Stimulatory effect of dietary alpha-lipoic acid on growth performance, antioxidant capacity, liver enzymes, immunity and protection of African catfish, Clarias gariepinus (B.), Edwardsiella tarda infection.

Edwardsiella tarda is one of the most common causes of fish diseases that hinder aquaculture. Oxidative stress in farm animals can induce a number of pathological disorders, production and general animal welfare. The use of exogenous dietary nonenzymatic antioxidants such as alpha-lipoic acid (ALA) can stop a pro-oxidant state and thus appears to have the potential to modulate the immune system and protect fish from bacterial infection. Thus, this study investigates the stimulatory effect of dietary ALA on growth performance, antioxidant capacity, liver enzymes, immunity and protection of African catfish, Clarias gariepinus (B.), against an infection with E. tarda. Five isonitrogenous and isocaloric diets (400 g/kg of crude protein) containing ALA at doses of 0.0 (control), 500, 1000, 1500 or 2000 mg/kg diet were served to 300 juveniles of African catfish (mean weight = 8.2 ± 0.2 g) adequately thrice per day for 12 weeks. Thereafter, 0.1 mL of E. tarda (ATCC 15947; 1.0 × 108 CFU/mL) was intraperitoneally injected into 10 fish from each tank and was monitored for 14 days. The results showed that ALA-fortified diets significantly boosted the fish growth, feed consumption and utilization and feed conversion ratio but no did not affect fish survival rate. The highest final fish weight (g), weight growth (g) and weight gain (%) were all considerably higher in fish fed with ALA-fortified diets (p < 0.05), especially from 1000 to 200 mg/kg ALA than the control group. Also, an enhanced hemato-biochemical, antioxidant and immune indices were noticed in African catfish-fed ALA-enriched diets. In a dose-dependent order, the levels of haematological indices such Ht, Hb, RBCs, WBCs and platelets were markedly increased (p < 0.05). Additionally, fish fed with ALA-based diets showed substantial (p < 0.05) declines in aspartate and alanine aminotransferase values, with the lowest values being found in the 2000 mg/kg diet while control group had highest values. Further, African catfish fed the feed fortified with 2000 mg ALA/kg diet showed the highest levels of lysozyme, respiratory burst, proteases and esterase activities (p < 0.05). Following exposure of fish to E. tarda infection, a significant reduction in the mortality was obtained in African catfish fed with ALA-based diets, especially from 1500 to 2000 mg ALA/kg diet (3.3%); while fish fed with the control diet had highest mortality (86.7%). Therefore, diets supplemented with ALA evoked fish growth performance, antioxidants and nonspecific immunity of African catfish. Also, resistance of African catfish to E. Tarda infection were raised when fed ALA-fortified diets at optimum inclusion rate of 1300 mg ALA/kg diet.

Fulminant necrotizing fasciitis by Edwardsiella tarda in a patient with alcoholic liver cirrhosis: A case report.

We herein present a unique and extremely rare fulminant case of Edwardsiella tarda infection-related necrotizing fasciitis. The patient had alcoholic cirrhosis and preferred to consume raw fish. He experienced painful swelling of the right forearm one day after he got a minor injury when falling from the ladder, and visited our hospital. His accompanied symptoms were diarrhea and general fatigue. His consciousness got deteriorated after the admission. The lesion of the right forearm had spread and the color had deteriorated with epidermolysis in a few hours. Necrotizing soft-tissue infection was suspected, and emergency debridement of the swollen forearm was performed 4 hours after the admission. However, unfortunately, he died of sepsis approximately 5 hours later. Histological examination of the biopsy specimen revealed features consistent with those of necrotizing fasciitis. The bacterial cultures of blood and the wound identified E. tarda. Since this microorganism is usually isolated from aquatic environments and can cause intestinal infection, sometimes followed by bacteremia especially in immunocompromised hosts, two possible infection routes were suspected. One route was from the skin injury, leading to bacteremia. Another possible route was per oral: orally taken E. tarda invaded deeper tissues from the intestine and reach the bloodstream, leading to extraintestinal infections, although direct evidence remains elusive. Raw fish eaten 1 week prior is considered to be the most possible contaminated food. Overall mortality rate of E. tarda bacteremia is very high and the clinician should pay attention on characteristic clinical findings of E. tarda infection on cirrhotic patients.

