- Email: [email protected]
Vaccination of Atlantic salmon, Salmo salar L., with particulate lipopolysaccharide antigens from Vibrio salmonicida and Vibrio anguillarum
Fish & Shellfish Immunology (1992) 2, 251-261
V a c c i n a t i o n o f A t l a n t i c s a l m o n , S a l m o s a l a r L., w i t h particulate
lipopolysaccharide antigens from salmonicida and Vibrio anguillarum
J. BOGWALD*$,K.
Vibrio
STENSV/~G*,J. HOFFMAN*,K. O. HOLM~ANDT. O. JORGENSEN*
*The Foundation of Applied Research at the University of Tromso, Tromso and ~Apothekernes Laboratorium A.S., Tromso, Norway (Received 22 July 1991, accepted in revised form 19 September 1991) Atlantic salmon were vaccinated by intraperitoneal injection of particulate lipopolysaccharide (LPS) antigens of the two fish pathogens Vibrio salmonicida and Vibrio anguillarum. Particulate LPS from V. salmonicida and V. anguillarum serotype 01 failed to demonstrate a protection against disease after intraperitoneal challenge with live bacteria. However, fish vaccinated with particulate LPS preparations from V. anguillarum serotype 02 acquired a high protection and the LPS-protein complex surface layer antigen VS-P1 from V. salmonicida was seen to give a protection which was superior to purified LPS alone. Vaccination with LPS particles modified by precoating with bovine serum albumin or oleic acid resulted in a slightly better protection compared to the unmodified LPS particle. Key words:
Vibrio salmonicida, Vibrio anguillarum, LPS vaccine. I.
Introduction
Lipopolysaccharides (LPS) which are also known as endotoxins, are essential components of the outer membrane of Gram-negative bacteria. The so-called O antigen of the LPS molecule carries the antigenic determinants that constitute the serological specificity. At the same time LPS carries the endotoxic properties associated with Gram-negative infections. LPS molecules may also induce a number of beneficial effects, and good examples are the non-specific activation ofmacrophages, the helper function for antigens of weak immunogenicity (adjuvant), and recognition of Gram-negative bacteria by the immun e system leads to the production of antibacterial antibodies which are predominantly directed against LPS determinants. Most of the biological activities of LPS reside in the Lipid A moiety (Galanos et al., 1977). Activities like lethal toxicity, pyrogenicity, complement activation, adjuvanticity and B l~zmphocyte mitogenicity were demonstrated with free isolated Lipid A. Toxic effects of LPS obtained from Escher.ichia coli by the phenol-water method of Westphal & J a n n (1965) have been investigated in several animal +Addresscorrespondenceto: J. Bogwald,The FoundationofAppliedResearchat the Universityof Tromso(FORUT),P.O. Box 2806Elverhoy,9001Troms~,Norway. 251 1050-4648192/040251 + 11$08.00/0 9 1992AcademicPress Limited
252
J. BOGWALD E T A L .
species (Berczi et al., 1966). The mammalian species examined displayed a wide range of sensitivity to LPS; calves were extremely sensitive whereas mice and rats were rather resistant. However, fish (carp) and frogs showed an extreme resistance against lethal effects of LPS. It is a general view that a vaccine should contain an antigen, a carrier and an adjuvant. It has been shown that particulate or encapsulated antigens are far more effective than soluble ones, and most antigens need an adjuvant for optimal stimulation of the host immune system (Altman & Dixon, 1989). Adjuvants are substances that will increase both non-specific disease resistance and the specific immune response and thus increase the duration of protection against disease, whereas adsorbed (particulate) antigens are reported to act as an antigen depot and to enhance the antigen uptake by antigen presenting cells (Altman & Dixon, 1989). Several kinds of particles have been used as carriers] adjuvants including polymethyl methacrylate particles (Kreuter et al., 1988), latex particles (Litwin.& Singer, 1965) and mineral particles (Hennessen, 1965; Gallily & Garvey, 1968; Joo, 1973). Liposomes (Allison & Gregoriadis, 1974), aluminium salts (Glenny et al., 1926; Bomford, 1989) and saponins (Dalsgaard, 1977) have also been shown to serve as adjuvants. The first attempts 0f vaccination offish against bacterial diseases took place 50 years ago (Duff, 1942). Today, effective vaccines against vibriosis, coldwater vibriosis and enteric red mouth disease have been developed (Johnson et al., 1982; Cossarini-Dunier, 1986; Holm & Jorgensen, 1987; Thorburn & Jansson, 1988). Vaccination of fish by formalin killed bacteria like Vibrio salmonicida, Vibrio al~guillarum and Yersinia ruckerii will protect effectively against disease. However, there is still much that is not known about the protective mechanisms involved. There are reports of induction of protective immunity in fish by LPS from V. anguillarum (Kawai & Kusuda, 1983) and Aeromonas hydrophila (Baba et al., 1988). We have previously shown that LPS of V. salmonicida is an important antigen for production of specific antibodies, both in the mouse and in fish (Bogwald et al., 1990), and in line with this finding it was of interest to search for antigens giving protective immunity. Preliminary results (unpubl.) indicated that the surface layer protein-lipopolysaccharide complex VS-P1 (Hjelmeland et al., 1988) induced protection against infection with V. salmonicida and that the purified LPS did not. The aim of the present study was to investigate if protection could be induced by particularising the LPS on a mineral carrier and in addition, it was of interest to investigate whether the formulation (LPS-carrier complex) was important for inducing protective immunity in the fish. II. Materials and Methods BACTERIA
Bacteria used in this study were V. salmonicida (NCMB 2262) and V. anguillarum serotype 01 (LF16012) and 02 (LF16004) represented by Norwegian isolates of the respective serotypes. Cultures were grown in 2~/o Marine Broth (Difco) with 2~/o peptone (Marine Biochemicals Peptone Powder, Tromso, Norway) at 12~ C in an orbital shaker.
V A C C I N A T I O N OF A T L A N T I C S A L M O N
253
Cells were killed by 0"5~/o formalin, centrifuged and washed twice in 10 mM phosphate buffer pH 7.3 containing 2% sodium chloride before being used to immunise salmon or in the ELISAs.
LIPOPOLYSACCHARIDES
Lipopolysaccharides (LPS) from V. salmonicida were isolated according to the phenol:chloroform:petroleum ether extraction method of Galanos et al. (1969). Vibrio anguillarum LPS of both serotypes were isolated by the phenolwater procedure of Westphal & J a n n (1965), and recovered from the phenol phase. P r o t e i n s in the LPS preparations were determined by the BCA method (Pierce, U.S.A.) as described by Smith et al. (1985).
