Neonatal sepsis with meningitis, ventriculitis and brain abscess caused by Edwardsiella tarda.

A case of neonatal sepsis caused by Edwardsiella tarda, an uncommon pathogen typically associated with aquatic lifeforms, is described. The infant presented in septic shock with seizures and respiratory failure and was found to have meningitis, ventriculitis and a brain abscess requiring drainage. Only a small number of case reports of neonatal E. tarda infection, several with sepsis with poor auditory or neurodevelopmental outcomes or meningitis, have been described in the literature. This case report suggests that E. tarda, while uncommon, can be a cause of serious central nervous system disease in the neonatal population and that an aggressive approach to pursuing and treating complications may lead to improved neurodevelopmental outcomes.

Virulent and Multi-drug-Resistant Edwardsiella tarda Infection in Oscar Fish: Unveiling the Threat of Mass Mortality and AMR Dissemination.

The Oscar fish (Astronotus ocellatus) is among the most commonly domesticated and exported ornamental fish species from Kerala. The ornamental fish industry faces a significant challenge with the emergence of diseases caused by multi-drug-resistant bacteria. In the present study, six isolates were resolved from the diseased Oscar fish showing haemorrhages, necrosis, and loss of pigmentation. After phenotypic and genotypic characterization, the bacteria were identified as Edwardsiella tarda, Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli, Brevibacillus borstelensis, and Staphylococcus hominis. Experimental challenge studies in healthy Oscar fish showed that E. tarda caused 100% mortality within 240 h with 6.99 × 106 CFU/fish as LD50 and histopathology revealed the typical signs of infection. The pathogen was re-recovered from the moribund fish thereby confirming Koch's postulates. E. tarda was confirmed through the positive amplification of tarda-specific gene and virulence genes viz., etfD and escB were also detected using PCR. Antibiotic susceptibility tests using disc diffusion displayed that the pathogen is multi-drug-resistant towards antibiotics belonging to aminoglycosides, tetracyclines, and quinolones categories with a MAR index of 0.32, which implicated the antibiotic pressure in the farm. Plasmid curing studies showed a paradigm shift in the resistance pattern with MAR index of 0.04, highlighting the resistance genes are plasmid-borne except for the chromosome-borne tetracycline resistance gene (tetA). This study is the first of its kind in detecting mass mortality caused by E. tarda in Oscar fish. Vigilant surveillance and strategic actions are crucial for the precise detection of pathogens and AMR in aquaculture.

Effects of adding Bacillus subtilis natto NTU-18 in paste feed on growth, intestinal morphology, gastrointestinal microbiota diversity, immunity, and disease resistance of Anguilla japonica glass eels.

Japanese eel, Anguilla japonica, holds significant importance in Taiwanese aquaculture. With the intensification of eel farming, the impact of Edwardsiella tarda has become increasingly severe. Consequently, the abusive use of antibiotics has risen. Bacillus subtilis natto NTU-18, a strain of Bacillus with a high survival rate in feed processing, plays a crucial role in promoting intestinal health through competitive rejection, enhancing immune responses against bacterial pathogens, and improving intestinal health by modulating gastrointestinal microbiota to produce beneficial metabolites of mice and grass carp, Ctenopharyngodon idella. This study investigated the effects of different proportions (control, 0.25 %, 0.5 %, 1 %, and 2 %) of B. subtilis natto NTU-18 added to paste feed on the growth performance, intestinal morphology, and microbiota, expression of immune-related genes, and resistance to E. tarda in Japanese glass eel. The results indicated that the growth performance of all groups with B. subtilis natto NTU-18 added was significantly higher than that of the control group and did not impact the villi morphology. The expression of immune-related genes in the kidney, specifically HSP70 and SOD, was significantly higher from 0.5 % and above than the control; however, no significant differences were observed in CAT, POD, and HSP90. In the liver, significant differences were found in HSP70 and IgM above 0.25 % compared to the control group, with no significant differences in SOD, CAT, POD, and HSP90 among all groups. Additionally, intestinal microbiota analysis revealed that the 2 % additional group had significantly lower diversity than other groups, with Cetobacterium as the dominant species. The challenge test observed that the survival rates of the 0.5 % and 1 % groups were significantly higher. This research suggests that adding 0.5 % and 1 % of B. subtilis natto NTU-18 to the diet is beneficial for Japanese glass eel's immunity, growth performance, and disease resistance.