Role of TRAF6 from obscure puffer (Takifugu obscurus) in immune response against Edwardsiella tarda infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a vital molecule of inflammatory signaling pathways in innate immune response against pathogens. To elucidate its role in defense against Edwardsiella tarda infection in teleost fish, TRAF6 homologue was identified from obscure puffer (Takifugu obscurus) and functionally analyzed in this study. The obscure puffer TRAF6 (ToTRAF6) is a protein of 565 amino acids containing conserved RING domain, zinc finger-TRAF and MATH_TRAF6 domain. ToTRAF6 mRNA distributed in various healthy tissues of obscure puffer and was upregulated in the immune related tissues after E. tarda infection. ToTRAF6 protein was localized in the cytoplasm and aggregate as dots around the nuclei in FHM cells. The overexpression of ToTRAF6 in FHM cells decreased the quantity of E. tarda and induced the significant upregulation of downstream MAPK signaling pathway genes. These data suggest that ToTRAF6 is a key molecule of MAPK signaling pathway in defense against E. tarda infection.

Adjuvant effect of allogeneic blood in vaccines against edwardsiellosis in ginbuna crucian carp Carassius auratus langsdorfii.

Edwardsiella tarda (E. tarda), an intracellular pathogen, has caused severe economic losses in aquaculture. Effective vaccine development for E. tarda prevention is urgently needed. A previous study indicates that cell-mediated immunity (CMI) might play an important role in E. tarda infection. We believe that the involvement of allograft rejection and CMI has now been well documented in mammals and some fishes. However, there is still little research on the application of blood allograft rejection in vaccine development. In the current study, we investigate the immune response and vaccine effect in fish vaccinated with allogeneic blood + formalin-killed cells vaccine (FKC), allogeneic blood + phosphate-buffered saline (PBS), PBS + FKC and PBS + PBS. In the challenge test, the relative percentage survival (RPS) of the allogeneic + FKC, the allogeneic blood + PBS and the PBS + FKC group was 61.46, 35.41, and 30.63 % respectively. The up-regulated expression of Th1-related genes IFN-γ 1, IFN-γ 1rel2, IL-12p35 and T-bet suggests the protection is via CMI induction. Only in the allogeneic + FKC group, gene expression of IFN-γ 1, IL-12p35 and T-bet is significantly higher, indicating synergy between the two substances. Furthermore, among the fish injected with the allogeneic blood cells, syngeneic blood cells and PBS group, only in the fish of the allogenic blood cells injection group, did expression of IFN-γ 1, IFN-γ 2 and IFN-γ rel2 gene expression significantly increased. The results indicate that the rejection was induced by allogeneic components. Thus, our findings might provide essential information and insights into vaccine development in aquaculture.

The regulatory function of GATA3 on immune response in Japanese flounder (Paralichthys olivaceus).

GATA3 belongs to the GATA family, and it could interact with the target gene promoter. It has been reported to play a central role in regulating lymphocyte differentiation. In this study, the GATA3 cDNA sequence was identified by a homologous clone and the RACE technology from Japanese flounder (Paralichthys olivaceus). The full-length of the GATA3 cDNA sequence was 2904 bp, including 1332 bp open reading frame (ORF), 265 bp 5 '-untranslated region (5' UTR), and 1308 bp 3 '-UTR, encoding 443 amino acids. GATA3 protein sequence was conserved in vertebrates and invertebrates, including two zinc finger domains. qRT-PCR showed that the expression of GATA3 was high in the gill, kidney, and spleen. Expression of GATA3 slowly increased at the earlier stages and culminated at the late gastrula and somatic stages. Immunohistochemistry (IHC) results showed that the GATA3 protein was expressed in lymphocyte cells, undifferentiated basal and pillar cells of the gills, as well as lymphocyte cells and melanin macrophages of the kidney. The expression of GATA3 was significantly regulated in tissues and different types of lymphocytes after stimulation with Edwardsiella tarda. Dual-luciferase reporter assay indicated that the GATA3 protein could directly interact with promoters of target genes involved in the immune response. These findings suggested that GATA3 plays a major role in regulating the immune response. This study provided a theoretical basis for the immune response mechanism of teleost and a useful reference for later research on fish immunology.

