Comparison of antibody responses in Atlantic cod (Gadus morhua L.) to Vibrio anguillarum, Aeromonas salmonicida and Francisella sp.

Fish & Shellfish Immunology 27 (2009) 112–119

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Comparison of antibody responses in Atlantic cod (Gadus morhua L.) to Vibrio anguillarum, Aeromonas salmonicida and Francisella sp. Merete Bjørgan Schrøder a, *, Terje Ellingsen a, Helene Mikkelsen b, Edel Anne Norderhus c, Vera Lund b a

Norwegian College of Fishery Science, University of Tromsø, Breivika, Tromso N-9037, Norway Nofima Akvaforsk-Fiskeriforskning, P.O. Box 6122, N-9291 Tromsø, Norway c PHARMAQ AS, P.O. Box 267, N-0213 Oslo, Norway b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 April 2008 Received in revised form 3 November 2008 Accepted 23 November 2008 Available online 6 December 2008

Bacterial diseases such as vibriosis, atypical furunculosis and francisellosis, are registered as an increasing problem in cod farming in Norway. In order to develop efficient vaccines against diseases it is of interest to investigate if the cod immune system differentiates between various serotypes of Vibrio anguillarum and variants of Aeromonas salmonicida associated with the diseases by raising specific antibody responses. Cod of the same origin were shown to raise significant responses to V. anguillarum, A. salmonicida and the intracellular bacteria Francisella sp. Individual responses to V. anguillarum or A. salmonicida varied from none to high responses, while all individuals immunised with Francisella revealed a significant response. The cod immune system appeared in some degree to distinguish between V. anguillarum serotypes and A. salmonicida variants. Although all bacteria had induced significant antibody responses detectable in whole cell ELISA, only some had induced antibodies with specificity to linear O-polysaccharide epitopes on blot. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Atlantic cod Vibriosis Atypical furunculosis Francisellosis Immunisation Antibody responses

1. Introduction Although farming of Atlantic cod (Gadus morhua L.) in Norway is still in its infancy, bacterial diseases are registered as an increasing problem [1]. Vibriosis has been the main bacterial disease problem since the start of cod farming and Vibrio anguillarum serotype O2a and O2b are most commonly associated with vibriosis in cod [2,3]. However, in recent years deviating sero-subtypes other than O2a and O2b have been isolated from diseased cod [4]. In Iceland atypical furunculosis is the main problem in cod farms [5], while the disease is an increasing problem in Norway. Atypical Aeromonas salmonicida cause various conditions of ulcer diseases or atypical furunculosis in both salmonids and marine fish species. They comprise a heterogeneous group of bacteria varying in phenotypic and molecular characteristics (reviewed in Ref. [6]), including important surface components like the A-layer protein [7,8] and the O-polysaccharide structure [9]. So far atypical A. salmonicida isolated from diseased cod appear to be of the subsp. achromogenes both in Iceland [5] and Norway (unpublished). In 2004 a disease characterised by white granulomas/cysts in the visceral organs and skin was for the first time reported in

* Corresponding author. Tel.: þ47 77646000; fax: þ47 77646020. E-mail address: [email protected] (M.B. Schrøder). 1050-4648/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2008.11.016

farmed cod in Norway. The disease was associated with a bacterium belonging to the genus Francisella [10,11]. Further characterisation showed that the bacterium was closely related to the intracellular Francisella philomiragia, but could phenotypically and genetically be distinguished from a F. philomiragia reference strain. Thus, the isolates from cod which appear very homogenous have been suggested as a novel species with the proposed name Francisella piscicida sp. nov. [12] or a novel subspecies F. philomiragia subsp. noatunensis subsp. nov. [13]. In order to develop efficient vaccines against the various serotypes of V. anguillarum and variants of A. salmonicida associated with diseased cod, it is of interest to investigate if the cod immune system differentiates between them by raising specific antibody responses. Such information is important for selecting isolates to be tested as vaccine antigens against homologous and heterologous challenge. Previously, we have reported that most cod individuals immunised with A. salmonicida were able to raise antibody responses comparable to what is seen in Atlantic salmon. Moreover, cod appeared to differentiate between typical and atypical isolates [14,15]. On the other hand, only a few cod individuals immunised with V. anguillarum serotype O2b revealed antibody responses, which were shown to be directed to lipopolysaccharide (LPS) epitopes [16]. Furthermore, these sera did not cross-react with other serotypes on immunoblot. So far, studies of cod antibody responses to Francisella sp. have not been reported.

