Specific and Natural Antibody Response of Cod Juveniles Vaccinated Against Vibrio Anguillarum

Fish & Shellﬁsh Immunology 26 (2009) 619–624

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Fish & Shellﬁsh Immunology

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Speciﬁc and natural antibody response of cod juveniles vaccinated against Vibrio anguillarum

S. Gudmundsdo´ ttir*, B. Magnado´ ttir, B. Bjo¨rnsdo´ ttir, H. A´ rnado´ ttir, B.K. Gudmundsdo´ ttir

Department of Fish Diseases, Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, 112 Reykjavı´k, Iceland article info abstract

Article history: The purpose of the present study was to study specific and natural antibody levels in individual cod Received 30 July 2008 juveniles before and after being vaccinated against Vibrio anguillarum. Different vaccine preparations and Received in revised form vaccination regimes, i.e. bathing, dipping, i.p. injection or combination of treatments were employed and 19 September 2008 the performance of different groups to bath challenge by the bacterium tested. Antibody responses to Accepted 26 September 2008 V. anguillarum antigens in groups vaccinated by bathing and/or dipping were negligible, while responses Available online 14 October 2008 were observed in i.p. injected fish. Fish receiving i.p. injection in addition to bathing, showed significant antibody response. Both groups showed increased levels of natural antibodies while levels were low in Keywords: Cod other groups. Fish bathed or dipped showed higher mortality when challenged than untreated fish, while Fry fish that received a second vaccination showed the best protection. It was not ascertained whether there Vaccinations is a long term difference between the effects of immersion versus i.p. injection as a booster method. Vibrio anguillarum Levels of antibodies against V. anguillarum antigens or natural antibodies in groups with the lowest Antibody response mortalities show that neither could have been used to predict protection given by the vaccines tested. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction environmental factors such as temperature [13] and size [14]. Cod mounts specific response to a limited number of antigens following Cod has for centuries been one of the most important species for infections and immunizations and typically individuals in a group fishing communities around the North Atlantic [1]. Today, with show a wide range of responses [10,15,16]. Studies on cod immu- catches rapidly declining, cod is probably the marine species with noglobulins were reviewed recently and the authors concluded the highest potential for sustainable cold water aquaculture [2]. that the explanation for the lack of specificity does not appear to be Intensive rearing of fish, suffers from outbreaks of diseases. In due to deficiencies in their structure, organization, diversity or cultivated cod, vibriosis, caused by Vibrio anguillarum, is one of the expression [17]. major bacterial diseases [3,4]. Vibriosis in cod is often associated Natural antibodies are important in the first line of defence with serotypes O2a and O2b [5,6] while serotypes O1 and O2a are against infections [18]. They react to common antigens of patho- most commonly isolated from salmonids. Vaccinations are the gens and various self-antigens. They are present in sera of normal, most effective way to avoid bacterial diseases and different fish non-immunized individuals, where they are proposed to form species may need different vaccines, vaccination methods and a link between innate and adaptive immunity [19]. Studies on vaccination schemes [7,8]. Cod has been successfully vaccinated natural antibodies have been carried out in a number of fish species against vibriosis and recently, vaccines adopted for use in cod, [20] and high levels of natural or non-specific antibodies have been containing serotypes O1, O2a and O2b, have become available reported in cod [13,14]. These antibodies reacted to a variety of commercially. Vaccine trials have included cod of various sizes, antigens and the strongest response was towards haptenated using bathing, immersion and injection and different challenge bovine serum albumin or TNP–BSA. Subsequently it was shown methods [9–12]. that i.p. injections with Freund’s complete adjuvant (FCA) led to Several studies of antibodies and antibody responses in cod increased activity against this antigen [16]. In a vaccination trial have been carried out during the last two decades. Cod blood has with V. anguillarum in adult cod some of the pre-immune sera high concentration of IgM in serum that increases by influence of contained antibodies that bound to antigens in the bacterial membrane [9] and all non-immunized control fish had antibodies that reacted with V. anguillarum in another trial [10]. A study in * Corresponding author. Tel.: þ354 5855100; fax: þ354 5673914. goldfish has shown that natural antibodies can influence results of E-mail address: [email protected] (S. Gudmundsdo´ ttir). vaccination [21].