Proteomics analysis of differentially abundant proteins in the rohu kidney infected with Edwardsiella tarda.

Edwardsiella tarda (Et) is a zoonotic gram-negative pathogen with a diverse host range, including fish. However, the in-depth molecular mechanisms underlying the response of Labeo rohita (rohu) kidney to Et are poorly understood. A proteomic and histopathological analysis was performed for the rohu kidney after Et infection. The histopathology of the infected rohu kidney showed vacuolation and necrosis. After LC-MS/MS analysis, ~1240 proteins were identified with ≥2 unique peptides. A total of 96 differentially abundant proteins (DAPs) were observed between the control and Et infected group (ET). Metascape and STRING analysis were used for the gene ontology (GO), and protein-protein interaction network (PPI) for the significant pathways of DAPs. In PPI, low-abundant proteins were mapped to metabolic pathways and oxidative phosphorylation (cox5ab, uqcrfs1). High-abundance proteins were mapped to ribosomes (rplp2), protein process in the ER (hspa8), and immune system (ptgdsb.1, muc2). Our label-free proteomic approach in the rohu kidney revealed abundant enriched proteins involved in vesicle coat (ehd4), complement activation (c3a.1, c9, c7a), phagosome (thbs4, mapk1), metabolic reprogramming (hao1, glud1a), wound healing (vim, alox5), and the immune system (psap) after Et infection. A targeted proteomics approach of multiple reaction monitoring (MRM) validated the DAPs (nprl3, ambp, vmo1a, hspg2, muc2, hao1 and glud1a) between control and ET. Overall, the current analysis of histology and proteome in the rohu kidney provides comprehensive data on pathogenicity and the potential immune proteins against Et.

Intestinal epithelial cells of Japanese flounder (Paralichthys olivaceus) as an in vitro model for studying intestine immune function based on transcriptome analysis.

Japanese flounder (Paralichthys olivaceus) is an economically crucial marine species, but diseases like hemorrhagic septicemia caused by Edwardsiella tarda have resulted in significant economic losses. E. tarda infects various hosts, and its pathogenicity in fish is not fully understood. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and are representative of typical PAMP molecules that cause activation of the immune system. The PoIEC cell line is a newly established intestinal epithelial cell line from P. olivaceus. In order to investigate whether it can be used as an in vitro model for studying the pathogenesis of E. tarda and LPS stimulation, we conducted RNA-seq experiments for the PoIECs model of E. tarda infection and LPS stimulation. In this study, transcriptome sequencing was carried out in the PoIEC cell line after treatment with LPS and E. tarda. A total of 62.52G of high-quality data from transcriptome sequencing results were obtained in nine libraries, of which an average of 87.96% data could be aligned to the P. olivaceus genome. Data analysis showed that 283 and 414 differentially expressed genes (DEGs) in the LPS versus Control (LPS-vs-Con) and E. tarda versus Control groups (Et-vs-Con), respectively, of which 60 DEGs were shared in two comparation groups. The GO terms were predominantly enriched in the extracellular space, inflammatory response, and cytokine activity in the LPS-vs-Con group, whereas GO terms were predominantly enriched in nucleus and positive regulation of transcription by RNA polymerase II in the Et-vs-Con group. KEGG analysis revealed that three immune-related pathways were co-enriched in both comparison groups, including the Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and Cytokine-cytokine receptor interaction. Five genes were randomly screened to confirm the validity and accuracy of the transcriptome data. These results suggest that PoIEC cell line can be an ideal in vitro model for studies of marine fish gut immunity and pathogenesis of Edwardsiellosis.

Adjuvant effects of ß-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus).