Genome-wide identification of TNF receptor-associated factors in Japanese flounder (Paralichthys olivaceus) and functional analysis of resistance to temperature and Edwardsiella tarda stress.

Tumor necrosis factor receptor-associated factors (TRAFs), as the signaling mediators of the tumor necrosis factor (TNFR) superfamily, toll-like receptors (TLR) and interleukin-1 receptor (IL-1R) superfamily, can activate downstream signal transduction pathways and play an important role in the body's immune process. In this study, six TRAF genes, namely PoTRAF2a, PoTRAF2b, PoTRAF3, PoTRAF4, PoTRAF6 and PoTRAF7, were identified and annotated in Japanese flounder by using bioinformatics methods. Phylogenetic analysis confirmed that TRAF genes can be divided into seven groups. Analysis of motif composition and gene structure demonstrated that all PoTRAF members were evolutionarily conserved. The expression patterns of PoTRAF genes were then further investigated in six different developmental stages and eleven tissues of healthy fish, and it was found that there were spatial and tissue specificities among the members. To investigate the immune response of Japanese flounder to abiotic and biotic stresses, we further analyzed the expression profile of PoTRAFs after temperature stress and pathogen challenge. The result showed that PoTRAF3 and PoTRAF4 were observably differentially expressed under temperature stress, indicating that they were involved in the immune response after temperature stress. The expression of PoTRAF2a, PoTRAF2b and PoTRAF4 was significantly different after E. tarda infection, suggesting that they might have antibacterial effects. These results would help to clarify the molecular roles of PoTRAF genes in the regulation of immune and inflammatory responses in Japanese flounder.

Type I interferon induced by polyinosinic-polycytidylic acid does not contribute to the efficacy of a formalin-killed cell vaccine against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus).

Polyinosinic-polycytidylic acid (poly I:C) is a type of pathogen-associated molecular pattern that can strongly induce the expression of type I interferon (I-IFN). Our previous study has demonstrated that the combination of poly I:C with a recombinant protein antigen not only stimulated the expression of I-IFN but also conferred protection against Edwardsiella piscicida in the Japanese flounder (Paralichthys olivaceus). In this study, our aim was to develop a better immunogenic and protective fish vaccine, for which we intraperitoneally coinjected P. olivaceus with poly I:C and formalin-killed cells (FKCs) of E. piscicida and compared the efficiency of protection against E. piscicida infection with that of FKC vaccine alone. Results showed that the expression levels of I-IFN, IFN-γ, interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and the interferon-stimulated genes (ISGs) ISG15 and Mx were significantly increased in the spleen of fish inoculated with poly I:C + FKC. The results of ELISA showed that the levels of specific serum antibodies in the FKC and FKC + poly I:C groups were gradually increased until 28 days postvaccination and were significantly higher than those in the PBS and poly I:C groups. At 3 weeks after vaccination in the challenge test, the respective cumulative mortality rates of fish in the PBS, FKC, poly I:C, and poly I:C + FKC groups were 46.7%, 20.0%, 33.3%, and 13.3% under low-concentration challenge and 93.3%, 46.7%, 78.6%, and 53.3% under high-concentration challenge. This study showed that poly I:C may not provide an effective adjuvant effect with FKC vaccine for intracellular bacterial infections.

Cathepsin H and cathepsin B of Cynoglossus semilaevis are involved in anti-bacterial immunity against Edwardsiella tarda.

Cathepsin H and Cathepsin B are two lysosomal cysteine proteases participating in various physiological processes including immune responses. In fish, the functional roles of Cathepsin H and Cathepsin B during bacterial infection are less understood. In a previous work, we characterized a Cathepsin B homologue (CsCatB) of half-smooth tongue sole (Cynoglossus semilaevis), an economically valuable fish species in China. In this report, we identified a Cathepsin H homologue (CsCatH) from C. semilaevis. In healthy tongue sole, the transcriptional expression of CsCatH was detected in nine different tissues. Laser scanning confocal microscopic analysis showed that ectopically expressed CsCatH and CsCatB were co-localized with the lysosome. Upon infection by Edwardsiella tarda, a significant fish pathogen which caused a severe fish disease termed edwardsiellosis, the expressions of CsCatH and CsCatB were remarkedly upregulated. The knockdown of CsCatH and CsCatB significantly increased the replication of E. tarda and mitigated E. tarda-induced apoptosis in tongue sole tissues. These findings revealed the importance of CsCatH and CsCatB in anti-bacterial immunity of tongue sole.

