The transforming growth factor beta-activated kinase 1 (TAK1)/c-Jun N-terminal kinase (JNK) axis is an essential MAPK upstream mediator and regulates immune signaling pathways. However, whether the TAK1/JNK axis harnesses the strength in regulation of signal transduction in early vertebrate adaptive immunity is unclear. In this study, by modeling on Nile tilapia (Oreochromis niloticus), we investigated the potential regulatory function of TAK1/JNK axis on lymphocyte-mediated adaptive immune response. Both OnTAK1 and OnJNK exhibited highly conserved sequences and structures relative to their counterparts in other vertebrates. Their mRNA was widely expressed in the immune-associated tissues, while phosphorylation levels in splenic lymphocytes were significantly enhanced on the 4th day post-infection by Edwardsiella piscicida. In addition, OnTAK1 and OnJNK were significantly up-regulated in transcriptional level after activation of lymphocytes in vitro by phorbol 12-myristate 13-acetate plus ionomycin (P + I) or PHA, accompanied by a predominant increase in phosphorylation level. More importantly, inhibition of OnTAK1 activity by specific inhibitor NG25 led to a significant decrease in the phosphorylation level of OnJNK. Furthermore, blocking the activity of OnJNK with specific inhibitor SP600125 resulted in a marked reduction in the expression of T-cell activation markers including IFN-γ, CD122, IL-2, and CD44 during PHA-induced T-cell activation. In summary, these findings indicated that the conserved TAK1/JNK axis in Nile tilapia was involved in adaptive immune responses by regulating the activation of lymphocytes. This study enriched the current knowledge of adaptive immunity in teleost and provided a new perspective for understanding the regulatory mechanism of fish immunity.

Takifugu rubripes is a highly valued cultured fish in Asia, while pathogen infections can result in severe diseases and lead to substantial economic losses. Toll-like receptors (TLRs), as pattern recognition receptors, play a crucial role on recognition pathogens and initiation innate immune response. However, the immunological properties of teleost-specific TLR23 remain largely unknown. In this study, we investigated the biological functions of TLR23 (TrTLR23) from T. rubripes, found that TrTLR23 existed in various organs. Following bacterial pathogen challenge, the expression levels of TrTLR23 were significantly increased in immune related organs. TrTLR23 located on the cellular membrane and specifically recognized pathogenic microorganism. Co-immunoprecipitation and antibody blocking analysis revealed that TrTLR23 recruited myeloid differentiation primary response protein (MyD88), thereby mediating the activation of the ERK signaling pathway. Furthermore, in vivo showed that, when TrTLR23 is overexpressed in T. rubripes, bacterial replication in fish tissues is significantly inhibited. Consistently, when TrTLR23 expression in T. rubripes is knocked down, bacterial replication is significantly enhanced. In conclusion, these findings suggested that TrTLR23 played a critical role on mediation TLR23-MyD88-ERK axis against bacterial infection. This study revealed that TLR23 involved in the innate immune mechanism, and provided the foundation for development disease control strategies in teleost.

Edwardsiella anguillarum, a highly virulent species within the Edwardsiella genus, causes significant mortality in milkfish farms in Taiwan. This study aimed to investigate the comparison of milkfish susceptibility, a newly identified host species in Taiwanese aquaculture, with other species Nile tilapia (Oreochromis niloticus) and Asian seabass (Lates calcarifer), to E. anguillarum, elucidating its pathogenicity across both seawater and freshwater aquaculture environments. The results showed milkfish exhibited the highest mortality rate of 85% within 48 h of infection, whereas Nile tilapia exhibited a mortality rate of 70% between the second- and tenth-day post challenge, and seabass exhibited a mortality rate of 25% between the second- and sixth-day post challenge. Gross lesions observed in milkfish included splenomegaly and haemorrhage, whereas Nile tilapia exhibited signs of ascites, exophthalmia and brain haemorrhage. Seabass displayed spleen granulomas and haemorrhage at the injection site. Histopathological analysis revealed common features across all three species, including multifocal necrosis, bacterial presence in the necrotic areas, serositis and oedema. Asian seabass also exhibited chronic lesions in the form of splenic granulomas. This study highlights the high susceptibility of milkfish and Nile tilapia to E. anguillarum, emphasizing the urgent need for further investigation into targeted vaccine development for these fish species. These results not only deepen our understanding of the differing levels of pathogenicity among the three species but also offer valuable insights for improving disease prevention and management strategies in aquaculture, including those applied within polyculture systems and for the maintenance of aquaculture water environments.

