BACKGROUND
zebrafish  is a well-established vertebrate model of human development and hematopoiesis  <cit> .
however, as the aquaculture industry grows to meet the needs of an expanding global population, there has been a rapid increase in the research of teleost physiology and immunology.
the zebrafish model system has proven to be a useful tool for studying infectious diseases that are natural threats to fish species of important human food sources, such as rock bream  and turbot   <cit> .
several studies of the zebrafish immune system have provided initial insights into host-microbe interactions with both pathogens and commensals  <cit> , and the molecular processes mediating clearance of pathogenic infections  <cit> .
not surprisingly, many features of the zebrafish defense responses following pathogen invasion resembled those of other mammals, including humans  <cit> .
the adult zebrafish has been used in a few studies to investigate new vaccines against specific pathogenic species.
for example, novoa et al.
<cit>  studied the efficacy of a vaccine derived by reverse genetics against viral hemorrhagic septicemia virus in fish by using the zebrafish as a model system, and revealed that the vaccine was protective even at low temperatures.
in addition, cui et al.
<cit>  used zebrafish to study an attenuated live mycobacterium marinum vaccine aimed at reducing mycobacteriosis in freshwater and marine fish.
finally, xiao et al.
<cit>  developed an edwardsiellosis zebrafish model to screen attenuated live edwardsiella tarda vaccine candidates in order to identify those most highly effective for subsequent development for industry use.
however, so far, no work involves the immune-related pathways underlying the zebrafish response to vaccination.
in order to design a novel and effective vaccine, it is essential to gain a comprehensive understanding of the immune responses elicited in host upon vaccination.
to date, most of the studies of the teleost immune system have focused on head kidney or/and spleen  <cit> .
however, the vertebrate liver has recently been recognized as an essential immune organ  <cit> , accommodating a variety of cell types  <cit> , including those primarily involved in immune activities.
since the liver receives blood from both the systemic circulation and the intestine, it is exposed to a wide array of antigens.
therefore, its immune-related cellular components can manifest a broad range of immune reactions  <cit> .
for example, the liver lymphocyte population includes both innate immune cells  and adaptive immune cells -i or -ii  <cit> ).
as such, different infectious pathogens would be expected to induce distinctive profiles of immune responses in the liver  <cit> , which might be manipulated to create specific and effective therapeutic strategies.
several methods exist by which to determine the comprehensive transcriptomic profile of a pathogen-specific immune response, including microarray and quantitative real-time pcr  <cit> .
however, the high-throughput rna sequencing  technology offers several advantages over the other profiling applications.
not only is rna-seq independent on predefined probes, which facilitates the discovery of new transcript variants, but the sequence platform also produces low background noise, which allow for distinction between closely homologous genes and detection of weakly expressed transcripts  <cit> .
in addition, concurrent advances in the bioinformatic algorithms used to analyze the rna-seq data have allowed for better interpretation of the whole transcriptomic profile and provided further insights into complex molecular processes.
the rna-seq approach has already been successfully applied to several infectious disease models of zebrafish  <cit> , including zebrafish embryo infected with salmonella <cit> , and adult zebrafish and embryos infected by mycobacteria <cit> .
in addition, other fish species infection models have been subjected to rna-seq analysis, including large yellow croaker  infected by aeromonas hydrophila <cit>  and japanese seabass  infected by vibrio harveyi <cit> , but the overall immune-related transcription profiles have differed among species  <cit> .
no reports exist in the literature of rna-seq technology used to analyze the changes in an infected fish transcriptome profile induced upon vaccine treatment.
edwardsiellosis, caused by the gram-negative edwardsiella tarda, is currently one of the most economically disastrous infectious diseases affecting the global aquaculture industry  <cit> .
e. tarda displays polymorphic phenotypes and has a broad range of hosts from aquatic invertebrates to higher vertebrates, including birds, reptiles, mammals, and even humans  <cit> .
in developing a putative live attenuated vaccine against edwardsiellosis, xiao et al.
<cit>  constructed an e. tarda mutant  with low residual virulence.
although the mutant was capable of inducing robust protection in zebrafish and turbot, the antibody titers detected in sera were relatively low.
by thoroughly understanding the immune-mechanism of zebrafish induced by the putative live attenuated vaccine, a more immunogenic vaccine may be able to be generated.
to this end, we performed a comparative gene transcription analysis of livers from mock-immunized and wed-immunized zebrafish using rna-seq technology to investigate their differential transcriptsomic profile.
furthermore,  <dig> genes associated with mhc antigen processing were analyzed by qpcr and the results revealed an activated mhc-i pathway and an inhibited mhc-ii pathway during the early stage of vaccine immunization.
it was prompted that wed conferred a robust protection in zebrafish by eliciting an effective cell immunity via the mhc-i pathway.
RESULTS
rna-seq of liver transcriptome
to better understand the early stage immune response of zebrafish immunized with wed, six solexa cdna libraries were constructed from the livers of mock-immunized and wed-immunized zebrafish .
biological replicates were pooled to make representative samples for deep sequencing analysis.
across the two groups of triplicate data, after normalization of the generated 95 bp pe raw reads,  <dig> , <dig>   <dig> , <dig> and  <dig> , <dig> reads were obtained from c1-c <dig>  and  <dig> , <dig>   <dig> , <dig> and  <dig> , <dig> reads from v1-v <dig>  respectively.
to assess the quality of sequencing, the reads were mapped to the zebrafish reference genome.
from the reads of each group, successful mapping occurred for  <dig> , <dig> ,  <dig> , <dig> ,  <dig> , <dig> ,  <dig> , <dig> ,  <dig> , <dig>  and  <dig> , <dig>  reads.
however,  <dig> , <dig>   <dig> , <dig> and  <dig> , <dig> unmapped reads were generated from c1-c <dig>  while  <dig> , <dig>   <dig> , <dig> and  <dig> , <dig> unmapped reads were found in v1-v <dig>  respectively; we plan to conduct de novo analysis of these unmapped reads to generate a better reference of immune-relevant genes in zebrafish.
analysis of differential expression among wed- and mock-immunized zebrafish liver
to identify the differentially expressed genes, the transcriptome data of zebrafish liver from two days after wed immunization and mock immunization were analyzed by using the deseq package in r software  <cit> .
the criteria of a two-fold or greater change in expression and p-value< <dig>   were chosen to determine significantly up-regulated or down-regulated genes following immunization.
the magnitude distribution of the significantly changed genes was illustrated by ma plot analysis .
