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.

Chitosan and Oregano Oil Treatments, Individually or in Combination, Used To Increase the Shelf Life of Vacuum-Packaged, Refrigerated European Eel (Anguilla anguilla) Fillets.

We investigated the impact of chitosan and oregano essential oil (EO) individually or in combination on the quality of eel fillets in vacuum packaging (VP) and stored under refrigeration (4°C). Treatments studied were (i) control eel fillets stored in VP (E), (ii) eel fillets treated with 0.3% (v/w) oregano EO and stored in VP (E-OR), (iii) eel fillets treated with 2.0% (w/v) chitosan and stored in VP (E-CH), and (iv) eel fillets treated with 2.0% (w/v) chitosan and 0.3% (v/w) oregano EO and stored in VP (E-CH-OR). Treatments E-CH-OR and E-CH significantly reduced counts of mesophilic bacteria, Pseudomonas, Shewanella, and yeasts and molds during storage. Use of chitosan alone or in combination with oregano EO led to a significant reduction in concentrations of trimethylamine nitrogen and total volatile basic nitrogen in fillets, which led to lower concentrations of thiobarbituric acid reactive substances compared with the control samples. The eel samples in the E-CH and E-CH-OR groups were sensorially acceptable during the entire refrigerated storage period of 18 days. Presence of chitosan in the E-CH and E-CH-OR fillets did not negatively affect the taste of the fillets. E-CH fillets received a higher taste score than did E-CH-OR fillets probably because of the distinct and "spicy" lemon taste of chitosan, which was well received by the sensory panel. Based on overall sensory data (based on mean sensory scores of odor and taste), the shelf life was 6 days for the control fillets, 10 days for the E-OR fillets, and >18 days for the E-CH and E-CH-OR fillets stored in VP at 4°C. Overall, chitosan-treated eel fillets had lower microbial loads and a longer shelf life compared with the controls. Chitosan-treated eel fillets were preferred over oregano-treated fillets. Chitosan alone or in combination with oregano could be used as a preservative treatment and shelf-life extender for other seafoods.

Hemerythrin is required for Aeromonas hydraphlia to survive in the macrophages of Anguilla japonica.

Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host macrophages, a library of mini-Tn10 transposon insertion mutants was constructed. The M85 mutant, whose survival in host macrophages was only 23.1% of that of the wild-type (WT) strain, was utilized for further study. Molecular analysis showed that a 756-bp open reading frame (ORF) (GenBank accession No. CP007576) in the M85 mutant was interrupted by mini-Tn10. This ORF encodes for a 183-amino acid protein and displays the highest sequence identity (99%) with the hemerythrin (Hr) protein of A. hydrophila subspecies hydrophila ATCC 7966. The survival of the WT, M85 mutant, and complemented M85 (Hr) strains were compared in host macrophages in vitro, and the results showed that M85 exhibited defective survival, while that of M85 (Hr) was restored. To investigate the possible mechanisms of A. hydrophila survival in host macrophages, the expression of Hr under hyperoxic and hypoxic conditions was evaluated. The results revealed that the expression of this protein was higher under hyperoxic conditions than under hypoxic conditions, which indicates that Hr protein expression is sensitive to O2 concentration. Hydrogen peroxide sensitivity tests further suggested that the M85 mutant was more sensitive to oxidative stress than the WT and M85 (Hr) strains. Taken together, these results suggest that the Hr protein may act as an O2 sensor and as a detoxifier of reactive oxygen species, and is required for A. hydrophila survival within host macrophages.

Immunomodulation of Lactobacillus pentosus PL11 against Edwardsiella tarda infection in the head kidney cells of the Japanese eel (Anguilla japonica).

