BIRC7 gene in channel catfish (Ictalurus punctatus): identification and expression analysis in response to Edwardsiella tarda, Streptococcus iniae and Channel catfish Hemorrhage Reovirus

A family member of inhibitor of apoptosis protein (IAP) termed baculoviral IAP repeat-containing 7 (BIRC7) from channel catfish (Ictalurus punctatus) was identified, the full length cDNA sequence of channel catfish BIRC7 (CcBIRC7) was 1686?bp, containing a 5'UTR of 93?bp, a 3'UTR of 399?bp with a poly (A) tail and an ORF of 1194?bp encoding a putative protein of 398 amino acids. The putative CcBIRC7 protein contains two BIR super-family conservative domains and a C-terminal RING finger motif. Phylogenetic analysis showed that catfish CcBIRC7 was moderately conserved with other BIRC7. Quantitative real-time PCR was conducted to examine the expression profiles of CcBIRC7 in healthy tissues and responding to different pathogens (Edwardsiella tarda, Streptococcus iniae and Channel catfish Hemorrhage Reovirus (CCRV)). CcBIRC7 was widely expressed in healthy tissues of channel catfish and with the highest 37.28-fold expression in blood. E.?tarda and S.?iniae could induce CcBIRC7 gene expression drastically in head kidney, liver and spleen, which the peak value reached 31.6-fold, 613.9-fold and 34.4-fold increase by E.?tarda infection, and 248.3-fold, 1540.3-fold and 120.4-fold increase post S.?iniae challenge, respectively. While, CCRV virus could slightly induce CcBIRC7 expression in head kidney and liver but reduce it in spleen. The result suggested BIRC7 may play a potential role in channel catfish innate immune system against bacterial and virus infections, especially as the anti-bacteria immune gene. This is the first report of BIRC7 gene identification and its expression in fish.     

First molecular cloning and gene expression analysis of a teleost CD200 (OX-2) glycoprotein from rock bream,Oplegnathus fasciatus

CD200 plays an important role in delivering an immunoregulatory signal to the immune system through interaction with its receptor. However, CD200 has not been characterized and its function in teleosts is unknown. In this study, the rock bream (Oplegnathus fasciatus) CD200 gene (RbCD200) was cloned and its expression profile was analyzed after infection with Edwardsiella tarda, Streptococcus iniae or red seabream iridovirus (RSIV). The coding region of RbCD200 cDNA was 855 bp, encoding 284 amino acid residues. The gene consisted of two extracellular Ig-like domains and a transmembrane domain. RbCD200 was highly expressed in the brain, erythrocytes, intestine and stomach of healthy rock bream. In the spleen, RbCD200 gene expression was down-regulated until 48 h after E. tarda exposure, except at 12 h RbCD200 gene expression was down-regulated then up-regulated at 12 h and 24 h after infection with S. iniae and RSIV, respectively. In the whole kidney, the RbCD200 gene was down-regulated in response to infection with E. tarda and S. iniae. However, RSIV infection increased RbCD200 gene expression in whole kidney until 48 h. These results suggest that RbCD200 is differentially expressed in the spleen and whole kidney after infection with different pathogens.     

Molecular cloning and characterization of Toll-like receptor 14 in Japanese flounder,Paralichthys olivaceus

Toll-like receptors (TLRs) are essential for activation of the innate immune system in response to invading pathogens. TLR14, which is unique to fish, has been identified in several fish species, but its function is unclear. In this study, Japanese flounder (Paralichthys olivaceus) TLR14 gene (JfTLR14) was cloned and its expression profiles were analyzed after infection with viral hemorrhagic septicemia virus, gram-positive Streptococcus iniae and gram-negative Edwardsiella tarda. The coding region of JfTLR14 cDNA was 2,607 bp, encoding 878 amino acid residues. JfTLR14 was highly expressed in head kidney of healthy flounder. In response to infection with VHSV and S. iniae, the JfTLR14 gene was up-regulated at only 1 day post-infection (dpi). However, E. tarda infection increased JfTLR14 gene expression from 1 to 6 dpi. These results imply that JfTLR14 participates more in the immune response against E. tarda infection than in the immune responses to other pathogen infections.     