ß-defensin of flounder plays an important role in immunomodulation by recruiting immune cells and has a potential vaccine adjuvant effect in addition to its bactericidal activity. In this study, adjuvant effects of ß-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus) were investigated. The bicistronic eukaryotic expression plasmid pBudCE4.1 plasmid vector with two independent coding regions was selected to construct DNA vaccine of p-OmpC which express only the gene for the outer membrane protein of Edwardsiella tarda and the vaccine of p-OmpC-ßdefensin which express both the outer membrane protein of the bacterium and ß-defensin of flounder. In vitro and in vivo studies have shown that the constructed plasmids can be expressed in flounder embryonic cell lines and injection sites of muscles. After vaccination by intramuscular injection, both p-OmpC and p-OmpC-ßdefensin groups showed significant upregulation of immune-response. Compared to the pBbudCE4.1 and the p-OmpC vaccinated groups, the p-OmpC-ßdefensin vaccinated group showed significantly more cell aggregation at the injection site and intense immune response. The proportion of sIgM+ cells, as well as the CD4-1+ and CD4-2+ cells in both spleen and kidney was significantly higher in the p-OmpC-ßdefensin vaccinated group at peak time point than in the control groups. The relative survival rate of the p-OmpC-ßdefensin vaccine was 74.17%, which was significantly higher than that of the p-OmpC vaccinated group 48.33%. The results in this study determined that ß-defensin enhances the responses in cellular and humoral immunity and evokes a high degree of protection against E. tarda, which is a promising candidate for vaccine adjuvant.

Elevated proton motive force is a tetracycline resistance mechanism that leads to the sensitivity to gentamicin in Edwardsiella tarda.

Tetracycline is a commonly used human and veterinary antibiotic that is mostly discharged into environment and thereby tetracycline-resistant bacteria are widely isolated. To combat these resistant bacteria, further understanding for tetracycline resistance mechanisms is needed. Here, GC-MS based untargeted metabolomics with biochemistry and molecular biology techniques was used to explore tetracycline resistance mechanisms of Edwardsiella tarda. Tetracycline-resistant E. tarda (LTB4-RTET ) exhibited a globally repressed metabolism against elevated proton motive force (PMF) as the most characteristic feature. The elevated PMF contributed to the resistance, which was supported by the three results: (i) viability was decreased with increasing PMF inhibitor carbonylcyanide-3-chlorophenylhydrazone; (ii) survival is related to PMF regulated by pH; (iii) LTB4-RTET were sensitive to gentamicin, an antibiotic that is dependent upon PMF to kill bacteria. Meanwhile, gentamicin-resistant E. tarda with low PMF are sensitive to tetracycline is also demonstrated. These results together indicate that the combination of tetracycline with gentamycin will effectively kill both gentamycin and tetracycline resistant bacteria. Therefore, the present study reveals a PMF-enhanced tetracycline resistance mechanism in LTB4-RTET and provides an effective approach to combat resistant bacteria.

Edwardsiella tarda Causing Septicemia in a Wild Crested Ibis (Nipponia nippon).

An adult Crested Ibis (Nipponia nippon) was found moribund in the Qinling area of China. Postmortem examination and histopathological analysis revealed lung inflammation and multi-organ hemorrhage. Bacterial isolation and whole-genome sequencing confirmed Edwardsiella tarda infection.

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.

Case Report: Disseminated Edwardsiella tarda infection in an immunocompromised patient.

Human infection caused by bacteria of the Edwardsiella genus is rare and most often presents with gastroenteritis that rarely requires antibiotics. Our case report describes a medically complex patient with chronic steroid use contributing to an immunocompromised state, who presented with fever and abdominal pain. The patient was later found to have Edwardsiella tarda (E. tarda) bacteremia and underwent paracentesis confirming E. tarda bacterial peritonitis requiring a prolonged antibiotic course. This case report aims to illustrate the presentation, diagnosis, and management of an uncommon infection that can have severe complications especially among immunocompromised patients.

Two novel teleost calreticulins PoCrt-1/2, with bacterial binding and agglutination activity, are involved in antibacterial immunity.

Calreticulin (Crt), a conserved lectin-like pleiotropic protein, plays crucial roles in mammalian immune response. In fish, the immunological function of Crt is limited investigated. Herein, we studied the antibacterial immunity of two type of Crt homologues (i.e. PoCrt-1 and PoCrt-2) in Japanese flounder (Paralichthys olivaceus). PoCrt-1 and PoCrt-2 are composed of 419 and 427 amino acid residues respectively, with 69.09% overall sequence identities with each other. Both PoCrt-1 and PoCrt-2 contain a signal peptide and three functional domains i.e. N-, P- and C-domains. Both PoCrt-1 and PoCrt-2 were constitutively expressed at various tissues with highest expression level in liver, and obviously regulated by Edwardsiella tarda and Vibrio harveyi. Furthermore, recombinant PoCrt-1 and PoCrt-2 (rPoCrt-1 and rPoCrt-2) could bind to different Gram-negative bacteria with highest binding index with E. tarda. At same time, in vitro rPoCrt-1 and rPoCrt-2 could agglutinate E. tarda, V. harveyi, and Vibrio anguillarum, and inhibit the bacterial growth. Similarly, in vivo rPoCrt-1 and rPoCrt-2 could significantly suppress the dissemination of E. tarda. Overall, these observations add new insights into the antibacterial immunity of Crt in P. olivaceus.

