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The primary and secondary antibody responses to IROMP antigens in Atlantic salmon (Salmo salarL) immunised with A+and A−Aeromonas salmonicidabacterins
Fish & Shellfish Immunology (1999) 9, 125–138 Article No. fsim.1998.0182 Available online at http://www.idealibrary.com on
The primary and secondary antibody responses to IROMP antigens in Atlantic salmon (Salmo salar L) immunised with A + and A Aeromonas salmonicida bacterins A. M. O’DOWD1†, I. R. BRICKNELL1 C. J. SECOMBES2 1
AND
A. E. ELLIS1*
Marine Laboratory, Victoria Rd., Aberdeen AB11 9DB, Scotland, U.K. 2 University of Aberdeen, Aberdeen AB24 2TZ, Scotland, U.K. (Received 4 June 1998, accepted after revision 3 November 1998)
The aim of this work was to investigate if the presence of Aeromonas salmonicida A-layer had a suppressive e#ect on the development of antibodies in Atlantic salmon to outer membrane protein antigens expressed when A. salmonicida is grown under iron-restricting conditions. Atlantic salmon were immunised with an A-layer positive (A + ) or A-layer negative (A ) A. salmonicida iron-restricted whole cell bacterin, and were boosted 8 weeks later with homologous bacterin, heterologous bacterin, or phosphate bu#ered saline. Serum antibody titres to iron-restricted outer membrane protein antigens were measured for a period of 8 months. It was found that the presence of A-layer at the time of priming, or the introduction of A-layer 8 weeks post-prime, had no significant e#ect on the development of antibody responses to iron-restricted outer membrane protein antigens. In addition, it was found that boosting fish when antibody levels are high gave an enhanced antibody response compared to unboosted fish, and this response was characterised by both an increase in specific antibody titres and an increase in the percentage of responding fish. 1999 Academic Press Key words: Aeromonas salmonicida, A-layer, Atlantic salmon, secondary responses, iron-regulated outer membrane proteins.
I. Introduction Growth of Aeromonas salmonicida under iron-restricting conditions in vitro induces the expression of four novel outer membrane proteins, which are defined as iron-regulated outer membrane proteins (IROMPs). IROMPs are of considerable interest because active and passive immunisation of Atlantic salmon with IROMPs or anti-IROMP antisera respectively has been shown to confer better protection against A. salmonicida challenge than outer membrane proteins from bacteria grown under iron-replete conditions (Hirst & Ellis, 1994). In the following paper (pp. 139–151), Bricknell et al. describe the antibody responses of Atlantic salmon primed with an A-layer negative (A ) A. salmonicida iron-restricted whole cell bacterin, and challenged with a live virulent A-layer positive (A + ) iron-restricted bacterin. A single immunisation *Corresponding author. †Present address: School of Biomedical Sciences, University of St. Andrews, North Street, St. Andrews, Fife KY16 9AL, Scotland, U.K. 1050–4648/99/020125+14 $30.00/0
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of fish with the iron-restricted A bacterin induced significant levels of antibodies to antigens present in a sarkosyl extraction of outer membrane proteins from iron-restricted A A. salmonicida (termed A IROMP antigens). However, these antibody responses decreased rapidly after a challenge with a virulent A + A. salmonicida strain, while antibody responses in unchallenged fish continued to increase. As the fish were immunised with an A strain and challenged with an A + strain, it may be postulated that A-layer protein is a suppressive or dominant antigen, suppressing antibody responses to other outer membrane protein antigens by the mechanism of antigen competition. Antigenic competition has been described in fish by O’Neill (1985) and Killie (1995), who found antibody responses to haptens were suppressed in the presence of carriers. It was also found that rabbits immunised with A. salmonicida extracellular products (ECP) were capable of producing antibodies to most of the ECP components, but rainbow trout immunised with the same preparation could only produce antibodies to a fraction of constituents (Hastings & Ellis, 1988, 1990). These authors found no antibodies present to either the haemolysin (glycerophospholipid:cholesterol acetyltransferase) or the serine protease, despite other reports that both these antigens are immunogenic in fish (Arnesen et al., 1993; Thuvander et al., 1993). These data suggest that a complex mixture of antigens may subvert antibody responses in fish away from certain individual components, thus it is possible that antibody responses to multideterminant molecules may be restricted. An alternative explanation for the post-challenge reduction in antibody levels described by Bricknell et al. is that the introduction of antigen to an environment where specific antibodies are already in existence supports in vivo immune complex formation, which may a#ect the subsequent detection or formation of antibodies. In mammals, when specific antibodies are in excess in vivo, they combine with antigens, masking epitopes and preventing further stimulation of B cell sIg receptors by competitive inhibition (Schwartz, 1971). Moreover, the antibodies may not be available for detection as they are complexed to antigen, thus the antibody levels appear to be decreased. In addition, under certain conditions immune complexes can specifically downregulate the formation of antibodies by cross-linking Fc receptors and sIg on B cells (Uhr & Baumann, 1961; Sinclair & Panoskaltsis, 1987; Klaus, 1988; Heyman, 1990). Scant attention has been paid to the e#ect of immune complexes on fish antibody responses (Rijkers et al., 1981; Secombes & Resink, 1984), but it is possible that immune complexes specifically inhibit antibody formation as found in mammals. If antibody-mediated feedback inhibition mechanisms operate in fish, then the administration of a second dose of antigen before the antibody levels have subsided to basal levels could a#ect the secondary antibody response. Although a number of studies describe secondary responses in fish, particularly teleosts, the results have been variable and equivocal, which may be due in part to the use of di#erent fish species, antigen types (Cossarini-Dunier, 1986), routes of administration (Lamers & Muiswinkel, 1984; Lamers et al., 1985a,b) and use of adjuvant (Tatner et al., 1987). One other major source of variation occurs because in general, the
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initial antibody response to the priming antigen has not subsided at the time of boosting. Thus the variable e$ciencies of boosting may be less to do with the actual interval between primary and secondary immunisation, and more related to the antibody status of the animal at the time of boosting. Indeed, some authors have drawn attention to an inverse relationship between thé existing antibody levels and a heightened immune response (Lamers & Muiswinkel, 1984). Taking into consideration the results of Bricknell et al. and the possibility that antigen competition or antibody-mediated feedback inhibition may exist in fish, it was considered worthwhile to investigate the e#ect of an A. salmonicida A + iron-restricted bacterin on developing antibody responses to outer membrane antigens present in an A iron-restricted bacterin. In contrast to the previous experiments performed by Bricknell et al., in this study Atlantic salmon experiencing a primary antibody response to an A bacterin were further exposed to killed bacteria rather than live bacteria. II. Materials and methods BACTERIAL STRAINS
A. salmonicida MT423 is a typical A + virulent strain, and A. salmonicida MT004 is a typical strain which was originally A + when isolated from Atlantic salmon parr, but lost its A-layer through culturing to become A (Ellis et al., 1988). Strains were stored in glycerol at 20 C, or on tryptone soya agar (TSA, Oxoid) plates at 4 C. PREPARATION OF A. SALMONICIDA WHOLE CELL BACTERINS
A. salmonicida MT004 and MT423 strains were grown under iron-restricting conditions by inoculation into tryptone soy broths (TSB, Oxoid) supplemented with 100 ìM dipyridyl (Sigma), followed by incubation at 22 C for 48 h with agitation. Cultures were inactivated with 0·5% formalin (BDH) overnight at 4 C. The formalised broths were centrifuged at 6000g for 30 min, and the pellet resuspended in phosphate bu#ered saline pH7·4 (PBS) containing 0·5% formalin to an absorbance of 1·0 at optical density 540 nm (corresponding to 109 cells ml 1). Presence of IROMPs was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described by Hirst & Ellis (1994). IMMUNISATION OF FISH WITH BACTERINS
Atlantic salmon of the S2 grade, weighing 5–10 g, were acclimatised for 2 weeks in 1 m diameter tanks supplied with continuously flowing fresh water at ambient temperature (temperature profile is described in Table 1). Fish were fed on a commercial pelleted diet at 3% body weight per day. Fish were anaesthetised with benzocaine (ethyl p-aminobenzoate, Sigma, dissolved in ethanol), and injected intraperitoneally with 100 ìl of bacterin or PBS. Fish in groups 1–3 and 4–6 were injected with the A and A + bacterin respectively, and group 7 fish were injected with PBS. Eight weeks after immunisation,
