Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum

Fish & Shellfish Immunology 23 (2007) 316e326 www.elsevier.com/locate/fsi

Protective efficiency of DNA vaccination in Asian seabass (Lates calcarifer) against Vibrio anguillarum S. Rajesh Kumar, V. Parameswaran, V.P. Ishaq Ahmed, S. Syed Musthaq, A.S. Sahul Hameed* Aquaculture Biotechnology Division, Department of Zoology, C. Abdul Hakeem College, Melvisharam 632 509, Vellore Dt., Tamil Nadu, India Received 10 August 2006; revised 1 November 2006; accepted 10 November 2006 Available online 22 November 2006

Abstract Vibriosis is one of the most prevalent fish diseases caused by bacteria belonging to the genus Vibrio. Vibriosis caused by Vibrio anguillarum produces a 38-kDa major outer membrane porin protein (OMP) for biofilm formation and bile resistant activity. The gene encoding the porin was used to construct DNA vaccine. The protective efficiency of such vaccine against V. anguillarum causing acute vibrio haemorrhagic septicaemia was evaluated in Asian seabass (Lates calcarifer Bloch), a common species of the Indian coast and a potential resource for the aquaculture industry. In vitro protein expression of porin gene was determined by fluorescent microscopy after transfection of seabass kidney cell line (SISK). Fish immunized with a single intramuscular injection of 20 mg of the OMP38 DNA vaccine showed significant serum antibody levels in 5th and 7th weeks after vaccination, compared to fish vaccinated with the control eukaryotic expression vector pcDNA3.1. Asian seabass vaccinated with the OMP38 DNA vaccine was challenged with pathogenic V. anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 55.6% was recorded. Bacterial agglutination and serum complement activity was analysed by using DNA vaccinated seabass serum above 80% of analysed strain was killed at the highest agglutination titre. Histopathological signs of V. anguillarum challenged fish were observed in around 45% of pVAOMP38, 90% of PBS and 87% of pcDNA3.1-vaccinated control fish. The results indicate that L. calcarifer vaccinated with a single dose of DNA plasmid encoding the major outer membrane protein shows moderate protection against acute haemorrhagic septicaemia and mortality by V. anguillarum experimental infection. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Vibrio anguillarum; Outer membrane protein (OMP38); pVAOMP38; Seabass (Lates calcarifer); Seabass kidney cell line (SISK)

1. Introduction Vibrio anguillarum is a Gram-negative, facultatively anaerobic, and rod shaped bacteria. It is the causative agent of acute haemorrhagic septicaemia in marine and estuarine fishes [1]. It is a primary pathogen of fishes, which causes a systemic infection leading to disease and death. The development of aquaculture has seen considerable economic losses due to pathogens such as V. anguillarum [2]. * Corresponding author. E-mail address: [email protected] (A.S.S. Hameed). 1050-4648/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2006.11.005

