Preparation of valine-curcumin conjugate and its in vitro antibacterial and antitumor activity and in vivo biological effects on American eels (Anguilla rostrata).

Curcumin (Cur) exhibits diverse natural pharmacological activities, despite its limited water solubility (hydrophobicity) and low bioavailability. In this investigation, a valine-curcumin conjugate (Val-Cur) was synthesized through amino acid side chain modification, and its solubility increased to 1.78 mg/mL. In vitro experimental findings demonstrated that the antibacterial activity of Val-Cur against Escherichia coli, Staphylococcus aureus, Aeromonas hydrophila, and Vibrio parahaemolyticus was significantly superior to that of Cur. The inhibition rate of Val-Cur against HepG2 (human hepatocellular carcinoma) cells was higher than that of Cur at low concentrations (below 25 µmol/L), although the IC50 value of Val-Cur did not differ significantly from that of Cur. In vivo biological effects of Val-Cur were assessed by adding it into the feed (150 mg/kg) of American eels (Anguilla rostrata). Val-Cur significantly improved the growth performance (↑weight gain rate, ↑specific growth rate, and ↓feed conversion rate) and activities of intestinal digestive enzymes (amylase and lipase) and antioxidant enzymes (superoxide dismutase) in American eels. Additionally, Val-Cur significantly improved serum biochemical indices (↑high-density lipoprotein cholesterol, ↓low-density lipoprotein cholesterol, ↓aspartate and alanine aminotransferases). Furthermore, Val-Cur increased intestinal microbial diversity, reduced the abundance of potentially pathogenic bacteria (Spiroplasma, Clostridium, and Pseudomonas), and elevated the abundance of beneficial digestion-promoting bacteria (Romboutsia, Phyllobacterium, Romboutsia sedimentorum, and Clostridium butyricum) conducive to glucose metabolism (P < 0.05). To the best of our knowledge, this study is the first to explore water-soluble curcumin in aquaculture, and the findings will lay the groundwork for the potential application of water-soluble curcumin in the field of aquaculture.

Δfur mutant as a potential live attenuated vaccine (LAV) candidate protects American eels (Anguilla rostrata) from Vibrio harveyi infection.

The eel farming industry is highly susceptible to Vibriosis. Although various types of vaccines against Vibriosis have been investigated, there is limited research on decreasing the virulence of Vibrions through gene knockout and utilizing it as live attenuated vaccines (LAV). In this study, we aim to develop a LAV candidate against Vibrio harveyi infection in American eels (Anguilla rostrata) using a ferric uptake regulator (fur) gene mutant strain of V. harveyi (Δfur mutant). After the eels were administrated with the Δfur mutant at the dose of 4 × 102 cfu/g body weight, the phagocytic activity of the leucocytes, plasma IgM antibody titers, activity of lysozyme and Superoxide Dismutase (SOD) enzyme, and gene expression levels of 18 immune related proteins were detected to evaluate the protection effect of the LAV. Preliminary findings suggest that the LAV achieved over 60% relative percent survival (RPS) after the American eels were challenged by a wild-type strain of V. harveyi infection on 28 and 42 days post the immunization (dpi). The protection was mainly attributed to increased plasma IgM antibody titers, higher levels of lysozyme, enhanced activity of SOD and some regulated genes encoded immune related proteins. Together, the Δfur mutant strain of V. harveyi, as a novel LAV vaccine, demonstrates promising protective effects against V. harveyi infection in American eels, thus presenting a potential candidate vaccine for fish farming.

Comparative phenotype and transcriptome analysis revealed the role of ferric uptake regulator (Fur) in the virulence of Vibrio harveyi isolated from diseased American eel (Anguilla rostrata).

