Chinese Journal of Oceanology and Limnology   2016, Vol. 34 issue(6): 1247-1257     PDF       
http://dx.doi.org/10.1007/s00343-016-5096-z
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

LUO Zhang(罗璋), LIU Zhixin(刘志新), FU Jianping(付建平), ZHANG Qiusheng(张秋胜), HUANG Bei(黄贝), NIE Pin(聂品)
Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella
Chinese Journal of Oceanology and Limnology, 34(6): 1247-1257
http://dx.doi.org/10.1007/s00343-016-5096-z

Article History

Received Mar. 28, 2015
accepted for publication Jun. 8, 2015
accepted in principle Aug. 25, 2015
Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella
LUO Zhang(罗璋)1,2,3, LIU Zhixin(刘志新)2, FU Jianping(付建平)2, ZHANG Qiusheng(张秋胜)2, HUANG Bei(黄贝)4, NIE Pin(聂品)2        
1 College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China;
2 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China;
3 Tianjin Fishery Research Institute, Tianjin 300221, China;
4 College of Fisheries, Jimei University, Xiamen 361021, China
ABSTRACT: Flavobacterium columnare causes columnaris disease in freshwater fish. In the present study, the antigenic regions of five outer membrane proteins (OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 kDa, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purified recombinant protein was used to vaccinate the grass carp, Ctenopharyngodon idella. Following vaccination of the fish their IgM antibody levels were examined, as was the expression of IgM, IgD and IgZ immunoglobulin genes and other genes such as MHC Iα and MHC IIβ, which are also involved in adaptive immunity. Interleukin genes (IL), including IL-1β, IL-8 and IL-10, and type I and type II interferon (IFN) genes were also examined. At 3 and 4 weeks post-vaccination (wpv), significant increases in IgM antibody levels were observed in the fish vaccinated with the recombinant fusion protein, and an increase in the expression levels of IgM, IgD and IgZ genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fish. At four wpv, the fish were challenged with F. columnare, and the vaccinated fish showed a good level of protection against this pathogen, with 39% relative percent survival (RPS) compared with the control group. It can be concluded, therefore, that the five OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fish and protection against F. columnare.
Key words: Flavobacterium columnare     outer membrane protein     antigen     immunogenicity     vaccine     immune response     grass carp    
1 INTRODUCTION

Flavobacterium columnare, a Gram-negative bacterium, is the causative agent of columnaris disease of freshwater fish in worldwide aquaculture (Decostere et al., 1999 ; Liu et al., 2008). A wide range of fish species can become infected with this bacterium, including cold-water fish such as the rainbow trout, Oncorhynchus mykiss (Barnes et al., 2009), tropical fish such as the Nile tilapia, Oreochromis niloticus (Figueiredo et al., 2005), and warm-water fish, as such the channel catfish, Ictalurus punctatus (Darwish and Mitchell, 2009) and the grass carp, Ctenopharyngodon idella (Liu et al., 2012). Columnaris disease often causes high mortality resulting in severe economic losses in the fish farming industry (Liu et al., 2012). Over the last two or three decades, research on F. columnare as a bacterial pathogen has been related mainly to its diagnosis, epidemiology, pathology and preventive measures against infection with it (see Declercq et al., 2013 for a review). In contrast, identification of potential virulence factors in this bacterium has attracted the attention of fewer studies (Xie et al., 2004 ; Li et al., 2010), while the development of a vaccine against columnaris disease has been the focus of some research over the last a few years. Using formalinkilled bacteria, Leal et al.(2010) found that intraperitoneally and intramuscularly injected bacteria provoked a specific antibody response in Nile tilapia fingerlings, but a protective effect was not observed in challenge infections. Shoemaker et al.(2011) developed an attenuated live vaccine by passing the bacterium through rifampicin-supplemented culture, and found that this vaccine was effective at protecting channel catfish and largemouth bass (Micropterus salmoides) from F. columnare infections. Meanwhile, Olivares-Fuster et al.(2010) tried to develop a recombinant vaccine, and observed a specific antibody response in channel catfish injected with the recombinant protein of a heat shock protein encoded by dnaJ of F. columnare although no protection was detected in the immunized fish when challenged with F. columnare. However, vaccination trials of other recombinant proteins based on F. columnare proteins are not available in the scientific literature in relation to the development of possible vaccines against columnaris disease.