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Table 1. Mean, minimum and maximum temperatures ( C) for each month of the experiment. Temperatures were recorded daily Month
May June July August September October November December January
Week post-
Temperature ( C)
Prime
Mean
Minimum
Maximum
0–4 4–8 8–12 12–16 16–20 20–24 24–28 28–32 32–34
14·4 14·4 13·5 13 12·5 9·6 6·6 3·8 2·2
13·3 13·5 11·9 11·6 11·6 6·6 5·7 2·9 1·6
15 15·6 14·8 13·6 13·8 12·1 7·3 5·1 3·9
10 fish per group were sampled by dead bleeding, and the remaining fish received a second ‘boost’ injection of either homologous or heterologous bacterin, or PBS. SAMPLING REGIME AND SERUM HANDLING
Ten fish per group were sacrificed at weeks 0, 8, 12, 16, 20, 26, 30 and 34 post-immunisation, and bled from the caudal vein. The blood was allowed to clot overnight at 4 C, centrifuged at 1500g for 15 min, and the serum was removed and stored at 20 C. ISOLATION OF A A. SALMONICIDA IROMP ANTIGENS
A. salmonicida MT004 strain was grown under iron-restricted conditions, and outer membrane proteins were sarkosyl-extracted as previously described by Hirst & Ellis (1994). This extraction procedure yielded a mixture of constitutive and iron-regulated outer membrane proteins (as shown by SDSPAGE). Lipopolysaccharide (LPS) has been associated with the extracted proteins (Hirst & Ellis, 1994), thus the term IROMP antigens is used to describe the antigens present in an A A. salmonicida iron-restricted outer membrane protein extract. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
ELISA plates were coated with IROMP antigens prepared as described above, and the ELISA performed as previously described by Bricknell et al. In order to determine antibody titres by end-point dilution, the absorbance values of each test serum dilution were compared with a standard pool of normal salmon serum (prepared as described in O’Dowd et al., 1998) diluted 1:10, which was included on each ELISA plate. Test sera with an absorbance greater than twice that of the standard normal serum was considered positive (absorbances of normal serum diluted 1:10 were generally 0·1), and the antibody titre is defined as the reciprocal of the last serum dilution to yield a
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positive absorbance. Fish with titres <10 (log2 titre of 3·32) were defined as non-responders, i.e. specific antibody titres were negligible. By contrast, fish with titres d10 (log2 titre of 3·32) were defined as responders, as their serum contained more specific antibody activity than the standard normal serum. WESTERN BLOTTING
A. salmonicida MT423 was grown in TSB for 48 h at 22 C with agitation. Cells were pelleted by centrifugation for 30 min at 6000g, treated with 2 ìg phenyl methane sulphonyl fluoride (Sigma) ml 1 PBS for 1 h, and re-centrifuged at 6000g. The pelleted cells were subjected to SDS-PAGE as described by Hirst & Ellis (1994). The proteins were transferred to Immobilon P blotting membrane (Biorad) using the Biorad wet blotting system, and blocked for 1 h at 37 C with 5% dried milk in PBS/0·05% Tween 20 (Sigma). The membrane was then incubated with salmon serum diluted 1:10, followed by monoclonal anti-trout Ig (4C10, Thuvander et al., 1990) conjugated to horseradish peroxidase (Hudson & Hay, 1989) and diluted 1:2000. Both incubation steps were carried out for 2 h at room temperature. The blotting membrane was developed with diaminobenzidine substrate (DAB Sigma Fast, Sigma) according to the suppliers’ instructions. STATISTICAL ANALYSIS
Inter-group comparisons of titres by Kruskal–Wallis analysis of variance (K-W AOV) were performed using SYSTAT. Pairwise comparisons were made using Tukey’s multiple comparison test, corrected for unequal sample sizes and tied rank data by using a standard error and test statistic proposed by Dunn, as described by Zar (1984) (referred to as Dunn’s Multiple Comparison Test in this paper). Statistical significance was accepted at P<0·05. Mean antibody titres presented in tables and graphs were calculated from titres which were transformed to log2. III. Results EFFECT OF A-LAYER ON ANTIBODY RESPONSES TO A. SALMONICIDA A IROMP ANTIGENS FOLLOWING PRIMARY IMMUNISATION
Fish were immunised with A or A + A. salmonicida iron-restricted whole cell bacterins at week 0, and serum antibody levels to A. salmonicida A IROMP antigens were detected by ELISA 8 weeks later. There were no significant di#erences between the titres of groups 1, 2 and 3, which had all been injected with the A bacterin at week 0 (P=0·096, K-W AOV). Additionally, there were no significant di#erences between the titres of groups 4, 5 and 6, which were injected with the A + bacterin at week 0 (P=0·923, K-W AOV). Therefore, the data from groups 1–3 and groups 4–6 were pooled to form two data sets representing fish injected with the A and A + bacterin respectively. Injection with an A or A + bacterin induced similar levels of specific antibodies in high percentages of fish (Table 2), and there were no significant
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Table 2. Antibody responses of Atlantic salmon immunised once with an A or A + A. salmonicida iron-restricted whole cell bacterin, or PBS. Antibody titres to IROMP antigens were assayed by ELISA 8 weeks postimmunisation. Data are expressed as mean antibody titres (log2) SE, and percentage of responding fish [i.e. the percentage of fish with specific antibody titres d 10 (log2 titre of 3·32)] Priming Mean titre % of Sample treatment (log2) SE responders size (n) A A+ PBS
4·820·65 5·720·51 00
72 90 0
29 30 10
di#erences between the titres of these two groups (P<0·5, Dunn’s Multiple Comparison Test). By contrast, there were no specific antibodies detectable in sera of control fish injected with PBS, and the titres of this group were significantly di#erent from the titres of the groups injected with an A or A + bacterin (P<0·001 for both comparisons, Dunn’s Multiple Comparison Test). These results show that the introduction of A-layer at the time of priming had no significant e#ect on the induction of antibody responses to A. salmonicida A IROMP antigens when measured 8 weeks post-immunisation. EFFECT OF A-LAYER ON ANTIBODY RESPONSES TO A. SALMONICIDA A IROMP ANTIGENS FOLLOWING TWO IMMUNISATIONS
Fish were immunised with A or A + A. salmonicida iron-restricted whole cell bacterins at week 0, and boosted 8 weeks later with homologous or heterologous bacterin, or with PBS. Serum antibody levels to A. salmonicida A IROMP antigens were assayed by ELISA at 4 week intervals from weeks 12 to 34. Data from all groups sampled from weeks 12 to 34 were treated separately, as the introduction of a boost at week 8 resulted in a unique immunisation pattern for each group. Comparison of the titres of bacterin-injected groups versus the titres of PBSinjected control groups At week 12, there were significantly higher mean antibody titres in each of the four boosted groups, group 1 (A /A ), group 2 (A /A + ), group 4 (A + /A ) and group 5 (A + /A + ) compared to the control group (Figure 1; Table 3 [Dunn’s Multiple Comparison Test]). At week 16, the control group titres were significantly di#erent only from the boosted group 2, and from weeks 20 to 34 there were no significant di#erences between the titres of the boosted groups and the control group (Table 3). The lack of significant di#erences between the boosted groups and the control group after week 12 coincided with the
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10 9
log2 antibody titre
8 7 6 5 4
positive
3
negative
2 1 0
4
prime
8
12
20 24 28 32 16 Weeks post immunisation
36
boost A–/A–
A–/A+
A–/PBS
A+/PBS
A+/A+
A+/A–
PBS/PBS
Fig. 1. Serum antibody titres to A. salmonicida A IROMP antigens from Atlantic salmon primed and boosted with various combinations of an A or A + A. salmonicida iron-restricted bacterin, or PBS. Each datum point represents the mean of log2 antibody titres, n=10. Standard error bars are omitted for clarity, and the straight line bisecting the graph indicates the border between positive and negative titres. Table 3. Pairwise comparisons of titres from bacterin-injected group (groups 1–6) with the PBS-injected control group (group 7) at each sampling point following boosting. p values were calculated by Dunn’s Multiple Comparison Test. A test of data generated at week 26 was not carried out as ANOVA indicated there was no significant di#erence between titres from all groups at this time point (data not shown) Group
Week 12
Week 16
Week 20
Week 30
Week 34
1 2 3 4 5 6
<0·05* <0·005* >0·05 <0·002* <0·001* >0·2
>0·5 <0·005* >0·5 >0·5 >0·1 >0·5
>0·5 >0·2 >0·5 >0·5 >0·5 >0·5
>0·5 >0·2 >0·5 >0·5 >0·5 >0·5
>0·5 >0·5 >0·5 >0·2 >0·05 >0·5
*= titres are significantly di#erent to titres of the PBS-injected control group.