The aim of the present study was to study the levels of specific Table 1 and natural antibody responses in individual cod juveniles before Experimental set up. Vaccinations by bathing, dipping, intra-peritoneal injection or combinations thereof (V), kidney samples for bacteriology (K) blood samples (S), and and after being vaccinated against V. anguillarum. Two different bath challenge (X). vaccine preparations and six vaccination regimes were employed. Further, the aim was to compare the performance of different Vaccine administration Weeks from start of experiment. Vaccinations (V), sampling (K, S) and challenge (X) groups to bath challenge by the bacterium. 0 weeks 6 weeks 15 weeks (I)a 24 weeks (II)b 2. Material and methods C: PBS control K, S K, S K, S, X B: bath V K K, S K, S, X D: dip V K, S K, S, X 2.1. Fish BD: bath and dip V V K, S K, S, X A: AJ-oil adjuvant i.p. V K, S, X The cod, of wild parentage from Icelandic waters, was hatched VA: adjuvated vaccine i.p. V K, S, X and reared at the Marine Institute’s Experimental farm, Stadur, B and VA V V K, S, X Grindavı´k [22]. Mean weight standard deviation was 1.2 0.4 g at Mean weight of fish at bathing 1.2 g; dipping 10.3 g; i.p. injection 25.7 g; bath the time of bath vaccination, 10.3 1.1 g at dipping, 25.7 7.2 g at challenge 61.5 g. a i.p. injection and 61.5 12.7 g when challenged. From the start of I: blood sampling before i.p. vaccination. b II: blood sampling before bath challenge. the experiment and until i.p. vaccination, different groups were kept separately in 350 l tanks, 120 individuals in each. After i.p. injection and individual marking of all fish along fin margins with 2.4. Bath challenge Visible Implant Fluorescent Elastomer dye (Northwest Marine Technology, Inc. (Salisbury, Great Britain)), all groups were trans- Sixty fish from each group were divided between 6 tanks. Two 1 ferred to one 3500 l tank. Tricaine methanesulphonate, 50 mg ml tanks were used for each of three bacterial concentrations tested by seawater, was used for light anaesthetization during tagging, i.p. bath challenge, 2 105 CFU ml1,2 106 CFU ml1 and immunization, weighing and blood sampling. Nine weeks post-i.p. 2 107 CFU ml1. The fish were bathed in 200 l seawater containing vaccination, the fish were transported to another facility (Sandgerdi bacteria in the concentrations as stated above. After 1 h, 150 l of Research Center). There, 30 fish from each group to be bath chal- fresh seawater were added to each tank and bating continued for an lenged were evenly distributed between six 350 l tanks, i.e. two additional hour. The seawater was oxygenated during these 2 h and tanks for each bath challenge and acclimatized for two weeks subsequently the flow rate was restored to normal. The fish were before challenge. Aerated borehole seawater of temperature observed for four weeks, hand fed twice daily, deaths recorded and 9 2 C and salinity 32& was used in both facilities and the fish dead individuals frozen. were fed commercial dry pellets.

2.2. Bacterium for challenge 2.5. Kidney samples for bacteriology

A frozen stock, of an Icelandic isolate of V. anguillarum (F-139- A total of 130 kidney samples for bacterial isolation on BA-NaCl 03) serotype O2b recently passaged three times through cod, was agar were collected. Sixty samples were collected during the inoculated onto blood agar with 1.5% NaCl (BA-NaCl) and incubated vaccination period at the indicated times and a total of 70 samples, at 16 C for 3 days. Growth was verified with BioNor agglutinating 10 from each group before challenge, see Table 1. Fish that died antibodies against V. anguillarum. A loopful of the growth was during challenge were frozen on the day of death and kidney suspended in 1.5 ml1 phosphate buffered saline with 1.1% peptone samples inoculated on BA-NaCl agar at the end of the experiment. and 1.5% NaCl (PBS-P-S) and distributed evenly on 150 mm BA-NaCl agar plates and incubated at 16 C for 19 h. Then, 4.5 ml of ice cold 2.6. Blood sampling PBS-P-S was poured over each agar plate, the growth scraped into the buffer, harvested and placed on ice. Density measured 2.0 at On three occasions, as shown in Table 1, a total of 120 blood 11 590 nm in spectrophotometer, or close to 10 colony forming units samples were taken from the caudal vein of 10 fish in each exper- (CFUs) according to previous measurements. The solution was imental group. The blood was allowed to clot overnight at 4 C, diluted by tenfold steps to the concentrations needed to do the bath spun and the serum harvested and kept at 20 C until tested. challenge. Doses were chosen according to results from previous challenge tests. CFUs were confirmed by plate counting after 2.7. Serology incubation for 3 days at 16 C.