Cloning, DNA sequence, and expression of flagellins from high and low virulence strains of Edwardsiella tarda and their macrophage-stimulating activities.

Edwardsiella tarda is a causative pathogen of edwardsiellosis in fish. Our previous studies on high (NUF251) and low (NUF194) virulent strains of E. tarda demonstrated that NUF251 strain induced significantly higher levels of NO and TNF-α from fish and mouse macrophages than NUF194 strain. Subsequent studies suggested that a flagellin-like protein secreted from E. tarda might be a responsible factor for the macrophage-stimulating activities. To evaluate the activities of flagellins of E. tarda, in this study, the flagellin genes of NUF251 and NUF194 strains were isolated by PCR and cloned into pQE-30 and pCold I expression vectors, and then the recombinant flagellins of two strains were overexpressed in E. coli JM109 and pG-Tf/BL21, respectively. The molecular weight of the purified recombinant flagellins of NUF251 and NUF194 strains were estimated to be 45 kDa and 37 kDa, respectively on SDS-PAGE analysis. Referring the three-dimensional structure of Salmonella flagellin, which has been reported to have 4 domains (D0, D1, D2, and D3), high sequence homology between two flagellins of E. tarda was observed at conservative domain (D0 and D1) regions, whereas the sequences equivalent to D2 and D3 domains were different, and even equivalent to 57 amino acids were deleted in NUF194. Both recombinant flagellins induced NO production, mRNA expression level of inducible NO synthase (iNOS), and intercellular ROS production in mouse macrophage cell line RAW264.7 cells. Also, the secretion of TNF-α and its mRNA expression level were increased by treatment of both recombinant flagellins. These results indicate that the recombinant flagellins from different virulent E. tarda strains can stimulate macrophages with nearly equal levels as judged by the parameters tested, even though they are differences in the structure and molecular weight, suggesting that conservative D0 and D1 domains are sufficient structural elements for the recombinant flagellins to induce a certain level of macrophage-stimulation in vitro. Further studies are necessary focusing on the role of D2 and D3 domain regions of the recombinant flagellins as macrophage-stimulating agent as well as their influence on host immune system in vivo.

Characteristics and prognosis of patients with Edwardsiella tarda bacteremia at a single institution, Japan, 2005-2022.

BACKGROUND: Edwardsiella tarda is a member of Enterobacteriaceae isolated from freshwater and sea. E. tarda infection in humans commonly causes gastroenteritis, but rarely causes bacteremia. However, few studies have described the clinical features of E. tarda bacteremia (ETB); therefore, we conducted a case review in our hospital. METHODS: We conducted a single-center, retrospective descriptive study using electronic medical records. Patient and microbial features were extracted and evaluated for 30- and 90-day mortality rates. RESULTS: From April 2005 to April 2022, the total set of blood cultures positive for any microorganisms was 9368, 38 of which were positive for E. tarda. Underlying cancer was observed in 65.8% of patients. The most common source of bacteremia was cholangitis, followed by cholecystitis, and endoscopic or surgical drainage was performed in almost all cases. Diarrhea was observed in only one patient, and there were no cases in which gastroenteritis was the source of bacteremia. All cases, except for one, were susceptible to all ß-lactams, such as ampicillin. The 30- and 90-day overall mortality rates were 8.6% (3/35) and 25.8% (8/31). Of these, 75% patients died because of cancer progression after successful ETB treatment. CONCLUSION: ETB may occur in patients with malignant underlying conditions. Biliary tract infections are common in ETB cases, whereas gastroenteritis may be an atypical cause of bacteremia. This study suggests that although the mortality rate for ETB at 30 day was low, it increased at 90 day as a result of the development of unfavorable underlying conditions.