detection of specific antibodies in the serum of a percentage of PBS-injected control fish, and a decline in the antibody responses in the boosted groups following a peak at week 16 (Figure 1). In contrast to the boosted groups, the titres from the unboosted groups (groups 3 and 6) were not significantly di#erent from titres of control fish between weeks 12 and 34 (for p values see (Table 3).
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A. M. O’DOWD ET AL.
Effect of A-layer on post-boost antibody responses to A. salmonicida A IROMP antigens There were no significant di#erences between the titres of fish primed/ boosted with A /A + (Group 2), or A + /A (Group 4) or A + /A + (Group 5) bacterins compared to titres of fish injected with A /A (Group 1) bacterins at any sampling point from weeks 12 to 34 (P>0·5 for all pairwise comparisons, Dunns Multiple Comparison Test). Likewise, there were no significant di#erences between the titres of unboosted groups receiving a single dose of A (Group 3) or A + bacterin (Group 6) from weeks 12 to 34 post-immunisation (P>0·5 for all pairwise comparisons, Dunn’s Multiple Comparison Test). These results indicate that the introduction of A-layer at the time of priming, boosting, or on both occasions, had no significant e#ect on post-boost antibody responses to A IROMP antigens. DETECTION OF ANTIBODIES TO A-PROTEIN IN THE SERUM OF FISH IMMUNISED WITH AN A + OR A IRON-RESTRICTED WHOLE CELL BACTERIN
Sera from fish primed and boosted with either A. salmonicida A or A + iron-restricted bacterins (A /A or A + /A + ), shown by ELISA to contain antibodies for A IROMP antigens, were selected to probe a blot of A. salmonicida A + whole cell preparation. Sera from fish injected twice with the A + bacterin stained a band located beside the 49·5 kDa marker (Figure 2, lane 2), which is identical to the molecular weight of the A-protein (Kay & Trust, 1997). By contrast, sera from the fish injected twice with the A bacterin showed no reaction with the A-protein (Figure 2, lane 3). Thus it is confirmed that individual fish which had been primed and boosted with an A + ironrestricted bacterin simultaneously produced serum antibodies to both the A-protein and A IROMP antigens. THE EFFECT OF BOOSTING ON ANTIBODY RESPONSES: A COMPARISON OF TITRES FROM FISH RECEIVING ONE OR TWO IMMUNISATIONS
There were no significant di#erences between the titres of the four boosted groups 1, 2, 4 and 5, or between the two unboosted groups, 3 and 6, on any sampling occasion (see previous section). Therefore, data from the unboosted groups and boosted groups were pooled to give two larger data sets, representing fish injected once (unboosted, n=20) or twice (boosted, n=40). The PBS-injected fish were used as a control group (n=10). There were significant di#erences between group titres on each sampling occasion from weeks 12 to 34, with the exception of week 26 post-immunisation (K-W AOV, p values not shown). Titres of the boosted group were significantly higher than those of the unboosted and control groups at weeks 12 (P<0·001 in both cases), 16 (P<0·001 in both cases) and 20 (P<0·001 and P<0·05 respectively) post-immunisation (Dunn’s Multiple Comparison Test, Figure 3). After a decrease in detectable titres in the boosted group at week 26, there was a subsequent increase in titres at weeks 30 and 34 (Figure 3). However, on these last two sampling occasions, there were no significant di#erences between titres of the boosted group and the control group (P>0·2 in both cases), but the boosted group did
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kDa
208 115 79 A
49.5 34.8 28.3 20.4 7.2 1
2
3
Fig. 2. Presence of serum antibodies to A. salmonicida A-protein as shown by Western blotting. A+ A. salmonicida cells were separated by gel electrophoresis, transferred to nitrocellulose, and stained with sera from Atlantic salmon injected twice with an iron-restricted A+ whole cell bacterin (lane 2) or an iron-restricted A whole cell bacterin (lane 3). The arrow denoted A marks the location of the A-protein. Lane 1, MW markers.
di#er significantly from the unboosted group (P<0·02 at week 30 and P<0·05 at week 34). There were no significant di#erences between the titres of the unboosted and control group at any time from weeks 12 to 34 (P>0·5 on all occasions), and the mean antibody titres of these two groups followed a very similar pattern, oscillating above and below the cut-o# line delineating positive and negative responses (Figure 3). When the percentage of responder fish, i.e. fish with serum titres d10 (log2 titre of 3·32), and the mean antibody titres of the responder fish are observed, it is apparent that boosting had a two fold e#ect, serving to increase both the percentage of fish showing an antibody response (Figure 4), and to enhance the magnitude of titres among the responder population (Figure 5). To summarise, two immunisations of A. salmonicida whole cell bacterin administered 8 weeks apart induced a higher mean antibody response than a single injection, and this e#ect was significant for three months post-boost. IV. Discussion One of the primary questions to be addressed by this work was what e#ect A-layer has on antibodies to other antigens present on the cell surface of A. salmonicida grown under iron-restricting conditions, i.e. constitutive and iron-regulated outer membrane proteins (IROMP antigens). The introduction of an iron-restricted A + bacterin, containing A-layer and IROMP antigens, at the time of priming and/or boosting had no significant e#ect on the subsequent antibody responses to IROMP antigens when compared to the responses of fish
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9 8 log2 antibody titre
7 6 5 4 positive 3
negative
2 1 0 prime
4
8
12
20 24 28 32 16 Weeks post immunisation
36
boost Boosted
Unboosted
PBS control
Fig. 3. Serum antibody titres to A. salmonicida IROMP antigens from Atlantic salmon injected with one (unboosted) or two (boosted) doses of A. salmonicida ironrestricted bacterin, or PBS. Each datum point represents the mean of log2 antibody titres SE, n=20 (unboosted), 40 (boosted) or 10 (PBS). The straight line bisecting the graph indicates the border between positive and negative titres.
primed and boosted with an iron-restricted A bacterin, which contained IROMP antigens but no A-layer. These results indicate that the presence of A-layer did not lead to suppression or inhibition of antibody formation to IROMP antigens. By Western blotting it was confirmed that the A-layer present in the A + bacterin was capable of inducing a specific antibody response, and serum containing these anti-A-layer antibodies was shown by ELISA to also contain antibodies to IROMP antigens. The combination of the ELISA and Western blot data suggest that the introduction of A-layer, and subsequent induction of A-layer-specific antibodies, did not suppress antibody responses to IROMP antigens. The interval between priming and boosting was 8 weeks, and when the ‘boosted’ group received a second immunisation of bacterin, this population still had high levels of specific serum antibodies. Such a situation has been proposed to diminish the di#erence between primary and secondary responses in fish (Lamers & Muiswinkel, 1984). It was expected that boosting when serum antibody levels are high would allow antigen and antibodies to complex in vivo. Immune complex formation in mammals can augument or suppress antibody responses depending on the ratio of antibody: antigen in the complex (Uhr & Baumann, 1961; Uhr & Moller, 1968), although a similar e#ect on antibody responses in fish is equivocal (Secombes & Resink, 1984). Despite the high levels of serum antibodies in the immunised population at the time of boosting, a second immunisation served to further increase antibody titres, and this contrasts with the arresting of the antibody response following exposure of immunised fish to live virulent A. salmonicida reported in Bricknell et al. accompanying paper.