ELISA tests. Cod sera were diluted 1:100 and added to 96 well 2.3. Vaccines and immunizations microtrays (Maxisorp, Nunc) coated with sonicated preparation of V. anguillarum, protein concentration 10 mgml1, followed by mouse The vaccines tested were a generous gift from Pharmaq in antibodies to cod IgM, alkaline phosphatase conjugated goat anti- Norway, where they were already licensed. A special permit was mouse Ig and the color developed [23]. When measuring natural issued by The Veterinary Officer for Fish Diseases in Iceland for the antibodies, the plates were coated with 5 mgml1 TNP–BSA con- use of these vaccines in the current experiment. The vaccines were taining 15.8 residues per BSA molecule. In both ELISAs the value of V. anguillarum bacterins for use in cod, containing serotypes O1a, the blank was detracted from the reading and the final value for O2a and O2b. A water-based preparation was used for bathing or each sample was the mean of OD readings from two wells. dipping and a preparation containing mineral oil adjuvant formula (AJ-oil) for injection. The immunizations were carried out as described by the manufacturers, i.e. bathing for 30 min in vaccine 2.8. Statistics diluted 1:200 in seawater, dipping for 30 s into vaccine diluted 1:10, injection with 0.1 ml i.p. or a combination thereof, see Table 1. The Kruskal–Wallis test was used to test for normality of the Control fish were subjected to bathing, dipping and injection with distribution of ELISA values and a t-test for comparisons of means PBS-P-S and one group received AJ-oil only. between groups. The programmes used were InStat and StatView. S. Gudmundsdo´ttir et al. / Fish & Shellfish Immunology 26 (2009) 619–624 621

3. Results Table 2 Accumulated death in % following bath challenge and antibody responses against V. anguillarum and TNP–BSA shown as mean ELISA values in vaccinated groups and 3.1. Challenge controls before challenge.

Two of three bath challenge doses induced very little death. For Vaccine-groups Accumulated death % Mean ELISA values of antibodies against fish challenged with the highest dose, 2 107 CFU ml1, accumulated death in the PBS control group was 20%, Fig. 1. In groups either V. anguillarum TNP–BSA bathed or dipped there was 50% accumulated death, with bathed C: PBS control 20 0.126 0.281 fish dying off faster than dipped. Accumulated death was 15% or B: bath 50 0.131 0.203 D: dip 50 0.094 0.252 less in other groups, Fig. 1. Means of antibody responses to BD: bath and dip 5 0.058 0.173 V. anguillarum and TNP–BSA before challenge are shown in Table 2 A: AJ-oil adjuvant i.p. 10 0.270 0.221 for comparison to challenge results. Evidently neither antibody VA: adjuvated vaccine i.p. 15 0.474 0.454 responses against V. anguillarum nor TNP–BSA ensured survival B and VA 0 0.846 0.499 after challenge since group BD with 5% mortality showed negligible antibody responses while in group B–VA with no mortality the highest antibody responses were observed. receiving i.p. vaccination showed moderate responses (OD405 0.5–1.0) to V. anguillarum antigen preparation, while the same number of fish bathed and vaccinated i.p. showed high responses 3.2. Bacterial isolations (OD405 > 1.0). Order of samples on the X-axis is always the same so individual responses to either antigen can be compared within each V. anguillarum was not isolated from any of the 130 samples group. Antibodies to TNP–BSA in majority of individuals in these taken at various times before challenge, but the bacterium was two groups showed raised levels. isolated from 83% of samples from fish that died during challenge.

4. Discussion 3.3. Antibody responses following vaccinations In the present study, levels of specific and natural antibody Antibody responses to V. anguillarum antigens in controls and responses in individual cod fry from groups subjected to different groups receiving bathing, dipping, or both treatments were negligible both at 15 or 24 weeks after the start of the experiment 2 (Fig. 2a). Fish receiving vaccination i.p. showed raised antibody a levels but comparison with controls did not show significant differences. On the other hand samples from fish receiving i.p. 1,5 injection in addition to bathing, II-B–VA, showed significantly raised mean, p < 0.05, when compared to controls before challenge. Adjuvant alone raised the antibody mean slightly but insignifi- 1 OD cantly compared to controls. Groups receiving i.p. vaccination 405 showed raised antibody responses to TNP–BSA while other groups showed negligible changes in their response to TNP–BSA during the ,5 time of the experiment (Fig. 2b). In Fig. 3, responses to V. anguillarum and TNP–BSA are shown for every individual in four groups. There are no examples of high 0 responses in controls and bathed individuals. Four out of 10 fish I-B I-C I-D II-B II-A II-C II-D I-BD II-VA II-BD

Bath Vaccine i.p. Bath and dip II-B-VA 70 Dip Adjuvant i.p. Bath and -vacc. i.p. 2 b Control 60

1,5 50

40 1 OD 405 30

,5 20

Accumulated in % death 0 I-B I-C I-D II-B II-A 0 II-C II-D I-BD II-VA II-BD II-B-VA