M.B. Schrøder et al. / Fish & Shellfish Immunology 27 (2009) 112–119

In this study, antibody responses to several serotypes of V. anguillarum, variants of A. salmonicida differing in surface components such as the A-protein and O-polysaccharide structure and to Francisella sp. were investigated in cod. The reactivity of individual immune sera was compared in whole cell ELISA and the specificity of the antibodies was characterised using immunoblotting. 2. Material and methods 2.1. Fish Atlantic cod were purchased from Sagafjord Sea Farm AS (2006) as juveniles of approximately 2 g and transported to the Aquaculture Research Station (Tromsø, Norway) for grow-out in seawater of 3.4% salinity at 10  C and with continuously feeding (Dana Feed AS, Denmark). Fish of the same origin were used in 2 separate immunisation experiments (Table 2), where the first started when the fish had a mean weight of 30 g (Exp. I) and the second 8 weeks later at a mean weight of 60 g (Exp. II). Prior to individual pit-tagging (passive integrated transponder), immunisation and blood sampling, the fish were anaesthetized with Metacainum (70 mg l1, Norsk Medisinaldepot). The fish were held in 500 l tanks at 10  C throughout the immunisation periods. 2.2. Bacteria

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The bacterin of the Francisella isolate Fr 1130 used as testing antigen in ELISA and immunoblotting was provided by PHARMAQ AS. 2.3. Immunisation The fish were individually pit-tagged 2 weeks prior to immunisation (Table 2). In Exp. I groups of fish were immunised with various A. salmonicida isolates and with Francisella sp. from cod and in Exp. II groups were immunised with various serotypes of V. anguillarum. Bacterins were centrifuged and the pellet suspended in 0.9% NaCl (saline) to appropriate concentrations before emulsified in the mineral oil used in ALPHA JECT vaccines (PHARMAQ AS) resulting in approximately 1 109 bacterial cells ml1, whereby OD600 nm ¼ 1 corresponds to 109 cells ml1. Experimental oil adjuvanted Francisella vaccines, one monovalent and one multivalent also including a V. anguillarum serotype O2b isolate, were provided by PHARMAQ AS. The fish were intraperitoneally (ip) immunised with 0.1 ml fish1, while the control fish received 0.1 ml saline in mineral oil. Blood sampling was performed 8 and 9 weeks post-immunisation (wpi) in Exp. I and II, respectively. Blood samples were withdrawn from the caudal vein using syringes and allowed to coagulate overnight at 4  C, before the sera were collected by centrifugation. Individual sera and serum pools for each group were stored at 20  C until use. 2.4. Enzyme-linked immunosorbent assay (ELISA)

The V. anguillarum and A. salmonicida isolates used for immunisation are described in Table 1. The V. anguillarum (Va) isolates represent various serotypes and sero-subtypes associated with vibriosis in cod [4]. In addition a V. anguillarum serotype O1 type strain (Va 2129) was included for comparison. The A. salmonicida isolates from marine fish species were received as atypical isolates (aAs) and in previous studies these isolates have been shown to differ in the surface A-protein [8,15] and O-polysaccharide structure [9]. Included in the study were an A-layer negative A. salmonicida subsp. achromogenes type strain (Asa 4036(A-)) also referred to as atypical and the A. salmonicida subsp. salmonicida (Ass 4017) isolate referred to as typical. The bacterial isolates stored at 80  C were inoculated on Tryptic Soya Agar (Oxoid) supplemented with 5% human blood and 1.5% NaCl and incubated for 3 days at 12  C. Further, V. anguillarum were grown in Marine Broth (MB-2216, Difco) at 12  C for 24 h, whereas A. salmonicida were grown in Brain Heart Infusion broth (BHI, Difco) at 12  C for 24–30 h. The bacterial cultures were inactivated by adding formaldehyde solution (37%) to a final concentration of 0.5% (vol/vol), before used for either immunisation, enzyme-linked immunosorbent assay (ELISA) or immunoblotting.