using these criteria, a total of  <dig> genes were significantly differentially expressed greater than two-fold, including  <dig> up-regulated genes and  <dig> down-regulated genes .
annotation of the differentially expressed genes was achieved through blastn similarity searches against the ensembl zebrafish refseq mrna database .
to perform an unbiased annotation of the functions of the differentially expressed genes identified by deseq analysis, go analysis of differentially expressed genes was carried out by two bioinformatics tools, david and bingo plugin .
among the  <dig> differentially expressed genes, david provided functional annotation for  <dig> genes.
go annotated differentially expressed genes mainly belonged to the three functional clusters , and were distributed among more than  <dig> categories .
the differentially expressed genes in the cluster of biological processes were found to be mainly related to stimulus response, immune response, regulation of immune system process, and regulation of development process .
to identify the biological pathways that are active in the zebrafish at the early stages of wed immunization,  <dig> differentially expressed genes were mapped to canonical signaling pathways found in kegg.
a total of  <dig> genes were mapped to  <dig> statistically significant categories .
protein processing in er was represented by  <dig> up-regulated and  <dig> down-regulated genes.
there were also a statistically significant amount of mapped genes for other major antigen processing-related pathways, such as those mediated by “proteasome” and “protein export” pathway, to indicate the vital role of antigen processing and presentation activated by wed immunization at the early stage in zebrafish liver, which would elicit the specific immune responses required in the restoration of homeostasis.
p
* indicates the percentage of genes in each pathway from  <dig> genes mapped to kegg.
in general, based on the results from go analysis  and kegg pathway analysis, the up- and down-regulated genes that were highly related to immune response of fish after wed immunization, significantly grouped into acute phase response , complement activation, immune/defense response, and antigen processing and presentation pathway.
the acute phase response is conserved in zebrafish liver following wed immunization
most of the conserved acute phase response genes were significantly differentially expressed following wed immunization .
this set of genes encoded the major apps , c-reactive protein , and serum amyloid p ), the minor and intermediate apps , the negative apps , several complement components, and ion-binding and transporting proteins.
many of the apps were up- or down-regulated greater than five-fold, suggesting that induction of apps in zebrafish liver likely plays an important role in host defense stimulated by wed vaccine.
similar subcategories of apps were also found to be differentially expressed in previous microarray-based studies of early stage immune response to bacterial infection in rainbow trout  <cit>  and catfish  <cit> , indicating the conservation of the vast majority of apps among teleost fish.
the major apps, two crp-like proteins  and saa, were induced to up-regulated their expressions by  <dig> -fold,  <dig> -fold and 883-fold, respectively, in the wed-immunized zebrafish liver, emphasizing their importance in teleost innate immune response.
in wed-immunized zebrafish, both the apolipoprotein a-iv, a fatty acid binding protein involved in extracellular and intracellular lipid transporting, and alpha-2-hs-glycoprotein were decreased by  <dig> -fold and  <dig> -fold, respectively.
the decrease of apolipoprotein a-iv was consistent to previous report in starved zebrafish liver  <cit> .
however, the functions of these two negative apps in immune response remain unknown.
p
identification of all differentially expressed genes was based on p <  <dig> .
a p-value <  <dig>  indicated that the gene was significantly altered in wed immunized fish with respect to mock-immunized fish.
the absolute value of “fold-change” is the magnitude of up- or down-regulation for each gene/homolog after wed immunization.
fold-change >  <dig> indicates up-regulation, and < − <dig> indicates down-regulation.
traditionally, complement has been considered as a supportive first line of defense against microbial intruders  <cit> .
in wed-immunized zebrafish liver, three isoforms of complement c <dig>  and the complement c <dig>  c <dig>  c <dig> and c <dig> were remarkably up-regulated.
the c <dig> inhibitor and the c1q were up-regulated and down-regulated, respectively .
hepatocytes, which account for 80% of the liver mass  <cit> , are the primary site of synthesis for all the genes involved in ion-binding and transporting  <cit> .
in the rna-seq data, more than  <dig> differentially expressed genes involved in ion-binding and transporting were strongly induced in zebrafish liver upon wed immunization .
these included haptoglobin, hemopexin, ceruloplasmin, transferrin receptor  <dig>  atpase, and cu2+ transporting alpha polypeptide.
intelectin  <cit> , which is involved in iron homeostasis, binding and transport, was one of the most up-regulated genes  in the ion-binding and transporting category; however, the functions of intelectin in the contexts of normal iron metabolism and disease defense in zebrafish need to be further clarified.
members of the transferrin and ferritin families were significantly affected to result in obvious up- and down-regulation in zebrafish liver by wed immunization .
leukocyte cell–derived chemotaxin  <dig>   <cit> , originally named for its possible neutrophil chemotactic activity in vitro, was strongly induced by  <dig> -fold in wed-immunized group, but its function in zebrafish remains unknown.
wed immunization induces defense responses and signaling transduction pathways
functional annotation of significantly differentially expressed genes in zebrafish liver was performed to define the transcriptome profile more precisely.
go classification  indicated that immune/defense response-related genes were enriched, specifically under go terms “response to chemical stimulus”, “regulation of immune system process”, and “immune response”.
toll-like receptors  detected the presence of pathogens and triggered an innate immune response, and several of the differentially expressed genes from wed-immunized liver mapped to the tlr signaling pathways .
tlr signaling has been remarkably conserved throughout evolution, and it can mediate immune responses to all types of pathogens and promote secondary disease.
in zebrafish, the pathogenesis of m. marinumstaphylococcus aureus, and aeromonas salmonicida has been shown to involve tlr signaling  <cit> .
to further investigate the function of tlr <dig> a/b that elicited the immune response in zebrafish embryo, assessment of tlr5a and tlr5b by morpholino-mediated knockdown followed by flagellin stimulation clearly demonstrated tlr5-dependent gene activation of mmp <dig>  cxcl-c1c, and irak <dig>  which suggested that the activation of tlr <dig> pathway can induce the expression of inflammatory mediators as well as the feedback control of the innate immune response  <cit> .
the functional investigation of tlr <dig> was also performed in a zebrafish embryo model, which suggested that the zebrafish tlr <dig> orthologs would negatively regulate the myd88-dependent nf-κb activation by sequestering the tlr adaptors and indicated that the existence of a tlr would negatively regulate tlr signaling upon engagement with its specific ligand  <cit> .