Fig. 2. Antibodies against a) V. anguillarum and b) TNP–BSA in groups of cod following 0 5 10 15 20 25 30 one or two vaccinations against V. anguillarum, showing median and range of ELISA Days readings of cod sera, 10 ﬁsh in each group. The line within each box is the median the boxes show 25–75% percentiles, the whiskers 10–90% percentiles and circles the Fig. 1. Accumulated death percentage in seven groups of vaccinated cod juveniles and extreme values. I and II represent sampling times as shown in Table 1; C: PBS control controls receiving PBS, subjected to bath challenge with 2 107 CFU ml1 V. anguil- group; B: bathed group; D: dipped group; BD: group bathed and dipped; A: adjuvant larum of serotype O2b for 2 h and observed for four weeks. administered i.p.; VA: adjuvated vaccine administered i.p. 622 S. Gudmundsdo´ttir et al. / Fish & Shellﬁsh Immunology 26 (2009) 619–624

Antibodies to V.anguillarum Antibodies to TNP-BSA a Control group 1,5 1,5

1 1

,5 ,5

0 0 Serum no. 1-10 Serum no. 1-10

b Bath vaccinated group 1,5 1,5

1 1

,5 ,5

0 0 Serum no. 11-20 Serum no. 11-20

c I.p. vaccinated group 1,5 1,5

1 1

,5 ,5

0 0 Serum no. 21-30 Serum no. 21-30

d Bath and i.p. vaccinated group

1,5 1,5

1 1

,5 II-B-VA ,5

0 0 Serum no. 31-40 Serum no. 31-40

Fig. 3. Antibodies against V. anguillarum and TNP–BSA in individual cod sera, 10 sera in each of four groups, sampled before challenge: a) control group; b) bath vaccinated; c) i.p. vaccinated; d) bath and i.p. vaccinated.

vaccination schemes were measured. This is the first study vaccinations are in danger of being underestimated while too low addressing antibody profiles of cod groups where vaccination starts accumulated death of untreated controls can obscure a real differ- with small fry. Further, performance of the different groups when ence between types of vaccines and vaccination modes [24]. The subjected to bath challenge was assessed. manufacturers of the vaccines tested recommended two vaccina- In terms of survival, the strongest bath challenge dose used in tions and according to our results, bathing followed by either the present study was suboptimal, since accumulated death in the dipping or injection, can give good results. AJ-oil alone did not control group was only 20%. Interestingly the groups bathed or induce significant protection in the current experiment. This is in dipped showed less survival than the control group with bathed agreement with another study where frys receiving AJ-oil only individuals dying faster than dipped and with both groups reaching were not protected against bath challenge while i.p. challenge of 50% accumulated death on day 21 after challenge. The cod juveniles the same group resulted in significant protection [12]. Due to the in the current study were 1.2 0.4 g at the time of bathing which is set up of the experiment, there were 24 weeks between bathing too small for a long time protection to be established according to and challenge, 18 weeks between dipping and challenge while 9 a recent paper [11]. In that same paper dipping at 5 g resulted in weeks passed between injection and challenge. long-term protection in an experiment where 95% of controls died, Antibody responses in the vaccinated groups demonstrate that which is in contrast with the results reported here. Difficulties in only individuals receiving adjuvated vaccine i.p. show raised anti- the establishment of bath-challenge models are well known. bodies to V. anguillarum. There were higher individual readings as Sometimes observed death of controls is too high and benefits of well as higher mean in the B–VA group (bathed prior to i.p. S. Gudmundsdo´ttir et al. / Fish & Shellfish Immunology 26 (2009) 619–624 623 vaccination) than in the group receiving single i.p. injection (VA) Acknowledgements suggesting secondary response. In a study where frys of 55–60 g were inoculated with bacterins, suspended either in FIA or AJ-oil Thanks are due to the staff at the Marine Institute’s Experi- [10], only the latter group showed specific antibody response in mental farm at Stadur and at Sandgerdi Research Center for help a pooled serum sample. A closer look revealed that the titer of the during vaccination and challenge periods. Further to Gudmundur pool was mainly due to serum from one individual. In that trial i.p. Logi Norddahl and Harpa Lind Bjo¨rnsdo´ ttir for technical help and vaccinated groups also showed high levels of protection. Western finally to Kjersti Gravningen and her colleagues at Pharmaq for blots done on selected sera in the present study (results not shown) providing the vaccines. The work was funded by The Icelandic showed antibodies directed mainly at LPS antigens of sizes ca. 43 Research Council, grant no.: 0308000003. and 34 kDa. In a study on specificity and durability of antibodies to different V. anguillarum antigens in two adult cod immunized with a bacterin in AJ-oil, antibodies against LPS were prominent and References lasted at least 44 weeks post-vaccination [25]. In that study, pre- immune sera and controls did not show elevated levels of anti- [1] Kurlanski M. Cod: a biography of the fish that changed the world. U.K.: bodies to V. anguillarum. 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