Intracellular bacteria such as those belonging to the genus Edwardsiella can survive and proliferate within macrophages. However, the detailed mechanisms underlying the host macrophage immune response and pathogen evasion strategies remain unknown. To advance the field of host macrophage research, we successfully established transgenic (Tg) Japanese medaka Oryzias latipes that possesses fluorescently visualized macrophages. As a macrophage marker, the macrophage-expressed gene 1.1 (mpeg1.1) was selected because of its predominant expression across various tissues in medaka. To validate the macrophage characteristics of the fluorescently labeled cells, May-Grünwald Giemsa staining and peroxidase staining were conducted. The labeled cells exhibited morphological features consistent with those of monocyte/macrophage-like cells and tested negative for peroxidase activity. Through co-localization studies, the fluorescently labeled cells co-localized with E. piscicida in the intestines and kidneys of infected medaka larvae, confirming the ingestion of bacteria through phagocytosis. In addition, the labeled cells expressed macrophage markers but lacked a neutrophil marker. These results suggested that the fluorescently labeled cells of Tg[mpeg1.1:mCherry/mAG] medaka were monocytes/macrophages, which will be useful for future studies aimed at understanding the mechanisms of macrophage-mediated bacterial infections.

The genus Edwardsiella, previously residing in the family Enterobacteriaceae and now a member of the family Hafniaceae, is currently composed of five species, although the taxonomy of this genus is still unsettled. The genus can primarily be divided into two pathogenic groups: E. tarda strains are responsible for almost all human infections, and two other species (E. ictaluri, E. piscicida) cause diseases in fish. Human infections predominate in subtropical habitats of the world and in specific geospatial regions with gastrointestinal disease, bloodborne infections, and wound infections, the most common clinical presentations in decreasing order. Gastroenteritis can present in many different forms and mimic other intestinal disturbances. Chronic gastroenteritis is not uncommon. Septicemia is primarily found in persons with comorbid conditions including malignancies and liver disease. Mortality rates range from 9% to 28%. Most human infections are linked to one of several risk factors associated with freshwater or marine environments such as seafood consumption. In contrast, edwardsiellosis in fish is caused by two other species, in particular E. ictaluri. Both E. ictaluri and E. piscicida can cause massive outbreaks of disease in aquaculture systems worldwide, including enteric septicemia in channel catfish and tilapia. Collectively, these species are increasingly being recognized as important pathogens in clinical and veterinary medicine. This article highlights and provides a current perspective on the taxonomy, microbiology, epidemiology, and pathogenicity of this increasingly important group.

Edwardsiella piscicida is an acute marine pathogen that causes severe damage to the aquaculture industry worldwide. The pathogenesis of E. piscicida is dependent mainly on the type III secretion system (T3SS) and type VI secretion system (T6SS), both of which are critically regulated by EsrB and EsrC. In this study, we revealed that fatty acids influence T3SS expression. Unsaturated fatty acids (UFAs), but not saturated fatty acids (SFAs), directly interact with EsrC, which abolishes the function of EsrC and results in the turn-off of T3/T6SS. Moreover, during the in vivo colonization of E. piscicida, host fatty acids were observed to be transported into E. piscicida through FadL and to modulate the expression of T3/T6SS. Furthermore, the esrCR38G mutant blocked the interaction between EsrC and UFAs, leading to dramatic growth defects in DMEM and impaired colonization in HeLa cells and zebrafish. In conclusion, this study revealed that the interaction between UFAs and EsrC to turn off T3/T6SS expression is essential for E. piscicida infection.

