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ABSTRACT Spring viremia of carp virus (SVCV), that can infect cyprinoid and koi causing an acute hemorrhagic symptom, with a feature of widespread epidemic, which poses a severe threat to the global aquaculture industry. Given the absence of specific effective medicine or vaccines, the early, accurate diagnosis is critical in the disease prevention. Many SVCV detection methods have been established, but most require specialized equipment and skilled operators, which limits their field application. In this study, we established a recombinase‐mediated isothermal amplification (RAA) in conjunction with the CRISPR/Cas12a system targeting the screened conserved region of the G gene in the genome to identify SVCV. The optimal combination of the primer pairs and crRNA was screened via the fluorescence intensity. In addition, the test exhibited high sensitivity and specificity, which could detect at the limit of 1.243 × 10 ¹ copies/μL and show no cross‐reactivity with other common freshwater fish pathogens. Furthermore, the result could be visible by excitation of fluorophores by blue light at 39°C within 30 min. The integrated method combines the advantages of RAA and CRISPR‐based signal transduction, offering a diagnosis project in resource‐limited aquaculture environments and a field‐applicable early warning of SVCV.

Infectious spleen and kidney necrosis virus (ISKNV), a member of the genus Megalocytivirus, threatens ornamental and farmed aquaculture because of its broad host range and environmental persistence. This study investigated ISKNV infection dynamics and stage-dependent horizontal transmission in angelfish (Pterophyllum scalare). Immersion challenge experiments demonstrated that angelfish were highly susceptible to ISKNV infection, with cumulative mortality reaching 95% at 106 TCID50/mL. The estimated LD50 and ID50 values indicated that infection occurred even at sublethal viral concentrations. Viral loads peaked at 7 days post-immersion and were positively correlated with the severity of histopathological lesions in the spleen and kidney, including multifocal necrosis and enlarged basophilic cells. To assess horizontal transmission, naive fish were cohabited with infected donors for 24 h at different infection stages. These findings indicate that ISKNV transmission can occur following short-term cohabitation and that transmission outcomes may vary according to the infection stage of donor fish under the experimental conditions examined, providing insights into ISKNV transmission dynamics and the potential implications for disease management in the ornamental fish industry.

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ABSTRACT Tenacibaculosis, caused by Tenacibaculum dicentrarchi , is an emerging bacterial disease affecting salmonid aquaculture worldwide; however, host–pathogen interactions and the immune response of Atlantic salmon ( Salmo salar ) remain poorly understood. This study evaluated the expression of 20 immune‐related genes in Atlantic salmon experimentally infected by immersion with T. dicentrarchi and in uninfected controls under identical conditions. The 10‐day challenge trial included sampling every two days (skin/muscle, gills, spleen and head kidney) for gene expression analyses. Koch's postulates were fulfilled in the challenge with the T. dicentrarchi TdCh06, which induced 40% cumulative mortality; deaths began at 2 days post‐infection and continued until Day 7. Of the 20 genes analysed by RT‐qPCR, seven (MHC‐I, MHC‐II, Arg‐1, IL‐1β, IL‐2, IL‐10 and IFN‐α1) were consistently detected across tissues and used for comparative analysis. The results show that T. dicentrarchi induces a rapid but heterogeneous immune response in Atlantic salmon: the spleen exhibits an early, transient systemic response; the head kidney shows delayed but strong transcriptional activation; skin/muscle displays early local activation with sustained IL‐1β and IFN‐α1 expression; and gills show early mucosal activation with persistent IL‐1β induction. Overall, infection triggers a tissue‐specific response involving inflammatory, antigen‐presentation, interferon‐associated and macrophage‐related pathways, suggesting sustained immune stimulation rather than effective bacterial clearance. These findings shed light on the host–pathogen interaction during tenacibaculosis and identify key immunological pathways involved in disease progression. Notably, the genes IL‐1β, MHC‐I, MHC‐II, IFN‐α1, IL‐2, IL‐10 and Arg‐1 are proposed as potential transcriptional markers of infection, offering valuable tools to characterise host responses. Importantly, these markers may also be useful for evaluating vaccine efficacy against T. dicentrarchi in future studies.