Innate immunity and protective effects of orally administered betaine against viral and bacterial diseases in the olive flounder Paralichthys olivaceus (Temminck & Schlegel).

Sustainable methods that increase farmed fish yield while controlling infections are required to prevent economic losses in aquaculture farms. In this study, we evaluated the effects of betaine-supplemented (0%, 0.1%, 0.5%, and 1.0%) feed on the growth and immunity of the olive flounder Paralichthys olivaceus. Feed conversion ratios, post-infection cumulative mortality rates and innate immune responses were monitored. Weight gain was significantly higher with 0.5% and 1.0% than with 0% and 0.1% betaine-supplemented feed. Lysozyme activity was highest with 1.0% betaine. Respiratory burst activity was highest with 0.5% and 1.0% betaine. Serum bactericidal activity against Edwardsiella tarda was highest with 1.0% betaine (40% increase in survival rates compared with those in the control). Furthermore, serum virucidal activity against the viral haemorrhagic septicaemia virus (VHSV) was higher with 1.0% betaine than with other concentrations. With 0.5% and 1.0% betaine, the survival rates against VHSV were higher than those in the control until day 11, after which they declined. Our study suggests that betaine is a promising agent for promoting the growth of and enhancing immunity against E. tarda in olive flounders. Our findings may further contribute to developing necessary alternatives to conventional antibiotics in fish farming.

Phenotypic differences between Edwardsiella piscicida and Edwardsiella anguillarum isolates in Japan.

OBJECTIVE: Edwardsiella tarda has been regarded as the causative agent of edwardsiellosis in cultured marine and freshwater fish species in Japan. Our previous study genetically classified an E. tarda-like isolate from diseased Olive Flounder Paralichthys olivaceus as E. piscicida and that from diseased Red Seabream Pagrus major as E. anguillarum. This study aimed to understand the phenotypic differences between E. piscicida and E. anguillarum. METHODS: Fourteen E. piscicida and seven E. anguillarum isolates were used in this study. The colonies of each isolate were grown on brain-heart infusion agar plates and then subjected to DNA extraction. The extracted DNA was amplified using PCR. carbohydrate fermentation of the isolates was examined using API 50 CH test kits. Moreover, the growth of the two species was examined in defined media. Also, free amino acids in Olive Flounder and Red Seabream sera were detected and quantified via high-performance liquid chromatography-mass spectrometry. Statistical differences in the concentrations of free amino acids were analyzed using Welch's t-tests. RESULT: The API 50 CH test revealed that L-arabinose and D-mannitol were fermented by E. anguillarum isolates but not E. piscicida isolates. Furthermore, the growth of E. piscicida and E. anguillarum was reduced in the defined medium without methionine and iron sulfate. The growth of E. piscicida was reduced in the defined medium without phenylalanine, tyrosine, alanine, or nicotinic acid, whereas the growth of E. anguillarum was reduced in the defined medium without serine, cysteine, leucine, threonine, or isoleucine. Tyrosine and alanine were present in higher concentrations in the Olive Flounder serum, whereas threonine and isoleucine were present in higher concentrations in the Red Seabream serum, suggesting favorable growth conditions for E. piscicida and E. anguillarum. CONCLUSION: This study characterizes a minimal defined medium that can be used for developing vaccines against E. piscicida and E. anguillarum.

A case report and review of acute cholangitis with septic shock induced by Edwardsiella tarda.

BACKGROUND: Edwardsiella tarda (E. tarda) is a gram-negative facultative anaerobic bacterium. Gastroenteritis is the most common manifestation of E. tarda infection. However, parenteral infections can occur in immunodeficient hosts, as well as hepatobiliary diseases, malignancies, and/or diabetes. The prognosis of sepsis caused by E. tarda is very worse, with a mortality rate of 38%. We report the occurrence of acute cholecystitis with septic shock and E. tarda bloodstream infection. CASE PRESENTATION: A 64-year-old male with acute cholecystitis secondary to hepatitis B virus infection showed fever and sudden upper abdominal pain. On arrival, right upper abdominal pain, nausea, vomiting, fever, and jaundice were observed. Computed tomography showed common bile duct stones and gallbladder stones. Choledocholithiasis with acute cholangitis was diagnosed and treated surgically. Due to septic shock, a blood culture was assessed showing E. tarda as the main pathogen. Choledocholithotomy, T-tube drainage, cholecystectomy, and intravenous antibiotic treatment after the operation. The patient recovered smoothly after the operation. CONCLUSIONS: Although E. tarda infection is extremely rare, it can cause rapid episodes of rapidly progressive and life-threatening disease, as well as intestinal and parenteral infections. If necessary, early surgical treatment of parenteral infection should be considered and antibiotics should be used in time.

