Fish & shellfish immunology最新文献

Grass carp hemorrhagic disease, caused by genotype II grass carp reovirus (GCRV-II), is a devastating disease that leads to high mortality in farmed grass carp. However, the absence of a standardized, high-virulence challenge strain that reliably recapitulates severe disease has hindered pathogenesis and vaccine research. Here, we report the isolation and characterization of a novel GCRV-II strain, GCRV-XT256. This strain induces consistent hemorrhagic pathology and high mortality, enabling the establishment of a robust infection model. We successfully purified GCRV-II virions, confirming their classic reoviral morphology, with a diameter of approximately 75 nm. Genomic analysis revealed high similarity to epidemic GCRV-II strains. The model using this strain elicited a potent immune response, marked by upregulation of genes such as nf-κb1, irf3, irf7, and il1β. Moreover, an inactivated vaccine prepared from GCRV-XT256 conferred up to ∼80% relative immune protection in grass carp. The GCRV-XT256 strain and the associated infection model thus serve as a foundational resource for future studies on GCRV-II virology and vaccine development.

Vibrio harveyi has emerged as one of the primary bacterial pathogens affecting grouper (Epinephelus spp.) aquaculture in recent years. However, effective control measures remain elusive. Gentiana macrophylla Pall. (GM), a traditional Chinese medicinal herb with over 2,000 years of documented use exhibits anti-inflammatory, antioxidant, antibacterial and antiviral properties. However, its application for controlling aquaculture diseases remains unexplored. Therefore, this study aimed to investigate the anti-Vibrio harveyi activity of GM extract (GME). Results indicated that GME inhibited Vibrio harveyi growth by suppressing protein synthesis and disrupting cell membrane integrity, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of 62.5 mg/mL and 125 mg/mL, respectively. Furthermore, 1% and 1.5% GME supplements significantly reduced mortality rates in vitro during Vibrio harveyi infections and alleviated inflammatory responses and pathological damage to major organs in vivo. These findings provide scientific support for developing GME as a fisheries drug or feed additive to prevent or treat infectious diseases in aquaculture.

Intracellular survival is a key virulence trait of Pseudomonas plecoglossicida, the agent of visceral granulomas disease in large yellow croaker (Larimichthys crocea). However, the bacterial determinants that support persistence within host phagocytes remain incompletely defined. Here, we investigated the contribution of the type VI secretion system-1 (T6SS-1) to macrophage infection using a ΔtssD-1 mutant and a head kidney-derived macrophage-like cell line (LYC-hk). Gentamicin protection assays showed that ΔtssD-1 bacteria entered macrophages at levels comparable to wild type at 0 h post-infection, but failed to undergo sustained intracellular replication at later time points, whereas a chromosomally complemented strain restored this phenotype. Differential permeabilization microscopy indicated that T6SS-1 is required for phagosomal rupture and exposure of bacteria to the host cytosol, and transmission electron microscopy confirmed cytosolic localization of wild-type and complemented bacteria but not the mutant. Time-resolved transcriptomic profiling revealed a shared early inflammatory signature in response to both strains, followed by divergence at 4-8 h post-infection. Wild-type infection was associated with distinct expression patterns, including strong induction of the osmotic stress marker lrrc8a, which was supported by RT-qPCR validation. Together, these results identify T6SS-1 as a key determinant of phagosomal rupture and cytosolic access in P. plecoglossicida macrophage infection and link this intracellular transition to distinct host transcriptional signatures.

The scaleless striped catfish (Pangasianodon hypophthalmus) is an economically important aquaculture species but is frequently infected by Aeromonas hydrophila (AH), causing substantial economic losses. Fish skin mucosa plays a protective role by initiating inflammatory responses to AH challenge. However, how the skin epithelial cells, the outermost defensive layer of skin tissue, respond and interact with AH remains unknown. This study aims to establish a skin epithelial cell line from striped catfish and investigate the response of skin epithelial cells under AH challenge. Skin epithelial cells were isolated and subcultured at optimal temperature and serum concentration, and epithelial properties were confirmed by the epithelial marker cytokeratin. The established skin epithelial cell line was inoculated with or without AH to evaluate survival and immune responses. We found cells survived at a tissue-damaging concentration of AH and showed an extremely higher immune response. Moreover, the presence of mucus and its microbiome may regulate AH-induced immune response, suggesting a complexity of skin-AH interaction in vivo. In conclusion, this study successfully established a striped catfish skin epithelial cell line and utilized it to characterize the outermost skin epithelial responses to AH in the presence of the mucus microbiome. By employing this cell line, new insights into epithelial-level immune interactions with pathogens were provided.