PRIMARY AND SECONDARY ANTI A. SALMONICIDA ANTIBODY RESPONSES
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100 90 80
Percentage
70 60 50 40 30 20 10 0
4
8 12 16 20 24 28 Weeks post immunisation Boosted PBS control
32
36
Unboosted
Fig. 4. The percentage of responder Atlantic salmon injected with one (unboosted) or two (boosted) doses of A. salmonicida iron-restricted bacterin, or PBS. Responder fish are defined as those fish with serum titres to A. salmonicida IROMP antigens d10; n=20 (unboosted), 40 (boosted) or 10 (PBS).
Thus, the mechanism of arresting the antibody response by exposure to live virulent A. salmonicida cells reported by Bricknell et al. does not appear to have a basis in antigen competition or immune complex suppression. This suggests that some factor(s) associated with the live pathogen may be able to inhibit antibody production following infection of the fish. Indeed, it has been found previously that when A. salmonicida extracellular products were co-administered with the non-related antigen MS2 phage at priming and boosting, the development of anti-MS2 antibodies was inhibited in Atlantic salmon. It is hypothesised that the immunosuppression was mediated by the 70 kDa serine protease released by A. salmonicida, which may act at the level of B cell proliferation (Noor, 1996). This immunosuppressive mechanism may be responsible for the down-regulation of antibody responses to IROMP antigens when Atlantic salmon are challenged with live A. salmonicida as reported by Bricknell et al. Having established that the A-layer was not immunodominant over other IROMP antigens, interest was focused on the kinetics of the antibody responses. The lack of significant di#erence between titres of fish injected twice with various combinations of A + and A bacterins allowed the pooling of data from all fish receiving two doses of A. salmonicida bacterin (boosted fish), and the same principle was applied to the data from fish injected only once with the A + or A bacterin (unboosted fish). The mean antibody titres of the boosted fish were significantly enhanced compared to those of unboosted fish for 3 months post-boost. In almost every group at every sampling time, a percentage of the vaccinated fish failed to respond to treatment, which allowed a delineation of
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A. M. O’DOWD ET AL.
9 8
log2 antibody titre
7 6 5 4
positive
3
negative
2 1 0
4
8
12
Boosted
16 20 24 28 32 36 Weeks post immunisation Unboosted
PBS control
Fig. 5. Serum antibody titres to A. salmonicida IROMP antigens from responder Atlantic salmon injected with one (unboosted) or two (boosted) doses of A. salmonicida iron-restricted bacterin, or PBS. Each datum point represents the mean of log2 antibody titres SE. The straight line bisecting the graph indicates the border between positive and negative titres. Responder fish are defined as those fish with serum titres to A. salmonicida A IROMP antigens d 10.
fish into responder and non-responder groups. A second immunisation of bacterin 8 weeks after the first resulted in an increased percentage of fish producing specific antibodies when compared to fish receiving PBS as a second injection. The term non-responders was employed to describe fish whose serum did not contain specific antibody levels above those present in a standard pool of normal serum from non-immunised fish. As the mean antibody titres are a reflection of the percentage of responders (i.e. an increased frequency of non-responders reduces the mean antibody titres), it was interesting to note that the mean antibody titres of responding fish were significantly higher in the boosted group compared to the unboosted group, indicating that the second exposure to antigen increased the antibody responses of the responder fish in addition to increasing the percentage of responder fish. At weeks 8 and 12, all PBS-injected control fish had negative antibody titres by ELISA, but at week 16 a number of fish sampled had positive titres, and this pattern continued until the trial was terminated. The pattern of mean antibody titres of the unboosted group and the PBS-injected control group was identical from week 12 onwards, indicating that the positive antibody titres detected in the sera of unboosted fish at week 12 may not be a result of bacterin immunisation. The responsiveness of sera from PBS-injected fish to A. salmonicida IROMP antigens may be a result of the development of natural antibodies in PBS-injected fish which cross-react with antigens coated on the ELISA plate. Development of low-a$nity antibodies which cross-react with
PRIMARY AND SECONDARY ANTI A. SALMONICIDA ANTIBODY RESPONSES
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various specific antigens following PBS immunisation of fish has been previously reported (Michel et al., 1990). In summary, the results presented here support other findings that the A-layer does not prevent the development of antibody responses to other outer membrane antigens present on the surface of iron-restricted A. salmonicida. The results also show that a second immunisation with antigen can enhance the magnitude of an ongoing specific antibody response by increasing the titres of responding fish and the percentage of responding fish. This may have important implications for levels of protection a vaccine confers on a population. This study was supported by a grant from the Scottish O$ce Agriculture, Environment and Fisheries Department (SOAFED awarded to A.M.O’D.
References Arnesen, J. A., Bjornsdottir, R., Jorgensen, T. & Eggset, G. (1993). Immunological responses in Atlantic salmon, Salmo salar L., against purified serine protease and haemolysins from Aeromonas salmonicida. Journal of Fish Diseases 16, 409–423. Cossarini-Dunier, M. (1986). Secondary response of rainbow trout (Salmo gairdneri Richardson) to DNP-haemocyanin and Yersinia ruckeri. Aquaculture 52, 81–86. Ellis, A. E., Burrows, A. S. & Stapleton, K. (1988). Lack of relationship between virulence of Aeromonas salmonicida and the putative virulence factors: A-layer, extracellular proteases and extracellular haemolysins. Journal of Fish Diseases 11, 309–323. Hastings, T. S. & Ellis, A. E. (1988). The humoral immune response of rainbow trout, Salmo gairdneri Richardson, and rabbits to Aeromonas salmonicida extracellular products. Journal of Fish Diseases 11, 147–160. Hastings, T. S. & Ellis, A. E. (1990). Detection of antibodies induced in rainbow trout by di#erent Aeromonas salmonicida vaccine preparations. Journal of Aquatic Animal Health 2, 135–140. Heyman, B. (1990). The immune complex: possible ways of regulating the antibody response. Immunology Today 11, 310–313. Hirst, I. D. & Ellis, A. E. (1994). Iron-regulated outer membrane proteins of Aeromonas salmonicida are important protective antigens in Atlantic salmon against furunculosis. Fish & Shellfish Immunology 4, 29–45. Hudson, L. & Hay, F. C. (1989). In Practical Immunology. pp. 33–34. Oxford: Blackwell Scientific. Kay, W. W. & Trust, T. J. (1997). The surface of Aeromonas salmonicida: What does it look like and what does it do? In Furunculosis: Multidisciplinary Fish Disease Research (E.-M. Bernoth, A. E. Ellis, P. J. Midtlyng, G. Olivier & P. Smith, eds) pp. 235–247. London: Academic Press. Killie, J-E. A. (1995). Immunoregulation of antibody responses in teleost fish. An in vivo study on antigen-induced regulation and suppression of antibody responses against haptenated antigens in Atlantic salmon (Salmo salar L.). Ph.D. Thesis, University of Tromso, Norway. Klaus, G. G. B. (1988). Transmembrane signalling by B lymphocyte receptors. Immunology Supplement 1, 35–37. Lamers, C. H. J. & van Muiswinkel, W. B. (1984). Prim ` ary and secondary immune responses in carp (Cyprinus carpio) after administration of Yersinia ruckeri O-antigen. In Fish Diseases (Ed. Acuigrup) pp. 119–127. Madrid: ATP. Lamers, C. H. J., de Haas, M. J. N. & van Muiswinkel, W. B. (1985a). The reaction of the immune system of fish to vaccination: Development of immunological memory in