Pathology and virulence of Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella anguillarum in channel (Ictalurus punctatus), blue (Ictalurus furcatus), and channel × blue hybrid catfish.

In the mid-2010s, Edwardsiella tarda was reaffiliated into three discrete taxa (E. anguillarum, E. piscicida, and E. tarda), obscuring previous descriptions of E. tarda-induced pathology in fish. To clarify ambiguity regarding the pathology of E. tarda, E. piscicida, and E. anguillarum infections in US farm-raised catfish, channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and channel × blue catfish hybrids were challenged with comparable doses of each bacterium. The most severe pathology and mortality occurred in fish challenged with E. piscicida, supporting previous reports of increased pathogenicity in commercially important ictalurids, while E. anguillarum and E. tarda warrant only minimal concern. Acute pathologic lesions among bacterial species were predominantly necrotizing and characteristic of gram-negative sepsis but became progressively granulomatous over time. After 100 days, survivors were exposed to the approximate median lethal doses of E. piscicida and E. ictaluri, revealing some cross-protective effects among E. piscicida, E. anguillarum, and E. ictaluri. In contrast, no fish that survived E. tarda challenge demonstrated any protection against E. piscicida or E. ictaluri. This work supports reports of increased susceptibility of channel, blue, and hybrid catfish to E. piscicida, while highlighting potential cross-protective affects among fish associated Edwardsiella spp.

Chronic Diarrhea Associated with Edwardsiella Tarda Gastroenteritis: A Case Report and Literature Review.

Edwardsiella tarda , a Gram-negative bacterium classified into the genus Enterobacteriaceae, causes self-limited gastroenteritis. Here, we report a case of E. tarda gastroenteritis in a previously healthy 12-year-old boy in whom inflammatory bowel disease was precluded by endoscopy and tissue biopsy due to 3-month history of diarrhea, abdominal pain and weight loss.

Phenotypic and molecular characterization of the causative agents of edwardsiellosis causing Nile tilapia (Oreochromis niloticus) summer mortalities.

Edwardsiellosis is a serious bacterial disease affecting Nile tilapia (Oreochromis niloticus), causing septicemia and mortalities. Edwardsiella tarda and Edwardsiella anguillarum were isolated from Nile tilapia summer mortality events in Egypt. Diseased fish showed hemorrhagic septicemia, skin erosions, and eye opacity. A total of 24 Edwardsiella spp. isolates were retrieved from the investigated fish specimens. Phenotypic and biochemical characteristics grouped isolates into typical Ed. tarda (n = 14 strains) and atypical Ed. tarda (n = 10 strains). The BLAST analysis of sodB gene sequencing confirmed the conventional identification of typical Ed. tarda strains (n = 14) and reidentified all the atypical strains (n = 10) as Ed. anguillarum. Isolates showed a combination of virulence factors, including biofilm formation (66.6%), hemolysis (100%), chondroitinase (50%), and proteolytic activity (20.8%). The major part of isolates showed high resistance to ampicillin, amoxicillin, gentamycin antibiotics and harbored tetA, blaCTX-M, and aadA1 resistance genes. Pathogenicity testing of isolates in O. niloticus confirmed their virulence. Challenged fish exhibited septicemic signs similar to naturally diseased fish. Infections in naturally infected tilapia triggered acute and chronic histopathological alterations. Degenerative and necrotic changes were noticed in hematopoietic organs. Granulomas were noticed in between the hepatic parenchyma. The data extracted from the study confirm that accurate identification of the causative agents of edwardsiellosis should be reliant on genetic-based approaches. Analysis of the bacterium virulence properties offers insights into establishing novel therapeutics for edwardsiellosis control. The findings refer to the need for antimicrobial sensitivity testing to minimize antimicrobial resistance and increase therapy efficacy.