Vibrio harveyi is commonly found in salt and brackish water and is recognized as a serious bacterial pathogen in aquaculture worldwide. In this study, we cloned the ferric uptake regulator (fur) gene from V. harveyi wild-type strain HA_1, which was isolated from diseased American eels (Anguilla rostrata) and has a length of 450 bp, encoding 149 amino acids. Then, a mutant strain, HA_1-Δfur, was constructed through homologous recombination of a suicide plasmid (pCVD442). The HA_1-Δfur mutant exhibited weaker biofilm formation and swarming motility, and 18-fold decrease (5.5%) in virulence to the American eels; compared to the wild-type strain, the mutant strain showed time and diameter differences in growth and haemolysis, respectively. Additionally, the adhesion ability of the mutant strain was significantly decreased. Moreover, there were 15 different biochemical indicators observed between the two strains. Transcriptome analysis revealed that 875 genes were differentially expressed in the Δfur mutant, with 385 up-regulated and 490 down-regulated DEGs. GO and KEGG enrichment analysis revealed that, compared to the wild-type strain, the type II and type VI secretion systems (T2SS and T6SS), amino acid synthesis and transport and energy metabolism pathways were significantly down-regulated, but the ABC transporters and biosynthesis of siderophore group non-ribosomal peptides pathways were up-regulated in the Δfur strain. The qRT-PCR results further confirmed that DEGs responsible for amino acid transport and energy metabolism were positively regulated, but DEGs involved in iron acquisition were negatively regulated in the Δfur strain. These findings suggest that the virulence of the Δfur strain was significantly decreased, which is closely related to phenotype changing and gene transcript regulation.

Effects of White Fish Meal Replaced by Low-Quality Brown Fish Meal with Compound Additives on Growth Performance and Intestinal Health of Juvenile American Eel (Anguilla rostrata).

With a reduced supply and increased price of white fish meal (WFM), the exploration of a practical strategy to replace WFM is urgent for sustainable eel culture. A 70-day feeding trial was conducted to evaluate the effects of replacing WFM with low-quality brown fish meal (LQBFM) with compound additives (CAs) on the growth performance and intestinal health of juvenile American eels (Anguilla rostrata). The 300 fish (11.02 ± 0.02 g/fish) were randomly distributed in triplicate to four groups (control group, LQBFM20+CAs group, LQBFM30+CAs group and LQBFM40+CAs group). They were fed the diets with LQBFM replacing WFM at 0, 20%, 30% and 40%, respectively. The CAs were a mixture of Macleaya cordata extract, grape seed proanthocyanidins and compound acidifiers; its level in the diets of the trial groups was 0.50%. No significant differences were found in the growth performance between the control and LQBFM20+CAs groups (p > 0.05), whereas those values were significantly decreased in LQBFM30+CAs and LQBFM40+CAs groups (p < 0.05). Compared to the control group, the activity of glutamic-pyruvic transaminase was significantly increased in LQBFM30+CAs and LQBFM40+CAs groups, while lysozyme activity and complement 3 level were significantly decreased in those two groups (p < 0.05). There were decreased antioxidant potential and intestinal morphological indexes in the LQBFM30+CAs and LQBFM40+CAs groups, and no significant differences in those parameters were observed between the control group and LQBFM20+CAs group (p > 0.05). The intestinal microbiota at the phylum level or genus level was beneficially regulated in the LQBFM20+CAs group; similar results were not shown in the LQBFM40+CAs group. In conclusion, with 0.50% CA supplementation in the diet, LQBFM could replace 20% of WFM without detrimental effects on the growth and intestinal health of juvenile American eels and replacing 30% and 40%WFM with LQBFM might exert negative effects on this fish species.

Pathogenicity of Edwardsiella anguillarum to American eels (Anguilla rostrata) and RNA-seq analysis of host immune response to the E. anguillarum infection.

Edwardsiella anguillarum is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-E. anguillarum infection remains uncertain. In this study, LD50 of E. anguillarum to American eels was determined and bacterial load in the liver and kidney of eels was assessed post the LD50 of E. anguillarum infection. The results showed that LD50 of E. anguillarum to American eels was determined to be 2.5 × 105 cfu/g body weight, and the bacterial load peaked at 36 and 72 h post the infection (hpi) in the kidney and liver, respectively. Then, the histopathology was highlighted by congestion in splenic blood vessels, atrophied glomeruli, and necrotic hepatocytes, as well as ultrastructural pathology in the kidney were charactered by acute nephritis, showing necrosis of the renal tubular epithelial cells, glomerular capillaries dilate, mitochondria swelling and ribosomes separate from the endoplasmic reticulum. Furthermore, the results of qRT-PCR revealed that 12 host immune-related genes showed significantly up or downregulated post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 6 hub DEGs play essential role to the anti-E. anguillarum infection in American eels. Pathogenicity of E. anguillarum to American eels and hub genes related host anti- E. anguillarum infection were firstly reported in this study, shedding new light on our understanding of the E. anguillarum pathogenesis and the host immune response to the E. anguillarum infection strategies in gene transcript.