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.

Functional Proteomics Analysis of Norfloxacin-Resistant Edwardsiella tarda.

Multidrug-resistant Edwardsiella tarda threatens both sustainable aquaculture and human health, but the control measure is still lacking. In this study, we adopted functional proteomics to investigate the molecular mechanism underlying norfloxacin (NOR) resistance in E. tarda. We found that E. tarda had a global proteomic shift upon acquisition of NOR resistance, featured with increased expression of siderophore biosynthesis and Fe3+-hydroxamate transport. Thus, either inhibition of siderophore biosynthesis with salicyl-AMS or treatment with another antibiotic, kitasamycin (Kit), which was uptake through Fe3+-hydroxamate transport, enhanced NOR killing of NOR-resistant E. tarda both in vivo and in vitro. Moreover, the combination of NOR, salicyl-AMS, and Kit had the highest efficacy in promoting the killing effects of NOR than any drug alone. Such synergistic effect not only confirmed in vitro and in vivo bacterial killing assays but also applicable to other clinic E. tarda isolates. Thus, our data suggest a proteomic-based approach to identify potential targets to enhance antibiotic killing and propose an alternative way to control infection of multidrug-resistant E. tarda.

Aspartate metabolic flux promotes nitric oxide to eliminate both antibiotic-sensitive and -resistant Edwardsiella tarda in zebrafish.

Introduction: Metabolic reprogramming potentiates host protection against antibiotic-sensitive or -resistant bacteria. However, it remains unclear whether a single reprogramming metabolite is effective enough to combat both antibiotic-sensitive and -resistant bacteria. This knowledge is key for implementing an antibiotic-free approach. Methods: The reprogramming metabolome approach was adopted to characterize the metabolic state of zebrafish infected with tetracycline-sensitive and -resistant Edwardsiella tarda and to identify overlapping depressed metabolite in dying zebrafish as a reprogramming metabolite. Results: Aspartate was identify overlapping depressed metabolite in dying zebrafish as a reprogramming metabolite. Exogenous aspartate protects zebrafish against infection caused by tetracycline-sensitive and -resistant E. tarda. Mechanistically, exogenous aspartate promotes nitric oxide (NO) biosynthesis. NO is a well-documented factor of promoting innate immunity against bacteria, but whether it can play a role in eliminating both tetracycline-sensitive and -resistant E. tarda is unknown. Thus, in this study, aspartate was replaced with sodium nitroprusside to provide NO, which led to similar aspartate-induced protection against tetracycline-sensitive and -resistant E. tarda. Discussion: These findings support the conclusion that aspartate plays an important protective role through NO against both types of E. tarda. Importantly, we found that tetracycline-sensitive and -resistant E. tarda are sensitive to NO. Therefore, aspartate is an effective reprogramming metabolite that allows implementation of an antibiotic-free approach against bacterial pathogens.

Transcriptome analysis of turbot (Scophthalmus maximus) kidney responses to inactivated bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda.

Turbot (Scophthalmus maximus) is a commercially important marine flatfish for global aquaculture. With intensive farming, turbot production is limited by several diseases, in which Aeromonas salmonicida and Edwardsiella tarda are two main causative agents. Vaccination is an effective and safe alternative to disease prevention compared to antibiotic treatment. In the previous study, we developed an inactivated bivalent vaccine against A. salmonicida and E. tarda with relative percent survival (RPS) of 77.1 %. To understand the protection mechanism in molecular basis of the inactivated bivalent vaccine against A. salmonicida and E. tarda, we use RNA-seq to analyze the transcriptomic profile of the kidney tissue after immunization. A total of 391,721,176 clean reads were generated in nine libraries by RNA-seq, and 96.35 % of the clean reads were mapped to the reference genome of S. maximus. 1458 (866 upregulated and 592 downregulated) and 2220 (1131 upregulated and 1089 downregulated) differentially expressed genes (DEGs) were obtained at 2 and 4 weeks post-vaccination, respectively. The DEGs were enriched in several important immune-related GO terms, including cytokine activity, immune response, and defense response. In addition, the analysis of several immune-related genes showed upregulation and downregulation, including pattern recognition receptors, complement system, cytokines, chemokines and immune cell surface markers. Eight DEGs (ccr10, calr, casr, mybpha, cd28, thr18, cd20a.3 and c5) were randomly selected for qRT-PCR analysis, which confirmed the validity of the RNA-seq. Our results provide valuable insight into the immune mechanism of inactivated bivalent vaccine against A. salmonicida and E. tarda in Scophthalmus maximus.