In our previous study, five proteins, including a zinc metalloprotease, a prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, which are all components of outer-membrane proteins (OMPs) in F. columnare, were screened by employing an immune-related screening method (Xie et al., 2004). As these proteins were immune-reactive with host sera, it is hypothesized that they could be used as possible immunogens for the development of recombinant protein vaccines. In the present study, the antigen regions of the five proteins were bioinformatically analyzed, and all such regions in these proteins were cloned into a pET-32a vector to express them as a single fusion protein. The potential immune protection of this fusion protein was then examined in the grass carp, C. idella, a freshwater fish species cultured widely and one that suffers severely from columnaris disease in China (Wang et al., 2010). The immune response, at the mRNA level, was also examined in the immunized fish in terms of the expression of IgM, IgD and IgZ genes, and MHC and MHC II β genes, as well as some innate immunity genes including type I and type II interferon (IFN) genes and interleukin (IL) genes.

2 MATERIAL AND METHOD 2.1 Bacterial strains and culture conditions

The G 4 strain of F. columnare, isolated originally from the outbreak of gill-rot disease in grass carp in 1972(Lu et al., 1975), was used in the present study. It has been continuously employed in our research and was cultivated at 25℃ with gentle agitation in Shieh broth as recommended by Decostere et al.(1997). Escherichia coli TOP10 and DE3 strains (TaKaRa, Dalian, China), used for gene cloning and expression studies, respectively, were cultivated at 37℃ in Luria-Bertani broth.

2.2 Amplification and fusion of the antigenic regions of the OMP genes

The sequences coding for zinc metalloprotease (GenBank Accession No. AY387596), prolyl oligopeptidase (AY387598), thermolysin (EF140791), collagenase (EF501979) and chondroitin AC lyase (AY912281) were retrieved from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The TMHMM Server v.2.0(http://www.cbs.dtu.dk/services/TMHMM/) and DNAStar software were used to evaluate transmembrane, hydrophilic and antigenic regions in the aforementioned proteins. Based on the results, five pairs of primers with appropriate restriction endonuclease sites were designed to amplify the antigenic regions of the five OMP genes. All primers used in this study are listed in Table 1. Genomic DNA prepared from F. columnare was used as the PCR template. Reactions were run in a PTC-100 thermocycler (MJ Research, USA) by heating the tubes at 94℃ for 4 min followed by 35 cycles at 94℃ for 30 s, 55℃ for 30 s and 72℃ for 1 min, and then a final extension at 72℃ for 10 min. After restriction enzyme digestion, the PCR products were ligated with T4 DNA ligase. Subsequently, the ligated product was used as the PCR template for amplifying the series of ligated OMP gene segments using the Maz-f and ClsA-r primer pair designed for this purpose. The PCR program used to amplify the gene segments was as follows: 5 min at 94℃, 40 cycles of 1 min at 94℃, 1 min at 55℃, and 2 min at 72℃ and a 10 min final extension at 72℃. The resultant PCR product was inserted into the pMD18T vector (Tiangen, Beijing, China) and then transformed into E. coli TOP10 competent cells. The recombinant transformant was confirmed by DNA sequencing.