ADSORPTION OFOLEICACIDTOSORBAL
Sorbal (a mineral particle of average diameter 0.2-2 llm, I g, Apothekernes Laboratorium, Norway) was suspended in 10 ml oleic acid (Fluka). The suspension was vortexed and sonicated to ensure a homogenous mixture. The adsorption was carried out for 30 mi{~at room temperature. To remove excess oleic acid 1 ml of the suspension was mixed with 1 ml of ethanol (96~o) and centrifuged at 11 000 x g for 10 min. The pellet was resuspended in 20 mM Tris-HCl buffer pH 7-3 containing 0"1 M NaC1 and centrifuged for 10 rain at 11 000 xg. The pellet was washed once and then resuspended in 1 ml buffer. This suspension was further incubated with LPS to make a LPS-oleic acid-Sorbal conjugate. ADSORPTION OF BOVINE SERUM ALBUMIN TO SORBAL
The Sorbal suspension (10 mg m1-1, 20 ml) was centrifuged and resuspended in 80 ml of 4% bovine serum albumin (BSA) solution containing 0"02~/o sodium azide. The suspension was incubated with constant mixing overnight at room temperature. The BSA-Sorbal conjugate was washed twice in distilled water and then in 0-1 ~, acetate buffer pH 5"0 till the supernatant no longer contained any detectable protein (using the BCA method above). This BSA-Sorbal conjugate was further incubated with LPS to make a LPS-BSA-Sorbal conjugate.
ADSORPTION OF LPS TOSORBAL
Purified LPS of V. salmonicida and V. anguillarum were slightly soluble in water or phosphate buffered saline at neutral pH. However, at pH 8-9 or 5 the LPS was soluble, but at pH 8-9 the LPS did not adsorb to Sorbal. Thus, the suspension of Sorbal was incubated with LPS in 0.1 Macetate buffer, pH 5"0 with constant mixing overnight at 4 ~ C. This procedure was also used to adsorb LPS to BSA-Sorbal and oleic acid coated-Sorbal. Adsorbed LPS was determined by the colorimetric method detecting neutral hexoses described by Dubois et al. (1956). VS-P1 (a protein-LPS complex isolat.ed from V. salmonicida, Hjelmeland
254
J. BOGWALDETAL.
et al., 1988 was incubated with Sorbal in phosphate buffered saline (PBS), 10H 7.4 at 4 ~ C with constant mixing for 24 h.
IMMUNOFLUORESCENCE
Adherence of LPS and VS-P1 to the particles was checked by indirect immunofluorescent staining. The antigen coated particles were incubated in 5% normal rabbit serum diluted in phosphate buffered saline (PBS) to avoid nonspecific binding of antibodies. After washing, the particles were incubated with monoclonal antibodies directed against the LPS determinants of V. salmonicida (Espelid et al., 1988; Bogwald et al., 1990) for 1 h at room temperature. After washing in PBS, fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse IgG (Dakopatts, Denmark), diluted in normal rabbit serum (5% in PBS) was added. The incubation was continued for 1 h at room temperature. The particles were thoroughly washed before examination in a Nikon fluorescence microscope.
VACCINATION
Atlantic salmon, Salmo salar L., weighing about 50 g each and reared in circular tanks in a mixture of fresh and salt (sea) water (final salinity 1-5%) at 12~ C and fed commercial salmon feed pellets (Ewos, Norway), were vaccinated by intraperitoneal (i.p.) injection with 0.1 ml of the various vaccine preparations (50 individuals in each group). Control fish received 10s formalin-killed bacteria of V. salmonicida or V. anguillarum serotype 01 and 02 respectively. In the LPS immunised groups, each fish was injected with 251lg purified LPS adsorbed to Sorbal particles or VS-P1 in an amount corresponding to a LPS content of 25 pg, adsorbed to Sorbal particles. All quantitations of LPS were done by the method of Dubois et al. (1956).
CHALLENGE
Fish were kept in circular tanks in a mixture of fresh and salt water at 10~ C (salinity 1.5%). Prior to challenge, the infectious dose to be used was determined in a prechallenge test. As a result of this test, vaccinated fish were challenged with 1 x 106, 5 • 104 and 1 x 104 bacteria of V. salmonicida or V. anguillarum 01 and 02 respectively 3 months after vaccination. The concentration and viability of each strain was controlled by counting the colony forming units (CFU) on Tryptic Soy Agar (Difco) plates supplemented with 1.5% NaC1 and 3% human red blood cells.
STATISTICAL ANALYSIS
To evaluate the significance of the differences in mortality between the control group (unvaccinated) and the vaccination groups, an approximation to standard normal distribution test was used.
VACCINATION OF ATLANTIC SALMON
255
100 ..-~- . t . . - - ~
L~" ...
,"
J
80
60
o~
4~ .s
20
-u
O
/
I0
5
2 " " ..........
15
20
Days after challenge
Fig. 1. Mortalities in Atlantic salmon vaccinated with various antigens of Vibrio salmonicida, after challenge with the homologous bacteria. RPS values are given in parentheses. (.. A.-), Unvaccinated; (-'+--), Sorbal (4"2); (--,--), whole cells (77-1); (--E]--), VS-Pl~.qorbal (44.8); (--O--), LPS~Sorbal (2"1). III. R e s u l t s CHALLENGE WITH VlBRIO SALMONIC1DA
As shown in Fig. 1 the accumulated mortality in the control group (unvaccinated fish) reached 95% 22 days after challenge with V. salmonicida. Fish injected with Sorbal particles alone or LPS adsorbed to the particles (126 pg LPS mg -1 Sorbal) resulted in the same mortality as fish in the control group (95%). However, fish injected with VS-P1 adsorbed to the particle (47pg LPS mg -1 Sorbal) gave a partial protection against infection (mortality 50~/o at day 22, P < 0.001). Washed formalin killed whole cells constituted the most effective vaccine with a mortality of only 20~/o (P<0.001). The relative percentage survival (RPS) values are indicated in Fig. 1 in parentheses. In another set of experiments, LPS was adsorbed to Sorbal particles precoated with protein (BSA) or lipid (oleic acid). The amount of LPS coated mg -1 Sorbal was 144 pg and 114 pg respectively. As shown in Table 1 the accumulated mortality in the control group (fish injected with.Sorbal particles alone) reached nearly 70~/o at day 20 after challenge. As observed in the former experiment, fish injected with LPS adsorbed to particles resulted in no protection against infection. In fish injected with particles precoated with BSA and then coated
256
J. BOGWALDETAL.
Table 1. Mortalities in Atlantic salmon vaccinated with modified LPS~Sorbal particles of Vibrio salmonicida 20 days after infection with the homologous bacteria
Preparation
Accumulated mortality (%)
RPS
61"9 54"8 16"7 0 47'6 40-5 53"7 39"0
11-3 72-6 100 22"6 35"5 12.9 37.1
Sorbal particle (negative control) LPS~orbal particle VS-P1-Sorbal particle Whole cell bacteria BSA-Sorbal particle LPS-BSA-Sorbal particle Oleic acid-Sorbal particle LPS-oleic acid-Sorbal particle RPS = 100% • f l '
\
% mortality in vaccinate~ _~ % mortality in unvaccinatcd]'
with LPS, a partial protection was seen (mortality 40% at day 20 after infection, P<0-01). However, injection of the BSA~Sorbal particle itself resulted in reduced mortality (RPS 22"6, P<0"05). Vaccination with Sorbal particles precoated with oleic acid (Cls:l) and then further coated with LPS, resulted in a partial protection against disease (mortality 39"0%, P<0"005). The reduction in mortality after injection of oleic acid coated Sorbal particles was not significant. The VS-P1 adsorbed to particles resulted in good protection (c. 20% mortality at day 20, P<0"001), while fish injected with whole cells showed no sign of disease (100% survival). The accumulated mortalities (%) and RPS values of these experiments are shown in Table 1.