Effects of weaning American glass eels (Anguilla rostrata) with the formula diet on intestinal microbiota and inflammatory cytokines genes expression.

This study aimed to investigate the effects of weaning American glass eels (Anguilla rostrata) with the formula diet on intestinal microbiota and the expression of inflammatory cytokines genes. During the feeding trial, the control group (termed IF group) was fed with initial feed for 34 days, and the experimental group (termed FF group) was fed with initial feed for 30 days, and then weaned with the formula diet for 4 days. After feeding trial, intestines were subjected to microbiota analysis using 16S rDNA high-throughput sequencing, and expression of three inflammatory cytokines genes in gut were examined by qPCR. The results indicated that the species richness and diversity of intestinal microbiota exhibited significantly higher in FF group than that in IF group (P < 0.05). At the phylum level, the core intestinal microflora was the same for two groups. The most abundant phylum was Firmicutes in IF group, while it was Proteobacteria in FF group. Five genera were significantly higher in the IF group compared with the FF group, and Bacillus was the most major enriched biomarker at genus level. Nine genera were significantly higher in the FF group compared with the IF group, and Acidovorax was the most major enriched biomarker. Weaning from initial feeding diet to formula feeding diet enhanced the expression levels of TNF-α and IL-8, and there was no significant change in IL-1ß expression between the two groups. These findings would be very useful to improve the diet formulation for weaning stage of American glass eels.

Evaluation of Methanotroph (Methylococcus capsulatus, Bath) Bacteria Protein as an Alternative to Fish Meal in the Diet of Juvenile American Eel (Anguilla rostrata).

This study was conducted to evaluate the effects of replacing fish meal (FM) with methanotroph (Methylococcus capsulatus, Bath) bacteria protein (MBP) in the diets of the juvenile American eel (Anguilla rostrata). Trial fish were randomly divided into the MBP0 group, MBP6 group, MBP12 group, and MBP18 group fed the diets with MBP replacing FM at levels of 0, 6%, 12%, and 18%, respectively. The trial lasted for ten weeks. There were no significant differences in weight gain or feed utilization among the MBP0, MBP6, and MBP12 groups (except for the feeding rate in the MBP12 group). Compared with the MBP0 group, the D-lactate level and diamine oxidase activity in the serum were significantly elevated in the MBP12 and MBP18 groups. In terms of non-specific immunity parameters in serum, the alkaline phosphatase activity was significantly decreased in the MBP18 group, and the complement 3 level was significantly elevated in the MBP12 and MBP18 groups. The activities of lipase and protease in the intestine were significantly decreased in the MBP12 and MBP18 groups. Compared with the MBP0 group, the total antioxidant capacity and activities of superoxide dismutase, catalase, and glutathione peroxidase in the intestine were significantly decreased in the MBP18 group, while the malondialdehyde level was significantly increased. The villus height, muscular thickness, and microvillus density were significantly decreased in the MBP12 and MBP18 groups. There were no significant differences in the foresaid parameters between the MBP0 group and the MBP6 group. The intestinal microbiota of the MBP6 group was beneficially regulated to maintain similar growth and health status with the MBP0 group. The adverse effects on the intestinal microbiota were reflected in the MBP18 group. In conclusion, MBP could successfully replace 6% of FM in the diet without adversely affecting the growth performance, serum biochemical parameters, and intestinal health of juvenile American eels.

Macleaya cordata extract improves the growth performance and intestinal health of American eels (Anguilla rostrata) farmed in intensive system.