Fish populations provide valuable genetic resources for sustainable breeding programs. For species like the European perch (Perca fluviatilis), which are early in domestication, systematic characterization is key to developing strong breeding lines. This pilot study assessed the immune response to the bacterium Aeromonas hydrophila in six different perch populations. Bacterial injection triggered two distinct patterns of immune reactions. Fish originating from Finland (FIN) and Slovakia (SVK-LM and SVK-RV) exhibited a rapid immune response, with a dramatic influx of cells, including 4 million myeloid cells, into the peritoneal cavity during the first 24 h. Conversely, fish from the Czech Republic (CZE), Italy (ITA), and Poland (POL) displayed a weaker response, with only 1-2 million myeloid cells detected. By 3 days postinoculation, the number of myeloid cells in the FIN and SVK groups had declined to about 500,000, whereas levels in the POL, ITA, and CZE groups remained elevated at 1-1.5 million cells. Nanofluidic Real-Time PCR identified the genes encoding interleukin-1 beta (il1b) and immunoglobulin heavy (igh) chain as robust indicators of peritoneal immune activation in perch. Expression of il1b was rapidly induced across most populations, while igh expression increased more gradually, consistent with early B-cell-mediated responses. Gene-expression and flow-cytometry data suggested divergent immune strategies among perch from different ecological and evolutionary backgrounds, highlighting a central role for myeloid and B cells, while canonical T -cell-associated markers were not strongly induced during the early infection stage. These pilot findings demonstrate how environmental conditions shape immune responsiveness, providing a starting point for future breeding programs.

An outbreak of disease in farmed Asian sea bass (Lates calcarifer) was investigated. A Bacillus cereus group strain, SB01, was consistently isolated from internal organs and identified by groEL PCR. Transmission electron microscopy (TEM) revealed rod-shaped cells with prominent flagella. Experimental infection demonstrated dose-dependent mortality with an LD₅₀ of 5.6 × 10⁶ CFU/fish and pathological signs including ascites, splenomegaly, and multi-organ necrosis. Whole-genome sequencing revealed a 5.26 Mb chromosome and a 240 kb plasmid (pSB01) encoding anthrax toxin-related genes, including protective antigen (PA), edema toxin, and a lethal factor-related metalloprotease. Multilocus sequence typing assigned SB01 to B. cereus sequence type (ST) 234. Given that PA is a well-established vaccine target in Bacillus anthracis, its presence and characteristics were examined in ST234 strains. Several amino acid substitutions were identified within PA domain 4 (PD4), corresponding to receptor-binding and major neutralizing epitope regions, compared with B. anthracis, leading to the selection of PD4 as a candidate subunit vaccine. In parallel, the prominent flagella observed by TEM prompted inclusion of flagellin as a secondary antigen. Formalin-killed cells (FKC), recombinant thioredoxin (rTrx), Trx-tagged PD4 (rPD4), and flagellin (rFla) were expressed, purified, formulated with Montanide ISA 763A, and evaluated in vaccination trials. Relative percent survival values were 0% for rTrx, 100% for rPD4 and FKC, and 75.6% for rFla. Vaccination induced early innate immune responses and elevated antigen-specific IgM levels. Overall, SB01 represents a highly virulent ST234 B. cereus strain, and both whole-cell and PD4/flagellin subunit vaccines conferred effective protection in Asian sea bass.

In mammals, interferon-gamma (IFN-γ) represents the only member of the type II interferon family; however, teleost fish possess an additional subtype known as ifn-gamma-related gene (ifn-γrel). While IFN-γrel has been shown to exert antiviral effects through activation of downstream signaling pathways, its roles and underlying mechanisms in antibacterial immunity remain poorly understood in teleosts. In this study, an IFN-γrel homolog (Caifn-γrel) was identified and characterized in the humpback grouper (Cromileptes altivelis). The open reading frame (ORF) of Caifn-γrel spans 564 bp, encoding a 187-amino acid protein, with the first 21 residues predicted to be a signal peptide. Sequence analysis revealed 83.66% identity between CaIFN-γrel and its counterpart from Epinephelus coioides. Phylogenetic analysis further confirmed close evolutionary relationships, with CaIFN-γrel clustering together with E. coioides IFN-γrel. Constitutive expression of Caifn-γrel was detected in all examined tissues, with particularly high levels observed in blood, intestine, gill, and liver. Upon Vibrio harveyi challenge, Caifn-γrel transcript levels were significantly upregulated. Recombinant CaIFN-γrel (rCaIFN-γrel) enhanced macrophage phagocytic activity, induced STAT1 phosphorylation, and promoted the expression of key cytokine genes. Moreover, CaIFN-γrel was found to activate downstream gene expression via the transcription factor CaIRF1. Functional studies demonstrated that overexpression of CaIFN-γrel suppressed V. harveyi replication in vivo, whereas knockdown of CaIFN-γrel increased host susceptibility to bacterial infection. Collectively, these findings provide new insights into the functional versatility and immunological significance of IFN-γrel in teleost fish.