CHALLENGEWITH V I B R I O A N G U I L L A R U M S E R O T Y P E 01
Mortality and RPS data are shown in Fig. 2. The mortality in the unvaccinated group reached 55%, 15 days after infection. A somewhat higher mortality (65%) was observed in the group which received the Sorbal particles alone without antigen. Fish injected with LPS adsorbed to the particles were slightly more resistant to infection (40% mortality, P=0"052). Those injected with whole bacterial cells showed a mortality of 10% (positive controls, P < 0.001).
CHALLENGEWITH VIBRIO A N G U I L L A R U M S E R O T Y P E 02
Unvaccinated salmon infected with V. a n g u i l l a r u m serotype 02, showed a mortality of 80% 19 days after infection as shown in Fig. 3. Fish injected with Sorbal particles alone, showed a non-specific protection against disease (55% mortality, P<0.01). In this case, vaccination of fish with the particulate LPS preparation resulted in a high protection against disease (10% mortality, P<0.001). The highest protection was obtained after vaccination with whole
257
VACCINATION OF ATLANTIC SALMON I00
80-
..-I-- .4:"
60-
..-I-.. 4:
.~. ,4:" ,~.. ~-.-~.-.~-
o~ ..~ .: ~.
9
.~."
.,/"
,!~::. a'" +
40
..::-
20
.'~
/~--~J~
i--i--i--i--~--i
0
w
i
,
5
i
I I0
I
I
1
i
I 15
Days after challenge
Fig. 2. Mortalities in Atlantic salmon vaccinated with antigens of Vibrio anguillarum serotype 01, after challenge with the homologous bacteria. RPS values are given in parentheses. (--A-'), Unvaccinated; (" "+" "), Sorbal (-); (--,--), whole cells (78"6); (--[7--), LPS-Sorbal (28'6).
formalin killed-bacteria (mortality 8%, P<0.001). RPS values are indicated in Fig. 3 in parentheses.
IV. D i s c u s s i o n Recognition of V. salmonicida cells by the Atlantic salmon immune system leads to production of antibodies predominantly directed against the LPS structures (Bogwald et al., 1991) and LPS is reported to be the protective antigen of V. anguillarum in ayu (Kawai & Kusuda, 1983) and A. hydrophila in carp (Baba et al., 1988). Results obtained in this study show that salmon vaccinated by i.p. injection of the particulate protein-LPS complex VS-P1 of V. salmonicida elicited an enhanced resistance against infection compared to fish immunised with a purified (i.e. protein free) preparation of LPS.(adsorbed to Sorbal particles). Although the protection seen after immunisation with VS--P1 was less than that with whole V. salmonicida cells, this result indicates an essential role of protein antigens in order to develop protective immunity against V. salmonicida. One
J. BOGWALDETAL.
258 I00
80-
~..~.-~-.~.-~.-~-.~.
~-~.~.~.~"
60-
+.+ .+.+.+. +.+.+.+.+.+.+
t~ 40-
ZO : §
.-l0
~_m_m_n=_=_i.-~-T-~-~ 0
5
~
,
,
,
I0
,
~
.
.
.
.
15
Doys ofter chollenge
Fig. 3. Mortalities in Atlantic salmon vaccinated with antigens of Vibrio anguillarum serotype 02, after challenge with the homologous bacteria. RPS values are given in parentheses. (.. A" "), Unvaccinated; (-" +-'), Sorbal (29-6); (---,--), whole cells (90-1); (--[:]~), LPS-Sorbal (85"2).
explanation for enhanced protection due to immunisation with VS-P1 is that the 40 kDa protein moiety of this complex (Hjelmeland et al., 1988) may stimulate helper cells in a T lymphocyte dependent anti-LPS response. Alternatively, the protein moiety of the protein-LPS antigen is needed for a proper presentation of the LPS structures. Intraperitoneal vaccination of fish with modified Sorbal particles precoated with BSA or oleic acid and further coated with LPS resulted in a reduced mortality compared to vaccination with unmodified LPS-Sorbal particles when infected with V. salmonicida. These results support the idea above concerning the need for a protein/lipid component for a proper stimulation of the immune system. Fish vaccinated with V. anguillarum serotype 01 LPS adsorbed to the Sorbal particles showed some protection against infection with the homologous bacterium. However, fish vaccinated with V. anguillarum serotype 02 LPS adsorbed to particles displayed a far better resistance against infection. It is difficult to explain the difference in protective capacity of the two LPS preparations although they show physio-chemical differences. In a previous report (Bogwald et al., 1990) we described certain characteristics of the two LPSs. LPS
VACCINATIONOF ATLANTICSALMON
259
from V. anguillarum serotypes 01 and 02 were found to differ with respect to the length of their O-specific side chains, i.e. serotype 02 expressed LPS with a more heterogenous chain length compared to serotype 01, as characterised by SDS-PAGE. While serotype 01 displayed three closely spaced bands, serotype 02 demonstrated five to nine clearly resolved bands. Also, the low molecular weight region (SDS-PAGE) of serotype 02 showed two broad bands whereas serotype 01 showed just one, and the relative amount of the low molecular fraction was higher in serotype 02 compared to serotype 01. In addition to physical differences, chemical differences between the two serotypes have also been observed (unpubl. res.). There seems to be no correlation between protective capacity and antibody production against the various bacteria (Bogwald et al., 1991). The antibody response in salmon after immunisation with whole bacterial cells of V. anguillarum serotype 02 was low compared to V. anguillarum serotype 01, when tested in ELISA using homologous whole cells as in vitro antigens. Western blots, using outer membrane preparations or purified LPS as antigens, showed that the antibody response is directed against epitopes on LPS (Bogwald et al., 1991), although the same sera tested against purified LPS antigens in ELISA, showed almost background activities. Concerning V. salmonicida, protection against infection was negligible when purified LPS was used for vaccination (adsorbed to the Sorbal particles), but LPS complexed with a 40 kDa molecule in the VS-P1 antigen elicited a substantial protection. Although the 40 kDa protein itself does not stimulate antibody production, this antigen may function as a T cell stimulating carrier and provide immunological memorY (T memory cells) and T cell help towards the B cells. Precoating of the Sorbal particles with BSA or oleic acid did not significantly enhance the amount of LPS bound per particle. In fact, the amount of LPS bound per particle in the VS-P1 complex was less compared to the purified LPS. In addition to the specific stimulation with the VS-P1 complex mentioned above, the amount and probably also the orientation of the LPS molecules bound to the particles are important for the establishment of immunity. It should be mentioned that in addition to the dominating antigenicity of the LPS antigens, a highly immunogenic 24 kDa surface protein can also be detected in Western blot experiments (Bogwald et al., 1991). The effect of this molecule in vaccination experiments is not ruled out, but it seems likely that the high protection of the whole cell bacterins demonstrated in Figs 1-3 and Table 1 also involve antigens other than LPS or LPS-protein complexes, like the 24 kDa molecule of V. salmonicida. There are some reports on the humoral and protective immunity in mammals by Vibrio cholera antigens of both protein and LPS nature (Neoh & Rowley, 1970, Manning et al., 1986). Antibodies to the protein antigen were reported to be more protective on a weight basis, but the LPS was by far the more potent immunogen. The resuIts obtained in this study do not definitely clarify the relation between the nature of antigens and protective capacity. However, a closer examination of the chemical structure of the LPSs of the two V. anguillarum serotypes would hopefully yield more exact information of the questions raised. Also, a more penetrating study must be done to elucidate the function of the protein carrier in the VS-P1 complex.