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Asian seabass (Lates calcarifer, Bloch) is an important species for aquaculture in Asia-Pacific region. This fish species is susceptible to various pathogens of parasitic, bacterial and viral origin [3e7]. Bacterial diseases mainly caused by Vibrio anguillarum are a major problem in seabass farming industries [8]. Although several studies have shown that different vaccine formulation against V. anguillarum by formalin killed bacteria, heat-inactivated V. anguillarum cells and V. anguillarum bacterin [9e11] may provide protection against V. anguillarum infections, there are currently no effective vaccines available against this important pathogen. Attenuated live vaccines stimulate an effective immune response, so these vaccines may represent a risk and revert to virulence. On the other hand, inactivated vaccines are safer, but they are usually poorer immunogens. Current vaccine research is oriented to replace conventional vaccines with new more effective and safer approaches, such as DNA vaccines. Immunization with antigen-encoding plasmid DNA can elicit very strong and long-lasting humoral and cellular immune responses. This approach also offers economic, environmental and safety advantages, which are particularly attractive for the aquaculture industry [12,13]. The role of 38 kDa major Outer membrane porin (OMP) protein of Vibrio anguillarum is bile resistant and stimulates biofilm formation [14]. Resistance to bile is important for bacterium to colonize intestine of the fish host [15e17] and biofilm formation provides an adaptive and survival advantage for bacteria in the aquatic environment [18,19]. It is a highly immunodominant bacterial antigen [11]. The major outer membrane porin protein may be very useful as a component of a V. anguillarum vaccine and also for diagnostic purposes [20,21]. Several reports have demonstrated the effectiveness of DNA vaccination in fish against viral infections, including Infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHS) [22,23]. To date there is only one report of DNAvaccination against extracellular bacterial pathogen Aeromonas veroni [24] in sandbass. In the present study we describe the construction of DNA vaccine using OMP38 gene of V. anguillarum and its efficiency in protective immune response in Asian seabass, against V. anguillarum by experimental infection. 2. Materials and methods 2.1. Bacterial strains Vibrio anguillarum NB10 (serotype O1) was isolated from the Gulf of Bothnia outside the Norrby Laboratory, Umea, Sweden. It was kindly provided by Dr Debra Milton. TCBS (Trisodium Citrate Bile salts sucrose) agar and Nutrient broth with 2% NaCl medium were used for growing V. anguillarum at 28  C. Escherichia coli DH5a (Bangalore Genei, India) grown at 37  C using LuriaeBertani (LB) medium was used as a host for DNA manipulations. 2.2. Fish Healthy juveniles of seabass (Lates calcarifer) (10 g in weight) were collected from grow-out ponds of CIBA (Central Institute of Brackishwater Aquaculture), Chennai and transported to the laboratory. In the laboratory, the animals were maintained in 500 L tanks supplied with aerated and UV-treated seawater at 24  C (salinity 30 ppm). The collected fish were confirmed free from V. anguillarum and fish were fed twice a day with boiled fish meat during the acclimatization and the experimental periods. 2.3. Construction and preparation of DNA vaccine The gene coding for OMP38 was amplified from V. anguillarum genomic DNA by the polymerase chain reaction (PCR) with specific primer set (Table 1) and cloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen). This vector carries the human cytomegalovirus (CMV) immediate early promoter, the bovine growth hormone (BGH) polyadenylation signal for transcription termination, the Ampicillin resistance gene and the ColE1 origin of replication for maintenance in E. coli. The forward primer contained a BamHI restriction site, and a Kozak sequence (CCACC) just before the start codon to ensure proper translation of prokaryotic genes in eukaryotic cells. The reverse primer contained an EcoRI restriction site and the C-terminal sequence of OMP38 including the translation stop codon (Table 1). Appropriate primer was used to amplify the coding sequence for the primary (including the amino acid leader sequences) and mature forms of OMP38 protein. PCR reaction was performed with Taq DNA polymerase

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Table 1 PCR primers used for amplification of OMP38 Primer

Sequence

VAOMP-F VAOMP-R

50 -CGCGGATCCACCGATGAACAAAACTCTGATTGC-30 50 -CCGGAATTCTTAGAAGTCGTAACGTAGAC-30

Sequences were taken from accession number AY 157299. Start codon is indicated in boldface and the Kozak sequence is italicized. Underlined section in VAOMP-F is BamHI and in VAOMP-R is EcoRI.