Screening with tuberculin remains widely used in humans and livestock for detecting mycobacterial immune sensitization, but tuberculin skin test (TST) interpretation is influenced by Bacillus Calmette-Guérin vaccination, environmental non-tuberculous mycobacteria (NTM), cutoff selection, and epidemiological context. The TST uses purified protein derivative (PPD), a complex mycobacterial antigen preparation, to elicit delayed-type hypersensitivity measured as local skin induration. Aquaculture vaccination is established for farmed fish, whereas vaccines targeting fish mycobacteriosis remain largely experimental, including BCG, attenuated or heat-inactivated mycobacteria, DNA vaccines, mycolic acids, and defined antigen combinations. This Perspective asks whether experimental mycobacterial vaccination of fish could plausibly influence human TST interpretation in occupationally exposed aquaculture workers. Such an interaction would require antigenic overlap with PPD, environmental release or persistence of mycobacterial material linked to vaccination, repeated worker exposure through water, biofilms, fish tissues, surfaces, or skin microtrauma, immune sensitization or amplification, and an induration shift sufficient to alter classification at operational cutoffs. The article defines this as a testable One Health interface and proposes construct-level antigen mapping, environmental monitoring around vaccination events, and prospective worker studies using continuous TST measurement with parallel IGRA testing.

Largemouth bass virus (LMBV) is the most prevalent and threatening virus in largemouth bass aquaculture. To establish effective control measures of LMBV, this study vaccinated broodstock with an LMBV MCP subunit vaccine to assess whether the subunit vaccine could activate maternal immunity to block vertical transmission of LMBV. Fourteen days post-immunization, significant upregulation of IgM, IgT, IL-6, TNF-α, and CD4 gene expression was observed in the relevant tissues of broodstocks. Serum titers reached their peak at 21 days post-immunization as detected by ELISA within 28 days. After challenge, the subunit vaccine achieved a relative protection rate of 87.4%. The MCP+LMBV group showed a significant reduction in viral load in the relevant tissues and a marked alleviation of histopathological damage compared to the Control + LMBV group. Compared to the Control group, vaccinated broodstocks exhibited normal spawning behavior, with no significant differences in spawning trends or total egg production, while egg production in the Control + LMBV group was significantly reduced. The fertilization and hatching rates in the MCP + LMBV group were 94.06% and 94.50%, respectively, with the hatching rate 27.77% higher than the Control + LMBV group. In the MCP + LMBV group, the virus positivity rate for both gonads and fertilized eggs were 0.0%. This study confirms that LMBV can be transmitted vertically from infected broodstock to their offspring, however, the MCP subunit vaccine can activate maternal immunity to block vertical transmission of LMBV, which provides a new approach to protect largemouth bass offspring from LMBV infection.

Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that catalyzes the desaturation of saturated fatty acids to monounsaturated fatty acids. Although lipid metabolism is increasingly recognized to influence antiviral innate immunity, the role of SCD1 in host defense, particularly in teleost fish, has not been fully explored. Here, we show that viral infection markedly suppresses SCD1 expression in zebrafish. Overexpression of zebrafish SCD1 significantly suppresses antiviral gene expression and enhances viral replication. Pharmacological inhibition of SCD1 desaturase activity enhances antiviral gene expression, reduces viral replication, and protects zebrafish larvae from viral lethality, indicating that SCD1 suppresses antiviral immunity in an enzymatic activity-dependent manner. Mechanistically, palmitic acid (PA), the saturated fatty acid substrate of SCD1, acts as a potent enhancer of antiviral innate immunity. PA treatment upregulates antiviral gene expression in vivo and in vitro, and restricts viral replication in ZFL cells. We demonstrate that PA exerts its immunostimulatory effects by inhibiting the two zebrafish isoforms of peroxisome proliferator-activated receptor α, PPARαa and PPARαb, as overexpression of either isoform attenuates PA-induced antiviral responses while a PPARα antagonist mimics the effects of PA. Furthermore, we show that PPARαa and PPARαb physically interact with interferon regulatory factor 3 (IRF3) and suppress IRF3-induced antiviral gene expression, thereby inhibiting type I interferon signaling. Collectively, our findings establish an SCD1-PA-PPARα-IRF3 regulatory axis in which SCD1 consumes its substrate PA to sustain PPARα-mediated negative regulation of IRF3-dependent antiviral gene expression, thereby restraining antiviral innate immunity.