Lipocalins play crucial roles in immune defense in vertebrates; however, their functional mechanisms in teleost fish remain poorly understood. In this study, the economically important marine fish large yellow croaker (Larimichthys crocea) was used as a model to investigate a novel lipocalin-like protein, LcApoM_AGPRP, and to elucidate its role in immune responses. A putative antibacterial protein, LcApoM_AGPRP, which lacks functional annotation in the genome, was identified in large yellow croaker. LcApoM_AGPRP was highly constitutively expressed in the liver, head kidney, ovary, gill and spermary of healthy large yellow croaker. It expression was significantly upregulated following LPS and Poly(I:C) stimulation, indicating a potential role in innate immune responses. Subcellular localization analysis demonstrated that LcApoM_AGPRP is predominantly localized in lysosomes, mainly distributed in the Golgi apparatus and mitochondria and partially distributed in the endoplasmic reticulum, implying its involvement in organelle-mediated immune defense. Functional assays showed that LcApoM_AGPRP possesses broad-spectrum antimicrobial activity. The recombinant protein exerts its antibacterial effects through multiple mechanisms, including disruption of bacterial membrane integrity, inhibition of bacterial motility, and interference with biofilm formation. Stability assays further confirmed that its antibacterial activity is maintained over a wide range of temperatures (25-100 °C) and salinities (0-40 ‰), highlighting its strong environmental adaptability. These properties suggest that LcApoM_AGPRP has considerable potential for application as an antibacterial agent, aquaculture feed additive, or food preservative. This study provides the first systematic identification and functional characterization of LcApoM_AGPRP, offering new insights into the innate immune system of teleost fish.

Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates immune responses, but its role in fish intestinal immunity remains poorly understood. In this study, we produced recombinant grass carp IL-21 (rCiIL-21) and investigated its protective effects against Aeromonas hydrophila-induced enteritis. Administration of rCiIL-21 significantly reduced intestinal bacterial load and mitigated histopathological damage caused by A. hydrophila infection. Furthermore, rCiIL-21 treatment enhanced intestinal barrier integrity by increasing mucus layer thickness, promoting goblet cell proliferation, and upregulating the expression of mucin-related genes (agr2, muc2, and klf4) and tight junction proteins (zo-1, claudin-3, and occludin) after infection. Additionally, rCiIL-21 attenuated apoptosis and oxidative stress while promoting the expression of antimicrobial factors (c-lysozyme, β-defensin 1 and β-defensin 2) and anti-inflammatory factor (il-10), collectively leading to reduced bacterial load and improved survival rates. Collectively, these results demonstrate that rCiIL-21 plays a multiple role in alleviating bacterial enteritis by reinforcing intestinal barrier integrity, modulating inflammatory responses, and enhancing innate antimicrobial defense, highlighting its potential as a therapeutic agent for bacterial enteritis in aquaculture.

Bursicon (burs) is a neuropeptide hormone composing of α and β heterodimeric subunits (bursα and bursβ) that regulates various physiological processes during development and reproduction in insects and crustaceans. Recently, the homodimeric form of bursicon has been demonstrated for its possible function in immune response by activating several antimicrobial peptide (AMP) genes in crustaceans. This study aims to investigate the function of bursα and bursβ homodimers in the Pacific white shrimp, Litopenaeus vannamei with a hypothesis that Lvbursα and Lvbursβ function in the activation of AMP gene expression and inhibit viral replication in L. vannamei. The cDNAs encoding Lvbursα and Lvbursβ were amplified and characterized. Lvbursα and Lvbursβ were specifically expressed in neuronal tissues with the high expression level in thoracic ganglia, and were upregulated during white spot syndrome virus (WSSV) challenge. Functional analysis of recombinant Lvbursα and Lvbursβ revealed that both proteins significantly activate the transcription of key genes involved in multiple immune-related signaling pathways and AMP genes, including Dorsal, Relish, IKK-β, ProPO1, Crustin, ALF, and Pen3. Pre-treatment with either Lvbursα or Lvbursβ prior to WSSV challenge exhibited a delay in cumulative mortality, indicating their antiviral potential. These results suggested that Lvbursα and Lvbursβ homodimers play a significant role in innate immune response by enhancing multiple immune-related pathways. These findings provide evidence supporting the role of homodimeric bursicon in innate immune modulation and highlight its potential relevance in enhancing resistance to viral infection in crustaceans.