A whole cell ELISA was used to determine serum reactivity to homologous and heterologous bacterial isolates [14]. Briefly, microtiter plates (Maxisorb, Nunc) were coated overnight at 4  C with 5 mg ml1 of poly-L-lysine (PLL, Sigma) in 50 mM carbonate buffer, pH 9.6. Further, 100 ml inactivated bacteria in saline (OD600 nm approx. 0.5) were added per well before the plates were centrifuged and incubated at room temperature for 30 min. Thereafter, all plates were saturated with 5% skimmed milk in PBS (phosphate buffered saline, pH 7.3), incubated overnight at 4  C and stored at 20  C until use. Cod sera were diluted 2-folds in 5% skimmed milk in PBS, 100 ml well1, and incubated overnight at 4  C. Bound antibodies were detected by incubations at room temperature with polyclonal rabbit-anti-cod Ig antiserum produced in our laboratory (2 h), followed by goat-anti-rabbit Ig conjugated with alkaline phosphatase (Sigma) in 0.5% skimmed milk in PBS (1 h). Finally, the substrate p-nitrophenyl-phosphate (Sigma) in ethanolamine buffer (1 mg ml1) pH 9.6, containing 1 mM MgCl2 was added and the colour reaction was read after

Table 1 Bacterial isolates used for immunisation of Atlantic cod.

Table 2 Atlantic cod immunised with Aeromonas salmonicida, Vibrio anguillarum and Francisella sp. in oil adjuvant.

Isolate code

Bacterial species

Characteristics

Designation

Host

Va 1275 Va 4299 Va 1282 Va 5022 Va 2129 aAs 4099

Vibrio anguillarum Vibrio anguillarum Vibrio anguillarum Vibrio anguillarum Vibrio anguillarum Aeromonas salmonicida Aeromonas salmonicida Aeromonas salmonicida Aeromonas salmonicida Aeromonas salmonicida Francisella sp.

Serotype Serotype Serotype Serotype Serotype atypical

1275 2001/09/700 1282 5022 NCIMB 2129 93/09/914

Atlantic cod Atlantic cod Atlantic cod Atlantic cod Rainbow trout Atlantic cod

subsp. achromogenes atypical

ATCC 33659

Brook trout

483/03

Atlantic Halibut

atypical

K-9/98

Spotted wolffish

subsp. salmonicida –

3329/89

Atlantic salmon

Fr 1130

Atlantic cod

Asa 4036(A-) aAs 4153 aAs 4067 Ass 4017 Fr 1130

O2a O2b O2 O2 O1

Experiment

Group

Bacterial antigen

N

Exp. I

1 2 3 4 5 6 7 8

aAs 4099 Asa 4036(A-) aAs 4153 aAs 4067 Ass 4017 Fr 1130 Fr 1130 þ Va O2b1 Saline

12 10 11 12 11 14 14 12

1 2 3 4 5 6

Va 1275 O2a Va 4299 O2b Va 1282 O2 Va 5022 O2 Va 2129 O1 Saline

21 23 22 22 18 19

Exp. II

1

Isolate identity not known.

Additional information

Immunised at mean weight of 30 g Blood sampling 8 wpi at 60 g

Immunised at mean weight of 60 g Blood sampling 9 wpi at 116 g

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1,40

45 min at Abs405 nm. The plates were washed 3 times with PBS containing 0.05% Tween 20 (Sigma) between each incubation step.

1,20

2.5. Statistical analysis

Va 5022 O2 Va 2129 O1 Saline

1,00

Abs405nm

Individual serum reactivity to homologous and heterologous bacterial isolates was read in ELISA as Abs405 nm at dilution 1:400 for the Vibrio and Aeromonas antisera and 1:800 for the Francisella antisera, and presented as box plots. Immunisation groups were compared using Mann–Whitney U non-parametric test (MinitabÒ 15.1.1.0, 2007). The line in the middle of the box represents the median (the middle of the data), the bottom of the box represents the first quartile (25% of the data are less or equal to this value) and the top of the box the third quartile (75% of the data are less or equal to this value). The upper and lower whiskers extend to the highest and lowest data value, respectively, and values beyond the whiskers are outliers (unusual large or small observations).

Va 1275 O2a Va 4299 O2b Va 1282 O2

0,80 0,60 0,40 0,20 0,00

Va 1275

Va 4299

Va 1282

Va 5022

Va 2129

Fig. 1. Antibody responses in Atlantic cod to various serotypes of Vibrio anguillarum measured in ELISA as antibody reactivity in serum pools (N ¼ 18–23) diluted 1:400 to homologous and heterologous isolates as indicated in the figure.