in humans and rodents, tlr-mediated signals in liver are associated with infection-induced granulomatous inflammation and ischemia-reperfusion injury, and can mediate liver regeneration processes  <cit> .
in wed-immunized zebrafish liver,  <dig> differentially expressed genes mapped to various tlr pathways , including the tlr <dig>  tlr <dig>  tlr <dig> and tlr <dig> subcategories, which are not only expressed on the outer membrane of immune cells but also on endosome-lysosome membranes.
tlr <dig> expression was up-regulated by  <dig> -fold, indicating that it played a key role during the early stage of wed vaccination.
immunity is a complex process of tightly controlled signals that involve a broad array of receptors, cytokines, enzymes, signal transducers, transcription factors, and other functional proteins.
in our study, wed immunization increased dramatically the expression of cytokine genes related to the jak-stat, mapk, tgf-β, apoptosis and vegf signaling pathways.
therefore, wed-induced gene expression in zebrafish liver might facilitate protection against e. tarda by activating these pathways.
similar results were obtained in a previous study of large yellow croaker spleen during a. hydrophila infection  <cit> .
since the majority of the differentially expressed genes in these signaling pathways were up-regulated in our study, it is possible that the wed immunization of zebrafish is capable of triggering a vigorous adaptive immune response.
wed immunization induces the antigen processing and presentation pathway
a large number of differentially expressed genes with functions in protein transportation, modification and degradation were up- or down-regulated in the zebrafish liver following wed immunization , indicating these genes were likely connected to the degradation and processing of antigens for mhc class i and ii molecules.
most of the differentially expressed genes related to mhc-i antigen processing pathways were significantly up-regulated, including the er-resident chaperone calreticulin, calnexin, endoplasmin , tap binding protein, proteasome activator , the heat shock proteins superfamilies, and cathepsin l. meanwhile, the typical mhc-ii processing pathway component, lysosomal membrane glycoprotein  <dig> , was down-regulated  in zebrafish liver after live attenuated vaccine immunization.
p
identification of all differentially expressed genes was based on p< <dig> .
a p-value< <dig>  indicated that the gene was significantly altered in wed-immunized fish with respect to mock-immunized fish.
the absolute value of “fold-change” is the magnitude of up- or down-regulation for each gene/homolog after wed immunization.
fold-change >  <dig> indicates up-regulation, and < − <dig> indicates down-regulation.
up-regulated genes with established roles in immune responses comprised another large functional category, indicating that active immune-surveillance, immune signaling, and immune cell activation were triggered in the wed-immunized zebrafish liver, like the mhc-i ze protein .
however, the mhc class ii dab, mhc class ii beta chain, mhc class ii invariant chain , mhc class ii transactivator , and cathepsin s were down-regulated at this stage .
to further explore the immune response profiles induced by wed immunization to the level of a single pathway, we used the kaas web-based pathway analysis program.
kegg analysis was performed to identify genes involved in phagosome and antigen processing and presentation pathways .
in the phagosome pathway,  <dig> genes were identified as strongly up-regulated upon wed immunization, while  <dig> genes were strongly down-regulated .
in the antigen processing and presentation signaling pathway, most of the up-regulated genes were found to be interrelated with the mhc-i processing pathway, while most of the down-regulated genes were related to the mhc-ii processing pathway.
in mammals, antigen processing and presentation are essential for triggering the downstream cellular and/or humoral immune responses  <cit> .
the kegg results revealed that eight genes involved in the mhc-i pathway were up-regulated, and six genes involved in the mhc-ii pathway were down-regulated by two days after wed immunization .
these results suggested that the mhc-i related pathways were co-induced following wed immunization, while the mhc-ii related pathways were co-depressed.
this unique perspective should be further clarified.
qpcr analysis of mhc processing pathways
we next sought to further clarify the strength of the correlations of up-regulated genes to mhc-i processing pathway and down-regulated genes to mhc-ii processing pathway in zebrafish during early stage following wed immunization.
differential expression of  <dig> genes associated with mhc antigen processing was analyzed by qpcr to confirm the hypothesis that antigen processing and presentation pathways elicit an adaptive immune response following immunization.
the assay was performed with both spleen and liver samples collected over the first five days post immunization.
most of the results were consistent with those of the rna-seq analysis .
mhc-i processing pathway related gene expression in liver tissues from wed-immunized groups were significantly up-regulated relative to mock-immunized groups .
the up-regulations of differentially expressed genes in liver mostly reappeared in spleen, except for hsp90α, hspa4a and calnexin.
the down-regulation expression of the three genes in spleen might reflect their different functions in two immune-associated organs.
in contrast, the mhc-ii processing pathway related gene expression was all down-regulated and completely coordinated in liver and spleen during the early stage following wed immunization .
this showed that mhc-ii processing pathway was inhibited in two immune organs by wed immunization.
thus, cd4+ t cells activation could be depressed following immunization.
the implications of this finding should be further investigated in teleost, specifically in zebrafish as model of e. tarda infection.
in addition, the qpcr data also revealed that antigen processing in liver possesses a comparatively dominant role to that in spleen.
the relatively intense expression in liver showed that antigen processing plays an essential role in wed-immunized zebrafish liver.
discussion
at present, molecular studies on the immune response to pathogens in fish models are mainly focused on infectious disease pathogenesis.
rna-seq and microarray-based transcriptome profiling studies have revealed that the teleosts are useful in vivo models for identifying host determinants of responses to bacterial infection  <cit> .
furthermore, the rna-seq approach has already been successfully applied to several infectious disease models of zebrafish  <cit> .
however, none have applied the rna-seq technology to elucidate the immune-related pathways underlying the zebrafish response to vaccination for more effective vaccine evaluation.
in this work, in order to gain comprehensive insight into the immunogenetics of zebrafish following immunization with the putative e. tarda live attenuated vaccine, a high-throughput deep sequencing-by-synthesis technology was used to investigate the immunization-related gene expression patterns.
deseq analysis identified  <dig> significantly differentially expressed genes in the zebrafish liver following wed immunization.
go and kegg analysis revealed that the genes involved in the er protein processing as well as the phagosome and antigen processing and presentation pathways are regulated at the early stage following wed immunization .
significantly, two class mhc pathways were found to be reversely regulated upon immunization, and the mhc class i pathway was activated and the mhc class ii pathway was inhibited .
both the rna-seq results and qpcr data from our study of zebrafish liver during the early stage after wed immunization indicated that activation of the mhc-i processing pathway in teleosts could elicit cellular immune responses for protection.