ABSTRACT This study optimised the dietary supplementation of Lactococcus lactis and Schizochytrium limacinum in genetically improved farmed tilapia (GIFT, Oreochromis niloticus ) to improve immunity and resistance to Streptococcus agalactiae infection. Single‑factor experiments and central composite design combined with response surface methodology (CCD‑RSM) were employed for sequential optimisation. A desirability function was developed as the optimisation criterion, integrating percentage survival of S. agalactiae ‑infected tilapia, non‑specific immune and antioxidant parameters (total antioxidant capacity, superoxide dismutase, catalase, alkaline phosphatase, and acid phosphatase), and liver health indices (reduced glutathione, alanine aminotransferase, and aspartate aminotransferase). Single‑factor experiments determined optimal individual supplementation levels of 1.5% for S. limacinum and 1.0 × 10 ⁷ CFU/g for L. lactis . CCD‑RSM revealed that L. lactis significantly affected total antioxidant capacity, catalase, and alanine aminotransferase, while S. limacinum significantly influenced percentage survival of S. agalactiae ‑infected tilapia, superoxide dismutase, catalase, alkaline phosphatase, acid phosphatase, alanine aminotransferase, and reduced glutathione, with significant interactive effects between the two supplements on multiple parameters. The RSM‑optimised combination was 5.45 × 10 ⁷ CFU/g L. lactis plus 0.91% S. limacinum . Tilapia were fed for 28 days with four dietary treatments: the RSM‑optimised combination, L. lactis alone, S. limacinum alone, and a control. The RSM group showed higher superoxide dismutase, catalase, and acid phosphatase activities, lower alanine aminotransferase and aspartate aminotransferase levels, and fewer hepatic vacuoles than other groups. Following S. agalactiae challenge (3 × 10 ⁸ CFU/kg), the RSM group achieved the highest relative per cent survival (44.12%), followed by L. lactis alone (35.29%) and S. limacinum alone (26.47%). Collectively, the RSM‑optimised combination demonstrates promising potential for enhancing resistance to S. agalactiae infection in GIFT tilapia.

In innate immunity, effective activation of the NF-κB signaling pathway is crucial for initiating inflammatory responses and eliminating invading bacteria, and its precise regulation relies heavily on the ubiquitin-proteasome system. Ubiquitination, a pivotal post-translational modification, is specifically catalyzed by E3 ubiquitin ligases that determine substrate selection. Ubiquitination of the adaptor protein MyD88 is critical for immune signaling; however, its regulatory mechanism in teleost fish, especially regarding the involvement of E3 ubiquitin ligases remains unclear. Among these E3 ubiquitin ligases, SMAD ubiquitylation regulatory factor 1 (Smurf1), while important in multiple signaling pathways, has not been reported to regulate antibacterial immunity in teleost fish. Thus, we systematically investigated Smurf1's role and molecular mechanism in this immune process, using Miichthys miiuy as a model. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) analysis in M. miiuy kidney cells (MKC) cells revealed that Smurf1 overexpression suppressed pro-inflammatory cytokine expression and enhanced Vibrio anguillarum proliferation and adhesion, while Smurf1 knockdown exerted the opposite effects. Mechanistically, Smurf1 directly interacts with MyD88, promoting MyD88 proteasomal degradation through K48-linked polyubiquitination, which are dependent on Smurf1's E3 ligase activity (C704A) and HECT domain, as the C704A mutation or HECT domain deletion abolished this function. Luciferase reporter assays confirmed that Smurf1 mediated MyD88 degradation inhibits NF-κB signaling, thereby negatively regulating innate immunity and maintaining inflammatory homeostasis. Our findings reveal a mechanism by which Smurf1 balances immunity and homeostasis via MyD88 ubiquitination, providing valuable insights into vertebrate immune regulation.