Table 1 PCR primers used in this study
2.3 Expression and purification of recombinant protein

Plasmids extracted from the antigen-expressing E. coli TOP10 clone were digested with Nco I and Sac I restriction enzymes, and the digested fragment including the target fragment was inserted into a pET- 32a vector (Novagen, Germany). The sequenced plasmid was transformed into E. coli DE3 cells and the recombinant strain was induced with 0.5 mmol/L isopropyl β-d-1-thiogalactopyranoside (IPTG). After 6 h induction, the bacterial cells were harvested, resuspended in phosphate-buffered saline (PBS; pH, 7.4), and lysed by sonication on ice. Inclusion bodies were solubilized in denaturing solution and purified using Ni-NTA based affinity chromatography as described by Poobalane et al.(2010). The purified protein was subsequently analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and visualized after staining with Coomassie brilliant blue R-250. As a negative control, an induced culture of E. coli DE3 previously transformed with the empty pET-32a vector was also subjected to purification using the same procedure.

2.4 Fish vaccinations

The purified recombinant protein was quantified by the bicinchoninic acid method of Smith et al.(1985), and its concentration was adjusted to 0.5 mg/mL. Grass carp, weighing 80-100 g each, purchased from the experimental station of the College of Fisheries, Huazhong Agricultural University, China, were acclimatized at 25℃ in our laboratory for three weeks before commencing the experiments. One hundred and twenty fish were divided randomly into two groups: individual fish in group 1 were each injected intraperitoneally with 1 μg recombinant protein/g body weight, and those in group 2 were each injected intraperitoneally with 1 μg of the pET-32a vector tag protein as a control group.

2.5 Preparation of a monoclonal antibody (mAb) against grass carp IgM

The recombinant IgM heavy chain constant region from grass carp was expressed and purified as reported by Yan et al.(2012) and Liu et al.(2012). The primers used were as follows: F-5′-GGTACCGTCATCGCTGAGGCATCGG- 3′ and R-5′-AAGCTTTTAAC CGCTCTTCCACTCAGAATAACT-3′. pET-32a was induced to express a protein of about 31.2 kDa (Yan et al., 2012). Hybridomas-secreting the mAb against grass carp IgM were established by fusing SP2/0 with spleen cells from BALB/c mice immunized with the purified IgM protein. Immunization, fusion and cloning were performed according to the method described by Al-Harbi et al.(2000).

2.6 Western blot analysis of the recombinant protein

The recombinant protein was electrophoresed on a 5% stacking gel and a 10% separating gel in Trisglycine buffer, and then electroblotted onto a nitrocellulose membrane (Invitrogen, USA). After blocking with 5%(w/v) nonfat milk in PBS containing 0.1% Tween 20(PBST), the membrane was incubated overnight with the serum (1:100 diluted in PBST) collected from the grass carp vaccinated with the recombinant protein at 4℃. Afterwards, the mouse anti-grass-carp IgM monoclonal antibody (1:5 000 diluted in PBST) and goat anti-rat IgG conjugated with horse radish peroxidase (HRP; 1:5 000 diluted in PBST) were used as the second antibody and the bound antibody, respectively. Immunoreactive bands on the blot were detected using a SuperSignal West Pico Trial kit (Thermo, USA) and an ECL Western blot system (Bio-Rad, USA) according to manufacturers' instructions.

2.7 Analysis of the antibody response

At 1-week intervals, serum was collected from the vaccinated group and control group for a period of four weeks. On each occasion, three fish from each group were anaesthetized with ethyl carbamate, and blood samples were collected individually from the caudal vein with a syringe. The blood samples were allowed to clot overnight at 4℃. Serum from each blood sample was obtained by centrifugation (6 000 × g, 10 min), and the IgM antibody level was determined by enzyme-linked immunosorbent assay (ELISA) as described by Wang et al.(2013). Briefly, 96-well plates were coated with the purified recombinant protein at 10 μg/mL. Fish serum (1:100 diluted in PBST) and mouse anti-grass-carp IgM monoclonal antibody (1:5 000 diluted in PBST) were used as the primary antibody and secondary antibodies, respectively. HRP-conjugated goat antirat IgG (1:5 000 diluted in PBST) was added to plates and the color was developed with 3, 3', 5, 5'-tetramethylbenzidine substrate for 30 min. The reaction was stopped with 2.0 mol/L H2 SO4. Plates were read with a microplate reader (Thermo, USA) at 450 nm.