once bacterial vaccines are administrated into the animal host, they are often internalized by phagocytes via different entry mechanism.
however, the subsequent issues involved in microbial sensing and antigen processing are not well defined.
in the conventional paradigm, mhc class ii molecules present antigenic fragments acquired by the endocytic route to the immune system for recognition and activation of cd4+ t cells  <cit> .
mhc class i molecules, on the other hand, are restricted to surveying the cytosol for endogenous antigen from intracellular pathogens , tumors, or self-proteins, which are degraded into proteasomal products and then presented on mhc class i molecules to cd8+ t cells, thus exersting an irreplaceable role on cellular-mediated immuno-protection toward intracellular pathogens  <cit> .
e. tarda is believed to be an intracellular pathogen that can survive and replicate within large phagosomes in macrophages  <cit> .
since wed is an attenuated strain from wild type e. tarda, it could be assumed that wed bacteria possess the ability to survive in phagosomes of apc cells and the internalized bacteria are recognized as endogenous or exogenous antigen which would be presented or cross-presented by the mhc-i pathway, and finally evoking a cd8+ ctl-mediated response to achieve immune protection.
in mhc-i antigen processing pathway, antigenic peptides are degraded in the cytoplasm by proteasome, then translocated into the er and loaded onto mhc-i molecules with the help of several protein components.
pa <dig>  as an important proteasome activator, is a heterohexameric ring that binds to one or both ends of the 20 s proteasome  <cit> .
upon binding, it increases the catalytic activity of all three of the proteasome active sites, leads to changes in substrate cleavage, thereby generating more mhc class i-presented peptides  <cit> .
khan et al.
<cit>  reported that constitutive proteasomes were replaced with immune-proteasomes in mice livers starting at two days after listeria monocytogenes infection.
immuno-proteasomes support the generation of mhc class i epitopes and shape immune-dominance hierarchies of cd8+ t cells  <cit> .
in mice, this switch is marked by the up-regulation of proteasome activator pa <dig> subunits, which alter the fragmentation of polypeptides through the proteasome and are inducible by ifn-γ  <cit> .
the study of immune responses to e. ictaluri infection in blue catfish liver demonstrated that both the pa28α and pa28β were up-regulated  <cit> .
in the study described herein, the genes encoding pa <dig> subunit  <dig>  pa <dig> subunit  <dig> and pa <dig> subunit  <dig> were all up-regulated in zebrafish liver, which suggested a shift toward mhc class i antigen processing occurred at the early stage after wed immunization.
heat shock proteins  are a type of highly conserved and ubiquitously expressed proteins that play an essential role as molecular chaperones in protein folding and transport within the cell  <cit>  and possess the ability to stimulate mhc class i antigen processing  <cit> .
hsp/peptide complexes are taken up by apc via specific receptors, whose signaling leads to mhc-i presentation of hsp-associated peptides and the induction of specific cd8+ cytotoxic t cells  <cit> .
the antigenic peptides chaperoned by hsps are known to be more efficient, by orders of magnitude, than the free peptides for presentation by mhc-i  <cit> .
in our work, three heat shock proteins  were found to be up-regulated following wed immunization, and the activated hsps suggested that the internalized wed bacteria were processed and loaded onto mhc class i molecules, ultimately initiating initiate the ctls.
as cited above, mhc class i molecules present antigenic peptides on cell surface for recognition by cd8+ t cells  <cit> .
like other glycoproteins, the folding and assembly of mhc class i molecules require interactions with a number of chaperone molecules in the er, some of which are specific to mhc class i molecules  <cit> .
among the known er chaperones, endoplasmin  possesses the ability to bind peptides suitable for assembly on to mhc class i molecules together with calreticulin  <cit> .
calreticulin and calnexin are specialized er lectin-binding chaperones to bind transiently to newly-synthesized glycoproteins, but the calreticulin has been suggested as unique to interactions with the hsp/grp <dig> complex, which leads to recruitment of er protein  <dig>  <cit> .
the interaction between calnexin and mhc class i molecules is believed to stabilize the class i heavy chain and help it to associate with the β2m component  <cit> .
in this work, the three er chaperons, calreticulin, calnexin and endoplasmin , were all found to be induced in wed-immunized zebrafish liver, providing further evidence that an active mhc class i processing pathway was stimulated by wed immunization.
in addition, tap binding protein, another molecule involved in mhc class i antigen loading  <cit> , and mhc class i complex ze protein were also up-regulated in wed-immunized zebrafish liver, strongly suggesting a vigorous activation of the mhc-i processing pathway.
the mhc antigen processing-associated genes from zebrafish have been extensively characterized.
however, little is known about their expression patterns in zebrafish following vaccine immunization.
recently, the coordinated up-regulation of mhc class i-related components including mhc class i alpha chain, β2m, calreticulin, endoplasmin, pa28α and pa28β were reported in large yellow croaker following poly i:c injection  <cit>  and in catfish following an intracellular bacterial infection  <cit> .
in this work, the rna-seq data were given to show a coordinated down-regulation of several mhc class ii antigen processing and presentation components, including the mhc-ii dab, mhc-ii beta chain, mhc-ii invariant chain , mhc class ii transactivator , cathepsin b and lysosomal membrane glycoprotein  <dig> .
this complex process is illustrated in figure  <dig> and the differentially expressed genes are listed in table  <dig>  furthermore, qpcr data confirmed the co-inhibition of lamp <dig>  mhc-ii dab, cd <dig>  and ciita in zebrafish liver and spleen .
in previous researches, a remarkable inhibition of mhc-ii expression and antigen presentation was ever reported in some pathogen infection models, including brucella abortus <cit> , and mycobacterium tuberculosis <cit> .
for pathogens, an ability to impair the antigen processing and presentation of host has been proposed to facilitate chronic infection by decreasing t cell responses to microbial antigens.
for vaccines, however, the underlying significance of suppression of the mhc-ii expression and antigen presentation remains unknown.
CONCLUSIONS
in conclusion, in this work, zebrafish was used as a model to investigate the host immune mechanisms underlying the protective effects of the e. tarda live attenuated vaccine.
rna-seq data revealed that the coordinate up-regulation of mhc-i processing pathways and down-regulation of mhc-ii-associated pathways occurred at the early stage of vaccine immunization, providing insights into the molecular mechanisms of immune protection.
the successful application of rna-seq technology in the vaccine-zebrafish interaction model in this work established a new experimental platform for investigating the vaccine-specific host immune responses in a comprehensive and sensitive manner.
future studies using this approach will likely provide further significant insights into the detailed mechanisms of teleost immunity that will benefit the aquaculture industry, both from economic and human food source perspectives.