(Bangalore Genei, India) and PCR product was cloned into pcDNA3.1 (Invitrogen) and transformed into E. coli DH5a. Recombinant clone was selected by ampicillin resistance and confirmed by restriction analysis of plasmids and DNA sequencing. Plasmid was named as pVAOMP38. Plasmid was purified with the EndoFree Plasmid Mega purification kit (Qiagen) according to the manufacturer’s instructions, aliquoted at 1 mg ml1 in sterile endotoxinfree phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), and stored at 20  C until further use. 2.4. In vitro transfection of pVAOMP38 in Seabass kidney cell line (SISK) The SISK cells were grown on coverslips for 24 h, subconfluent monolayers were transfected with pVAOMP38 plasmid using lipofectamine 2000 (Invitrogen). After 48 h, the cells were fixed with 3.7% p-formaldehyde for 10 min at 4  C, washed with PBS, and permeabilized with 0.1% Triton X-100 at 4  C for 4 min, then blocked in PBS containing 1% bovine serum albumin (BSA) for 30 min at room temperature. Polyclonal antibody against Outer membrane porin protein (OMP38) was diluted (1:75) in PBS with 1% BSA and directly added to the fixed cells and kept for 2 h at room temperature. Then the cells were washed with wash buffer, followed by addition of the rabbit anti-mouse Ig secondary antibody (IgG) conjugated with FITC at a dilution of 1: 50 for 45 min at room temperature. The cells were washed and mounted with antifade 1,4-diazobicyclo-2,2,2-octanex (DABCO) in mounting medium (Sigma). The coverslips was observed under a confocal laser scanning microscope (Carl Zeiss, Germany) fitted with a CCD-4230 camera using a computer-based programmable image analyzer KS300 (Carl Zeiss, Germany). 2.5. In vivo expression of OMP38 by immunohistochemistry For in vivo expression of OMP38 by immunohistochemical analyses, a specimen was taken from the muscle tissue at the site of pVAOMP38-injected seabass and fixed in 10% phosphate-buffered formalin. After 48 h, the specimen was processed and embedded in paraffin wax by using Embedder (Leica, Germany), then sectioned into 4-mm thickness using a Rotary microtome (Leica, Germany), incubated at 37  C for 24 h and then incubated over night at 58  C. Paraffin section was deparaffinized in xylene, and hydrated through descending graded levels of alcohol to distilled water. The section was treated with trypsin (0.1% w/v in PBS, biochemical grade, Hi Media, India) for 10 min and washed twice with PBSeTween 20 (T-20, Hi Media, India; 0.01% v/v with PBS). Non-specific antibody binding sites were blocked for 30 min using 3% bovine serum albumin (in PBS, pH 7.4) and washed with PBST. The section was treated with the primary antibody (murine antiserum specifically raised against outer membrane protein (OMP38) of V. anguillarum) at a dilution of 1: 50 (sterile PBS, pH 7.4) for 1 h in a humid chamber. Section was washed with PBST and treated with rabbit anti-mouse FITC conjugate at a dilution of 1: 50 for 30 min at room temperature. After washing it was mounted using glycerol. The slide was observed under a fluorescent microscope (Carl Zeiss, Germany). 2.6. Fish vaccination Seabass were randomly divided into three groups of 10 fish and vaccinated as follows. Fish were anaesthetized by immersion in a 50 mg ml1 solution of tricaine methane sulfonate (MS-222) and intramuscularly injected at the base of the dorsal fin with 20 mg of the specified DNA vaccine solution (1 mg ml1). Three replica groups of 10 fish per treatment were assessed. The treatment groups tested for humoral immune response consisted of fish vaccinated with pVAOMP38, and two negative control groups including (ii) pcDNA3.1 and (iii) sterile PBS were assessed for efficiency of protective immunity.