2.8 Real time PCR quantification of immune genes following vaccination

Fish were sampled as described above, and each blood sample was used separately to extract total RNA using Trizol (Invitrogen, USA). The RNA was then dissolved in diethylpyrocarbonate-treated water, and digested with DNase I (Fermentas) to eliminate genomic DNA, before being used for cDNA synthesis with the RevertAid TM First Strand cDNA Synthesis Kit (Fermentas), which was used according to the manufacturer's instructions. Real time quantitative PCR was performed using the primers listed in Table 1. The 20 μL PCR reaction mixtures contained 1 μL of each primer (0.5 μmol/L), 2 μL template, 6 μL H2 O and 10 μL 2×iQ TM SYBR Green Supermix (Bio-Rad), on the Bio-Rad CFX96™ Real-Time System (Bio- Rad). The PCR conditions involved an initial denaturation cycle of 95℃ for 3 min, 45 cycles of 95℃ for 20 s, 55℃ for 20 s, and 72℃ for 30 s. Each sample was run in triplicate. Relative gene expression levels were determined by the comparative threshold cycle method (2-ΔΔ Ct) with β-actin used as the reference gene (Zhang et al., 2012).

2.9 Bacterial challenge experiment

Twenty-eight days post-immunization, thirty grass carp in each group were challenged by intra-peritoneal inoculation of 0.2 mL of an F. columnare G 4 suspension at a concentration of 5×108 colonyforming units/mL. Mortality was recorded daily for two weeks following the challenge, and cumulative mortality was calculated. Dead fish were removed and their gills and livers were streaked onto Shieh agar plates to confirm the presence of F. columnare. Relative percentage survival (RPS) was calculated as reported by Zhang et al.(2007) : RPS=(1-[mortality of vaccinated fish/mortality of control fish])×100%.

2.10 Statistical analysis

Data are expressed as the mean±S.D. and significant differences in the antibody titers and the transcript levels of immune genes were determined using the Student's t-test (P<0.05).

3 RESULT 3.1 Fusion of antigenic regions of five OMPs of F. columnare

Sequences encoding the antigenic regions of five the OMPs (i.e., zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase, and chondroitin AC lyase) were successfully PCR amplified, and the appropriate restriction sites were introduced into the PCR products. The five PCR products were fused by ligation with T4 DNA ligase, and the fused product was amplified using Maz-f and ClsA-r primers (Fig. 1).

Figure 1 PCR amplification of the antigenic regions encoded by the DNA sequences of the five outer membrane protein (OMP) genes in Flavobacterium columnare and the fused DNA sequence of the antigenic regions M indicates DL2000 markers at 2 000, 1 000, 750, 500, 250 and 100 bp; lane 1: antigen region sequence of zinc metalloprotease, 620 bp; lane 2: prolyl oligopeptidase, 393 bp; lane 3: thermolysin, 363 bp; lane 4: collagenase, 315 bp; lane 5: chondroitin AC lyase, 410 bp; lane 6: the full-length fused sequence containing the antigen regions of the five OMP genes, 2 053 bp.
3.2 Expression, purification and immunogenicity of the fusion protein

A novel protein band was detected by SDS-PAGE in induced E. coli containing the recombinant plasmid, while no protein band was found at the same position in uninduced E. coli containing the recombinant plasmid (Fig. 2). The molecular mass of the recombinant protein revealed by SDS-PAGE was about 95.6 kDa, which is in agreement with the molecular weight deduced from its amino acid sequence. The recombinant protein reached an optimal expression level after 6 h induction with 0.5 mmol/L IPTG. After sonication of the cells, the recombinant protein was purified by Ni-NTA affinity chromatography, and the purity was >90%, as determined by bandscan software. The immunogenicity of the recombinant protein was analyzed by Western blotting with sera from the grass carp immunized with the recombinant protein followed by incubation with the mouse anti-grass carp IgM monoclonal antibody (Fig. 3). The recombinant protein was recognized by the sera collected from the grass carp vaccinated with the recombinant protein (Fig. 2), while the sera collected from the control group fish did not react with the recombinant protein (data not shown).