 zebrafish is a well-established vertebrate model of human development and hematopoiesis. however, as the aquaculture industry grows to meet the needs of an expanding global population, there has been a rapid increase in the research of teleost physiology and immunology. the zebrafish model system has proven to be a useful tool for studying infectious diseases that are natural threats to fish species of important human food sources, such as rock bream and turbot. several studies of the zebrafish immune system have provided initial insights into host-microbe interactions with both pathogens and commensals, and the molecular processes mediating clearance of pathogenic infections. not surprisingly, many features of the zebrafish defense responses following pathogen invasion resembled those of other mammals, including humans. the adult zebrafish has been used in a few studies to investigate new vaccines against specific pathogenic species. for example, novoa et al. studied the efficacy of a vaccine derived by reverse genetics against viral hemorrhagic septicemia virus in fish by using the zebrafish as a model system, and revealed that the vaccine was protective even at low temperatures. in addition, cui et al. used zebrafish to study an attenuated live mycobacterium marinum vaccine aimed at reducing mycobacteriosis in freshwater and marine fish. finally, xiao et al. developed an edwardsiellosis zebrafish model to screen attenuated live edwardsiella tarda vaccine candidates in order to identify those most highly effective for subsequent development for industry use. however, so far, no work involves the immune-related pathways underlying the zebrafish response to vaccination. in order to design a novel and effective vaccine, it is essential to gain a comprehensive understanding of the immune responses elicited in host upon vaccination. to date, most of the studies of the teleost immune system have focused on head kidney or/and spleen. however, the vertebrate liver has recently been recognized as an essential immune organ, accommodating a variety of cell types, including those primarily involved in immune activities. since the liver receives blood from both the systemic circulation and the intestine, it is exposed to a wide array of antigens. therefore, its immune-related cellular components can manifest a broad range of immune reactions. for example, the liver lymphocyte population includes both innate immune cells and adaptive immune cells -i or -ii ). as such, different infectious pathogens would be expected to induce distinctive profiles of immune responses in the liver, which might be manipulated to create specific and effective therapeutic strategies. several methods exist by which to determine the comprehensive transcriptomic profile of a pathogen-specific immune response, including microarray and quantitative real-time pcr. however, the high-throughput rna sequencing technology offers several advantages over the other profiling applications. not only is rna-seq independent on predefined probes, which facilitates the discovery of new transcript variants, but the sequence platform also produces low background noise, which allow for distinction between closely homologous genes and detection of weakly expressed transcripts. in addition, concurrent advances in the bioinformatic algorithms used to analyze the rna-seq data have allowed for better interpretation of the whole transcriptomic profile and provided further insights into complex molecular processes. the rna-seq approach has already been successfully applied to several infectious disease models of zebrafish, including zebrafish embryo infected with salmonella, and adult zebrafish and embryos infected by mycobacteria. in addition, other fish species infection models have been subjected to rna-seq analysis, including large yellow croaker infected by aeromonas hydrophila and japanese seabass infected by vibrio harveyi, but the overall immune-related transcription profiles have differed among species. no reports exist in the literature of rna-seq technology used to analyze the changes in an infected fish transcriptome profile induced upon vaccine treatment. edwardsiellosis, caused by the gram-negative edwardsiella tarda, is currently one of the most economically disastrous infectious diseases affecting the global aquaculture industry. e. tarda displays polymorphic phenotypes and has a broad range of hosts from aquatic invertebrates to higher vertebrates, including birds, reptiles, mammals, and even humans. in developing a putative live attenuated vaccine against edwardsiellosis, xiao et al. constructed an e. tarda mutant with low residual virulence. although the mutant was capable of inducing robust protection in zebrafish and turbot, the antibody titers detected in sera were relatively low. by thoroughly understanding the immune-mechanism of zebrafish induced by the putative live attenuated vaccine, a more immunogenic vaccine may be able to be generated. to this end, we performed a comparative gene transcription analysis of livers from mock-immunized and wed-immunized zebrafish using rna-seq technology to investigate their differential transcriptsomic profile. furthermore, genes associated with mhc antigen processing were analyzed by qpcr and the results revealed an activated mhc-i pathway and an inhibited mhc-ii pathway during the early stage of vaccine immunization. it was prompted that wed conferred a robust protection in zebrafish by eliciting an effective cell immunity via the mhc-i pathway.  rna-seq of liver transcriptome to better understand the early stage immune response of zebrafish immunized with wed, six solexa cdna libraries were constructed from the livers of mock-immunized and wed-immunized zebrafish. biological replicates were pooled to make representative samples for deep sequencing analysis. across the two groups of triplicate data, after normalization of the generated 95 bp pe raw reads,,, and, reads were obtained from c1-c and,, and, reads from v1-v respectively. to assess the quality of sequencing, the reads were mapped to the zebrafish reference genome. from the reads of each group, successful mapping occurred for,,,,,,,,, and, reads. however,,, and, unmapped reads were generated from c1-c while,, and, unmapped reads were found in v1-v respectively; we plan to conduct de novo analysis of these unmapped reads to generate a better reference of immune-relevant genes in zebrafish. analysis of differential expression among wed- and mock-immunized zebrafish liver to identify the differentially expressed genes, the transcriptome data of zebrafish liver from two days after wed immunization and mock immunization were analyzed by using the deseq package in r software. the criteria of a two-fold or greater change in expression and p-value< were chosen to determine significantly up-regulated or down-regulated genes following immunization. the magnitude distribution of the significantly changed genes was illustrated by ma plot analysis. using these criteria, a total of genes were significantly differentially expressed greater than two-fold, including up-regulated genes and down-regulated genes. annotation of the differentially expressed genes was achieved through blastn similarity searches against the ensembl zebrafish refseq mrna database. to perform an unbiased annotation of the functions of the differentially expressed genes identified by deseq analysis, go analysis of differentially expressed genes was carried out by two bioinformatics tools, david and bingo plugin. among the differentially expressed genes, david provided functional annotation for genes. go annotated differentially expressed genes mainly belonged to the three functional clusters, and were distributed among more than categories. the differentially expressed genes in the cluster of biological processes were found to be mainly related to stimulus response, immune response, regulation of immune system process, and regulation of development process. to identify the biological pathways that are active in the zebrafish at the early stages of wed immunization, differentially