260
J. BOGWALDETAL.
This work was supported by A/S Apothekernes Laboratorium, Oslo]Tromso, Norway, and the Norwegian Research Council of Fisheries (NFFR). The assistance of Edvin Bredrup in carrying out the statistical analysis is highly appreciated.
References Allison, A. C. & Gregoriadis, G. (1974). Liposomes as immunological adjuvants. Nature 252, 252. Altman, A. & Dixon, F. J. (1989). Immunomodifiers in vaccines. In Vaccine Biotechnology. Advances in Veterinary Science and Comparative Medicine (J. L. Bittle & F. L. Murphy, eds) 33, pp. 301-343. San Diego, CA: Academic Press. Baba, T., Imamura, J., Izawa, K. & Ikeda, K. (1988). Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide. Journal offish Diseases 11,237-244. Berczi, I., Bertok, L. & Bereznai, T. (1966). Comparative studies on the toxicity of Escherichia coli lipopolysaccharide endotoxin in various animal species. Canadian Journal of Microbiology 12, 1070-1071. Bomford, R. (1989). Aluminium salts: perspectives in their use as adjuvants. In Immunological Adjuvants and Vaccines (G. Gregoriades, A. C. Allison & G. Poste, eds) pp. 35--41. New York: Plenum Press. Bogwald, J., Stensv~g, K., Hoffman, J., Espelid, S., Holm, K. O. & Jorgensen T. (1990). Electrophoretic and immunochemical analysis of surface antigens of the fish pathogen Vibrio salmonicida and Vibrio anguillarum. Journal of Fish Diseases 13,293-301. Bogwald, J., Stensv~g, K., Hoffman, J. & Jorgensen, T. (1991). Antibody specificities in Atlantic salmon, Salmo salar L., against the fish pathogens Vibrio salmonicida and Vibrio anguillarum. Journal offish Diseases 14, 79-87. Cossarinin-Dunier, M. (1986). Protection against enteric redmouth disease in rainbow trout, Salmo gairdneri Richardson, after vaccination with Yersinia ruckeri bacterin. Journal offish Diseases 9, 27-33. Dalsgaard, K. (1977). Evaluation of the adjuvant "Quil-A" in the vaccination of cattle against foot-and-mouth disease. Acta Veterinaria Scandinavica 18, 349-360. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956). Colorimertic method for determination of sugars and related substances. Analytical Chemistry 28, 350-356. Duff, D. C. B. (1942). The oral immunization of trout against Bacillus salmonicida. Journal of Immunology 44, 87-94. Espelid, S., Holm, K. O., Hjelmeland, K. & Jorgensen, T. (1988). Monoclonal antibodies against Vibrio salmonicida: the causative agent of coldwater vibriosis ("Hitra disease") in Atlantic salmon, Salmo salar L. Journal offish Diseases 11,207-214. Galanos, C., Luderitz, O., Rietschel, E. T. & Westphal, O. (1977). Newer aspects of the chemistry and biology of bacterial lipopolysaccharides, with special reference to their lipid A component. In International Review of Biochemistry, Biochemistry of Lipids 11, Vol. 14 (T. W. Goodwin, ed.) pp. 239-335. Baltimore, MD: University Park Press. Galanos, C., Luderitz, O. & Westphal, O. (1969). A new method for the extraction of R lipopolysaccharides. European Journal of Biochemistry 9, 245-249. Gallily, R. & Garvey, J. S. (1968). Primary stimulation of rats and mice with hemocyanin in solution and adsorbed on bentonite. The Journal of Immunology 101,924-929. Glenny, A. T., Pope, C. G., Waddington, H. & Wallace, U. (1926). The antigenic value of the toxin-antitoxin precipitate of Ramon. Journal of Pathology and Bacteriology 29, 31-40. Hennessen, W. (1965). The mode of action of mineral adjuvants. Progress in Immunobiological Standard 2, 71-79.
VACCINATION OF ATLANTIC SALMON
261
Hjelmeland, K., Stensv~g, K., Jorgensen, T. & Espelid S. (1988). Isolation and characterization of a surface layer antigen from Vibrio salmonicida. Journal of Fish Diseases 11,197-205. Holm, K. O. & JDrgensen, T. (1987). A successful vaccination of Atlantic salmon, Salmo salar L., against 'Hitra disease' or coldwater vibriosis. Journal of Fish Diseases 10, 85-90. Johnson, K. A., Flynn, J. K. & Amend, D. F. (1982). Duration of immunity in salmonids vaccinated by direct immersion with Yersinia ruckerii and Vibrio anguillarum bacterins. Journal offish Diseases 5, 207-213. Joo, I. (1973). Mineral carriers as adjuvants. Symposium Series of Immunobiological Standards 22, 123-130. Kawai, K. & Kusuda, R. (1983). Efficacy of the lipopolysaccharide vaccine against vibriosis in cultured ayu. Bulletin of the Japanese Society of Scientific Fisheries 49, 511-514. Kreuter, J., Liehl, E., Berg, U., Soliva, M. & Speiser, P. P. (1988). Influence of hydrophobicity on the adjuvant effect of particulate polymeric adjuvants. Vaccine 6, 253-256. Litwin, S. D. & Singer, J. M. (1965). The adjuvant action of latex particulate carriers. Journal of Immunology 95, 1147-1152. Manning, P. A., Heuzenroeder, M. W., Yeadon, J., Leavesley, D. I., Reeves, P. R. & Rowley, D. (1986). Molecular cloning and expression in Escherichia coli K-12 of the O-antigens of the Inaba and Ogawa serotypes of the Vibrio cholera 01 lipopolysaccharides and their potential for vaccine development. Infection and Immunity 53,272-277. Neoh, S. H. & Rowley, D. (1970). The antigens of Vibrio cholera involved in vibriocidal action of antibody and complement. Journal of Infectious Diseases 121,505-513. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150, 76-85. Thorburn, M. A. & Jansson, E. L. K. (1988). The effects of booster vaccination and fish size on survival and antibody production following Vibrio infection of bathvaccinated rainbow trout, Salmo gairdneri. Aquaculture 71,285-291. Westphal, O. & Jann, K. (1965). Bacterial lipopolysaccharides: extraction with phenolwater and further applications of the procedure. Methods in Carbohydrate Chemistry 5, 83-96.