Edwardsiella tarda DnaK: expression, activity, and the basis for the construction of a bivalent live vaccine against E. tarda and Streptococcus iniae

Edwardsiella tarda and Streptococcus iniae are important aquaculture pathogens that affect many species of farmed fish. In this study, we analyzed the expression, activity, and immunoprotective potential of E. tarda heat shock protein DnaK. We found that dnaK expression was upregulated under conditions of heat shock, oxidative stress, and infection of host cells. Recombinant DnaK (rDnaK) purified from Escherichia coli exhibited ATPase activity and induced protection in Japanese flounder (Paralichthys olivaceus) against lethal E. tarda challenge. On the basis of these results and our previous observation that a protective S. iniae antigen Sia10 which, when expressed heterogeneously in E. coli DH5α, is secreted into the extracellular milieu, we constructed a chimeric antigen by fusing DnaK to Sia10. The resulting fusion protein Sia10-DnaK was expressed in DH5α via the plasmid pTDK. Western blot analysis indicated that Sia10-DnaK was detected in the culture supernatant of DH5α/pTDK. When flounder were vaccinated with live DH5α/pTDK, strong protection was observed against both E. tarda and S. iniae. ELISA analysis detected specific serum antibody production in fish vaccinated with rDnaK and DH5α/pTDK. Taken together, these results indicate that rDnaK is an intrinsic ATPase with immunoprotective property and that Sia10-DnaK delivered by a live bacterial host is an effective bivalent vaccine candidate against E. tarda and S. iniae infection.     

Channel catfish (Ictalurus punctatus) protein disulphide isomerase, PDIA6: molecular characterization and expression regulated by bacteria and virus inoculation

Protein disulfide isomerases (PDIs) are thought to aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Currently, increasing evidence suggests PDIs play an important role in host cell invasion and they are relevant targets for the host immune response. However the roles of specific PDIs in teleosts are little known. Here, we characterized the Protein disulfide isomerase family A, member 6 (PDIA6) from channel catfish, Ictalurus punctatus (named as ccPDIA6). The catfish ccPDIA6 gene was homologous to those of other vertebrate species with 13 exons and 12 introns. The consensus full-length ccPDIA6 cDNA contained an ORF of 1320 bp encoding a putative protein of 439 amino acids. It had a 19 amino acid signal peptide and two active thioredoxin-like domains. Sequence of phylogenic analysis and multiple alignments showed that ccPDIA6 was conserved throughout vertebrate evolution. Southern blot analysis suggested the presence of one copy of the ccPDIA6 gene in the catfish genome. Tissue distribution shows that ccPDIA6 was expressed in all examined tissues at the mRNA level. When using the aquatic zoonotic pathogens such as Edwardsiella tara, Streptococcus iniae, and channel catfish reovirus （CCRV） to challenge channel catfish, ccPDIA6 expression was significant changed in immune-related tissues such as head kidney, intestine, liver and spleen. The results suggested that ccPDIA6 might play an important role in the immunity of channel catfish. This is the first report that the PDI gene may be involved in fish host defense against pathogen infection.     

A Streptococcus iniae DNA vaccine delivered by a live attenuated Edwardsiella tarda via natural infection induces cross‐genus protection

Edwardsiella tarda and Streptococcus iniae are important fish pathogens. We have reported previously a live E. tarda vaccine based on the attenuated strain TX5RM and a S. iniae DNA vaccine based on the antigen Sia10. In this study, we examined the possibility of constructing a cross‐genus vaccine by taking advantage of the residual infectivity of TX5RM and using it as a carrier host for the natural delivery of a S. iniae DNA vaccine. For this purpose, the recombinant TX5RM, TX5RMS10, was created, which harbours and retains stably the DNA vaccine plasmid pCS10 that expresses Sia10. When flounder were vaccinated with TX5RMS10 via oral and immersion routes, TX5RMS10 was detected in multiple tissues within 12–14 days postvaccination (p.v.). At 7 and 14 days p.v., expression of the DNA vaccine was detected in spleen, kidney and liver. Following E. tarda and S. iniae challenge at one and 2 months p.v., the vaccinated fish exhibited relative per cent survival rates of 69–83%. Immunological analysis indicated that TX5RMS10‐vaccinated fish produced specific serum antibodies and exhibited enhanced expression of a wide range of immune genes.     

Identification of Bacillus Strains for Biological Control of Catfish Pathogens

Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10⁷ CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.     