2.6. Immunoblotting The specificity of the antibody responses in cod to V. anguillarum and Francisella sp. was determined by immunostaining of bacterial cells on Western blot as previously described [14]. The Bio-Rad Criterion electrophoresis and blotting system including pre-cast 12% Bis–Tris gels, premixed running buffer and XT MOPS sample buffer with reducing agent, were used. Suspension of bacterial cells in 20 mM Tris pH 8 (OD600 nm approx. 1.0) was mixed with the sample buffer according to manufacturer’s protocol. The samples were boiled for 5 min prior to separation by SDS gel electrophoresis and subsequently transferred onto 0.45 mm nitrocellulose membranes (Bio-Rad). Gels were protein stained with coomassie (Imperial Protein Stain, Pierce). SDS-PAGE Standard Broad Range and Precision Plus Protein Standard All Blue (Bio-Rad) were used as molecular weight markers on gels and blots, respectively. The membranes were blocked with 5% skimmed milk in PBS with 0.05% Tween 20 (PBS-Tween) before immunostained with sera diluted in 1.0% skimmed milk in PBS-Tween. The V. anguillarum and A. salmonicida sera were diluted 1:200 and the Francisella sera 1:400. Bound antibodies were detected by 1 h stepwise incubations with rabbit-anti-cod Ig antiserum and goat-anti-rabbit Ig conjugated with alkaline phosphatase (DakoCytomation). The membranes were washed 3  10 min with PBS-Tween between each incubation step. Finally, the substrates nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3indolyl phosphate (BCIP) (Promega) in substrate buffer (0.1 M Tris pH 9.5, 0.1 M NaCl and 50 mM MgCl2), were added. The staining was stopped after 5 min by soaking the membranes in distilled H2O. A rabbit serum raised against a Francisella sp. isolated from cod provided by PHARMAQ AS was used for immunostaining of the Fr 1130 cells on blot. 3. Results The antibody responses to the different serotypes and serosubtypes of V. anguillarum, variants of A. salmonicida and Francisella sp. were measured in ELISA as the reactivity of serum pools and individual sera from each group to homologous and heterologous bacterial cells. The results were presented as box plots. The specificity of the antisera was investigated using immunoblotting. 3.1. Antibody responses to V. anguillarum Initially, serum pools from the various groups immunised with V. anguillarum (Table 2) were tested against homologous and heterologous isolates in ELISA (Fig. 1). The Va 1275 O2a serum and

the Va 1282 O2 serum reacted strongly with cells of isolate Va 1275 O2a, while the other serum pools revealed a reaction similar as the control serum. When tested against Va 4299 O2b cells, the Va 1275 O2a serum reacted more strongly than homologous serum, while sera from the other groups did not differ from the control serum. The serum pools from the groups immunised with the serotype O2 isolates Va 1282 and Va 5022 in addition to the Va 1275 O2a serum reacted strongly with the O2 isolates, while the Va 4299 O2b and the Va 2129 O1 sera did not differ from the control serum. Finally, only the Va 2129 O1 serum reacted significantly with the Va 2129 O1 cells in ELISA. The reactivity of individual sera to homologous and heterologous V. anguillarum isolates (Fig. 2) was only investigated for the groups which serum pools revealed a positive reaction (Fig. 1). Although, the responses varied between individuals from none or low to high responders in all groups, all immunisation groups revealed significant responses to homologous V. anguillarum serotypes. Furthermore, sera from the groups immunised with V. anguillarum serotypes O2a, O2b and O2 also revealed significant reactivity against heterologous serotypes. The Va 1275 O2a sera revealed a significantly higher reactivity to Va 1275 O2a cells compared to the Va 1282 O2 sera (Fig. 2A). However, the respective serum pools showed similar reactivity to Va 1275 O2a (Fig. 1), which is probably due to the 2 ‘‘high responders’’ indicated as outliers, thus contributing to the strong reactivity of the Va 1282 O2 serum pool. Although the reactivity of the Va 1275 O2a sera with Va 4299 O2b cells appeared stronger compared to that of the homologous sera, they did not differ significantly (p ¼ 0.5889) (Fig. 2B). The reactivity of the Va 1275 O2a, Va 1282 O2 and Va 5022 O2 sera to the O2 isolates did not differ significantly (Fig. 2C and D). Furthermore, the reactivity of the sera within each group varied more when tested against the Va 1282 O2 and Va 5022 O2 cells compared to when tested against Va 1275 O2a (Fig. 2A). Finally, the Va 2129 O1 sera were shown to react strongly with homologous cells (Fig. 2E). The specificity of the antibody responses to V. anguillarum serotypes was determined by reacting the serum pools from each group with a panel of the V. anguillarum isolates on blot (Fig. 3). Apparently, all the serum pools including the control serum contained antibodies that reacted with a component of approximately 75 kDa present in Va 2129 O1 and with 2 components of 20 and 25 kDa present in all isolates. The 20-kDa component of Va 5022 O2 was more strongly stained with all sera. While the Va 1275 O2a, 4299 O2b and 2129 O1 sera did not contain additional antibodies compared to the control serum (Fig. 3A, B, E and F), the Va 1282 and 5022 serotype O2 isolates had induced antibodies that reacted