Figure 2 SDS-PAGE and Western blot analysis of the recombinant fusion protein M indicates MK039 marker at 97.4, 66.2, 43.0, 31.0 and 20.1 kDa; lane 1: protein expression from recombinant plasmids in E. coli DE3 without IPTG; lane 2: protein expression from recombinant plasmids in E. coli DE3 with 0.5 mmol/L IPTG with a 6 h induction; lane 3: supernatant after sonication; lane 4: inclusion bodies; lane 5: purified recombinant fusion protein; lane 6: Western blot of the purified recombinant fusion protein.
Figure 3 Western blot of the purified recombinant IgM heavy chain constant region and the IgM molecule in whole serum from grass carp using a mouse anti-grass carp IgM monoclonal antibody for detection Lane 1: purified recombinant IgM heavy chain constant region with a mass of about 32 KDa, as reported by Yan et al.(2012) ; lane 2: IgM in whole serum from grass carp; the relative mobility is equivalent with that reported by Yan et al.(2012).
3.3 Analysis of the antibody response following fusion protein immunization

The IgM antibody levels in grass carp immunized with the recombinant fusion protein were measured with ELISA. As shown in Fig. 4, the antibody titers in the immunized fish rose gradually from 1 to 4 weeks post-vaccination (wpv), and significantly higher levels were detected in the fish at 3 and 4 wpv (P<0.05).

Figure 4 IgM antibody levels in grass carp vaccinated with the recombinant fusion protein as detected by using ELISA Data are expressed as the mean±S.D. of OD 450 values from the sera of three grass carp. Asterisks (*) indicate significant differences (P<0.05) among the different groups on the same sampling occasion.
3.4 Expression of immune genes following fusion protein immunization

The transcription profile of the immune genes was examined in the immunized fish; the relevant genes included IgM, IgD and IgZ immunoglobulin genes, two other genes, MHC and MHC II β, which are also involved in the adaptive immune response, type I and type II IFNs, and IL - 1β, IL - 8 and IL - 10 genes (Fig. 5). Expression of IgM, IgD and IgZ immunoglobulin genes showed an increasing trend over the four week period following immunization (Fig. 5a), and a significant increase was observed for all three Ig genes at 1 wpv, and for IgM at 3 wpv. The IL genes, IL - 1β, IL - 8 and IL - 10, showed significant increases at 1 wpv, but their levels declined gradually, although IL - 8 gene expression was significantly upregulated at 4 wpv (Fig. 5b). Type I IFN and type II IFN expression was also detected, but only significantly at 1 wpv for type I IFN (Fig. 5c). On other sampling occasions, IFN and IFN - γ expression levels were relatively low (Fig. 5c). A decreased trend was also observed for MHC and MHC IIβ expression, but MHC was expressed at a significantly high level on 1 wpv and 3 wpv

Figure 5 Expression of immune genes, IgM, IgD and IgZ (a), IL - 1β, IL - 8 and IL - 10(b), IFN and IFN - γ(c), and MHC and MHC IIβ(d) in grass carp, as quantified by real-time quantitative PCR following vaccination of fish with the recombinant fusion protein The mRNA level of each immune gene was normalized against β-actin. Data represent the mean±S.D. of three assays. Significant differences between the control and vaccination groups are indicated by * at P<0.05.
3.5 Protection of grass carp from F. columnare challenge

At 4 weeks post-immunization, the grass carp were challenged with F. columnare G 4. In the group vaccinated with the recombinant fusion protein, the cumulative percent mortality was 56.7%, while in the control group it was 93.3%(RPS=39%; Fig. 6). Death in the fish was observed on the second day postchallenge. The clinical signs were swelling at the injection site, and fin and gill necrosis in the morbid fish, and the pathogenic bacterium F. columnare was isolated from the gills and livers.