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carp (Cyprinus carpio L.), following direct immersion in Aeromonas hydrophila bacterin. Journal of Fish Diseases 8, 253–262. Lamers, C. H. J., de Haas, M. J. N. & van Muiswinkel, W. B. (1985b). Humoral response and memory formation in carp after injection of A. hydrophila bacterin. Developmental and Comparative Immunology 9, 65–75. Michel, C., Gonzalez, R., Bonjour, E. & Avrameas, S. (1990). A concurrent increasing of natural antibodies and enhancement of resistance to furunculosis in rainbow trout. Annales de Recherches Veterinaires 21, 311–218. Noor, I. (1996). Immunosuppression in Atlantic salmon by the serine protease of Aeromonas salmonicida. Ph. D. Thesis. Glasgow University. O’Dowd, A. M., Ellis, A. E. & Secombes, C. J. (1998). Binding of immune complexes to Atlantic salmon peripheral blood leucocytes. Developmental and Comparative Immunology 4, 439–448. O’Neill, J. G. (1985). Sequential antigenic competition in teleosts: A bacteriophageAeromonas salmonicida interaction. In Fish Immunology (M. J. Manning & M. F. Tatner, eds): pp. 141–155. London: Academic Press. Rijkers, G. T., Van Oosterom, R. & van Muiswinkel, W. B. (1981). The immune system of cyprinid fish. Oxytetracycline and the regulation of humoral immunity in carp (Cyprinus carpio). Veterinary Immunology and Immunopathology 2, 281–290. Schwartz, R. S. (1971). Immunoregulation by antibody. In Progress in Immunology (B. Amos, ed). Academic Press, N.Y. pp. 1081–1092. Secombes, C. J. & Resink, J. W. (1984). The immune responses of carp, Cyprinus carpio L., following injection of antigen-antibody complexes. Journal of Fish Biology 24, 193–200. Sinclair, N. R. StC. & Panoskaltsis, A. (1987). Immunoregulation by Fc signals. Immunology Today 8, 76–79. Tatner, M. F., Adams, A. & Leschen, W. (1987). An analysis of the primary and secondary antibody response in intact and thymectomized rainbow trout, Salmo gairdneri Richardson, to human gamma globulin and Aeromonas salmonicida. Journal of Fish Biology 31, 177–195. Thuvander, A., Fossum, C. & Lorenzen, N. (1990). Monoclonal antibodies to salmonid immunoglobulin, characterisation and applicability in immunoassays. Developmental & Comparative Immunology 14, 415–423. Thuvander, A., Wichardt, U. P. & Reitan, L. J. (1993). Humoral antibody response of brown trout Salmo trutta vaccinated against furunculosis. Diseases of Aquatic Organisms 17, 17–23. Uhr, J. W. & Baumann, J. B. (1961). Antibody formation. I. The suppression of antibody formation by passively administered antibody. Journal of Experimental Medicine 113, 935–957. Uhr, J. W. & Moller, G. (1968). Regulatory e#ect of antibody on the immune response. Advances in Immunology 8, 81–123. Zar, J. H. (1984). Biostatistical Analysis. Prentice-Hall International, Inc.
The primary and secondary antibody responses to IROMP antigens in Atlantic salmon (Salmo salar L) immunised with A + and A Aeromonas salmonicida bacterins A. M. O’DOWD1†, I. R. BRICKNELL1 C. J. SECOMBES2 1
AND
A. E. ELLIS1*
Marine Laboratory, Victoria Rd., Aberdeen AB11 9DB, Scotland, U.K. 2 University of Aberdeen, Aberdeen AB24 2TZ, Scotland, U.K. (Received 4 June 1998, accepted after revision 3 November 1998)
The aim of this work was to investigate if the presence of Aeromonas salmonicida A-layer had a suppressive e#ect on the development of antibodies in Atlantic salmon to outer membrane protein antigens expressed when A. salmonicida is grown under iron-restricting conditions. Atlantic salmon were immunised with an A-layer positive (A + ) or A-layer negative (A ) A. salmonicida iron-restricted whole cell bacterin, and were boosted 8 weeks later with homologous bacterin, heterologous bacterin, or phosphate bu#ered saline. Serum antibody titres to iron-restricted outer membrane protein antigens were measured for a period of 8 months. It was found that the presence of A-layer at the time of priming, or the introduction of A-layer 8 weeks post-prime, had no significant e#ect on the development of antibody responses to iron-restricted outer membrane protein antigens. In addition, it was found that boosting fish when antibody levels are high gave an enhanced antibody response compared to unboosted fish, and this response was characterised by both an increase in specific antibody titres and an increase in the percentage of responding fish. 1999 Academic Press Key words: Aeromonas salmonicida, A-layer, Atlantic salmon, secondary responses, iron-regulated outer membrane proteins.
I. Introduction Growth of Aeromonas salmonicida under iron-restricting conditions in vitro induces the expression of four novel outer membrane proteins, which are defined as iron-regulated outer membrane proteins (IROMPs). IROMPs are of considerable interest because active and passive immunisation of Atlantic salmon with IROMPs or anti-IROMP antisera respectively has been shown to confer better protection against A. salmonicida challenge than outer membrane proteins from bacteria grown under iron-replete conditions (Hirst & Ellis, 1994). In the following paper (pp. 139–151), Bricknell et al. describe the antibody responses of Atlantic salmon primed with an A-layer negative (A ) A. salmonicida iron-restricted whole cell bacterin, and challenged with a live virulent A-layer positive (A + ) iron-restricted bacterin. A single immunisation *Corresponding author. †Present address: School of Biomedical Sciences, University of St. Andrews, North Street, St. Andrews, Fife KY16 9AL, Scotland, U.K. 1050–4648/99/020125+14 $30.00/0
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of fish with the iron-restricted A bacterin induced significant levels of antibodies to antigens present in a sarkosyl extraction of outer membrane proteins from iron-restricted A A. salmonicida (termed A IROMP antigens). However, these antibody responses decreased rapidly after a challenge with a virulent A + A. salmonicida strain, while antibody responses in unchallenged fish continued to increase. As the fish were immunised with an A strain and challenged with an A + strain, it may be postulated that A-layer protein is a suppressive or dominant antigen, suppressing antibody responses to other outer membrane protein antigens by the mechanism of antigen competition. Antigenic competition has been described in fish by O’Neill (1985) and Killie (1995), who found antibody responses to haptens were suppressed in the presence of carriers. It was also found that rabbits immunised with A. salmonicida extracellular products (ECP) were capable of producing antibodies to most of the ECP components, but rainbow trout immunised with the same preparation could only produce antibodies to a fraction of constituents (Hastings & Ellis, 1988, 1990). These authors found no antibodies present to either the haemolysin (glycerophospholipid:cholesterol acetyltransferase) or the serine protease, despite other reports that both these antigens are immunogenic in fish (Arnesen et al., 1993; Thuvander et al., 1993). These data suggest that a complex mixture of antigens may subvert antibody responses in fish away from certain individual components, thus it is possible that antibody responses to multideterminant molecules may be restricted. An alternative explanation for the post-challenge reduction in antibody levels described by Bricknell et al. is that the introduction of antigen to an environment where specific antibodies are already in existence supports in vivo immune complex formation, which may a#ect the subsequent detection or formation of antibodies. In mammals, when specific antibodies are in excess in vivo, they combine with antigens, masking epitopes and preventing further stimulation of B cell sIg receptors by competitive inhibition (Schwartz, 1971). Moreover, the antibodies may not be available for detection as they are complexed to antigen, thus the antibody levels appear to be decreased. In addition, under certain conditions immune complexes can specifically downregulate the formation of antibodies by cross-linking Fc receptors and sIg on B cells (Uhr & Baumann, 1961; Sinclair & Panoskaltsis, 1987; Klaus, 1988; Heyman, 1990). Scant attention has been paid to the e#ect of immune complexes on fish antibody responses (Rijkers et al., 1981; Secombes & Resink, 1984), but it is possible that immune complexes specifically inhibit antibody formation as found in mammals. If antibody-mediated feedback inhibition mechanisms operate in fish, then the administration of a second dose of antigen before the antibody levels have subsided to basal levels could a#ect the secondary antibody response. Although a number of studies describe secondary responses in fish, particularly teleosts, the results have been variable and equivocal, which may be due in part to the use of di#erent fish species, antigen types (Cossarini-Dunier, 1986), routes of administration (Lamers & Muiswinkel, 1984; Lamers et al., 1985a,b) and use of adjuvant (Tatner et al., 1987). One other major source of variation occurs because in general, the