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2.7. Analysis of antibody response Thirty-five days post vaccination, six fish from each treatment group were assayed for antibody response against OMP38 by enzyme-linked immunosorbent assay (ELISA). Fish were euthanized by an overdose of anaesthetic (MS-222, 500 mg ml1) and the blood was collected directly from the heart. After coagulation, the blood was centrifuged and the serum was collected and stored at 80  C. An ELISA plate (96 wells) was coated overnight at 4  C with OMP38 protein of V. anguillarum (10 mg ml1) in 100 ml aliquots in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). The plate was washed with PBS containing 0.05% Tween 20 (PBSeTween) and then blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at 28  C. After washing with PBSeTween, serial dilutions of the sera (1:10 and 1:100) obtained from individually vaccinated fish were added to triplicate wells of the plate and incubated for 90 min at 37  C. The plate was then washed twice and rabbit anti-seabass immunoglobulins (1:1000) (kindly provided by Dr Roberto Carlos Vazquez-Juarez, Mexico) were added to the plate and incubated for 2 h at 37  C. After washing thrice, alkaline phosphate goat anti-rabbit IgG (1:2000) (Sigma Chemical Co., USA) was added to the wells and incubated for 2 h at 37  C. After washing, the reaction was developed by addition of pNPP (p-nitrophenyl phosphate). After 30 min incubation in darkness at room temperature (the reaction was stopped by the addition of 3 N NaOH) the plate was read with a microplate reader (Thermo Lab Systems) at 405 nm. 2.8. Bacterial agglutination and complement mediate response of immunized serum 2.8.1. Bacterial agglutination titre The levels of specific antibody in seabass were determined by agglutination test performed with 96-well microtitre plates. Immunized serum (IS) specific for V. anguillarum (NB10) was obtained from DNA vaccinated (pVAOMP38) seabass. Immunized serum was heat inactivated at 44  C for 20 min to inactivate the complement activity and stored at 20  C. Then Serum (50 ml) was serially diluted in PBS, and 50 ml of V. anguillarum cultures (5  108 bacteria ml1) was added to each well. After incubation for 2 h at 20  C and overnight at 5  C, titres were read as the highest serum dilution giving positive agglutination. 2.8.2. Extraction of complement source from normal serum Normal serum (NS) of complement source was obtained from Asian seabass. Serum was absorbed before use to remove potential natural Ab directed against V. anguillarum serogroup O1. For absorption each ml of NS was incubated with 1  108 live washed cells of V. anguillarum NB10, on ice. After 1.5 h of absorption, serum was centrifuged (13,800  g) and the supernatant of complement factor was filtered through a 0.22 mm membrane filter. 2.8.3. Bacterial survival in IS with NS as complement source Bacterial survival in immunized serum with normal serum as complement source was determined in round-bottom microtitre plates as for the agglutination test. IS (50 ml) was serially diluted in PBS, and 50 ml of V. anguillarum cultures (5  108 bacteria ml1) was added to each well. After incubation for 1 h at 20  C the bacteria were thoroughly resuspended by pipetting up and down, thereafter 80 ml of the suspension was removed from each well and replaced with 80 ml NS (or PBS as a control) so that the bacterium suspension-to-NS ratio was 1:4. After 1 h, serial dilutions in PBS were plated onto Nutrient agar with 2% NaCl and colony-forming units were counted after incubation at 25  C for 24 h. 2.9. Experimental Vibrio anguillarum challenge Thirty-five days post vaccination fish were anaesthetized and challenged by intramuscular injection of V. anguillarum (NB10) cell suspension. In a preliminary trial we determined the median lethal dose (LD50) to be approximately 1.2  106 cfu fish1. Cell suspension was prepared as follows. V. anguillarum was grown overnight in NB broth with 2% NaCl at 28  C (harvested by 2500 rpm for 10 min and washed with PBS buffer thrice). Bacteria were suspended in PBS buffer and adjusted to an absorbance value of 1 at 540 nm, which corresponds to a cell density of approximately 108 cfu ml1. To count the bacterial concentration, the number of cfu ml1 were counted by spreading 0.1 ml of 10-fold serial dilutions of bacterial suspension on TCBS agar plates, which were incubated overnight at 28  C. After post-challenging, cumulative mortality and clinical signs were recorded daily, and dead fish were autopsied to