Alterations in certain immunological parameters in the skin mucus of the carp, Cirrhinus mrigala, infected with the bacteria, Edwardsiella tarda.

The bacterial fish pathogen Edwardsiella tarda causes heavy stock mortality, severely hampering fish production, resulting in great economic loss to the farming industry. The first biological barriers that confer immune protection against pathogen entry are the fish mucosal surfaces. The present study was undertaken to investigate the influence of E. tarda on certain enzymatic and non-enzymatic parameters in the skin mucous secretions of the fish Cirrhinus mrigala using spectrophotometry and zymography. Fish were randomly divided into three groups: control, vehicle control, and infected. A sublethal dose of E. tarda (2.2 × 106 CFU/fish) suspended in 50 µL of PBS was injected intra-peritoneally at 0 day (d). Subsequently, mucus samples were collected at 2 d, 4 d, 6 d and 8 d post-infection. The activities of lysozyme (LYZ), protease (PROT), alkaline phosphatase (ALP), acid phosphatase (ACP), catalase (CAT), peroxidase (PER), superoxide dismutase (SOD), and glutathione S-transferase (GST) decreased significantly in the skin mucus of the challenged fish, indicating the suppressed immune system and decreased antioxidant capacity of C. mrigala to E. tarda infection. Lipid peroxidation (LPO) and total nitrate-nitrite were significantly higher at several time points post-infection, suggesting that physiological functions have been impaired following pathogen challenge. The present findings could be relevant for fish aquaculture and underline the importance of skin mucus not only for assessing fish immune status but also for identifying early warning signals of disease caused by pathogens.

Characterisation, evolution and expression analysis of the interferon regulatory factor (IRF) family from olive flounder (Paralichthys olivaceus) in response to Edwardsiella tarda infection and temperature stress.

Interferon regulatory factor (IRF) family involves in the transcriptional regulation of type I Interferons (IFNs) and IFN-stimulated genes (ISGs) and plays a critical role in cytokine signaling and immune response. However, systematic identification of the IRF gene family in teleost has been rarely reported. In this study, twelve IRF members, named PoIRF1, PoIRF2, PoIRF3, PoIRF4a, PoIRF4b, PoIRF5, PoIRF6, PoIRF7, PoIRF8, PoIRF9, PoIRF10 and PoIRF11, were identified from genome-wide data of olive flounder (Paralichthys olivaceus). Phylogenetic analysis indicated that PoIRFs could be classified into four clades, including IRF1 subfamily (PoIRF1, PoIRF11), IRF3 subfamily (PoIRF3, PoIRF7), IRF4 subfamily (PoIRF4a, PoIRF8, PoIRF9, PoIRF10) and IRF5 subfamily (PoIRF5, PoIRF6). They were evolutionarily related to their counterparts in other fish. Gene structure and motif analysis showed that PoIRFs protein sequences were highly conserved. Under normal physiological conditions, all PoIRFs were generally expressed in multiple developmental stages and healthy tissues. After E. tarda attack and temperature stress, twelve PoIRFs showed significant and different changes in mRNA levels. The expression of PoIRF1, PoIRF3, PoIRF4a, PoIRF5, PoIRF7, PoIRF8, PoIRF9, PoIRF10 and PoIRF11 could be markedly induced by E. tarda, indicating that they played a key role in the process of antibacterial immunity. Besides, temperature stress could significantly stimulate the expression of PoIRF3, PoIRF5, PoIRF6 and PoIRF7, indicating that they could transmit signals rapidly when the temperature changes. In conclusion, this study reported the molecular properties and expression analysis of PoIRFs, and explored their role in immune response, which laid a favorable foundation for further studies on the evolution and functional characteristics of the IRF family in teleost fish.

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.