The present study was conducted to evaluate the effects of dietary Macleaya cordata extract (MCE) supplementation on growth performance and intestinal health of American eels (Anguilla rostrata) farmed in intensive system. A total of six cement tanks of fish were randomly divided into a control group fed a commercial diet and an MCE group fed the commercial diet with 100 mg/kg MCE, respectively. There were three replicates in each group. The results suggested that 100 mg/kg MCE could improve the growth performance and intestinal health of the American eels by strengthening the barrier function and antioxidative ability in the intestine and beneficially modulating intestinal microbiota with the higher relative abundance and more species of the potential probiotics and the lower relative abundance of potentially pathogenic bacteria.

Feeding Strategies for Adapting Lake Sturgeon (Acipenser fulvescens) Larvae to Formulated Diets at Early Life Stages.

Cost-effective feeding management is required to support conservation hatcheries for lake sturgeon (Acipenser fulvescens), an ecologically important species in the Great Lakes region. This study investigated an approach to transition lake sturgeon larvae from live feed (Artemia) to formulated feed and its effect on growth performance, survival, and response to acute hypoxia stress. The first experiment showed that sturgeon had similar (p > 0.05) growth and survival when fed Artemia or the combined feeding of Artemia with the commercial diet (crude protein, 551 g/kg diet). Feeding solely on the commercial or lab-made (crude protein, 491 g/kg diet) diet significantly reduced growth and survival (p < 0.05). In the second experiment, the growth performance of sturgeon (14 days post-hatch, DPH) fed with either Artemia only or combined feeding different feeding durations of two, three, and four weeks followed by a complete transition to the commercial diet. At the end of six weeks, the 3- and 4-week combined feeding periods resulted in significantly higher body weight and survival compared to the 2-week combined and the Artemia only feeding treatments. In the last experiment, sturgeons (27 DPH) were fed only with Artemia or combined feeding of Artemia with the commercial diet for four weeks followed by the complete transition to the commercial diet for two weeks. Eighteen fish from each treatment were investigated the response to acute hypoxic conditions (gradual decrease in dissolved oxygen level from 8 to 2.3 mg/L at the rate of 1 mg/L per hour). When the dissolved oxygen was between 3 and 4 mg/L, the mortality rate of the combination-fed sturgeon (11.7%) was significantly lower than those fed only Artemia (83.3%). These results clearly demonstrate that a commercial diet can partially replace Artemia at early life stages to improve growth, survival, and hypoxia tolerance and thus its co-feeding with Artemia is recommended.

Comparative genomics analysis of the multidrug-resistant Aeromonas hydrophila MX16A providing insights into antibiotic resistance genes.

In this paper, the whole genome of the multidrug-resistant Aeromonas hydrophila MX16A was comprehensively analyzed and compared after sequencing by PacBio RS II. To shed light on the drug resistance mechanism of A. hydrophila MX16A, a Kirby-Bauer disk diffusion method was used to assess the phenotypic drug susceptibility. Importantly, resistance against ß-lactam, sulfonamides, rifamycins, macrolides, tetracyclines and chloramphenicols was largely consistent with the prediction analysis results of drug resistance genes in the CARD database. The varied types of resistance genes identified from A. hydrophila MX16A revealed multiple resistance mechanisms, including enzyme inactivation, gene mutation and active effusion. The publicly available complete genomes of 35 Aeromonas hydrophila strains on NCBI, including MX16A, were downloaded for genomic comparison and analysis. The analysis of 33 genomes with ANI greater than 95% showed that the pan-genome consisted of 9556 genes, and the core genes converged to 3485 genes. In summary, the obtained results showed that A. hydrophila exhibited a great genomic diversity as well as diverse metabolic function and it is believed that frequent exchanges between strains lead to the horizontal transfer of drug resistance genes.

Effects of Dietary Supplementation of Peanut Skin Proanthocyanidins on Growth Performance and Lipid Metabolism of the Juvenile American Eel (Anguilla rostrata).