expressed genes were mapped to canonical signaling pathways found in kegg. a total of genes were mapped to statistically significant categories. protein processing in er was represented by up-regulated and down-regulated genes. there were also a statistically significant amount of mapped genes for other major antigen processing-related pathways, such as those mediated by “proteasome” and “protein export” pathway, to indicate the vital role of antigen processing and presentation activated by wed immunization at the early stage in zebrafish liver, which would elicit the specific immune responses required in the restoration of homeostasis. p * indicates the percentage of genes in each pathway from genes mapped to kegg. in general, based on the results from go analysis and kegg pathway analysis, the up- and down-regulated genes that were highly related to immune response of fish after wed immunization, significantly grouped into acute phase response, complement activation, immune/defense response, and antigen processing and presentation pathway. the acute phase response is conserved in zebrafish liver following wed immunization most of the conserved acute phase response genes were significantly differentially expressed following wed immunization. this set of genes encoded the major apps, c-reactive protein, and serum amyloid p ), the minor and intermediate apps, the negative apps, several complement components, and ion-binding and transporting proteins. many of the apps were up- or down-regulated greater than five-fold, suggesting that induction of apps in zebrafish liver likely plays an important role in host defense stimulated by wed vaccine. similar subcategories of apps were also found to be differentially expressed in previous microarray-based studies of early stage immune response to bacterial infection in rainbow trout and catfish, indicating the conservation of the vast majority of apps among teleost fish. the major apps, two crp-like proteins and saa, were induced to up-regulated their expressions by -fold, -fold and 883-fold, respectively, in the wed-immunized zebrafish liver, emphasizing their importance in teleost innate immune response. in wed-immunized zebrafish, both the apolipoprotein a-iv, a fatty acid binding protein involved in extracellular and intracellular lipid transporting, and alpha-2-hs-glycoprotein were decreased by -fold and -fold, respectively. the decrease of apolipoprotein a-iv was consistent to previous report in starved zebrafish liver. however, the functions of these two negative apps in immune response remain unknown. p identification of all differentially expressed genes was based on p < . a p-value <  indicated that the gene was significantly altered in wed immunized fish with respect to mock-immunized fish. the absolute value of “fold-change” is the magnitude of up- or down-regulation for each gene/homolog after wed immunization. fold-change >  indicates up-regulation, and < − indicates down-regulation. traditionally, complement has been considered as a supportive first line of defense against microbial intruders. in wed-immunized zebrafish liver, three isoforms of complement c and the complement c c c and c were remarkably up-regulated. the c inhibitor and the c1q were up-regulated and down-regulated, respectively. hepatocytes, which account for 80% of the liver mass, are the primary site of synthesis for all the genes involved in ion-binding and transporting. in the rna-seq data, more than differentially expressed genes involved in ion-binding and transporting were strongly induced in zebrafish liver upon wed immunization. these included haptoglobin, hemopexin, ceruloplasmin, transferrin receptor atpase, and cu2+ transporting alpha polypeptide. intelectin, which is involved in iron homeostasis, binding and transport, was one of the most up-regulated genes in the ion-binding and transporting category; however, the functions of intelectin in the contexts of normal iron metabolism and disease defense in zebrafish need to be further clarified. members of the transferrin and ferritin families were significantly affected to result in obvious up- and down-regulation in zebrafish liver by wed immunization. leukocyte cell–derived chemotaxin, originally named for its possible neutrophil chemotactic activity in vitro, was strongly induced by -fold in wed-immunized group, but its function in zebrafish remains unknown. wed immunization induces defense responses and signaling transduction pathways functional annotation of significantly differentially expressed genes in zebrafish liver was performed to define the transcriptome profile more precisely. go classification indicated that immune/defense response-related genes were enriched, specifically under go terms “response to chemical stimulus”, “regulation of immune system process”, and “immune response”. toll-like receptors detected the presence of pathogens and triggered an innate immune response, and several of the differentially expressed genes from wed-immunized liver mapped to the tlr signaling pathways. tlr signaling has been remarkably conserved throughout evolution, and it can mediate immune responses to all types of pathogens and promote secondary disease. in zebrafish, the pathogenesis of m. marinumstaphylococcus aureus, and aeromonas salmonicida has been shown to involve tlr signaling. to further investigate the function of tlr a/b that elicited the immune response in zebrafish embryo, assessment of tlr5a and tlr5b by morpholino-mediated knockdown followed by flagellin stimulation clearly demonstrated tlr5-dependent gene activation of mmp cxcl-c1c, and irak which suggested that the activation of tlr pathway can induce the expression of inflammatory mediators as well as the feedback control of the innate immune response. the functional investigation of tlr was also performed in a zebrafish embryo model, which suggested that the zebrafish tlr orthologs would negatively regulate the myd88-dependent nf-κb activation by sequestering the tlr adaptors and indicated that the existence of a tlr would negatively regulate tlr signaling upon engagement with its specific ligand. in humans and rodents, tlr-mediated signals in liver are associated with infection-induced granulomatous inflammation and ischemia-reperfusion injury, and can mediate liver regeneration processes. in wed-immunized zebrafish liver, differentially expressed genes mapped to various tlr pathways, including the tlr tlr tlr and tlr subcategories, which are not only expressed on the outer membrane of immune cells but also on endosome-lysosome membranes. tlr expression was up-regulated by -fold, indicating that it played a key role during the early stage of wed vaccination. immunity is a complex process of tightly controlled signals that involve a broad array of receptors, cytokines, enzymes, signal transducers, transcription factors, and other functional proteins. in our study, wed immunization increased dramatically the expression of cytokine genes related to the jak-stat, mapk, tgf-β, apoptosis and vegf signaling pathways. therefore, wed-induced gene expression in zebrafish liver might facilitate protection against e. tarda by activating these pathways. similar results were obtained in a previous study of large yellow croaker spleen during a. hydrophila infection. since the majority of the differentially expressed genes in these signaling pathways were up-regulated in our study, it is possible that the wed immunization of zebrafish is capable of triggering a vigorous adaptive immune response. wed immunization induces the antigen processing and presentation pathway a large number of differentially expressed genes with functions in protein transportation, modification and degradation were up- or down-regulated in the zebrafish liver following wed immunization, indicating these genes were likely connected to the degradation and processing of antigens for mhc class i and ii molecules. most of the differentially expressed genes related to mhc-i antigen processing pathways were significantly up-regulated, including the er-resident chaperone calreticulin, calnexin, endoplasmin, tap