Transcriptome generation and analysis from spleen of Indian catfish, Clarias batrachus (Linnaeus, 1758) through normalized cDNA library

Catfishes are commercially important fish for both the fisheries and aquaculture industry. Clarias batrachus, an Indian catfish species is economically important owing to its high demand. A normalized cDNA library was constructed from spleen of the Indian catfish to identify genes associated with immune function. One thousand nine hundred thirty seven ESTs were submitted to the GenBank with an average read length of approximately 700 bp. Clustering analysis of ESTs yielded 1,698 unique sequences, including 184 contigs and 1,514 singletons. Significant homology to known genes was found by homology searches against data in GenBank in 576 (34 %) ESTs, including similarity to functionally annotated unigenes for 158 ESTs. Additionally, 433 ESTs revealed similarity to unigenes and ESTs in the dbEST but the remaining 658 EST sequences (39 %) did not match any sequence in GenBank. Of a total of 1,698 ESTs generated, 65 ESTs were found to be associated with immune functions. Gene Ontology and KEGG pathway analyses of C. batrachus ESTs collectively revealed a preponderance of immune relevant pathways apart from the presence of pathways involved in protein processing, localization, folding and protein degradation. This study constitutes first EST analysis of lymphoid organ in aquaculturally important Indian catfish species and could pave the way for further research of immune-related genes and functional genomics in this catfish.     

Molecular cloning,characterisation and expression of the translationally controlled tumor protein gene in rock bream (Oplegnathus fasciatus)

Translationally controlled tumor protein (TCTP) is an important immune regulator that has been implicated in a number of cellular processes, including cell growth, cell cycle progression, apoptosis regulation and protection of cells against various environmental stresses. In this study, we cloned and characterised TCTP from rock bream (Oplegnathus fasciatus), which is an economically important species in the Korean aquaculture industry. The full-length rock bream TCTP (RbTCTP) cDNA was of 1,041 bp and contained an open reading frame of 513 bp, which encoded 170 amino acids. The 5′ untranslated region (UTR) was 90 bp, while the 3′ UTR was 438 bp, containing a polyadenylation signal. RbTCTP showed 76, 75 and 74 % amino acid sequence identities to those of tilapia (Oreochromis niloticus), orange-spotted grouper (Epinephelus coioides) and Japanese sea perch (Lateolabrax japonicus), respectively. The positions of microtubule binding region, Ca⁺ binding region and TCTP signature regions in RbTCTP were similar to other fish species and mammals. RbTCTP mRNA expression level was highest in the gill compared to other tissues. The level of RbTCTP mRNA expression was significantly regulated by injection of red seabream iridovirus, Streptococcus iniae and Edwardsiella tarda.     

Molecular characterization and transcriptional analysis of non-mammalian type Toll like receptor (TLR21) from rock bream (Oplegnathus fasciatus)

Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919 bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112 kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.     

Molecular characterisation and biological activity of a novel CXC chemokine gene in rock bream (Oplegnathus fasciatus)

Chemokines are chemoattractant cytokines defined by the presence of four conserved cysteine residues. In mammals, these cytokines can be divided into four subfamilies depending on the arrangement of the first two conserved cysteines in the sequence, and include the CXC(α), CC(β), C(γ), and CX3C(δ) classes. We identified CXC chemokine cDNA, designated RbCXC, isolated using expressed sequence tag analysis of a lipopolysaccharide (LPS)-stimulated rock bream liver cDNA library. The full-length RbCXC cDNA (742 bp) contained an open reading frame of 342 bp encoding 114 amino acids. Results from phylogenetic analysis showed that RbCXC was strictly separated into a distinct clade compared to other known CXC chemokine subgroups. RbCXC was significantly expressed in the trunk kidney, liver, spleen, gill, peripheral blood leukocytes (PBLs), and head kidney. Rock bream PBLs were stimulated with several mitogens, including LPS and polyinosinic-polycytidylic acid (poly I:C), which significantly induced the expression of RbCXC mRNA. RbCXC mRNA expression was examined in several tissues under conditions of bacterial and viral challenge. Experimental challenges revealed that all examined tissues from fish infected with Edwardsiella tarda and red sea bream iridovirus showed significant increases in RbCXC expression compared to the control. In the case of Streptococcus iniae infection, RbCXC mRNA expression was markedly upregulated in the kidney, spleen, and liver. In addition, a maltose binding protein fusion recombinant RbCXC (～53 kDa) was produced in an Escherichia coli expression system and purified. Subsequently, the addition of purified recombinant RbCXC (rRbCXC) to kidney leukocytes was examined to investigate the impact of proliferative and chemotactic activity. The rRbCXC induced significant kidney leukocyte proliferation and attraction at concentrations ranging from 10 to 300 μg/mL, suggesting that it can be utilised as an immune stimulant and/or molecular adjuvant to enhance the immunological effects of vaccines.     