Figure 6 Cumulative mortality after challenge infection with Flavobacterium columnare G 4 in grass carp vaccinated 4 weeks previously with the recombinant fusion protein Fish were vaccinated with recombinant protein (■). Control fish were vaccinated with the pET-32a vector tag-protein (●).
4 DISCUSSION

Bacterial OMPs are regarded as candidate immunogens for the development of recombinant protein vaccines because their external existence on the bacterial cell surface may facilitate recognition by the host immune system (Lin et al., 2002). It is recognized that recombinant proteins of some OMPs from pathogenic bacteria such as Vibrio parahaemolyticus (Mao et al., 2007) and V. harveyi (Zhang et al., 2007), Streptococcus iniae (Cheng et al., 2010), and Edwardsiella tarda (Sun et al., 2011) can induce protective immunity in host species. In a previous study, Sun and Nie (2001) found that some F. columnare OMPs were immunogenic, as revealed by Western blotting with fish antiserum. Indeed, the present study has shown that F. columnare OMPs are immunogenic and have potential as candidate antigens for the development of vaccines against columnaris disease.

However, virulence factors, despite their pathogenicity, may also serve as promising vaccine candidates. For example, adhesin, which is a type of virulence factor in some pathogens, can be used readily as a target for vaccine development (Wells et al., 2007). Additionally, proteases, together with chondroitin AC lyases and adherence factors, have been reported as virulence factors in F. columnare (Newton et al., 1997 ; Decostere et al., 1999 ; Xie et al., 2005). In the present study, five possible virulence factors, including chondroitin AC lyase and four proteases (zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase), which are F. columnare OMPs, were analyzed to determine their antigenic regions, and these antigenic regions, when expressed as a recombinant fusion protein, induced adaptive as well as some innate immune responses in grass carp, thereby providing immune protection against infection with F. columnare.

Chondroitin AC lyase can degrade acidic polysaccharides such as hyaluronic acid and chondroitin sulfates, which are components of the extracellular matrix of host tissues (Newton et al., 1997), thus facilitating bacterial invasion (Xie et al., 2005). In F. columnare, lyase is highly related with virulence (Suomalainen et al., 2006). It has been shown that the other four OMPs are involved in pathogenicity in other bacterial species. Zinc metalloprotease is distributed widely in eukaryotes and in prokaryotes such as V. anguillarum and V. aestuarianus ; this protein was found to have roles in bacterial invasion and host lethality, and even in influencing host immune responses (Norqvist et al., 1990 ; Labreuche et al., 2010). Prolyl oligopeptidase, also known as prolyl endopeptidase and post-proline cleaving enzyme, is a member of the serine protease family, and is present in many pathogenic bacteria, such as A. hydrophila (Kanatani et al., 1993). Thermolysin, which is very stable in organic solutions and under conditions of high salt or high temperature, may be functional in the survival and reproduction of bacteria under certain conditions (Inouye et al., 1996 ; Muta and Inouye, 2002). Bacterial collagenases have a broad range of substrates and can degrade natural and denatured collagens (Peterkofsky, 1982). The type II collagenase of F. psychrophilum, for example, can degrade gelatin and type II collagen, thus facilitating bacterial invasion and colonization of host species (Ostland et al., 2000). Here, we described the preparation and fusion of the regions encoding the antigenic regions of the five OMPs from F. columnare. The expressed recombinant fusion protein had high immunogenicity in grass carp, and when used as a vaccine was able to protect the fish from challenge with F. columnare.

Recombinant fusion proteins have been reported to have desirable attributes when used as vaccines. Zhang et al.(2007) found that the recombinant fusion protein, r-OmpK-GAPDH, had a higher level of immunogenicity and provided greater protection against V. harveyi than r-OmpK or r-GAPDH alone in the large yellow croaker. The OmpH-like surface antigen can generate a protective immune response in rainbow trout against another bacterial pathogen also in the genus Flavobacterium, F. psychrophilum (Dumetz et al., 2006). In the present study, the recombinant fusion protein designed on the basis of antigenic regions of OMPs also had a high level of immunogenicity and provided significant protection against F. columnare infection in grass carp, although the protective effects of the five OMPs were not examined separately.