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determine the cause of death and to detect the presence of V. anguillarum in the tissues by bacterial culture in TCBS agar. The relative percent survival (RPS) was calculated according to Amend [25]   %Mortality in vaccinated fish RPS ¼ 1   100 %Mortality in control fish At the end of the experiment, fish that survived from the bacterial challenge were calculated as mentioned above and samples from kidney, intestine, liver, and spleen were dissected for histopathological analysis. Vaccine efficacy was determined by comparing the RPS and the histopathological damage among experimental treatments. 2.10. Statistical analysis Data are expressed as mean  SD. A Statistical analysis was performed using one-way ANOVA for antibody response, and a log rank test using the KaplaneMeier method was used for survival analysis. p < 0.05 was taken to indicate statistical significance. 3. Results 3.1. In vivo and in vitro transcription of recombinant OMP38 gene Transcription analyses of the OMP38 gene in injected muscle and SISK cell line were performed by a RTePCR reaction on DNaseI-treated RNA from fish injected cells transfected with pVAOMP38 and the empty plasmid pcDNA3.1. Amplification product of 993 bp was obtained (Fig. 1) from muscle samples taken at the site of injection 4 days after immunization with pVAOMP38 and 2 days after transfection with pVAOMP38 in SISK cell line using the specific primer set (Table 1). No amplification was observed in injected and transfected control pcDNA3.1. 3.2. In vitro and in vivo expression of OMP38 gene The ability of the constructed DNA vaccine to express OMP38 in a eukaryotic (SISK cell line) cells transfected with pVAOMP38, and the negative control pcDNA3.1 were studied. After 48 h of incubation after transfection, only pVAOMP38-transfected cells expressed protein OMP38, which was detected by confocal laser scanning microscope (Fig. 2A). In vivo expression of OMP38 protein could also be detected at the site of injection in fish vaccinated with pVAOMP38 by fluorescent microscopy (Fig. 2B). 3.3. Antibody response to DNA immunization The humoral immune response of Asian seabass to immunization with the OMP38 DNA vaccine was assessed by ELISA, at the time of 5th and 7th week after vaccination (Fig. 3). Fish injected intramuscularly with pVAOMP38P M

1

2

3

4

993bp 500bp 300bp

Fig. 1. Agarose gel showing transcription analysis of pVAOMP38 in SISK cell line (in vitro) and seabass fish muscle (in vivo) by RTePCR. M, DNA marker; Lane 1, pCDNA3.1 transfected seabass kidney cells; Lane 2, pVAOMP38 transfected SISK cell line PCR; Lane 3, pCDNA3.1 injected seabass muscle; Lane 4, pVAOMP38 injected seabass muscle.

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Fig. 2. (A) Immunofluorescence labelling of cells for the detection of expressed VAOMP38 in SISK cell line. Panel 1 shows the DAPI in the nucleus, panel 2 shows the fluorescent image of the expressed pVAOMP38, panel 3 shows phase image, and panel 4 shows the fluorescent images overlapped with the phase image of the cells, respectively. (B) Immunofluorescence detection of expressed plasmid encoding the Vibrio anguillarum outer membrane protein (pVAOMP38) in injected section of seabass muscle.

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Fig. 3. ELISA detection of anti-Omp38 antibody in serum from fish immunized with plasmid DNA vaccine (pVAOMP38) and control fish injected with PBS and plasmid control (pcDNA3.1). Each column represents the mean optical density (OD) among replica groups measure data at a serum dilution of 1:25 OD. Statistical differences were found between treatment and control. *Denotes significant difference of pVAOMP38, using one-way ANOVA. (At 5th week: F ¼ 270.75, p < 0.05 and 7th week, F ¼ 194.75, p < 0.05).

produced very low levels of anti-OMP38 antibody. Antibody levels against OMP38 were statistically significantly at the serum dilution 1:25 in vaccinated fish in relation to negative controls of PBS buffer and pcDNA3.1.

3.4. Protection of seabass from Vibrio anguillarum challenge Asian seabass immunized with the DNA vaccine encoding for OMP38 was assessed for protection against V. anguillarum intramuscular challenge. As shown in Fig. 4, mortalities following exposure to the bacteria were lower in fish vaccinated with pVAOMP38 compared to those of the control groups injected with PBS buffer and pcDNA3.1 alone. Cumulative mortality percent rate was also calculated (Table 2). The pVAOMP38 vaccine protected fish from the bacterium with 55.6% of RPS which was significant with survival analysis (Fig. 5). Histopathological analyses of vaccinated and non-vaccinated control fish that survived the bacterial challenges revealed signs of tissue lesions resulting from V. anguillarum infection (data not shown). Major lesions were observed in the kidney, liver and spleen, including severe necrosis and infiltration of macrophages and aggregation of melanomacrophage centres. Percentages of fish that presented the histological alterations were 92, 87 and 45 for fish vaccinated with PBS buffer, pcDNA3.1, and pVAOMP38P, respectively.