V a c c i n a t i o n o f A t l a n t i c s a l m o n , S a l m o s a l a r L., w i t h particulate
lipopolysaccharide antigens from salmonicida and Vibrio anguillarum
J. BOGWALD*$,K.
Vibrio
STENSV/~G*,J. HOFFMAN*,K. O. HOLM~ANDT. O. JORGENSEN*
*The Foundation of Applied Research at the University of Tromso, Tromso and ~Apothekernes Laboratorium A.S., Tromso, Norway (Received 22 July 1991, accepted in revised form 19 September 1991) Atlantic salmon were vaccinated by intraperitoneal injection of particulate lipopolysaccharide (LPS) antigens of the two fish pathogens Vibrio salmonicida and Vibrio anguillarum. Particulate LPS from V. salmonicida and V. anguillarum serotype 01 failed to demonstrate a protection against disease after intraperitoneal challenge with live bacteria. However, fish vaccinated with particulate LPS preparations from V. anguillarum serotype 02 acquired a high protection and the LPS-protein complex surface layer antigen VS-P1 from V. salmonicida was seen to give a protection which was superior to purified LPS alone. Vaccination with LPS particles modified by precoating with bovine serum albumin or oleic acid resulted in a slightly better protection compared to the unmodified LPS particle. Key words:
Vibrio salmonicida, Vibrio anguillarum, LPS vaccine. I.
Introduction
Lipopolysaccharides (LPS) which are also known as endotoxins, are essential components of the outer membrane of Gram-negative bacteria. The so-called O antigen of the LPS molecule carries the antigenic determinants that constitute the serological specificity. At the same time LPS carries the endotoxic properties associated with Gram-negative infections. LPS molecules may also induce a number of beneficial effects, and good examples are the non-specific activation ofmacrophages, the helper function for antigens of weak immunogenicity (adjuvant), and recognition of Gram-negative bacteria by the immun e system leads to the production of antibacterial antibodies which are predominantly directed against LPS determinants. Most of the biological activities of LPS reside in the Lipid A moiety (Galanos et al., 1977). Activities like lethal toxicity, pyrogenicity, complement activation, adjuvanticity and B l~zmphocyte mitogenicity were demonstrated with free isolated Lipid A. Toxic effects of LPS obtained from Escher.ichia coli by the phenol-water method of Westphal & J a n n (1965) have been investigated in several animal +Addresscorrespondenceto: J. Bogwald,The FoundationofAppliedResearchat the Universityof Tromso(FORUT),P.O. Box 2806Elverhoy,9001Troms~,Norway. 251 1050-4648192/040251 + 11$08.00/0 9 1992AcademicPress Limited
252
J. BOGWALD E T A L .
species (Berczi et al., 1966). The mammalian species examined displayed a wide range of sensitivity to LPS; calves were extremely sensitive whereas mice and rats were rather resistant. However, fish (carp) and frogs showed an extreme resistance against lethal effects of LPS. It is a general view that a vaccine should contain an antigen, a carrier and an adjuvant. It has been shown that particulate or encapsulated antigens are far more effective than soluble ones, and most antigens need an adjuvant for optimal stimulation of the host immune system (Altman & Dixon, 1989). Adjuvants are substances that will increase both non-specific disease resistance and the specific immune response and thus increase the duration of protection against disease, whereas adsorbed (particulate) antigens are reported to act as an antigen depot and to enhance the antigen uptake by antigen presenting cells (Altman & Dixon, 1989). Several kinds of particles have been used as carriers] adjuvants including polymethyl methacrylate particles (Kreuter et al., 1988), latex particles (Litwin.& Singer, 1965) and mineral particles (Hennessen, 1965; Gallily & Garvey, 1968; Joo, 1973). Liposomes (Allison & Gregoriadis, 1974), aluminium salts (Glenny et al., 1926; Bomford, 1989) and saponins (Dalsgaard, 1977) have also been shown to serve as adjuvants. The first attempts 0f vaccination offish against bacterial diseases took place 50 years ago (Duff, 1942). Today, effective vaccines against vibriosis, coldwater vibriosis and enteric red mouth disease have been developed (Johnson et al., 1982; Cossarini-Dunier, 1986; Holm & Jorgensen, 1987; Thorburn & Jansson, 1988). Vaccination of fish by formalin killed bacteria like Vibrio salmonicida, Vibrio al~guillarum and Yersinia ruckerii will protect effectively against disease. However, there is still much that is not known about the protective mechanisms involved. There are reports of induction of protective immunity in fish by LPS from V. anguillarum (Kawai & Kusuda, 1983) and Aeromonas hydrophila (Baba et al., 1988). We have previously shown that LPS of V. salmonicida is an important antigen for production of specific antibodies, both in the mouse and in fish (Bogwald et al., 1990), and in line with this finding it was of interest to search for antigens giving protective immunity. Preliminary results (unpubl.) indicated that the surface layer protein-lipopolysaccharide complex VS-P1 (Hjelmeland et al., 1988) induced protection against infection with V. salmonicida and that the purified LPS did not. The aim of the present study was to investigate if protection could be induced by particularising the LPS on a mineral carrier and in addition, it was of interest to investigate whether the formulation (LPS-carrier complex) was important for inducing protective immunity in the fish. II. Materials and Methods BACTERIA
Bacteria used in this study were V. salmonicida (NCMB 2262) and V. anguillarum serotype 01 (LF16012) and 02 (LF16004) represented by Norwegian isolates of the respective serotypes. Cultures were grown in 2~/o Marine Broth (Difco) with 2~/o peptone (Marine Biochemicals Peptone Powder, Tromso, Norway) at 12~ C in an orbital shaker.
V A C C I N A T I O N OF A T L A N T I C S A L M O N
253
Cells were killed by 0"5~/o formalin, centrifuged and washed twice in 10 mM phosphate buffer pH 7.3 containing 2% sodium chloride before being used to immunise salmon or in the ELISAs.
LIPOPOLYSACCHARIDES
Lipopolysaccharides (LPS) from V. salmonicida were isolated according to the phenol:chloroform:petroleum ether extraction method of Galanos et al. (1969). Vibrio anguillarum LPS of both serotypes were isolated by the phenolwater procedure of Westphal & J a n n (1965), and recovered from the phenol phase. P r o t e i n s in the LPS preparations were determined by the BCA method (Pierce, U.S.A.) as described by Smith et al. (1985).