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Fig. 2. Atlantic cod immunised with Vibrio anguillarum. Individual antibody responses in groups immunised with 1. Va 1275 O2a, 2. Va 4299 O2b, 3. Va 1282 O2, 4. Va 5022 O2, 5. Va 2129 O1 and 6. Saline, were measured in ELISA against homologous and heterologous cells and presented as box plots. Antisera diluted 1:400 were tested against the following isolates: A. Va 1275 O2a, B. Va 4299 O2b, C. Va 1282 O2, D. Va 5022 O2 and E. Va 2129 O1. The homologous immunisation group is underlined, and significant differences are indicated with different letters.

strongly with several bands forming a typical LPS ladder in both homologous and heterologous O2 isolates (Fig. 3C and D). However, only about 50% of the individual sera in these groups revealed the same reaction pattern on blot (not shown). In addition, the Va 5022

O2 and the Va 1282 O2 serum pools reacted faintly with a component of 15 kDa in both isolates (Fig. 3C and D), whereas the Va 1282 O2 serum pool also reacted faintly with a component of <15 kDa present in Va 1275 O2a as well (Fig. 3C).

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Fig. 3. Specificity of antibody responses in Atlantic cod to Vibrio anguillarum serotypes determined by immunoblotting. Western blots with a panel of bacterial cells: 1. Va 1275 O2a, 2. Va 4299 O2b, 3. Va 1282 O2, 4. Va 5022 O2 and 5. Va 2129 O1 immunostained with pooled antiserum (a-) to the various isolates as indicated (A–E), and with serum from saline group (F). Reaction with homologous isolate is marked with asterisks.

3.2. Antibody responses to Francisella sp. The individual responses in groups immunised with the monovalent Francisella vaccine or the multivalent vaccine including V. anguillarum serotype O2b were compared when reacted with Fr 1130 or Va 4299 O2b. All individuals in both groups had responded and revealed significant and equal reactivity to Fr 1130, while only the group immunised with the multivalent vaccine with V. anguillarum O2b revealed a low but significant reaction with Va 4299 O2b (Fig. 4). A rabbit antiserum to Francisella sp. was shown to recognise a component of approximately 20 kDa in Fr 1130 cells (Fig. 5B) that were not visible on a protein stained gel. Apparently, the same component was recognised by the cod serum pools from both groups (Fig. 5C) while the blot stained with the control serum was negative. Most individuals (9 of 12) from the group immunised with Fr 1130 alone had responded to this component (Fig. 5D). Similar was shown for individual sera from the group immunised with Fr 1130 and Va O2b (data not shown). 3.3. Antibody responses to A. salmonicida All A. salmonicida isolates had induced significant antibody responses to homologous isolate, but the individual responses within each group varied (Fig. 6). Significant reactivity to

heterologous cells was revealed for most serum groups, but some serum groups showed significantly lower reactivity compared to homologous groups. The reactivity of the aAs 4099 sera to homologous cells did not differ from that of the aAs 4067 sera, but was significantly higher compared to that of the Asa 4036(A-) and aAs 4153 sera (Fig. 6A). The reactivity of sera to the atypical isolates did not differ when tested against aAs 4153 cells (Fig. 6C), but when tested against Asa 4036(A-) and aAs 4067 cells the reactivity of the aAs 4153 sera was significantly lower (Fig. 6B and D). The typical Ass 4017 sera revealed low but significant reactivity to the homologous isolate only (Fig. 6E), but did not differ from the control sera when tested against the atypical isolates. Except for the aAs 4153 sera the antisera to atypical isolates reacted weakly but significantly with the typical isolate compared to the control group. 4. Discussion The immune system of Atlantic cod has been shown to differ from that of other bony fish species so far investigated in having high concentration of serum IgM [17–19], and that none or only very low increases in antibody levels are detected post-immunisation with Vibrio salmonicida [20], V. anguillarum [21] or protein antigens as well as hapten-carriers [22,23]. However, the cod immunoglobulin gene repertoire appears to be sufficient to raise