As a functional feed additive, grape seed proanthocyanidin extract has received a lot of attention due to its biological activity in the health of aquatic animals, but its high cost limits the application of this feed additive in the diet of many fish species. It is thus urgent to develop a new resource of proanthocyanidin extract. We aimed to investigate the effects of dietary supplementation with peanut skin proanthocyanidins (PSPc) on growth parameters and lipid metabolism of juvenile American eel (Anguilla rostrata). Four hundred and fifty juvenile eels were randomly divided into five groups fed diets with five PSPc supplementation levels. The trial lasted for 8 weeks. Dietary PSPc supplementation significantly improved weight gain and feed utilization, and the best growth performance was found in the group fed with 900 mg/kg PSPc. PSPc supplementation significantly affected the crude protein level of whole fish and serum lipid parameters, and the best lipid-lowering effect was found in the fish fed with 900 mg/kg PSPc. Dietary PSPc supplementation increased lipolytic enzyme activities and decrease lipid synthase levels in the liver. The lipid metabolites affected by 900 mg/kg PSPc in the liver were mainly upregulated phosphatidylethanolamine in autophagy, downregulated ceramides in sphingolipid metabolism, upregulated phosphatidylcholine and phosphatidylethanolamine, downregulated 2-lysophosphatidylcholine in glycerophospholipid metabolism, and upregulated phosphatidylcholine in linoleic acid metabolism. In conclusion, an appropriate level of PSPc might effectively improve growth performance and regulate the lipid metabolism of the juvenile American eel, and 900 mg/kg PSPc is recommended in the diet of this fish species.

Transcriptomic analysis using dual RNA sequencing revealed a Pathogen-Host interaction after Edwardsiella anguillarum infection in European eel (Anguilla anguilla).

Many studies have explored differentially expressed genes (DEGs) between some pathogens and hosts, but no study has focused on the interaction of DEGs between Edwardsiella anguillarum (Ea) and Anguilla anguilla (Aa). In this study, we examined the interactions of DEGs during Ea infection and Aa anti-infection processes by dual RNA sequencing. Total RNA from in vitro and in vivo (Aa liver) Ea culture was extracted. Using high-throughput transcriptomics, significant DEGs that were expressed between Ea cultured in vitro versus in vivo and those in the liver of the infected group versus control group were identified. Protein-protein interactions between the pathogen and host were explored using Cytoscape according to the HPIDB 3.0 interaction transcription database. The results showed that the liver in the infection group presented with severe bleeding and a large number of thrombi in the hepatic vessels. We found 490 upregulated and 398 downregulated DEGs of Ea in vivo versus Ea cultured in vitro, and 2177 upregulated and 970 downregulated genes in the liver of the infected eels. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the pathogen DEGs revealed that the upregulated genes were mainly enriched in migration, colonization, biofilm formation, and significantly enriched in ABC transport and quorum sensing; the downregulated genes were mainly involved in metabolism, information transduction, organelle formation, enzyme catalysis, molecular transport, and binding. GO of the host DEGs showed that metabolic process, catalytic activity, single organism metabolic process, small molecule binding, nucleotide binding, nucleotide phosphate binding, and anion binding were markedly enriched. Finally, we found that 79 Ea and 148 Aa proteins encoded by these DEGs were involved in an interaction network, and some pathogen (DegP, gcvP, infC, carB, rpoC, trpD, sthA, and FhuB) and host proteins (MANBA, STAT1, ETS2, ZEP1, TKT1, NMI and RBPMS) appear to play crucial roles in infection. Thus, determining the interaction networks revealed crucial molecular mechanisms underlying the process of pathogenic infection and host anti-infection.

Grape seed proanthocyanidin extract regulates lipid metabolism of the American eel (Anguilla rostrata).

The current study was aimed to examine the effect of grape seed proanthocyanidin extract (GSPE) on regulating lipid metabolism of American eels. A total of six cement tanks of fish were randomly divided into a control group fed with a commercial diet and a GSPE group fed with a commercial diet supplemented 400 mg/kg GSPE. There were three replicates in each group. Results suggested that GSPE could decrease the levels of total cholesterol, triglycerides, and low-density lipoprotein cholesterol, and increase the high-density lipoprotein cholesterol level in serum. GSPE might regulate lipid metabolism through upregulating linoleic acid metabolism and arachidonic acid metabolism along with downregulating metabolisms of phenylalanine, tyrosine, and tryptophan biosynthesis and valine, leucine, and isoleucine biosynthesis.