TNFR2 is a regulatory target of pol-miR-194a and promotes the antibacterial immunity of Japanese flounder Paralichthys olivaceus.

MicroRNAs (miRNAs) are regulatory RNAs that modulate target gene expression after transcription. Pol-miR-194a had been reported to be a miRNA of Japanese flounder (Paralichthys olivaceus) involved in Edwardsiella tarda infection. Here, we identified tumor necrosis factor receptor 2 (TNFR2) as a target gene of pol-miR-194a. Pol-miR-194a markedly repressed the protein expression of flounder TNFR2 (PoTNFR2) via specific interaction with the 3'UTR of PoTNFR2. PoTNFR2 responded to E. tarda infection in a manner that was opposite to that of pol-miR-194a and inhibited E. tarda invasion by activating the NF-κB pathway. Consistently, dysregulation of PoTNFR2 had a significant impact on E. tarda dissemination in flounder tissues. Together, these results add new insights into the regulation mechanism and immune function of fish TNFR2 and pol-miR-194a.

Transcriptome analysis and protein-protein interaction in resistant and susceptible families of Japanese flounder (Paralichthys olivaceus) to understand the mechanism against Edwardsiella tarda.

Edwardsiella tarda is one of the most harmful bacterial pathogens for aquaculture flatfish. After artificial infection of 47 Japanese flounder (Paralichthys olivaceus) families, resistant and susceptible families were identified in this study. High-throughput sequencing was performed on the liver transcriptome of uninfected groups (PoRU and PoSU) and infected groups (PoRC and PoSC). Through assembly and annotation, a total of 3012 and 1386 differentially expressed genes (DEGs) were identified in PoRU vs. PoSU and PoRC vs. PoSC. The significant enrichment pathways between PoRU and PoSU were mainly in metabolic and biosynthesis pathways. A total of thirty dominant enrichment pathways between PoRC and PoSC mainly focused on some immune-related pathways, including the hematopoietic cell lineage, cytokine-cytokine receptor interaction, complement and coagulation cascades, antigen processing and presentation, the intestinal immune network for immunoglobulin A (IgA) production and T/B cell receptor signaling pathway. Under the protein-protein interaction (PPI) analysis, hub genes, including CD molecules, complement component factors and chemokines, were identified in the network, and 16 core genes were differentially expressed in resistant and sustainable families in quantitative polymerase chain reaction (qPCR) validation. This study represents the first transcriptome analysis based on resistant and susceptible families and provides resistant genes to understand the potential molecular mechanisms of antibacterial function in marine fish. The results obtained in this study provide crucial information on gene markers for resistant breeding of Japanese flounder.

Preparation and characterization of glucose-conjugated super-paramagnetic iron oxide nanoparticles (G-SPIONs) for removal of Edwardsiella tarda and Aeromonas hydrophila from water.