ADSORPTION OFOLEICACIDTOSORBAL
Sorbal (a mineral particle of average diameter 0.2-2 llm, I g, Apothekernes Laboratorium, Norway) was suspended in 10 ml oleic acid (Fluka). The suspension was vortexed and sonicated to ensure a homogenous mixture. The adsorption was carried out for 30 mi{~at room temperature. To remove excess oleic acid 1 ml of the suspension was mixed with 1 ml of ethanol (96~o) and centrifuged at 11 000 x g for 10 min. The pellet was resuspended in 20 mM Tris-HCl buffer pH 7-3 containing 0"1 M NaC1 and centrifuged for 10 rain at 11 000 xg. The pellet was washed once and then resuspended in 1 ml buffer. This suspension was further incubated with LPS to make a LPS-oleic acid-Sorbal conjugate. ADSORPTION OF BOVINE SERUM ALBUMIN TO SORBAL
The Sorbal suspension (10 mg m1-1, 20 ml) was centrifuged and resuspended in 80 ml of 4% bovine serum albumin (BSA) solution containing 0"02~/o sodium azide. The suspension was incubated with constant mixing overnight at room temperature. The BSA-Sorbal conjugate was washed twice in distilled water and then in 0-1 ~, acetate buffer pH 5"0 till the supernatant no longer contained any detectable protein (using the BCA method above). This BSA-Sorbal conjugate was further incubated with LPS to make a LPS-BSA-Sorbal conjugate.
ADSORPTION OF LPS TOSORBAL
Purified LPS of V. salmonicida and V. anguillarum were slightly soluble in water or phosphate buffered saline at neutral pH. However, at pH 8-9 or 5 the LPS was soluble, but at pH 8-9 the LPS did not adsorb to Sorbal. Thus, the suspension of Sorbal was incubated with LPS in 0.1 Macetate buffer, pH 5"0 with constant mixing overnight at 4 ~ C. This procedure was also used to adsorb LPS to BSA-Sorbal and oleic acid coated-Sorbal. Adsorbed LPS was determined by the colorimetric method detecting neutral hexoses described by Dubois et al. (1956). VS-P1 (a protein-LPS complex isolat.ed from V. salmonicida, Hjelmeland
254
J. BOGWALDETAL.
et al., 1988 was incubated with Sorbal in phosphate buffered saline (PBS), 10H 7.4 at 4 ~ C with constant mixing for 24 h.
IMMUNOFLUORESCENCE
Adherence of LPS and VS-P1 to the particles was checked by indirect immunofluorescent staining. The antigen coated particles were incubated in 5% normal rabbit serum diluted in phosphate buffered saline (PBS) to avoid nonspecific binding of antibodies. After washing, the particles were incubated with monoclonal antibodies directed against the LPS determinants of V. salmonicida (Espelid et al., 1988; Bogwald et al., 1990) for 1 h at room temperature. After washing in PBS, fluorescein isothiocyanate (FITC)-conjugated rabbit antimouse IgG (Dakopatts, Denmark), diluted in normal rabbit serum (5% in PBS) was added. The incubation was continued for 1 h at room temperature. The particles were thoroughly washed before examination in a Nikon fluorescence microscope.
VACCINATION
Atlantic salmon, Salmo salar L., weighing about 50 g each and reared in circular tanks in a mixture of fresh and salt (sea) water (final salinity 1-5%) at 12~ C and fed commercial salmon feed pellets (Ewos, Norway), were vaccinated by intraperitoneal (i.p.) injection with 0.1 ml of the various vaccine preparations (50 individuals in each group). Control fish received 10s formalin-killed bacteria of V. salmonicida or V. anguillarum serotype 01 and 02 respectively. In the LPS immunised groups, each fish was injected with 251lg purified LPS adsorbed to Sorbal particles or VS-P1 in an amount corresponding to a LPS content of 25 pg, adsorbed to Sorbal particles. All quantitations of LPS were done by the method of Dubois et al. (1956).
CHALLENGE
Fish were kept in circular tanks in a mixture of fresh and salt water at 10~ C (salinity 1.5%). Prior to challenge, the infectious dose to be used was determined in a prechallenge test. As a result of this test, vaccinated fish were challenged with 1 x 106, 5 • 104 and 1 x 104 bacteria of V. salmonicida or V. anguillarum 01 and 02 respectively 3 months after vaccination. The concentration and viability of each strain was controlled by counting the colony forming units (CFU) on Tryptic Soy Agar (Difco) plates supplemented with 1.5% NaC1 and 3% human red blood cells.
STATISTICAL ANALYSIS
To evaluate the significance of the differences in mortality between the control group (unvaccinated) and the vaccination groups, an approximation to standard normal distribution test was used.
VACCINATION OF ATLANTIC SALMON
255
100 ..-~- . t . . - - ~
L~" ...
,"
J
80
60
o~
4~ .s
20
-u
O
/
I0
5
2 " " ..........
15
20
Days after challenge
Fig. 1. Mortalities in Atlantic salmon vaccinated with various antigens of Vibrio salmonicida, after challenge with the homologous bacteria. RPS values are given in parentheses. (.. A.-), Unvaccinated; (-'+--), Sorbal (4"2); (--,--), whole cells (77-1); (--E]--), VS-Pl~.qorbal (44.8); (--O--), LPS~Sorbal (2"1). III. R e s u l t s CHALLENGE WITH VlBRIO SALMONIC1DA
As shown in Fig. 1 the accumulated mortality in the control group (unvaccinated fish) reached 95% 22 days after challenge with V. salmonicida. Fish injected with Sorbal particles alone or LPS adsorbed to the particles (126 pg LPS mg -1 Sorbal) resulted in the same mortality as fish in the control group (95%). However, fish injected with VS-P1 adsorbed to the particle (47pg LPS mg -1 Sorbal) gave a partial protection against infection (mortality 50~/o at day 22, P < 0.001). Washed formalin killed whole cells constituted the most effective vaccine with a mortality of only 20~/o (P<0.001). The relative percentage survival (RPS) values are indicated in Fig. 1 in parentheses. In another set of experiments, LPS was adsorbed to Sorbal particles precoated with protein (BSA) or lipid (oleic acid). The amount of LPS coated mg -1 Sorbal was 144 pg and 114 pg respectively. As shown in Table 1 the accumulated mortality in the control group (fish injected with.Sorbal particles alone) reached nearly 70~/o at day 20 after challenge. As observed in the former experiment, fish injected with LPS adsorbed to particles resulted in no protection against infection. In fish injected with particles precoated with BSA and then coated
256
J. BOGWALDETAL.
Table 1. Mortalities in Atlantic salmon vaccinated with modified LPS~Sorbal particles of Vibrio salmonicida 20 days after infection with the homologous bacteria
Preparation
Accumulated mortality (%)
RPS
61"9 54"8 16"7 0 47'6 40-5 53"7 39"0
11-3 72-6 100 22"6 35"5 12.9 37.1
Sorbal particle (negative control) LPS~orbal particle VS-P1-Sorbal particle Whole cell bacteria BSA-Sorbal particle LPS-BSA-Sorbal particle Oleic acid-Sorbal particle LPS-oleic acid-Sorbal particle RPS = 100% • f l '
\
% mortality in vaccinate~ _~ % mortality in unvaccinatcd]'
with LPS, a partial protection was seen (mortality 40% at day 20 after infection, P<0-01). However, injection of the BSA~Sorbal particle itself resulted in reduced mortality (RPS 22"6, P<0"05). Vaccination with Sorbal particles precoated with oleic acid (Cls:l) and then further coated with LPS, resulted in a partial protection against disease (mortality 39"0%, P<0"005). The reduction in mortality after injection of oleic acid coated Sorbal particles was not significant. The VS-P1 adsorbed to particles resulted in good protection (c. 20% mortality at day 20, P<0"001), while fish injected with whole cells showed no sign of disease (100% survival). The accumulated mortalities (%) and RPS values of these experiments are shown in Table 1.