binding protein, proteasome activator, the heat shock proteins superfamilies, and cathepsin l. meanwhile, the typical mhc-ii processing pathway component, lysosomal membrane glycoprotein, was down-regulated in zebrafish liver after live attenuated vaccine immunization. p identification of all differentially expressed genes was based on p<. a p-value< indicated that the gene was significantly altered in wed-immunized fish with respect to mock-immunized fish. the absolute value of “fold-change” is the magnitude of up- or down-regulation for each gene/homolog after wed immunization. fold-change >  indicates up-regulation, and < − indicates down-regulation. up-regulated genes with established roles in immune responses comprised another large functional category, indicating that active immune-surveillance, immune signaling, and immune cell activation were triggered in the wed-immunized zebrafish liver, like the mhc-i ze protein. however, the mhc class ii dab, mhc class ii beta chain, mhc class ii invariant chain, mhc class ii transactivator, and cathepsin s were down-regulated at this stage. to further explore the immune response profiles induced by wed immunization to the level of a single pathway, we used the kaas web-based pathway analysis program. kegg analysis was performed to identify genes involved in phagosome and antigen processing and presentation pathways. in the phagosome pathway, genes were identified as strongly up-regulated upon wed immunization, while genes were strongly down-regulated. in the antigen processing and presentation signaling pathway, most of the up-regulated genes were found to be interrelated with the mhc-i processing pathway, while most of the down-regulated genes were related to the mhc-ii processing pathway. in mammals, antigen processing and presentation are essential for triggering the downstream cellular and/or humoral immune responses. the kegg results revealed that eight genes involved in the mhc-i pathway were up-regulated, and six genes involved in the mhc-ii pathway were down-regulated by two days after wed immunization. these results suggested that the mhc-i related pathways were co-induced following wed immunization, while the mhc-ii related pathways were co-depressed. this unique perspective should be further clarified. qpcr analysis of mhc processing pathways we next sought to further clarify the strength of the correlations of up-regulated genes to mhc-i processing pathway and down-regulated genes to mhc-ii processing pathway in zebrafish during early stage following wed immunization. differential expression of genes associated with mhc antigen processing was analyzed by qpcr to confirm the hypothesis that antigen processing and presentation pathways elicit an adaptive immune response following immunization. the assay was performed with both spleen and liver samples collected over the first five days post immunization. most of the results were consistent with those of the rna-seq analysis. mhc-i processing pathway related gene expression in liver tissues from wed-immunized groups were significantly up-regulated relative to mock-immunized groups. the up-regulations of differentially expressed genes in liver mostly reappeared in spleen, except for hsp90α, hspa4a and calnexin. the down-regulation expression of the three genes in spleen might reflect their different functions in two immune-associated organs. in contrast, the mhc-ii processing pathway related gene expression was all down-regulated and completely coordinated in liver and spleen during the early stage following wed immunization. this showed that mhc-ii processing pathway was inhibited in two immune organs by wed immunization. thus, cd4+ t cells activation could be depressed following immunization. the implications of this finding should be further investigated in teleost, specifically in zebrafish as model of e. tarda infection. in addition, the qpcr data also revealed that antigen processing in liver possesses a comparatively dominant role to that in spleen. the relatively intense expression in liver showed that antigen processing plays an essential role in wed-immunized zebrafish liver. discussion at present, molecular studies on the immune response to pathogens in fish models are mainly focused on infectious disease pathogenesis. rna-seq and microarray-based transcriptome profiling studies have revealed that the teleosts are useful in vivo models for identifying host determinants of responses to bacterial infection. furthermore, the rna-seq approach has already been successfully applied to several infectious disease models of zebrafish. however, none have applied the rna-seq technology to elucidate the immune-related pathways underlying the zebrafish response to vaccination for more effective vaccine evaluation. in this work, in order to gain comprehensive insight into the immunogenetics of zebrafish following immunization with the putative e. tarda live attenuated vaccine, a high-throughput deep sequencing-by-synthesis technology was used to investigate the immunization-related gene expression patterns. deseq analysis identified significantly differentially expressed genes in the zebrafish liver following wed immunization. go and kegg analysis revealed that the genes involved in the er protein processing as well as the phagosome and antigen processing and presentation pathways are regulated at the early stage following wed immunization. significantly, two class mhc pathways were found to be reversely regulated upon immunization, and the mhc class i pathway was activated and the mhc class ii pathway was inhibited. both the rna-seq results and qpcr data from our study of zebrafish liver during the early stage after wed immunization indicated that activation of the mhc-i processing pathway in teleosts could elicit cellular immune responses for protection. once bacterial vaccines are administrated into the animal host, they are often internalized by phagocytes via different entry mechanism. however, the subsequent issues involved in microbial sensing and antigen processing are not well defined. in the conventional paradigm, mhc class ii molecules present antigenic fragments acquired by the endocytic route to the immune system for recognition and activation of cd4+ t cells. mhc class i molecules, on the other hand, are restricted to surveying the cytosol for endogenous antigen from intracellular pathogens, tumors, or self-proteins, which are degraded into proteasomal products and then presented on mhc class i molecules to cd8+ t cells, thus exersting an irreplaceable role on cellular-mediated immuno-protection toward intracellular pathogens. e. tarda is believed to be an intracellular pathogen that can survive and replicate within large phagosomes in macrophages. since wed is an attenuated strain from wild type e. tarda, it could be assumed that wed bacteria possess the ability to survive in phagosomes of apc cells and the internalized bacteria are recognized as endogenous or exogenous antigen which would be presented or cross-presented by the mhc-i pathway, and finally evoking a cd8+ ctl-mediated response to achieve immune protection. in mhc-i antigen processing pathway, antigenic peptides are degraded in the cytoplasm by proteasome, then translocated into the er and loaded onto mhc-i molecules with the help of several protein components. pa as an important proteasome activator, is a heterohexameric ring that binds to one or both ends of the 20 s proteasome. upon binding, it increases the catalytic activity of all three of the proteasome active sites, leads to changes in substrate cleavage, thereby generating more mhc class i-presented peptides. khan et al. reported that constitutive proteasomes were replaced with immune-proteasomes in mice livers starting at two days after listeria monocytogenes infection. immuno-proteasomes support the generation of mhc class i epitopes and shape immune-dominance hierarchies of cd8+ t cells. in mice, this switch is marked by the up-regulation of proteasome activator pa subunits, which alter the fragmentation of polypeptides through the proteasome and are inducible