Wild and farm-raised fish can be simultaneously exposed to different types of pathogens in their habitats. Hence, it is important to study their effects, whether isolated or in combination. Therefore, the aim of this study was to evaluate the effects of Lactobacillus pentosus PL11 on the transcription of specific cytokine genes related to immune response, using Japanese eel macrophages as an in vitro model. Head kidney leukocytes were isolated from Japanese eels and cell viability was determined using an MTT reagent. In addition, the Griess reagent was used to determine the nitric oxide (NO) production while, an enzyme-linked immunosobent assay (ELISA) and a quantitative polymerase chain reaction (qPCR) were utilized to quantify the level of proinflammatory cytokines. The results of the study indicated that infection by Edwardsiella tarda alone causes a higher rate of cell death and an increase in the production of proinflammatory cytokines, such as interleukin-1ß (IL-1ß, 822.67 ± 29.48 pg mL(-1)), interleukin-6 (IL-6, 13.57 ± 0.55 pg mL(-1)), and tumor necrosis factor-α (TNF-α, 2033.67 ± 84.68 pg mL(-1)). However, co-culture with L. pentosus PL11 downregulates the production of NO and the related IL-1ß, IL-6, and TNF-α by 46%, 88.4%, 59%, and 77%, respectively. Quantification of the mRNA expression level revealed it to be consistent with the ELISA analysis. Hence, we infer that L. pentosus PL11 plays a significant role in the immunmodulation of the inflammatory responses that arise in fish owing to infection by pathogenic bacteria such as Edwardsiella tarda.

Effects of dietary supplementation of Lactobacillus pentosus PL11 on the growth performance, immune and antioxidant systems of Japanese eel Anguilla japonica challenged with Edwardsiella tarda.

The aim of this study was to determine the efficacy of dietary administration of Lactobacillus pentosus PL11 on growth performance and the immune and antioxidant systems in Japanese eel Anguilla japonica challenged with Edwardsiella tarda. A total of 75 Japanese eels (24.63±0.83 g) were grouped into 5 treatment diets which were a control diet (C) without E. tarda and 4 treatment diets with E. tarda challenge, including C for E. tarda challenge (NC), C plus L. pentosus PL11 supplemented diet (108 cfu g⁻¹) (T-PL11), C plus L. pentosus KCCM 40997 supplemented diet (108 cfu g⁻¹) (T-Lp) and C plus Weissella hellenica DS-12 supplemented diet (108 cfu g⁻¹) (T-Wh) for 5 weeks (4 week before and 1 week after challenge). The results showed enhanced growth performance in fish fed the diet containing L. pentosus PL11 compared to others. The growth performance parameters including specific growth rate (SGR) and weight gain (WG), feed intake (FI), feed conversion ratio (FCR) and survival were significantly (P<0.05) higher in fish maintained on L. pentosus PL11 supplemented diet compared to C and NC. T-PL11 group also shows a significant increase in the levels of plasma immunoglobulin M, CAT and SOD activities compared to NC. Hematological parameters and mieloperoxidase were significantly better in fish fed the L. pentosus PL11 supplemented diet than in the control. L. pentosus PL11 supplementation recover the reduced expression of SOD, CAT and heat shock protein 70 genes in liver and intestine in pathogen challenged fishes. In conclusion the result of the current study demonstrated L. pentosus PL11 potential as an alternative to antibiotic supplementation to improve the growth and health performance of Japanese eel (A. japonica).

Immunogenicity of pressure inactivated Edwardsiella tarda bacterin to Anguilla japonica (Japanese eel).