CHALLENGEWITH V I B R I O A N G U I L L A R U M S E R O T Y P E 01
Mortality and RPS data are shown in Fig. 2. The mortality in the unvaccinated group reached 55%, 15 days after infection. A somewhat higher mortality (65%) was observed in the group which received the Sorbal particles alone without antigen. Fish injected with LPS adsorbed to the particles were slightly more resistant to infection (40% mortality, P=0"052). Those injected with whole bacterial cells showed a mortality of 10% (positive controls, P < 0.001).
CHALLENGEWITH VIBRIO A N G U I L L A R U M S E R O T Y P E 02
Unvaccinated salmon infected with V. a n g u i l l a r u m serotype 02, showed a mortality of 80% 19 days after infection as shown in Fig. 3. Fish injected with Sorbal particles alone, showed a non-specific protection against disease (55% mortality, P<0.01). In this case, vaccination of fish with the particulate LPS preparation resulted in a high protection against disease (10% mortality, P<0.001). The highest protection was obtained after vaccination with whole
257
VACCINATION OF ATLANTIC SALMON I00
80-
..-I-- .4:"
60-
..-I-.. 4:
.~. ,4:" ,~.. ~-.-~.-.~-
o~ ..~ .: ~.
9
.~."
.,/"
,!~::. a'" +
40
..::-
20
.'~
/~--~J~
i--i--i--i--~--i
0
w
i
,
5
i
I I0
I
I
1
i
I 15
Days after challenge
Fig. 2. Mortalities in Atlantic salmon vaccinated with antigens of Vibrio anguillarum serotype 01, after challenge with the homologous bacteria. RPS values are given in parentheses. (--A-'), Unvaccinated; (" "+" "), Sorbal (-); (--,--), whole cells (78"6); (--[7--), LPS-Sorbal (28'6).
formalin killed-bacteria (mortality 8%, P<0.001). RPS values are indicated in Fig. 3 in parentheses.
IV. D i s c u s s i o n Recognition of V. salmonicida cells by the Atlantic salmon immune system leads to production of antibodies predominantly directed against the LPS structures (Bogwald et al., 1991) and LPS is reported to be the protective antigen of V. anguillarum in ayu (Kawai & Kusuda, 1983) and A. hydrophila in carp (Baba et al., 1988). Results obtained in this study show that salmon vaccinated by i.p. injection of the particulate protein-LPS complex VS-P1 of V. salmonicida elicited an enhanced resistance against infection compared to fish immunised with a purified (i.e. protein free) preparation of LPS.(adsorbed to Sorbal particles). Although the protection seen after immunisation with VS--P1 was less than that with whole V. salmonicida cells, this result indicates an essential role of protein antigens in order to develop protective immunity against V. salmonicida. One
J. BOGWALDETAL.
258 I00
80-
~..~.-~-.~.-~.-~-.~.
~-~.~.~.~"
60-
+.+ .+.+.+. +.+.+.+.+.+.+
t~ 40-
ZO : §
.-l0
~_m_m_n=_=_i.-~-T-~-~ 0
5
~
,
,
,
I0
,
~
.
.
.
.
15
Doys ofter chollenge
Fig. 3. Mortalities in Atlantic salmon vaccinated with antigens of Vibrio anguillarum serotype 02, after challenge with the homologous bacteria. RPS values are given in parentheses. (.. A" "), Unvaccinated; (-" +-'), Sorbal (29-6); (---,--), whole cells (90-1); (--[:]~), LPS-Sorbal (85"2).
explanation for enhanced protection due to immunisation with VS-P1 is that the 40 kDa protein moiety of this complex (Hjelmeland et al., 1988) may stimulate helper cells in a T lymphocyte dependent anti-LPS response. Alternatively, the protein moiety of the protein-LPS antigen is needed for a proper presentation of the LPS structures. Intraperitoneal vaccination of fish with modified Sorbal particles precoated with BSA or oleic acid and further coated with LPS resulted in a reduced mortality compared to vaccination with unmodified LPS-Sorbal particles when infected with V. salmonicida. These results support the idea above concerning the need for a protein/lipid component for a proper stimulation of the immune system. Fish vaccinated with V. anguillarum serotype 01 LPS adsorbed to the Sorbal particles showed some protection against infection with the homologous bacterium. However, fish vaccinated with V. anguillarum serotype 02 LPS adsorbed to particles displayed a far better resistance against infection. It is difficult to explain the difference in protective capacity of the two LPS preparations although they show physio-chemical differences. In a previous report (Bogwald et al., 1990) we described certain characteristics of the two LPSs. LPS
VACCINATIONOF ATLANTICSALMON
259
from V. anguillarum serotypes 01 and 02 were found to differ with respect to the length of their O-specific side chains, i.e. serotype 02 expressed LPS with a more heterogenous chain length compared to serotype 01, as characterised by SDS-PAGE. While serotype 01 displayed three closely spaced bands, serotype 02 demonstrated five to nine clearly resolved bands. Also, the low molecular weight region (SDS-PAGE) of serotype 02 showed two broad bands whereas serotype 01 showed just one, and the relative amount of the low molecular fraction was higher in serotype 02 compared to serotype 01. In addition to physical differences, chemical differences between the two serotypes have also been observed (unpubl. res.). There seems to be no correlation between protective capacity and antibody production against the various bacteria (Bogwald et al., 1991). The antibody response in salmon after immunisation with whole bacterial cells of V. anguillarum serotype 02 was low compared to V. anguillarum serotype 01, when tested in ELISA using homologous whole cells as in vitro antigens. Western blots, using outer membrane preparations or purified LPS as antigens, showed that the antibody response is directed against epitopes on LPS (Bogwald et al., 1991), although the same sera tested against purified LPS antigens in ELISA, showed almost background activities. Concerning V. salmonicida, protection against infection was negligible when purified LPS was used for vaccination (adsorbed to the Sorbal particles), but LPS complexed with a 40 kDa molecule in the VS-P1 antigen elicited a substantial protection. Although the 40 kDa protein itself does not stimulate antibody production, this antigen may function as a T cell stimulating carrier and provide immunological memorY (T memory cells) and T cell help towards the B cells. Precoating of the Sorbal particles with BSA or oleic acid did not significantly enhance the amount of LPS bound per particle. In fact, the amount of LPS bound per particle in the VS-P1 complex was less compared to the purified LPS. In addition to the specific stimulation with the VS-P1 complex mentioned above, the amount and probably also the orientation of the LPS molecules bound to the particles are important for the establishment of immunity. It should be mentioned that in addition to the dominating antigenicity of the LPS antigens, a highly immunogenic 24 kDa surface protein can also be detected in Western blot experiments (Bogwald et al., 1991). The effect of this molecule in vaccination experiments is not ruled out, but it seems likely that the high protection of the whole cell bacterins demonstrated in Figs 1-3 and Table 1 also involve antigens other than LPS or LPS-protein complexes, like the 24 kDa molecule of V. salmonicida. There are some reports on the humoral and protective immunity in mammals by Vibrio cholera antigens of both protein and LPS nature (Neoh & Rowley, 1970, Manning et al., 1986). Antibodies to the protein antigen were reported to be more protective on a weight basis, but the LPS was by far the more potent immunogen. The resuIts obtained in this study do not definitely clarify the relation between the nature of antigens and protective capacity. However, a closer examination of the chemical structure of the LPSs of the two V. anguillarum serotypes would hopefully yield more exact information of the questions raised. Also, a more penetrating study must be done to elucidate the function of the protein carrier in the VS-P1 complex.