The present research was conducted to prepare efficient G-SPIONs by co-precipitation to remove Edwardsiella tarda and Aeromonas hydrophila from the aqueous solution. The synthesized G-SPIONs were characterized by UV-Vis spectrophotometer, DLS, FEG-TEM, FT-IR, XRD, and VSM analysis. The results showed that the synthesized G-SPIONs had super-paramagnetic properties (58.31 emu/g) and spherical shape (16 ± 3 nm). The antibacterial activity was assessed in sterilized distilled water at different G-SPIONs concentrations viz. 0, 1.5, 3, 6, 12, 24, 48, 120, and 240 mg/L against E. tarda and A. hydrophila with various bacterial loads viz. 1 × 103 , 1 × 104 , 1 × 105 , 1 × 106 , and 1 × 107 CFU/ml at different time intervals 15, 30, 45, and 60 min. At a lower bacterial load of E. tarda and A. hydrophila 1 × 103 -1 × 104 CFU/ml, 100% bacterial load was removed by 15 min exposure with NPs concentration 6-48 mg/L and 1.5-6 mg/L, respectively. Cent percent bacterial removal was observed in both the bacterial species even at higher bacterial load (1 × 105 -1 × 107 CFU/ml) by increasing exposure time (15-60 min) and nanoparticle concentration as well (24-240 mg/L). At an initial bacterial load of E. tarda and A. hydrophila (1 × 103 -1 × 107 CFU/ml), the EC50 ranged between 0.01-6.51 mg/L and 0.02-3.84 mg/L, respectively, after 15-60 min exposure. Thus, it is concluded that the antibacterial effect of G-SPIONs depends on concentration and exposure time. Hence, G-SPIONs can be used as an antibacterial/biocidal agent to treat Edwardsiellosis and Aeromonosis disease in aquaculture. HIGHLIGHTS: The glucose-conjugated super-paramagnetic iron oxide nanoparticles (G-SPIONs) synthesized by rapid and high yield co-precipitation method shows bacterial removal efficiency against Edwardsiella tarda and Aeromonas hydrophila The bacterial removal efficiency of G-SPIONs depends on concentration and exposure time to both the bacterial species The 100% bacterial removal efficiency can be achieved even at the highest bacterial load (1 × 107 CFU/ml) against E. tarda and A. hydrophila after 45 and 30 min exposure Due to the high bacterial removal efficiency of G-SPIONs against E. tarda and A. hydrophila, it can be used for the treatment of Edwardsiellosis and Aeromonosis in fish.

Changes in immune genes expression, immune response, digestive enzymes -antioxidant status, and growth of catla (Catla catla) fed with Astragalus polysaccharides against edwardsiellosis disease.

The effect of four level of Astragalus polysaccharides (APs) supplementation diets, (CD: control diet and three experiment diet (E), EA: 100 mg kg-1 APs; EB: 200 mg kg-1 APs; EC: 300 mg kg-1 APs) on growth, changes in haemato-biochemical parameters and metabolic-digestive enzymes, enhancement of antioxidant activity, innate-adaptive immune response, and cytokine gene expression were studied in catla (Catla catla) against Edwardsiella tarda. The healthy and challenged groups fed the CD displayed no mortality, while fish fed EA or EC revealed 10% mortality, but the mortality was only 5% in diet EB. Fish fed diet EB and EC revealed significantly better growth rates and high RBC count during the experimental period. Albumin and globulin levels were significant improved when fish were fed the diet EB and EC from weeks 6-8. The superoxide dismutase (SOD) was significant ameliorated by EB feeding from weeks 4-8. In contrast, serum myeloperoxidase (MPO), catalase (CAT), malondialdehyde (MDA)/lipid peroxidation (LPO), glutathione peroxidase (GPx), respiratory burst activity (RBA), bactericidal action (BCA), serum lysozyme activity (SLA), nitric oxide synthase (NOS), head kidney leukocytes response proliferation (HKLP), hemolytic action (HLA), hydrogen peroxides (H2O2), and immunoglobulin (Ig) were significantly improved from week 6-8. Groups fed the APs enriched diets had significant ameliorated interleukin (IL)-1ß and interferon (IFN)-γ mRNA expression after 6 and 8 weeks of feeding. However, IL-10 and major histocompatibility complex (MHC)-1 mRNA expressions were significant enhanced in catla fed all APs diets on week 8. APs enriched diets revealed significant improved tumor necrosis factor (TNF)-α and TNF receptor-associated factor-6 (TRAF6) mRNA expression on week 4, but toll-like receptor-2 (TLR2) and TLR4 mRNA expression were significant enhanced by diet EB and EC after weeks 6 and 8. Similarly, the lysozyme (Lyz)-C and Lyz-G mRNA levels in the head kidney (HK) increased by APs feeding on weeks 6 and 8, whereas the EB diet, the expression of nucleotide binding oligomerization domain-1 (NOD1) was significantly improved on weeks 6 and 8, but NOD2 mRNA expression was only significant enhanced after 8 weeks of diet EB. By feeding healthy catla and E. tarda challenged fish fed diet EB, resulted in significantly increased growth, haemato-biochemical indices, metabolic-digestive enzymes, antioxidant activities, innate-adaptive immune responses, and cytokine gene expression mainly between 6 and 8 weeks.