Aquatic environments serve as critical repositories for pollutants and have significantly accumulated micro- and nanoplastics (MNPs) due to the extensive production and application of plastic products. While the disease resistance and immunity of fish are closely linked to the condition of their aquatic habitats, the specific effects of nanoplastics (NPs) and microplastics (MPs) within these environments on fish immune functions are still not fully understood. The present study utilized zebrafish (Danio rerio) embryos and larvae as model organisms to examine the impacts of polystyrene NPs (100 nm) and MPs (5 μm) on fish immune responses. Our findings reveal that NPs and MPs tend to accumulate on the surfaces of embryos and within the intestines of larvae, triggering oxidative stress and significantly increasing susceptibility to Edwardsiella piscicida infection in zebrafish larvae. Transmission electron microscopy examined that both NPs and MPs inflicted damage to the kidney, an essential immune organ, with NPs predominantly inducing endoplasmic reticulum stress and MPs causing lipid accumulation. Transcriptomic analysis further demonstrated that both NPs and MPs significantly suppress the expression of key innate immune pathways, notably the C-type lectin receptor signaling pathway and the cytosolic DNA-sensing pathway. Within these pathways, the immune factor interleukin-1 beta (il1b) was consistently downregulated in both exposure groups. Furthermore, exposure to E. piscicida resulted in restricted upregulation of il1b mRNA and protein levels, likely contributing to diminished disease resistance in zebrafish larvae exposed to MNPs. Our findings suggest that NPs and MPs similarly impair the innate immune function of zebrafish larvae and weaken their disease resistance, highlighting the significant environmental threat posed by these pollutants.

The production of endogenous hydrogen sulfide (H2S) is an important phenotype of bacteria. H2S plays an important role in bacterial resistance to ROS and antibiotics, which significantly contributes to bacterial pathogenicity. Edwardsiella piscicida, the Gram-negative pathogen causing fish edwardsiellosis, has been documented to produce hydrogen sulfide. In the study, we revealed that Ferric uptake regulator (Fur) controlled H2S synthesis by activating the expression of phsABC operon. Besides, Fur participated in the bacterial defense against ROS and cationic antimicrobial peptides and modulated T3SS expression. Furthermore, the disruption of fur exhibited a significant in vivo colonization defect. Collectively, our study demonstrated the regulation of Fur in H2S synthesis, stress response, and virulence, providing a new perspective for better understanding the pathogenesis of Edwardsiella.

Edwardsiella piscicida causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel Edwardsiella phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of Edwardsiella piscicida infection and could reduce the content of the floR resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth in vitro and significantly improved the zebrafish survival rate in vivo (P = 0.0035), achieving an efficacy comparable to that of florfenicol (P = 0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the floR gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.

Edwardsiella piscicida, a significant intracellular pathogen, is widely distributed in aquatic environments and causes systemic infection in various species. Therefore, it\'s essential to develop a rapid, uncomplicated and sensitive method for detection of E. piscicida in order to control the transmission of this pathogen effectively. The recombinase-aided amplification (RAA) assay is a newly developed, rapid detection method that has been utilized for various pathogens. In the present study, a real-time RAA (RT-RAA) assay, targeting the conserved positions of the EvpP gene, was successfully established for the detection of E. piscicida. This assay can be performed in a one-step single tube reaction at a temperature of 39°C within 20 min. The RT-RAA assay exhibited a sensitivity of 42 copies per reaction at a 95% probability, which was comparable to the sensitivity of real-time quantitative PCR (qPCR) assay. The specificity assay confirmed that the RT-RAA assay specifically targeted E. piscicida without any cross-reactivity with other important marine bacterial pathogens. Moreover, when clinical specimens were utilized, a perfect agreement of 100% was achieved between the RT-RAA and qPCR assays, resulting a kappa value of 1. These findings indicated that the established RT-RAA assay provided a viable alternative for the rapid, sensitive, and specific detection of E. piscicida.