Water-soluble natural products from seaweed have limited potential in controlling bacterial pathogens in fish aquaculture

Seaweeds producing natural products with in vitro efficacy against bacterial pathogens offer the opportunity for therapeutic services in fish aquaculture, for example inhibiting the growth of pathogens through the direct release of bioactive metabolites into the culture environment. The red alga Asparagopsis taxiformis was used to test this hypothesis because of its effective natural products and mechanisms to release these metabolites into the surrounding environment and because it can be cultivated intensively in land-based systems. Here we quantify the release, accumulation and residence time of the major halogenated metabolites from A. taxiformis in the cultivation medium and subsequently test the in vitro bioactivity of these water-soluble metabolites against multiple strains of the pathogenic bacterium Streptococcus iniae. The two major halogenated metabolites in A. taxiformis (bromoform and dibromoacetic acid) were released into the water and reaching steady-state concentrations of 27.7?±?1.5 μg L^?1 and 4.8?±?1.3 μg L^?1, respectively, under standard cultivation conditions. This water delayed the in vitro growth of the pathogenic bacteria S. iniae but did not prevent it. Increasing the levels of A. taxiformis metabolites in the water by three orders of magnitude with aqueous extracts of biomass inhibited the growth of S. iniae, but it was also toxic to fish. In an immersion challenge experiment, intermediate non-ichthyotoxic levels of A. taxiformis metabolites were ineffective as a treatment for barramundi infected by S. iniae. The evidence suggests that there is little potential for A. taxiformis bioactive metabolites to provide therapy services to fish infected with S. iniae.     

Restriction fragment length polymorphisms of 16S rDNA and of whole rRNA genes (ribotyping) of Streptococcus iniae strains from the United States and Israel

Streptococcus iniae (junior synonym S. shiloi) isolated from tilapia and trout in Israel and in the United States were subtyped by restriction length polymorphism (RFLP) based on PCR amplified 16S rDNA and by ribotyping. 16S rDNA RFLP discriminated between S. iniae and other fish pathogens but not between S. iniae strains. HindIII and EcoRI ribotypes of S. iniae discriminated American from Israeli strains rejecting the possibility of an epidemiological link between S. iniae infections in the two countries. Israeli strains isolated from tilapia and trout could not be completely differentiated. The S. iniae ATCC 29178^T (T=Type strain) strain, isolated from a freshwater dolphin belonged to a ribotype different from those of all the fish isolates.     

Miiuy croaker (Miichthys miiuy) Peroxiredoxin2: Molecular characterization,genomic structure and immune response against bacterial infection

Peroxiredoxin2 (Prx2) protein is an important member in cellular antioxidant protein superfamily. Prx2 exists widely in prokaryotes and eukaryotes, it not only plays a part in eliminate reactive oxygen, but also takes effect in many other metabolic activities, such as stimulate epithelial cell proliferation, participate in the signal transduction in cells and so on. After molecular cloning we got that the complete cDNA sequence of Prx2 consists 882 bp, including a 5′-UTR of 46 bp, an open reading frame (ORF) of 591 bp, and a 3′-UTR of 245 bp. The complete gene of miiuy croaker Prx2 has 5 exons and 4 introns. The deduced 197 amino acid residues of miiuy croaker Prx2 consists a Val-Cys-Pro (VCP) motifs. In order to better elucidate the immune mechanisms of the Prx2 in the lower vertebrates, we conducted a research about the Prx2 gene of miiuy croaker and its expression pattern after bacterial infection. Real-time PCR (RT-PCR) results showed that expression of Prx2 was up-regulated in kidney, liver and spleen under infection with Vibrio anguillarum, and expressed level differently in ten different uninjected tissues. Our results suggested that Prx2 might be involved in immune defence in miiuy croaker.     