Japanese eel Anguilla japonica were immunized with inactivated Edwardsiella tarda bacterin preparations (formalin-killed cells, FKC (0.4%), formalin with heat-killed cells, FHKC (0.1% and 70 degreesC for 10 min), heat-killed cells, HKC (70 degrees C for 15 min), potassium chloride-killed cells, KKC (0.6%), tannic acid-killed cells, TKC (0.9%), citric acid-killed cells, CAKC (0.9%), pressure-killed cells, PKC (600 psi for 5 min) and electric current-killed cells, ECKC (100 mA at 12 v DC for 5 sec) via intraperitoneal injection in order to develop adequate inactivating method. Immune parameters in the immunized eel were measured to compare responses to different bacterins. Generally, eel rose agglutinating antibody titer in the serum within 2 week and the maximum titer occurred at 6 weeks post immunization. Elevated and significantly higher titer was produced with the PKC of E. tarda than other bacterin preparations. An Enzyme Linked Immunosorbent Assay (ELISA), to determine specific anti-E. tarda antibody in the serum, also showed significantly higher antibody titer with PKC than the other antigen preparations. Bacteriostatic assay with serum and live E. tarda indicated significantly higher activity in the PKC-immunized fish. Immunization with PKC also showed the increased level ofphagocytosis. PKC-inactivated vaccine at an immunization dose of 10(6) cells/fish induced high protection against experimental infection. Coincident with higher immune parameters and protection in the fish immunized with the PKC bacterin strongly suggested that pressure-killing is an effective inactivating method to develop an effective vaccine against edwardsiellosis.

Identification of amino acid residues required for ferric-anguibactin transport in the outer-membrane receptor FatA of Vibrio anguillarum.

Vibrio anguillarum 775 is a fish pathogen that causes a disease characterized by a fatal haemorrhagic septicaemia. It harbours the 65 kbp pJM1 plasmid, which encodes an iron sequestering system specific for the siderophore anguibactin and is essential for virulence. The genes involved in the biosynthesis of anguibactin are located on both the pJM1 plasmid and the chromosome. However, the genes for the outer-membrane receptor FatA and the other transport proteins are only carried on the plasmid. With the aim of elucidating the mechanism of ferric-anguibactin transport mediated by FatA, this work focuses on the identification of FatA amino acid residues that play a role in the transport of ferric-anguibactin, by analysing the transport kinetics of site-directed mutants. The mutations studied were located in conserved residues of the lock region, which contains a cluster of ten residues belonging to the N-terminal and barrel domains, and of the channel region of FatA, which contains conserved glycines located in the beta5-beta6 loop and a conserved arginine located in strand 11 of the beta-barrel. In the case of the FatA lock region, it is clear that although the residues analysed in this work (R95, K130, E505 and E550) are conserved among various outer-membrane receptors, their involvement in the transport process might differ among receptors. Furthermore, it was determined that in the FatA channel region double substitutions of the conserved glycines 131 and 143 with alanine resulted in a variant receptor unable to transport ferric-anguibactin. It was also shown that the conserved arginine 428 located in strand 11 is essential for transport. The results suggest that a conformational change or partial unfolding of the plug domain occurs during ferric-anguibactin transport.

Antagonistic activity of Aeromonas media strain A199 against Saprolegnia sp., an opportunistic pathogen of the eel, Anguilla australis Richardson.

A bacteriocin-like inhibitory substance (BLIS) produced by Aeromonas media strain A199 inhibited the growth of Saprolegnia sp. in vitro, an opportunistic pathogen isolated from affected eels, Anguilla australis (Richardson). The presence of BLIS in solid media inhibited the growth of the vegetative state of the aquatic mould as well as the germination of cysts. Uninhibited growth was, however, observed in the presence of inactive BLIS, suggesting that the in vitro antagonism derived from the BLIS of A199. In four independent in vivo tank observations of fish affected with saprolegniosis, the daily addition of A199 to tank water contributed to the subsequent swift recovery of affected hosts from invasion by this opportunistic pathogen.

Morphogenesis and fine structure of eel virus (Berlin), a member of the proposed Birnavirus group.

Eel Virus (Berlin) is associated with the occurrence of skin tumors in European eels. The genome of the virus consists of two segments of double-stranded (ds) RNA. The agent is assembled exclusively in the cytoplasm. Isometric particles with a diameter of 61 nm and in addition tubular structures and smaller particles were observed. The virion has a single shell: its capsid is composed of 132 interconnected morphological units with T = 13 dextro symmetry. According to particle size and bipartite nature of the genome, this virus has to be assigned to the tentatively proposed group of bisegmented ds RNA animal viruses.