Transcriptome of Edwardsiella anguillarum in vivo and in vitro revealed two-component system, ABC transporter and flagellar assembly are three pathways pathogenic to European eel (Anguilla anguilla).

Edwardsiella anguillarum is one of the common bacterial pathogens for the cultivated eels in China. The aim of this study was to reveal the cause of E. anguillarum pathogenic to European eel (Anguilla anguilla) from the perspective of the transcriptome. In this study, we first prepared E. anguillarum cultured in vitro and analysed the whole transcriptome after extracting the total RNA. Then, eels were i.p injected with E. anguillarum, and total RNA were extracted from the liver of European eels 48 h after the infection. After sequencing the transcriptome, we obtained average 1.97 × 108 clean reads cultured in vitro and 1.36 × 105 clean reads located in vivo after annotating all reads into the genome of E. anguillarum. The whole transcriptome showed, compared to the E. anguillarum cultured in vitro, 503 significantly up and 657 significantly down-regulated different expressed genes (DEGs) were observed. KEGG analysis showed that 38 DEGs of Two-Component System, 41 DEGs of ABC transporter, and 10 DEGs flagellar assembly pathways were highly upregulated in E. anguillarum located in vivo. Then, we designed primers to analyse the up-regulated DEGs through qRT-PCR and confirmed some up-regulated DEGs. The results of this study provide important reference for the further study of pathogen-host interaction between E. anguillarum and European eel.

Molecular insight, expression profile and subcellular localization of two STAT family members, STAT1a and STAT2, from Japanese eel, Anguilla japonica.

Signal transducer and activator of transcription 1 (STAT1) and STAT2 are critical components of type I and type II IFNs signaling. To date, seven STAT family proteins have been identified from mammals. However, the information on STAT genes in teleost fish is still limited. In the present study, two STAT family genes (STAT1a and STAT2) were identified from Japanese eel, Anguilla japonica and designated as AjSTAT1a and AjSTAT2. The open reading frames of AjSTAT1a and AjSTAT2 are 2244 bp and 2421 bp, encoding for polypeptides of 747 aa and 806 aa, respectively. Both AjSTAT1a and AjSTAT2 contain the conserved domains of STAT proteins. Phylogenetic analysis was performed based on the STATs protein sequences, and showed that AjSTAT1a and AjSTAT2 shared the closest relationship with Oncorhynchus mykiss. Quantitative real-time PCR analysis revealed that AjSTAT1a and AjSTAT2 were expressed in most examined tissues, with the highest expression both in blood. Significantly up-regulated transcripts of AjSTAT1a and AjSTAT2 were detected in response to poly I:C stimulation, and Edwardsiella tarda induced increase in the expression of AjSTAT1a and AjSTAT2 genes. Subcellular localization analysis showed that in both IFNγ-stimulated and unstimulated EPC cells AjSTAT1a and AjSTAT2 were mainly distributed in the cytoplasm, but few AjSTAT1a was distributed in the nucleus. All these results suggested that AjSTAT1a and AjSTAT2 may be critical for regulating the host innate immune defense against pathogens invasion.

Construction expression and immunogenicity of a novel trivalent outer membrane protein (OmpU-A-II) from three bacterial pathogens in Japanese eels (Anguilla japonica).

Vibrio vulnificus, Edwardsiella anguillarum and Aeromonas hydrophila are three common bacterial pathogens in cultivated eels. To protect farming eels from infection by these pathogens, a trivalent outer membrane protein (OMP) containing partial sequences of OmpU from V. vulnificus, OmpA from E. anguillarum and OmpII from A. hydrophila was expressed and purified; then, the OMP was used as a vaccine to immunize Japanese eels (Anguilla japonica). Whole-blood cell proliferation, antibody titres and complement and lysozyme activities were detected at different days post-immunization (dpi), and the relative per cent survival (RPS) was determined after eels were infected with V. vulnificus, E. anguillarum or A. hydrophila at 28 dpi. The results showed that the OMP significantly stimulates the antibody titres. At 14 days after the challenge (i.e. at 28 dpi), the RPS of OMP against V. vulnificus, E. anguillarum and A. hydrophila was 20%, 70% and 11.1%, respectively. The construction, expression and immunogenicity of a trivalent Omp were reported for the first time, and this study will provide a valuable reference for the development of fish multiplex vaccines.