Fig. 4. Development of cumulated mortality in DNA vaccinated and control seabass after challenge with Vibrio anguillarum.

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Table 2 Cumulative percent mortality and relative percent survival (RPS) of vaccinated Seabass (Lates calcarifer) following intramuscular challenge with Vibrio anguillarum Fish injected with

Cumulative percent mortality (dead fish/total injected fish)

RPS

PBS Buffer pCDNA3.1 pVAOMP38

90.0% (27/30) 86.6% (26/30) 40.0% (12/30)

e e 55.60%

Values are cumulative percent of mortality recorded 3 weeks after challenge.

3.5. Bacterial agglutination and bacterial survival in immunized serum Different dilutions of seabass immunized serum with specificity to V. anguillarum NB 10 were tested for bacterial agglutination titre and ability to kill the bacterium in a microtitre plate. The tested strain of V. anguillarum was recognized by Ab. The highest IS dilution giving positive agglutination of 5  108 bacteria ml1 was 1:32. At this dilution the IS in the presence of normal serum as complement source killed more than 80% of bacterial survival. At the increased dilution bacterial survival percentages were increased. 4. Discussion Vaccination with outer membrane protein (OMP) has been shown to be effective against Vibrio vulnificus [26], Aeromonas salmonicida and Aeromonas hydrophila infections in fish. However antigens, which are purified and structurally mature forms are important for effective immunization. DNA vaccines offer several advantages over classical antigen vaccines (i.e., live attenuated, killed and subunit vaccines). DNA vaccines using OMP genes have already been shown to give partial protection against experimental challenge with Aeromonas veronii [24]. In this work, we investigated the potential for DNA Vaccine against Vibrio anguillarum using the gene encoding for major outer membrane protein (OMP38) from V. anguillarum OMP38. The present study shows that immunization of Asian seabass with DNA vaccine induced moderate protection against experimental challenge with V. anguillarum. Efficient protection could not be obtained when injected with OMP38 DNA vaccine because of low levels of protein expression in fish tissue due to the intrinsic features of the OMP38 gene, such as codon usage and mRNA stability. If the expression vector pcDNA3. I had mRNA stability and codon usage there would probably have been a high level of expression. The low antibody response is associated Survival Functions

1.2

Cum Survival

1.0

.8 Group 3

.6

.4

.2

Group 2 Group 1

0.0

3

4

5

6

7

8

9

10

11

DAYS Fig. 5. Log rank test for survival analysis. Group 1: PBS; Group 2: pcDNA3.1; Group 3: pVAOMP38. pVAOMP38 challenged fish survival is significantly increased compared to controls (PBS and pcDNA3.1). p < 0.00001.

