of 25%, whereas in the former group, about 1% of the transposon-generated mutants were found to be avirulent. The underlying molecular mechanisms leading to avirulence are at present unclear. It is likely that VAN00 has a defect in the regulation of virulence-associated molecules rather than being a structural-gene mutant, since the mutant most probably has a changed L12 ribosomal protein. Such mutants have been observed before, e.g., in Yersinia pseiudotiuberelulosis and Y. pestis (3, 4), and in these two cases the mutations leading to antibiotic resistance had a pleiotropic effect on the cells, resulting in avirulence. VAN20 and VAN70, on the other hand, are more likely to be

6 VOL. 55, 1989 structural-gene mutants of virulence-associated functions. The corresponding wild-type genes have been cloned, and these genes and their products are presently being analyzed in our laboratory. ACKNOWLEDGMENTS We thank Stina Olofsson and Ulla Eriksson for their skillful technical assistance. We also thank Trevor Trust for critical reading of and comments on the manuscript. This study was supported by The Swedish Council for Forestry and Agricultural Research and by The Swedish Board for Technical Development. LITERATURE CITED 1. Adams, A., N. Auchinachie, A. Bundy, M. F. Tatner, and M. T. Horne The potency of adjuvanted injected vaccines in rainbow trout (Salmo gairdneri Richardson) and bath vaccines in Atlantic salmon (Salmo salar L.) against furunculosis. Aquaculture 69: Agius, C., M. T. Horne, and P. D. Ward Immunization of rainbow trout, Salmo gairdneri Richardson, against vibriosis: comparison of an extract antigen with whole cell bacterins by oral and intraperitoneal routes. J. Fish Dis. 6: Bolin, I., D. A. Portnoy, and H. Wolf-Watz Expression of the temperature-inducible outer membrane proteins of yersiniae. Infect. Immun. 48: Brubaker, R. R., and M. J. Surgalla Genotypic alteration associated with avirulence in streptomycin-resistant Pasteurella pestis. J. Bacteriol. 84: Croy, T. R., and D. F. Amend Immunization of sockeye salmon, Oncorhynchus nerka, against vibriosis using the hyperosmotic infiltration technique. Aquaculture 12: Ely, B Vectors for transposon mutagenesis of nonenteric bacteria. Mol. Gen. Genet. 200: Filip, C., G. Fletcher, J. L. Wulif, and C. F. Earhart Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium lauryl sarcosinate. J. Bacteriol. 115: Hahn, H., and S. H. E. Kaufmann The role of cellmediated immunity in bacterial infections. Rev. Infect. Dis. 3: Hone, D., R. Morona, S. Attridge, and J. Hackett Con- VACCINATION OF RAINBOW TROUT 1405 struction of defined gale mutants of Salmonella for use as vaccines. J. Infect. Dis. 156: Horne, M. T., M. Tatner, S. McDerment, C. Agius, and P. Ward Vaccination of the rainbow trout, Salmo gairdneri Richardson, at low temperatures and the long-term persistence of protection. J. Fish Dis. 5: Johnson, K. A., J. K. Flynn, and D. F. Amend Duration of immunity in salmonids vaccinated by direct immersion with Yesinia ruckeri and Vibrio anguillarum bacterins. J. Fish Dis. 5: Kawano, K., T. Aoki, and T. Kitao Duration of protection against vibriosis in ayu, Plecoglossus altivelis, vaccinated by immersion and oral administration with Vibrio anguillarum. Bull. Jpn. Soc. Sci. Fish. 50: Laemmli, U. K., and M. Favre Maturation of the head of bacteriophage T4. J. Mol. Biol. 80: Reed, L. J., and H. Muench A simple method of estimating fifty per cent endpoints. Am. J. Hyg. 27: Sakai, M., T. Aoki, T. Kitao, J. S. Rohovec, and J. L. Fryer Comparison of the cellular immune response of fish vaccinated by immersion and injection of Vibrio anguillarum. Bull. Jpn. Soc. Sci. Fish. 50: Swanson, J., L. W. Mayer, and M. R. Tam Antigenicity of Neisseria gonorrhoeae outer membrane protein(s) III detected by immunoprecipitation and Western blot transfer with a monoclonal antibody. Infect. Immun. 38: Thuvander, A., T. Hongslo, E. Jansson, and B. Sundquist Duration of protective immunity and antibody titres measured by ELISA after vaccination of rainbow trout, Salmo gairdneri Richardson, against vibriosis. J. Fish Dis. : Wahdan, M. H., C. Serie, Y. Cerisier, S. Sallam, and R. Germanier A controlled field trial of live Salmonella typhi strain Ty 21a oral vaccine against typhoid: three-year results. J. Infect. Dis. 145: Walter, M. A., S. A. Potter, and J. H. Crosa Iron uptake system mediated by Vibrio anguillarum plasmid pjm1. J. Bacteriol. 156: Ward, P. D., M. F. Tatner, and M. T. Horne Factors influencing the efficacy of vaccines against vibriosis caused by Vibrio anguillarum, p In M. J. Manning and M. F. Tatner (ed.), Fish immunology. Academic Press, Inc. (London), Ltd., London.