by ifn-γ. the study of immune responses to e. ictaluri infection in blue catfish liver demonstrated that both the pa28α and pa28β were up-regulated. in the study described herein, the genes encoding pa subunit pa subunit and pa subunit were all up-regulated in zebrafish liver, which suggested a shift toward mhc class i antigen processing occurred at the early stage after wed immunization. heat shock proteins are a type of highly conserved and ubiquitously expressed proteins that play an essential role as molecular chaperones in protein folding and transport within the cell and possess the ability to stimulate mhc class i antigen processing. hsp/peptide complexes are taken up by apc via specific receptors, whose signaling leads to mhc-i presentation of hsp-associated peptides and the induction of specific cd8+ cytotoxic t cells. the antigenic peptides chaperoned by hsps are known to be more efficient, by orders of magnitude, than the free peptides for presentation by mhc-i. in our work, three heat shock proteins were found to be up-regulated following wed immunization, and the activated hsps suggested that the internalized wed bacteria were processed and loaded onto mhc class i molecules, ultimately initiating initiate the ctls. as cited above, mhc class i molecules present antigenic peptides on cell surface for recognition by cd8+ t cells. like other glycoproteins, the folding and assembly of mhc class i molecules require interactions with a number of chaperone molecules in the er, some of which are specific to mhc class i molecules. among the known er chaperones, endoplasmin possesses the ability to bind peptides suitable for assembly on to mhc class i molecules together with calreticulin. calreticulin and calnexin are specialized er lectin-binding chaperones to bind transiently to newly-synthesized glycoproteins, but the calreticulin has been suggested as unique to interactions with the hsp/grp complex, which leads to recruitment of er protein. the interaction between calnexin and mhc class i molecules is believed to stabilize the class i heavy chain and help it to associate with the β2m component. in this work, the three er chaperons, calreticulin, calnexin and endoplasmin, were all found to be induced in wed-immunized zebrafish liver, providing further evidence that an active mhc class i processing pathway was stimulated by wed immunization. in addition, tap binding protein, another molecule involved in mhc class i antigen loading, and mhc class i complex ze protein were also up-regulated in wed-immunized zebrafish liver, strongly suggesting a vigorous activation of the mhc-i processing pathway. the mhc antigen processing-associated genes from zebrafish have been extensively characterized. however, little is known about their expression patterns in zebrafish following vaccine immunization. recently, the coordinated up-regulation of mhc class i-related components including mhc class i alpha chain, β2m, calreticulin, endoplasmin, pa28α and pa28β were reported in large yellow croaker following poly i:c injection and in catfish following an intracellular bacterial infection. in this work, the rna-seq data were given to show a coordinated down-regulation of several mhc class ii antigen processing and presentation components, including the mhc-ii dab, mhc-ii beta chain, mhc-ii invariant chain, mhc class ii transactivator, cathepsin b and lysosomal membrane glycoprotein. this complex process is illustrated in figure and the differentially expressed genes are listed in table furthermore, qpcr data confirmed the co-inhibition of lamp mhc-ii dab, cd and ciita in zebrafish liver and spleen. in previous researches, a remarkable inhibition of mhc-ii expression and antigen presentation was ever reported in some pathogen infection models, including brucella abortus, and mycobacterium tuberculosis. for pathogens, an ability to impair the antigen processing and presentation of host has been proposed to facilitate chronic infection by decreasing t cell responses to microbial antigens. for vaccines, however, the underlying significance of suppression of the mhc-ii expression and antigen presentation remains unknown.  in conclusion, in this work, zebrafish was used as a model to investigate the host immune mechanisms underlying the protective effects of the e. tarda live attenuated vaccine. rna-seq data revealed that the coordinate up-regulation of mhc-i processing pathways and down-regulation of mhc-ii-associated pathways occurred at the early stage of vaccine immunization, providing insights into the molecular mechanisms of immune protection. the successful application of rna-seq technology in the vaccine-zebrafish interaction model in this work established a new experimental platform for investigating the vaccine-specific host immune responses in a comprehensive and sensitive manner. future studies using this approach will likely provide further significant insights into the detailed mechanisms of teleost immunity that will benefit the aquaculture industry, both from economic and human food source perspectives. 
further qpcr analysis confirmed that the genes encoding the factors involved in major histocompatibility complex -i processing pathway were up-regulated, while those involved in mhc-ii pathway were down-regulated. in this work, to clarify the host immune mechanisms underlying the protective effects of a putative vaccine and improve its immunogenicity in the future efforts, high-throughput rna sequencing technology was used to investigate the immunization-related gene expression patterns of zebrafish immunized with edwardsiella tarda live attenuated vaccine. these data provided insights into the molecular mechanisms underlying zebrafish immune response to wed immunization and might aid future studies to develop a highly immunogenic vaccine against gram-negative bacteria in teleosts. among those, functional classifications were found in the gene ontology database for and in the kyoto encyclopedia of genes and genomes database for several pathways involved in acute phase response, complement activation, immune/defense response, and antigen processing and presentation were remarkably affected at the early stage of wed immunization. zebrafish is a prominent vertebrate model of human development and pathogenic disease and has recently been utilized to study teleost immune responses to infectious agents threatening the aquaculture industry.
 zebrafish is a prominent vertebrate model of human development and pathogenic disease and has recently been utilized to study teleost immune responses to infectious agents threatening the aquaculture industry. in this work, to clarify the host immune mechanisms underlying the protective effects of a putative vaccine and improve its immunogenicity in the future efforts, high-throughput rna sequencing technology was used to investigate the immunization-related gene expression patterns of zebrafish immunized with edwardsiella tarda live attenuated vaccine.  average reads of million and million were obtained from livers of zebrafish immunized with phosphate buffered saline and e. tarda vaccine, respectively. the reads were annotated with the ensembl zebrafish database before differential expressed genes sequencing comparative analysis, which identified significantly differentially expressed genes. among those, functional classifications were found in the gene ontology database for and in the kyoto encyclopedia of genes and genomes database for several pathways involved in acute phase response, complement activation, immune/defense response, and antigen processing and presentation were remarkably affected at the early stage of wed immunization. further qpcr analysis confirmed that the genes encoding the factors involved in major histocompatibility complex -i processing pathway were up-regulated, while those involved in mhc-ii pathway were down-regulated.  these data provided insights into the molecular mechanisms underlying zebrafish immune response to wed immunization and might aid future studies to develop a highly immunogenic vaccine against gram-negative bacteria in teleosts. 