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initial antibody response to the priming antigen has not subsided at the time of boosting. Thus the variable e$ciencies of boosting may be less to do with the actual interval between primary and secondary immunisation, and more related to the antibody status of the animal at the time of boosting. Indeed, some authors have drawn attention to an inverse relationship between thé existing antibody levels and a heightened immune response (Lamers & Muiswinkel, 1984). Taking into consideration the results of Bricknell et al. and the possibility that antigen competition or antibody-mediated feedback inhibition may exist in fish, it was considered worthwhile to investigate the e#ect of an A. salmonicida A + iron-restricted bacterin on developing antibody responses to outer membrane antigens present in an A iron-restricted bacterin. In contrast to the previous experiments performed by Bricknell et al., in this study Atlantic salmon experiencing a primary antibody response to an A bacterin were further exposed to killed bacteria rather than live bacteria. II. Materials and methods BACTERIAL STRAINS
A. salmonicida MT423 is a typical A + virulent strain, and A. salmonicida MT004 is a typical strain which was originally A + when isolated from Atlantic salmon parr, but lost its A-layer through culturing to become A (Ellis et al., 1988). Strains were stored in glycerol at 20 C, or on tryptone soya agar (TSA, Oxoid) plates at 4 C. PREPARATION OF A. SALMONICIDA WHOLE CELL BACTERINS
A. salmonicida MT004 and MT423 strains were grown under iron-restricting conditions by inoculation into tryptone soy broths (TSB, Oxoid) supplemented with 100 ìM dipyridyl (Sigma), followed by incubation at 22 C for 48 h with agitation. Cultures were inactivated with 0·5% formalin (BDH) overnight at 4 C. The formalised broths were centrifuged at 6000g for 30 min, and the pellet resuspended in phosphate bu#ered saline pH7·4 (PBS) containing 0·5% formalin to an absorbance of 1·0 at optical density 540 nm (corresponding to 109 cells ml 1). Presence of IROMPs was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) as previously described by Hirst & Ellis (1994). IMMUNISATION OF FISH WITH BACTERINS
Atlantic salmon of the S2 grade, weighing 5–10 g, were acclimatised for 2 weeks in 1 m diameter tanks supplied with continuously flowing fresh water at ambient temperature (temperature profile is described in Table 1). Fish were fed on a commercial pelleted diet at 3% body weight per day. Fish were anaesthetised with benzocaine (ethyl p-aminobenzoate, Sigma, dissolved in ethanol), and injected intraperitoneally with 100 ìl of bacterin or PBS. Fish in groups 1–3 and 4–6 were injected with the A and A + bacterin respectively, and group 7 fish were injected with PBS. Eight weeks after immunisation,
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Table 1. Mean, minimum and maximum temperatures ( C) for each month of the experiment. Temperatures were recorded daily Month
May June July August September October November December January
Week post-
Temperature ( C)
Prime
Mean
Minimum
Maximum
0–4 4–8 8–12 12–16 16–20 20–24 24–28 28–32 32–34
14·4 14·4 13·5 13 12·5 9·6 6·6 3·8 2·2
13·3 13·5 11·9 11·6 11·6 6·6 5·7 2·9 1·6
15 15·6 14·8 13·6 13·8 12·1 7·3 5·1 3·9
10 fish per group were sampled by dead bleeding, and the remaining fish received a second ‘boost’ injection of either homologous or heterologous bacterin, or PBS. SAMPLING REGIME AND SERUM HANDLING
Ten fish per group were sacrificed at weeks 0, 8, 12, 16, 20, 26, 30 and 34 post-immunisation, and bled from the caudal vein. The blood was allowed to clot overnight at 4 C, centrifuged at 1500g for 15 min, and the serum was removed and stored at 20 C. ISOLATION OF A A. SALMONICIDA IROMP ANTIGENS
A. salmonicida MT004 strain was grown under iron-restricted conditions, and outer membrane proteins were sarkosyl-extracted as previously described by Hirst & Ellis (1994). This extraction procedure yielded a mixture of constitutive and iron-regulated outer membrane proteins (as shown by SDSPAGE). Lipopolysaccharide (LPS) has been associated with the extracted proteins (Hirst & Ellis, 1994), thus the term IROMP antigens is used to describe the antigens present in an A A. salmonicida iron-restricted outer membrane protein extract. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
ELISA plates were coated with IROMP antigens prepared as described above, and the ELISA performed as previously described by Bricknell et al. In order to determine antibody titres by end-point dilution, the absorbance values of each test serum dilution were compared with a standard pool of normal salmon serum (prepared as described in O’Dowd et al., 1998) diluted 1:10, which was included on each ELISA plate. Test sera with an absorbance greater than twice that of the standard normal serum was considered positive (absorbances of normal serum diluted 1:10 were generally 0·1), and the antibody titre is defined as the reciprocal of the last serum dilution to yield a
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positive absorbance. Fish with titres <10 (log2 titre of 3·32) were defined as non-responders, i.e. specific antibody titres were negligible. By contrast, fish with titres d10 (log2 titre of 3·32) were defined as responders, as their serum contained more specific antibody activity than the standard normal serum. WESTERN BLOTTING
A. salmonicida MT423 was grown in TSB for 48 h at 22 C with agitation. Cells were pelleted by centrifugation for 30 min at 6000g, treated with 2 ìg phenyl methane sulphonyl fluoride (Sigma) ml 1 PBS for 1 h, and re-centrifuged at 6000g. The pelleted cells were subjected to SDS-PAGE as described by Hirst & Ellis (1994). The proteins were transferred to Immobilon P blotting membrane (Biorad) using the Biorad wet blotting system, and blocked for 1 h at 37 C with 5% dried milk in PBS/0·05% Tween 20 (Sigma). The membrane was then incubated with salmon serum diluted 1:10, followed by monoclonal anti-trout Ig (4C10, Thuvander et al., 1990) conjugated to horseradish peroxidase (Hudson & Hay, 1989) and diluted 1:2000. Both incubation steps were carried out for 2 h at room temperature. The blotting membrane was developed with diaminobenzidine substrate (DAB Sigma Fast, Sigma) according to the suppliers’ instructions. STATISTICAL ANALYSIS
Inter-group comparisons of titres by Kruskal–Wallis analysis of variance (K-W AOV) were performed using SYSTAT. Pairwise comparisons were made using Tukey’s multiple comparison test, corrected for unequal sample sizes and tied rank data by using a standard error and test statistic proposed by Dunn, as described by Zar (1984) (referred to as Dunn’s Multiple Comparison Test in this paper). Statistical significance was accepted at P<0·05. Mean antibody titres presented in tables and graphs were calculated from titres which were transformed to log2. III. Results EFFECT OF A-LAYER ON ANTIBODY RESPONSES TO A. SALMONICIDA A IROMP ANTIGENS FOLLOWING PRIMARY IMMUNISATION
Fish were immunised with A or A + A. salmonicida iron-restricted whole cell bacterins at week 0, and serum antibody levels to A. salmonicida A IROMP antigens were detected by ELISA 8 weeks later. There were no significant di#erences between the titres of groups 1, 2 and 3, which had all been injected with the A bacterin at week 0 (P=0·096, K-W AOV). Additionally, there were no significant di#erences between the titres of groups 4, 5 and 6, which were injected with the A + bacterin at week 0 (P=0·923, K-W AOV). Therefore, the data from groups 1–3 and groups 4–6 were pooled to form two data sets representing fish injected with the A and A + bacterin respectively. Injection with an A or A + bacterin induced similar levels of specific antibodies in high percentages of fish (Table 2), and there were no significant