260
J. BOGWALDETAL.
This work was supported by A/S Apothekernes Laboratorium, Oslo]Tromso, Norway, and the Norwegian Research Council of Fisheries (NFFR). The assistance of Edvin Bredrup in carrying out the statistical analysis is highly appreciated.
References Allison, A. C. & Gregoriadis, G. (1974). Liposomes as immunological adjuvants. Nature 252, 252. Altman, A. & Dixon, F. J. (1989). Immunomodifiers in vaccines. In Vaccine Biotechnology. Advances in Veterinary Science and Comparative Medicine (J. L. Bittle & F. L. Murphy, eds) 33, pp. 301-343. San Diego, CA: Academic Press. Baba, T., Imamura, J., Izawa, K. & Ikeda, K. (1988). Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide. Journal offish Diseases 11,237-244. Berczi, I., Bertok, L. & Bereznai, T. (1966). Comparative studies on the toxicity of Escherichia coli lipopolysaccharide endotoxin in various animal species. Canadian Journal of Microbiology 12, 1070-1071. Bomford, R. (1989). Aluminium salts: perspectives in their use as adjuvants. In Immunological Adjuvants and Vaccines (G. Gregoriades, A. C. Allison & G. Poste, eds) pp. 35--41. New York: Plenum Press. Bogwald, J., Stensv~g, K., Hoffman, J., Espelid, S., Holm, K. O. & Jorgensen T. (1990). Electrophoretic and immunochemical analysis of surface antigens of the fish pathogen Vibrio salmonicida and Vibrio anguillarum. Journal of Fish Diseases 13,293-301. Bogwald, J., Stensv~g, K., Hoffman, J. & Jorgensen, T. (1991). Antibody specificities in Atlantic salmon, Salmo salar L., against the fish pathogens Vibrio salmonicida and Vibrio anguillarum. Journal offish Diseases 14, 79-87. Cossarinin-Dunier, M. (1986). Protection against enteric redmouth disease in rainbow trout, Salmo gairdneri Richardson, after vaccination with Yersinia ruckeri bacterin. Journal offish Diseases 9, 27-33. Dalsgaard, K. (1977). Evaluation of the adjuvant "Quil-A" in the vaccination of cattle against foot-and-mouth disease. Acta Veterinaria Scandinavica 18, 349-360. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. & Smith, F. (1956). Colorimertic method for determination of sugars and related substances. Analytical Chemistry 28, 350-356. Duff, D. C. B. (1942). The oral immunization of trout against Bacillus salmonicida. Journal of Immunology 44, 87-94. Espelid, S., Holm, K. O., Hjelmeland, K. & Jorgensen, T. (1988). Monoclonal antibodies against Vibrio salmonicida: the causative agent of coldwater vibriosis ("Hitra disease") in Atlantic salmon, Salmo salar L. Journal offish Diseases 11,207-214. Galanos, C., Luderitz, O., Rietschel, E. T. & Westphal, O. (1977). Newer aspects of the chemistry and biology of bacterial lipopolysaccharides, with special reference to their lipid A component. In International Review of Biochemistry, Biochemistry of Lipids 11, Vol. 14 (T. W. Goodwin, ed.) pp. 239-335. Baltimore, MD: University Park Press. Galanos, C., Luderitz, O. & Westphal, O. (1969). A new method for the extraction of R lipopolysaccharides. European Journal of Biochemistry 9, 245-249. Gallily, R. & Garvey, J. S. (1968). Primary stimulation of rats and mice with hemocyanin in solution and adsorbed on bentonite. The Journal of Immunology 101,924-929. Glenny, A. T., Pope, C. G., Waddington, H. & Wallace, U. (1926). The antigenic value of the toxin-antitoxin precipitate of Ramon. Journal of Pathology and Bacteriology 29, 31-40. Hennessen, W. (1965). The mode of action of mineral adjuvants. Progress in Immunobiological Standard 2, 71-79.
VACCINATION OF ATLANTIC SALMON
261
Hjelmeland, K., Stensv~g, K., Jorgensen, T. & Espelid S. (1988). Isolation and characterization of a surface layer antigen from Vibrio salmonicida. Journal of Fish Diseases 11,197-205. Holm, K. O. & JDrgensen, T. (1987). A successful vaccination of Atlantic salmon, Salmo salar L., against 'Hitra disease' or coldwater vibriosis. Journal of Fish Diseases 10, 85-90. Johnson, K. A., Flynn, J. K. & Amend, D. F. (1982). Duration of immunity in salmonids vaccinated by direct immersion with Yersinia ruckerii and Vibrio anguillarum bacterins. Journal offish Diseases 5, 207-213. Joo, I. (1973). Mineral carriers as adjuvants. Symposium Series of Immunobiological Standards 22, 123-130. Kawai, K. & Kusuda, R. (1983). Efficacy of the lipopolysaccharide vaccine against vibriosis in cultured ayu. Bulletin of the Japanese Society of Scientific Fisheries 49, 511-514. Kreuter, J., Liehl, E., Berg, U., Soliva, M. & Speiser, P. P. (1988). Influence of hydrophobicity on the adjuvant effect of particulate polymeric adjuvants. Vaccine 6, 253-256. Litwin, S. D. & Singer, J. M. (1965). The adjuvant action of latex particulate carriers. Journal of Immunology 95, 1147-1152. Manning, P. A., Heuzenroeder, M. W., Yeadon, J., Leavesley, D. I., Reeves, P. R. & Rowley, D. (1986). Molecular cloning and expression in Escherichia coli K-12 of the O-antigens of the Inaba and Ogawa serotypes of the Vibrio cholera 01 lipopolysaccharides and their potential for vaccine development. Infection and Immunity 53,272-277. Neoh, S. H. & Rowley, D. (1970). The antigens of Vibrio cholera involved in vibriocidal action of antibody and complement. Journal of Infectious Diseases 121,505-513. Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. & Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150, 76-85. Thorburn, M. A. & Jansson, E. L. K. (1988). The effects of booster vaccination and fish size on survival and antibody production following Vibrio infection of bathvaccinated rainbow trout, Salmo gairdneri. Aquaculture 71,285-291. Westphal, O. & Jann, K. (1965). Bacterial lipopolysaccharides: extraction with phenolwater and further applications of the procedure. Methods in Carbohydrate Chemistry 5, 83-96.