Fig. 4. Atlantic cod immunised with Francisella sp. and Vibrio anguillarum. Individual antibody responses in groups immunised with 6. Fr 1130, 7. Fr 1130 and Va O2b and 8. Saline, were measured in ELISA and presented as box plots. The antisera were diluted 1:800 when tested against Fr 1130 (A) and 1:400 when tested against Va 4299 O2b (B). The homologous immunisation group is underlined, and significant differences are indicated with different letters.

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Fig. 5. Specificity of antibody responses in Atlantic cod to Francisella sp. determined by immunoblotting. A. Coomassie stained gel with Fr 1130 cells. B. Fr 1130 cells on blot immunostained with rabbit anti-Francisella serum. C. Fr 1130 cells on blot immunostained with serum pools from groups immunised with 6. Fr 1130, 7. Fr 1120 and Va O2b and 8. Saline. D. Fr 1130 cells immunostained with individual sera from group 6.

specific antibody responses (reviewed in Ref. [24]), and the cod antibody responses to A. salmonicida have been shown to be comparable to that in salmon [14,15]. The inconsistency in reports on cod antibody responses may be due to differences in origin of cod, weight or age of the fish, type and concentration of antigens, in addition to type of adjuvant used for immunisation and ELISA protocols used for antibody detection. Some of these parameters may be crucial for detecting antibody responses in cod. To our knowledge A. salmonicida and Francisella have previously not been used for immunisation of cod by other research groups. The low reproducibility in ELISA detection of fish antibodies has been suggested to be due to high background caused by non-specific reactions between IgM and antigens [25–27]. Recently, non-specific adsorption of fish IgMs to blocking reagents was reported as a common property of fish, which could be one of the major problems for quantitative detection of fish IgM using the ELISA system [28]. The non-specific adsorption could be prevented by competitive blocking of the fish sera with 5% non-fat skimmed milk. Such treatment reduced background without inhibiting specific fish IgM immuno-reactivity. Thus, our success in detecting specific cod antibody responses to various fish pathogens in this and previous studies [14–16] may be explained by the use of 5% skimmed milk both as blocking agent and for dilution of cod sera. Specific humoral antibody responses in cod immunised with V. anguillarum have been reported to be poor and primarily directed to LPS. Cod immune sera to serotype O2a and O2b were shown to distinguish between antigenic differences in the two sero-subtypes [21] and another study indicated that cod did not produce antibodies against a protein antigen [22,23]. Previously, we have shown that cod raised a weak but specific antibody response to V. anguillarum O2b [14], but only a few of the immunised individuals had raised antibodies with O-polysaccharide specificities that did not cross-react with either Va 1275 O2a or the serotype O2 isolates Va 1282 and Va 5022 on blot [16]. In this study the cod appear to have responded more strongly to other V. anguillarum serotypes than O2b. The reactivity of the sera to both homologous and heterologous isolates in ELISA may indicate common conformational surface epitopes among the serotype O2a, O2b and O2 isolates, while immunoblotting clearly demonstrated differences in immunogenic O-polysaccharide epitopes. Compared to the control sera the V. anguillarum O1, O2a and O2b sera did not contain additional antibodies with specificity to epitopes on blot. In contrast, the serotype O2 sera contained antibodies reacting with a typical O-polysaccharide ladder that did not cross-react with the other serotypes tested. Apparently, the recently described Va O2 isolates [4] seem to be more immunogenic in cod than O2a and O2b and the results indicate that Va O2a is more immunogenic than Va O2b.