The immunogenicity and protective role of the recombinant fusion protein may be a result of the IgM antibody response detected and the expression of immune genes following the intraperitoneal injection of the recombinant protein followed by the challenge infection. In grass carp, as in other teleost fish, three Ig types have been reported (Xiao et al., 2010). In fish, IgM functions in systemic and mucosal immune responses, IgZ/T plays a role in mucosal responses (Zhang et al., 2010 ; Xu et al., 2013), while the role of IgD is not well-defined (Enholm et al., 2011). Additionally, IgZ in grass carp is very much similar to IgM in terms of its amino acid sequence in this fish (Xiao et al., 2010), and efforts to develop antibodies to recombinant IgZ in grass carp were unsuccessful (own unpublished data); this may have been caused by the high level of similarity between IgM and IgZ in grass carp (Xiao et al., 2010). In the present study, fish serum was examined for IgM only following immunization (Fig. 4). A significant increase in IgM antibody levels was observed at 3 and 4 wpv (Fig. 4), and the higher level of IgM gene expression also at 3 and 4 wpv (Fig. 5a) may be indicative of an adaptive immune response to the recombinant fusion protein. As fish that have not been exposed to pathogens are not available in aquaculture research, the early increase in the expression of IgD, IgM and IgZ genes at 1 wpv may be explained as a slightly provoked response in these fish. Nevertheless, it is not unreasonable to conclude that fish can develop an antibody response to the pathogenic bacterium, F. columnare. Increases in the expression of MHC and MHC IIβ genes, which are involved in antigen processing and presentation, followed by adaptive immune responses (Chen et al., 2010), may provide further evidence to support this conclusion. In teleost fish, it was once considered that endogenous and exogenous antigens were presented by MHC I and MHC II molecules, respectively (see Dijkstra et al., 2001 for a discussion on this). But, exogenous antigens can be offered by MHC class I molecules when they are packed into or connected to a structure that delivers them into the cytoplasm (Dijkstra et al., 2001). A significant increase in the expression of the MHC gene was found also in Japanese flounder vaccinated with recombinant Eta2, an exogenous protein (Sun et al., 2011). However, MHC I and MHC II antigen presentation in fish has received little attention in research, and it would be interesting to characterize the antigens presented by MHC I and MHC II molecules in teleost fish.

IL and IFN genes have also been observed to respond to stimulation by recombinant fusion proteins. IL - 1β, a typical pro-inflammatory cytokine, has roles in immune cell proliferation, and in leukocyte respiratory burst and phagocytosis (Secombes et al., 2001 ; Randelli et al., 2008). IL - 8, a member of the CXC chemokine subfamily, possesses potent neutrophil chemotactic activity (Oehlers et al., 2010). In mammals, IL - 10 is an immunosuppressive cytokine, and is considered to be an inflammatory cytokine in fish (Inoue et al., 2005). Fish have two IFN types (type I and II IFNs), which possibly have a wide range of effects in the immune system (Zou and Secombes, 2011). The increased expression of IL - 1β, IL - 8 and IL - 10, and IFN, IFN - γ genes we observed may imply that fish also have innate immunity against F. columnare.

The adaptive immune response has been reported in mandarin fish (Siniperca chuatsi) against stimulation with inactivated F. columnare, with a similar pattern of increase in the expression of IgM, IgD and IgZ genes as that observed in the present study (Tian et al., 2009). Recently, Makesh et al.(2015) examined IgM antibody levels, and IgM, IgD and IgT gene expression in the systemic and mucosal immune systems of rainbow trout following immunization with attenuated F. psychrophilum, and found an elevated level of IgM antibodies, and of IgM, IgD and IgT gene expression. To meet the challenge of developing a fish vaccine against F. columnare, further research should be carried out to examine other possible vaccine candidate molecules in F. columnare and to understand the global adaptive and innate immune response of fish in the process of vaccination and challenge infections.

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