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with low levels of antigen expression in the host tissue. A number of studies have demonstrated that there is a good correlation between the codon bias of genes encoding for viral, bacterial, and parasitic antigens and its level of expression [27e33]. Codon usage optimization has an enhancing effect on expression levels and immunogenicity of DNA candidate vaccines. Plasmid DNA vaccine tested in this work did not carry a signal sequence to express the antigen as extracellular protein. If the vector carries the appropriate signal sequence, considerable humoral response can be obtained against OMP antigen. It has been recently reported that the antibody response is dramatically increased when antigens are expressed as extracellular proteins from plasmid expression vectors carrying the appropriate signal sequence [34e38]. Therefore this should be considered for further vaccine development. Intramuscular injection of plasmid DNA enters the myocytes by an unknown mechanism. Once the antigen is expressed, it is released from the myocytes either as a secreted protein or due to cell damage and/or death and then it is internalized by antigen presenting cells [39]. Direct gene delivery to antigen presenting cells should be considered for extracellular pathogen for improving the immunological potency, particularly the antibody response to antigen. The targeted vaccine was shown to induce accelerated and increased antibody response (as compared with those receiving the non-targeted control). This approach may be useful in improving the protective efficacy of DNA vaccines. [40,41]. While designing DNA vaccines insertion of CpG motifs can improve immunogenicity of bacterial pathogen. CpG DNA can be used as an effective adjuvant for DNA vaccine and as a potent enhancer of antigen specific immune responses in numerous animal species [42e47], and is important for anti-bacterial defence mechanisms in fish. It minimizes the impact of fish diseases and enhances the efficacy of DNA vaccines [42]. OMP38 DNA vaccine when injected induces a significant antibody immune response. This DNA vaccine was able to moderate levels of protection to experimental challenge with V. anguillarum, including a reduction in the number of fish with histopathological alterations compared to control. This protection level is similar to those obtained in sandbass genetically immunized with OMP38 and OMP48 from A. veroni [24], and mice genetically immunized with the genes coding OMPs from Chlamydia pathogens [35,48], immunization with major outer membrane protein of V. vulnificus in a murine model [26] and Rainbow trout (Oncorhynchus mykiss) vaccinated with a 28 kDa OMP (porin) from A. salmonicida [49]. It has been stated that specific antibodies might play a role in the prevention of bacteria from adhering and penetrating the epithelium of the fish gills, skin and fins during the first stages of infection, and opsonization of the bacteria for elimination by phagocytosis [50]. The assay of agglutination titre and killing effect of V. anguillarum specific immunized serum shown to Ab can kill the bacterium if normal serum is present as a complement source. This suggests that small amounts of Ab are sufficient for activation of the classical pathway and give protective immunity. It is suggested that serum killing rather than agglutination of V. anguillarum by specific antiserum is more important for the protective effect of immunization against V. anguillarum. There are several conflicting reports regarding the correlation between protection of fish against bacterial infection and the level of serum specific antibody [51e53]. The protection responses elicited by the OMP38 DNA vaccine suggest that humoral and cellular immunity might be involved in the protection of seabass. In conclusion, the results presented here demonstrate that DNA vaccination in seabass, with the major OMP encoding gene from V. anguillarum, induces a significant humoral immune response and moderate protection against V. anguillarum experimental infection. Further vaccine designs and vaccination trials should focus on improving the levels of expression and antigenicity of this DNA vaccine. Four main factors are recommended for consideration. (1) codon usage optimization of the genes leads to efficient translation and stability of the mRNA. (2) Inclusion of a strong heterologous signal sequence enhances antigen secretion. (3) Consider fusion of gene targeting (antigen presenting cells) protein to DNA vaccine designing; the targeted vaccine is shown to induce an accelerated and increased antibody response. (4) Insertion of immunostimulatory motifs (CpG) can be important for designing of DNA vaccines used as potent enhancer of antigen specific immune responses. Acknowledgements The authors thank the management of C. Abdul Hakeem College for providing the facilities to carry out this work, The Director, Central Institute of Brackish water Aquaculture, for providing the experimental animals and Dr Debra

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Milton, Umea University, Sweden for providing Vibrio anguillarum NB10 Strain, The authors also thank Dr Roberto Carlos Vazquez-Juarez, Mexico, for providing Rabbit anti-seabass immunoglobulin. References [1] Rucker RR, Earp B, Ordal E. Infectious diseases of Pacific Salmon. Trans Am Fisheries Soc 1953;83:307e12. [2] Austin B, Austin DA. Bacterial fish pathogens. Disease of farmed and wild fish. Chichester: Springer-praxis; 1999. [3] Wong SY, Leong TS. A comparative study of Vibrio infections in healthy and diseases marine finfishes cultured in floating cages near Penang, Malaysia. Asian Fish Sci 1989;3:353e9 [special issue]. [4] Anderson IG, Norton JH. Diseases of barramundi in aquaculture. Aust Aquacult 1991;5:21e4. [5] Subhasinghe RP, Shariff M. Multiple bacteriosis, with special reference to spoilage bacterium Shewanell putrefaciens, in cage culture barramundi in Malaysia. J Aquat Anim Health 1992;4:309e11. [6] Soltani M, Munday BL, Burke CM. 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