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Table 2. Antibody responses of Atlantic salmon immunised once with an A or A + A. salmonicida iron-restricted whole cell bacterin, or PBS. Antibody titres to IROMP antigens were assayed by ELISA 8 weeks postimmunisation. Data are expressed as mean antibody titres (log2) SE, and percentage of responding fish [i.e. the percentage of fish with specific antibody titres d 10 (log2 titre of 3·32)] Priming Mean titre % of Sample treatment (log2) SE responders size (n) A A+ PBS
4·820·65 5·720·51 00
72 90 0
29 30 10
di#erences between the titres of these two groups (P<0·5, Dunn’s Multiple Comparison Test). By contrast, there were no specific antibodies detectable in sera of control fish injected with PBS, and the titres of this group were significantly di#erent from the titres of the groups injected with an A or A + bacterin (P<0·001 for both comparisons, Dunn’s Multiple Comparison Test). These results show that the introduction of A-layer at the time of priming had no significant e#ect on the induction of antibody responses to A. salmonicida A IROMP antigens when measured 8 weeks post-immunisation. EFFECT OF A-LAYER ON ANTIBODY RESPONSES TO A. SALMONICIDA A IROMP ANTIGENS FOLLOWING TWO IMMUNISATIONS
Fish were immunised with A or A + A. salmonicida iron-restricted whole cell bacterins at week 0, and boosted 8 weeks later with homologous or heterologous bacterin, or with PBS. Serum antibody levels to A. salmonicida A IROMP antigens were assayed by ELISA at 4 week intervals from weeks 12 to 34. Data from all groups sampled from weeks 12 to 34 were treated separately, as the introduction of a boost at week 8 resulted in a unique immunisation pattern for each group. Comparison of the titres of bacterin-injected groups versus the titres of PBSinjected control groups At week 12, there were significantly higher mean antibody titres in each of the four boosted groups, group 1 (A /A ), group 2 (A /A + ), group 4 (A + /A ) and group 5 (A + /A + ) compared to the control group (Figure 1; Table 3 [Dunn’s Multiple Comparison Test]). At week 16, the control group titres were significantly di#erent only from the boosted group 2, and from weeks 20 to 34 there were no significant di#erences between the titres of the boosted groups and the control group (Table 3). The lack of significant di#erences between the boosted groups and the control group after week 12 coincided with the
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10 9
log2 antibody titre
8 7 6 5 4
positive
3
negative
2 1 0
4
prime
8
12
20 24 28 32 16 Weeks post immunisation
36
boost A–/A–
A–/A+
A–/PBS
A+/PBS
A+/A+
A+/A–
PBS/PBS
Fig. 1. Serum antibody titres to A. salmonicida A IROMP antigens from Atlantic salmon primed and boosted with various combinations of an A or A + A. salmonicida iron-restricted bacterin, or PBS. Each datum point represents the mean of log2 antibody titres, n=10. Standard error bars are omitted for clarity, and the straight line bisecting the graph indicates the border between positive and negative titres. Table 3. Pairwise comparisons of titres from bacterin-injected group (groups 1–6) with the PBS-injected control group (group 7) at each sampling point following boosting. p values were calculated by Dunn’s Multiple Comparison Test. A test of data generated at week 26 was not carried out as ANOVA indicated there was no significant di#erence between titres from all groups at this time point (data not shown) Group
Week 12
Week 16
Week 20
Week 30
Week 34
1 2 3 4 5 6
<0·05* <0·005* >0·05 <0·002* <0·001* >0·2
>0·5 <0·005* >0·5 >0·5 >0·1 >0·5
>0·5 >0·2 >0·5 >0·5 >0·5 >0·5
>0·5 >0·2 >0·5 >0·5 >0·5 >0·5
>0·5 >0·5 >0·5 >0·2 >0·05 >0·5
*= titres are significantly di#erent to titres of the PBS-injected control group.
detection of specific antibodies in the serum of a percentage of PBS-injected control fish, and a decline in the antibody responses in the boosted groups following a peak at week 16 (Figure 1). In contrast to the boosted groups, the titres from the unboosted groups (groups 3 and 6) were not significantly di#erent from titres of control fish between weeks 12 and 34 (for p values see (Table 3).
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A. M. O’DOWD ET AL.
Effect of A-layer on post-boost antibody responses to A. salmonicida A IROMP antigens There were no significant di#erences between the titres of fish primed/ boosted with A /A + (Group 2), or A + /A (Group 4) or A + /A + (Group 5) bacterins compared to titres of fish injected with A /A (Group 1) bacterins at any sampling point from weeks 12 to 34 (P>0·5 for all pairwise comparisons, Dunns Multiple Comparison Test). Likewise, there were no significant di#erences between the titres of unboosted groups receiving a single dose of A (Group 3) or A + bacterin (Group 6) from weeks 12 to 34 post-immunisation (P>0·5 for all pairwise comparisons, Dunn’s Multiple Comparison Test). These results indicate that the introduction of A-layer at the time of priming, boosting, or on both occasions, had no significant e#ect on post-boost antibody responses to A IROMP antigens. DETECTION OF ANTIBODIES TO A-PROTEIN IN THE SERUM OF FISH IMMUNISED WITH AN A + OR A IRON-RESTRICTED WHOLE CELL BACTERIN
Sera from fish primed and boosted with either A. salmonicida A or A + iron-restricted bacterins (A /A or A + /A + ), shown by ELISA to contain antibodies for A IROMP antigens, were selected to probe a blot of A. salmonicida A + whole cell preparation. Sera from fish injected twice with the A + bacterin stained a band located beside the 49·5 kDa marker (Figure 2, lane 2), which is identical to the molecular weight of the A-protein (Kay & Trust, 1997). By contrast, sera from the fish injected twice with the A bacterin showed no reaction with the A-protein (Figure 2, lane 3). Thus it is confirmed that individual fish which had been primed and boosted with an A + ironrestricted bacterin simultaneously produced serum antibodies to both the A-protein and A IROMP antigens. THE EFFECT OF BOOSTING ON ANTIBODY RESPONSES: A COMPARISON OF TITRES FROM FISH RECEIVING ONE OR TWO IMMUNISATIONS
There were no significant di#erences between the titres of the four boosted groups 1, 2, 4 and 5, or between the two unboosted groups, 3 and 6, on any sampling occasion (see previous section). Therefore, data from the unboosted groups and boosted groups were pooled to give two larger data sets, representing fish injected once (unboosted, n=20) or twice (boosted, n=40). The PBS-injected fish were used as a control group (n=10). There were significant di#erences between group titres on each sampling occasion from weeks 12 to 34, with the exception of week 26 post-immunisation (K-W AOV, p values not shown). Titres of the boosted group were significantly higher than those of the unboosted and control groups at weeks 12 (P<0·001 in both cases), 16 (P<0·001 in both cases) and 20 (P<0·001 and P<0·05 respectively) post-immunisation (Dunn’s Multiple Comparison Test, Figure 3). After a decrease in detectable titres in the boosted group at week 26, there was a subsequent increase in titres at weeks 30 and 34 (Figure 3). However, on these last two sampling occasions, there were no significant di#erences between titres of the boosted group and the control group (P>0·2 in both cases), but the boosted group did
PRIMARY AND SECONDARY ANTI A. SALMONICIDA ANTIBODY RESPONSES
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kDa
208 115 79 A
49.5 34.8 28.3 20.4 7.2 1
2
3
Fig. 2. Presence of serum antibodies to A. salmonicida A-protein as shown by Western blotting. A+ A. salmonicida cells were separated by gel electrophoresis, transferred to nitrocellulose, and stained with sera from Atlantic salmon injected twice with an iron-restricted A+ whole cell bacterin (lane 2) or an iron-restricted A whole cell bacterin (lane 3). The arrow denoted A marks the location of the A-protein. Lane 1, MW markers.