Immunization of a novel bivalent outer membrane protein simultaneously resisting Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus infection in European eels (Angullia angullia).

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, European eels (Anguilla anguilla) were immunized by the bivalent expression products of the outer membrane protein (Omp) gene from A. hydrophila (OmpⅡ) and E. anguillarum (OmpA), and the effects of the bivalent protein (rOmpⅡ-A) on the immune function of the European eel were detected. Three hundred eels were divided average into three groups of PBS, adjuvant and rOmp. Eels of three goups were injected intraperitoneal with 0.2 mL of PBS (0.01 mol/L, pH7.4), PBS + F (PBS mixed equal volume of freund's uncomplete adjuvant) or rOmpⅡ-A (1 mg mL-1 rOmpⅡ-A mixed equal volume of freund's uncomplete adjuvant). Four immune-related genes expression, proliferation of whole blood cells, serum and skin mucus antibody titer, superoxide dismutase (SOD) activity and the relative percent of survival (RPS) were studied at different days (or hours) post the immunization. The results showed that the igm, lysC, mhc2 and sod gene in the liver, spleen, kidney and intestine tract were significant increased in the Omp group; On the 28 day post the immunization (dpi), blood cell proliferation was increased in the Omp group, and on the 14, 21, 28 and 42 dpi, antibody titers in serum and mucus of the Omp group were significantly higher than that of the PBS and adjuvant group, regardless of coating with bacteria or Omp antigen. The SOD activity of Omp group increased significantly in liver, kidney, skin mucus and serum from 14 to 42 dpi, especially in serum. Eels chanllenged by A. hydrophila, E. anguillarum and V. vulnificus in the bivalent Omp group showed the RPS were 83.33%, 55.56% and 44.44%, respectively. The results of this study showed that immunization of the bivalent Omp could effectively improve the immune function of European eels, and produced effectively protection to A. hydrophila and E. anguillarum infection. Simultaneously, the bivalent Omp also produced distinct cross-protection to the eels challenged by V. vulnificus.

Immunization of a novel outer membrane protein from Aeromonas hydrophila simultaneously resisting A. hydrophila and Edwardsiella anguillarum infection in European eels (Angullia angullia).

In cultivated European eels, Aeromonas hydrophila, Edwardsiella anguillarum and Vibrio vulnificus are three important bacterial pathogens. In this study, an expressed recombinant Outer membrane proteinⅡ (rOmpⅡ) from A. hydrophila was intraperitoneally injected into European eels (Angullia angullia). All examined eels were equally divided into three groups. One group was injected with PBS only (PBS group), one group was injected with 1:1 mixture of PBS and Freund's incomplete adjuvant (PBS + F, adjuvant group), and the third group was injected with 1:1 mixture of 1 mg mL-1 rOmpⅡ and Freund's incomplete adjuvant (rOmpⅡ+F, OmpⅡ group). The immunogenicity of OmpⅡ was studied by detecting the expression of 4 immune-related genes, stimulation index (SI) of the whole blood cell, serum antibody titer, lysozyme and Superoxide Dismutase (SOD) activity, and relative percent of survival (RPS) rate. The results showed that gene expression of MHC-Ⅱ, LysC, SOD and IgM in the OmpⅡ group significantly increased in liver, spleen, kidney and intestine. At 28 days post the immunization (dpi), the SI of whole blood cells in the OmpⅡ group increased significantly; at 14, 21, 28 and 42 dpi, the serum antibody titers against A. hydrophila and E. anguillarum in the OmpⅡ group were significantly higher than that of the PBS and the adjuvant group; the SOD in the OmpⅡ group was found increased significantly in liver, kidney, mucus and serum. On the 28 dpi, eels were challenged by A. hydrophila, E. anguillarum and V. vulnificus for cross protection study. The results showed that the RPS of the OmpⅡ group were 83.33%, 55.56% and 33.33% respectively. These results showed that the expressed OmpⅡ from A. hydrophila significantly improve the immune function of Europena eels and their resistance to the infection of A. hydrophila and E. anguillarum simultaneously.