Expressed sequence tags (ESTs) based identification of genes and expression analysis of leukocyte cell-derived chemotaxin-2 (LECT2) from Epinephelus bruneus

The kelp grouper, Epinephelus bruneus, is an economically important intensively cultured species in Southeast Asia. Despite the insatiable demand its large-scale production has been hindered by problems associated with water quality, nutrition, and diseases especially due to increased rearing density. It is generally accepted that in fish both innate and adaptive immune system provide protection from diseases. In the present study a cDNA library of Streptococcus iniae-challenged kelp grouper was constructed to identify the genes that reveal molecular mechanism, physiological functions, and gene expression in different tissues using expressed sequence tags (ESTs) and RT-PCR strategy. Of a total of 2170 ESTs examined 279 (12.9%) were identified as contig and 860 (39.6%) as singletons. A total of 190 important immune and enzyme related genes (16.7%) were identified in both contig and singletons. The key immune molecules identified comprise complement factors, chemotaxin, chemokine, Fas ligand, ferritins, hepcidin, lysozyme c, MHC, and TLR which are involved in the innate or adaptive immune system. Among the genes a full-length cDNA of leukocyte cell-derived chemotaxin-2 (EbLECT2) with 540 base pair (bp) was identified; it consists of a 5′-untranslated region (UTR) of 17 bp, a 3′-UTR of 76 bp, and a stop codon TAA in 3′-UTR. The EbLECT2 is an important molecule in the innate immunity. It is a multifunctional protein involved in cell growth, differentiation, and autoimmunity. The open reading frame (ORF) of the EbLECT2 encodes with 155 amino acid (aa) residues with a predicted molecular weight and isoelectric point (pI) of 17 kDa and 9, respectively. The close phylogenetic relationship of EbLECT2 shares the highest similarity with the already reported LECT2 from Epinephelus coioides (96%) and Epinephelus akaara (94%). EbLECT2 mRNA was expressed predominantly in liver, spleen, and kidney while the expression was moderate in gills, heart, and muscle in E. bruneus after being challenged with LPS from Escherichia coli and pathogenic bacterium Vibrio anguillarum both of which involve the immune defense system. Further, the recombinant mature EbLECT2 (rEbLECT2) was successfully expressed in E. coli BL21 (DE3), and the antiserum against EbLECT2 was obtained for further investigations. The significant number of ESTs genome results obtained constitutes a powerful resource for further investigation to establish the gene discovery, functional genomic research, molecular mechanisms, and development of microarrays for the gene expression studies in kelp grouper.     

Genomic mapping of the MHC transactivator CIITA using an integrated ChIP-seq and genetical genomics approach

Background

The master transactivator CIITA is essential to the regulation of Major Histocompatibility Complex (MHC) class II genes and an effective immune response. CIITA is known to modulate a small number of non-MHC genes involved in antigen presentation such as CD74 and B2M but its broader genome-wide function and relationship with underlying genetic diversity has not been resolved.

Results

We report the first genome-wide ChIP-seq map for CIITA and complement this by mapping inter-individual variation in CIITA expression as a quantitative trait. We analyse CIITA recruitment for pathophysiologically relevant primary human B cells and monocytes, resting and treated with interferon-gamma, in the context of the epigenomic regulatory landscape and DNA-binding proteins associated with the CIITA enhanceosome including RFX, CREB1/ATF1 and NFY. We confirm recruitment to proximal promoter sequences in MHC class II genes and more distally involving the canonical CIITA enhanceosome. Overall, we map 843 CIITA binding intervals involving 442 genes and find 95% of intervals are located outside the MHC and 60% not associated with RFX5 binding. Binding intervals are enriched for genes involved in immune function and infectious disease with novel loci including major histone gene clusters. We resolve differentially expressed genes associated in trans with a CIITA intronic sequence variant, integrate with CIITA recruitment and show how this is mediated by allele-specific recruitment of NF-kB.

Conclusions

Our results indicate a broader role for CIITA beyond the MHC involving immune-related genes. We provide new insights into allele-specific regulation of CIITA informative for understanding gene function and disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0494-z) contains supplementary material, which is available to authorized users.     

Evaluation of an in vitro cell assay to select attenuated bacterial mutants of Aeromonas hydrophila and Edwardsiella tarda to channel catfish

Aims: To evaluate the feasibility of using an in vitro cell assay to select attenuated bacterial mutants. Methods and Results: Using catfish gill cells G1B, the feasibility of using an in vitro assay instead of in vivo virulence assay using live fish to select attenuated bacterial mutants was evaluated in this study. Pearson correlation analysis between in vitro virulence to G1B cells and in vivo virulence of Aeromonas hydrophila and Edwardsiella tarda revealed that there was a significant correlation between the two (r = ?0·768, P value = 3·7 × 10^?16). Conclusions: The in vitro cell assay might be initially used to screen large quantities of bacteria to select attenuated mutants of catfish pathogens. Significance and Impact of the Study: The in vitro cell assay using catfish gill cells to identify attenuated mutants of catfish pathogens will reduce cost involved in the in vivo virulence assay that requires many fish and aquariums.