di#er significantly from the unboosted group (P<0·02 at week 30 and P<0·05 at week 34). There were no significant di#erences between the titres of the unboosted and control group at any time from weeks 12 to 34 (P>0·5 on all occasions), and the mean antibody titres of these two groups followed a very similar pattern, oscillating above and below the cut-o# line delineating positive and negative responses (Figure 3). When the percentage of responder fish, i.e. fish with serum titres d10 (log2 titre of 3·32), and the mean antibody titres of the responder fish are observed, it is apparent that boosting had a two fold e#ect, serving to increase both the percentage of fish showing an antibody response (Figure 4), and to enhance the magnitude of titres among the responder population (Figure 5). To summarise, two immunisations of A. salmonicida whole cell bacterin administered 8 weeks apart induced a higher mean antibody response than a single injection, and this e#ect was significant for three months post-boost. IV. Discussion One of the primary questions to be addressed by this work was what e#ect A-layer has on antibodies to other antigens present on the cell surface of A. salmonicida grown under iron-restricting conditions, i.e. constitutive and iron-regulated outer membrane proteins (IROMP antigens). The introduction of an iron-restricted A + bacterin, containing A-layer and IROMP antigens, at the time of priming and/or boosting had no significant e#ect on the subsequent antibody responses to IROMP antigens when compared to the responses of fish
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9 8 log2 antibody titre
7 6 5 4 positive 3
negative
2 1 0 prime
4
8
12
20 24 28 32 16 Weeks post immunisation
36
boost Boosted
Unboosted
PBS control
Fig. 3. Serum antibody titres to A. salmonicida IROMP antigens from Atlantic salmon injected with one (unboosted) or two (boosted) doses of A. salmonicida ironrestricted bacterin, or PBS. Each datum point represents the mean of log2 antibody titres SE, n=20 (unboosted), 40 (boosted) or 10 (PBS). The straight line bisecting the graph indicates the border between positive and negative titres.
primed and boosted with an iron-restricted A bacterin, which contained IROMP antigens but no A-layer. These results indicate that the presence of A-layer did not lead to suppression or inhibition of antibody formation to IROMP antigens. By Western blotting it was confirmed that the A-layer present in the A + bacterin was capable of inducing a specific antibody response, and serum containing these anti-A-layer antibodies was shown by ELISA to also contain antibodies to IROMP antigens. The combination of the ELISA and Western blot data suggest that the introduction of A-layer, and subsequent induction of A-layer-specific antibodies, did not suppress antibody responses to IROMP antigens. The interval between priming and boosting was 8 weeks, and when the ‘boosted’ group received a second immunisation of bacterin, this population still had high levels of specific serum antibodies. Such a situation has been proposed to diminish the di#erence between primary and secondary responses in fish (Lamers & Muiswinkel, 1984). It was expected that boosting when serum antibody levels are high would allow antigen and antibodies to complex in vivo. Immune complex formation in mammals can augument or suppress antibody responses depending on the ratio of antibody: antigen in the complex (Uhr & Baumann, 1961; Uhr & Moller, 1968), although a similar e#ect on antibody responses in fish is equivocal (Secombes & Resink, 1984). Despite the high levels of serum antibodies in the immunised population at the time of boosting, a second immunisation served to further increase antibody titres, and this contrasts with the arresting of the antibody response following exposure of immunised fish to live virulent A. salmonicida reported in Bricknell et al. accompanying paper.
PRIMARY AND SECONDARY ANTI A. SALMONICIDA ANTIBODY RESPONSES
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100 90 80
Percentage
70 60 50 40 30 20 10 0
4
8 12 16 20 24 28 Weeks post immunisation Boosted PBS control
32
36
Unboosted
Fig. 4. The percentage of responder Atlantic salmon injected with one (unboosted) or two (boosted) doses of A. salmonicida iron-restricted bacterin, or PBS. Responder fish are defined as those fish with serum titres to A. salmonicida IROMP antigens d10; n=20 (unboosted), 40 (boosted) or 10 (PBS).
Thus, the mechanism of arresting the antibody response by exposure to live virulent A. salmonicida cells reported by Bricknell et al. does not appear to have a basis in antigen competition or immune complex suppression. This suggests that some factor(s) associated with the live pathogen may be able to inhibit antibody production following infection of the fish. Indeed, it has been found previously that when A. salmonicida extracellular products were co-administered with the non-related antigen MS2 phage at priming and boosting, the development of anti-MS2 antibodies was inhibited in Atlantic salmon. It is hypothesised that the immunosuppression was mediated by the 70 kDa serine protease released by A. salmonicida, which may act at the level of B cell proliferation (Noor, 1996). This immunosuppressive mechanism may be responsible for the down-regulation of antibody responses to IROMP antigens when Atlantic salmon are challenged with live A. salmonicida as reported by Bricknell et al. Having established that the A-layer was not immunodominant over other IROMP antigens, interest was focused on the kinetics of the antibody responses. The lack of significant di#erence between titres of fish injected twice with various combinations of A + and A bacterins allowed the pooling of data from all fish receiving two doses of A. salmonicida bacterin (boosted fish), and the same principle was applied to the data from fish injected only once with the A + or A bacterin (unboosted fish). The mean antibody titres of the boosted fish were significantly enhanced compared to those of unboosted fish for 3 months post-boost. In almost every group at every sampling time, a percentage of the vaccinated fish failed to respond to treatment, which allowed a delineation of
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9 8
log2 antibody titre
7 6 5 4
positive
3
negative
2 1 0
4
8
12
Boosted
16 20 24 28 32 36 Weeks post immunisation Unboosted
PBS control
Fig. 5. Serum antibody titres to A. salmonicida IROMP antigens from responder Atlantic salmon injected with one (unboosted) or two (boosted) doses of A. salmonicida iron-restricted bacterin, or PBS. Each datum point represents the mean of log2 antibody titres SE. The straight line bisecting the graph indicates the border between positive and negative titres. Responder fish are defined as those fish with serum titres to A. salmonicida A IROMP antigens d 10.
fish into responder and non-responder groups. A second immunisation of bacterin 8 weeks after the first resulted in an increased percentage of fish producing specific antibodies when compared to fish receiving PBS as a second injection. The term non-responders was employed to describe fish whose serum did not contain specific antibody levels above those present in a standard pool of normal serum from non-immunised fish. As the mean antibody titres are a reflection of the percentage of responders (i.e. an increased frequency of non-responders reduces the mean antibody titres), it was interesting to note that the mean antibody titres of responding fish were significantly higher in the boosted group compared to the unboosted group, indicating that the second exposure to antigen increased the antibody responses of the responder fish in addition to increasing the percentage of responder fish. At weeks 8 and 12, all PBS-injected control fish had negative antibody titres by ELISA, but at week 16 a number of fish sampled had positive titres, and this pattern continued until the trial was terminated. The pattern of mean antibody titres of the unboosted group and the PBS-injected control group was identical from week 12 onwards, indicating that the positive antibody titres detected in the sera of unboosted fish at week 12 may not be a result of bacterin immunisation. The responsiveness of sera from PBS-injected fish to A. salmonicida IROMP antigens may be a result of the development of natural antibodies in PBS-injected fish which cross-react with antigens coated on the ELISA plate. Development of low-a$nity antibodies which cross-react with
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various specific antigens following PBS immunisation of fish has been previously reported (Michel et al., 1990). In summary, the results presented here support other findings that the A-layer does not prevent the development of antibody responses to other outer membrane antigens present on the surface of iron-restricted A. salmonicida. The results also show that a second immunisation with antigen can enhance the magnitude of an ongoing specific antibody response by increasing the titres of responding fish and the percentage of responding fish. This may have important implications for levels of protection a vaccine confers on a population. This study was supported by a grant from the Scottish O$ce Agriculture, Environment and Fisheries Department (SOAFED awarded to A.M.O’D.
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