However, the present work does not explain why, but one may speculate whether these differences are caused by antigenic differences among the isolates or if the antibody repertoire in cod is unable to recognise all epitopes on the O2b isolates. Interestingly, nearly all cod individuals revealed significant antibody responses to Francisella sp., as shown both by ELISA and immunoblotting. Seemingly, combined immunisation with Francisella and V. anguillarum O2b did not affect the cod responses to either of them. Most cod individuals had induced antibodies to a Francisella component of 20 kDa that was not detected on a coomassie stained gel. Apparently, this component also induced strong antibody responses in rabbit. Similarly, a component of approximately the same molecular weight from Francisella victoria isolated from tilapia was also highly immunogenic in rabbit and was characterised as a lipooligosaccharide [29]. Unfortunately, in the present study no attempts were done to characterise the Francisella sp. component. Cod appeared to respond more strongly to the atypical than to the typical A. salmonicida isolate. The reactivity of the sera from the various immunisation groups with homologous and heterologous isolates indicates similarities among the isolates. Clearly there are also differences such as the reactivity of the aAs 4153 sera that did not differ from sera to other atypical isolates when tested against homologous isolate, but revealed significantly lower reactivity when tested against heterologous isolates. The specificity of the A. salmonicida serum pools was investigated in a parallel study [15], and except for the sera to aAs 4153 and the typical Ass 4017 cells, they were shown to be towards O-polysaccharide epitopes on blot as previously reported [14]. In this and previous studies we have so far only been able to identify antibodies with LPS specificity in cod to V. anguillarum [16] and A. salmonicida [14], and probably to Francisella sp. However, all the bacterial isolates used had induced significant antibody responses detectable by whole cell ELISA, while immunoblotting revealed that only some of the serum pools reacted specifically with O-polysaccharide epitopes. Thus, it cannot be ruled out that antibodies with specificity to conformational and/or combined surface epitopes of both protein and/or LPS nature have been induced in cod. The role of antibodies in the cod immune system has not yet been elucidated. Our interest in investigating the cod antibody responses is with regard to vaccine development, as the success of vaccination relies on recognition of specific bacterial antigens/ epitopes by the immune system of the host. Although, correlation between antibody responses and vaccine efficacy is questioned, differentiated responses to various serotypes or variants of the same bacterial species may indicate that other parts of the immune

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Fig. 6. Atlantic cod immunised with Aeromonas salmonicida. Individual antibody responses in groups immunised with various variants of A. salmonicida measured in ELISA against homologous and heterologous cells and presented as box plots. Groups were immunised with 1. aAs 4099, 2. Asa 4036(A-), 3. aAs 4153, 4. aAs 4067, 5. Ass 4017 and 8. Saline, and antisera diluted 1:400 were tested against: A. aAs 4099, B. Asa 4036(A-), C. aAs 4153, D. aAs 4067 and E. Ass 4017. The homologous immunisation group is underlined, and significant differences are indicated with different letters.

system important for protective immunity also may discriminate between isolates. LPS is suggested as important protective antigens in vibriosis vaccines and correlation between V. anguillarum serotype and protection has been indicated [4], although antibodies with LPS specificity were only detected to serotype O2 isolates in this study. The present work is so far the only report on antibody responses in cod to the facultative intracellular Francisella isolate from cod, shown to be related to the pathogens F. philomiragia and Francisella tularensis [11]. Protective immunity to intracellular bacteria including F. tularensis is assumed to mainly rely on T-cell mediated

responses. Interestingly, passive immunisation with LPS antibodies to F. tularensis appear to be protective in mice models and it is speculated that LPS antibodies may play a critical role in protection against F. tularensis [30,31]. Thus, investigations on immune responses in cod to live Francisella are of high importance to elucidate how cod may be protected against this serious bacterial disease. In summary, cod of the same origin were shown to raise significant and specific antibody responses to V. anguillarum, A. salmonicida and the intracellular bacteria Francisella sp. Individual responses varied from none to high response in groups

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immunised with V. anguillarum or A. salmonicida, while in groups immunised with Francisella all individuals were responders compared to the control group. The cod immune system appeared in some degree to distinguish between V. anguillarum serotypes and A. salmonicida variants. Although all bacteria had induced significant antibody responses detectable in whole cell ELISA, only some had induced antibodies with specificity to linear O-polysaccharide epitopes on blot, indicating that the cod may also have produced antibodies to non-LPS epitopes. Acknowledgement The work was financially supported by The Research Council of Norway, Innovation Norway, Fiskeriforskning and Norwegian College of Fishery Science, University of Tromsø. We want to thank Susanna Børdal for assisting with the fish experiments and performing ELISA and Oddvar Dahl for helping with figure layout.

[13]

[14]

[15]

[16]

[17]

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