Enterococcus faecalis represents a potential public health threat and leads to significant economic losses in the fish producing sectors. To investigate the prevalence, multidrug resistance profiles, virulence characteristics, antibiotic resistance genes, and pathogenicity of E. faecalis, 240 fish samples, including 120 Oreochromis niloticus and 120 Mugil cephalus, were obtained from fish farms in Ismailia, Egypt, and subjected to clinical, postmortem, and bacteriological investigations. The total prevalence of E. faecalis in the examined fish was 14.17% (34/240), with rates of 16.67% (20/120) in O. niloticus and 11.67% (14/120) in M. cephalus. From these positive fish, 62 organ-specific isolates were obtained for further characterization. The liver was the most frequently infected organ. PCR analysis revealed that the predominant virulence-related genes in the recovered E. faecalis strains were gelE (100%), esp (88.70%), hyl (74.19%), and cylA (70.97%). Furthermore, 32.20% of E. faecalis isolates exhibited multidrug resistance (MDR) to seven classes of antimicrobials and harbored the ermB, tetM, vanA, blaZ, and aac(6')-Ie-aph(2″)-Ia genes. Additionally, 19.30% (12/62) of E. faecalis isolates were MDR across six antibiotic classes and harbored the ermB, tetM, vanA, and blaZ genes. The pathogenicity of the recovered E. faecalis isolates was evaluated by experimental challenge in O. niloticus and T. zillii. Kaplan-Meier survival analysis demonstrated significant differences in survival probabilities among the challenged groups, reflecting marked variation in isolate virulence. Mortality outcomes were associated with the distribution of virulence-associated genes, suggesting that the pathogenicity of E. faecalis is governed by isolate-specific virulence determinants and varies between host species. In summary, the study highlights the emergence of MDR E. faecalis in both O. niloticus and M. cephalus, underscoring a potential public health concern that warrants further investigation. MDR E. faecalis strains in fish increasingly possess the resistance genes ermB, tetM, vanA, blaZ, and aac(6')-Ie-aph(2″)-Ia, as well as the virulence genes gelE, esp, hyl, and cylA.

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为实现草鱼(Ctenopharyngodon idella)主要病原的快速、准确检测,本研究建立了一种基于RAA-LFD技术的草鱼呼肠孤病毒(Grass carp reovirus,GCRV)与嗜水气单胞菌(Aeromonas hydrophila)双重检测方法.以GCRV 的 vp2 基因和 A.hydrophila 的 gyrB 基因为靶标,设计特异性引物和探针,构建双重 RAA-LFD 检测体系,并对反应条件、灵敏度、特异性及临床样品适用性进行系统评价.结果表明,该方法在 37℃条件下反应 15 min 即可完成扩增,并通过试纸条实现结果的快速可视化判读.灵敏度分析显示,RAA-LFD 对 GCRV 和 A.hydrophila 的最低检测限分别为 1 ng/μL 和100 pg/μL,而 qPCR 对 GCRV 和 A.hydrophila 的最低检测限均为10 pg/μL.特异性分析表明,该方法对副溶血弧菌、迟缓爱德华氏菌、金黄色葡萄球菌及锦鲤疱疹病毒均无交叉反应.对 20 份临床样品检测结果显示,RAA-LFD 与 qPCR 具有较高一致性,GCRV 和 A.hydrophila 的 Kappa 值分别为 0.88 和 0.91.综上,本研究建立的 RAA-LFD 双重检测方法具有操作简便、检测快速和特异性强等优点,可为草鱼养殖中多病原的现场快速检测提供有效技术手段.