Grass carp reovirus (GCRV) poses a serious economic threat to grass carp aquaculture and oral vaccination has become the strategy of choice for its control. In this study, multiple sequence alignment was conducted on the sequences of the VP56 of seven GCRV-II strains. The most frequent amino acid at each site was assigned to the fusion sequence to obtain the sequences of GCRV-FUSION (VP561-512). Through antigenic index analysis, the full-length sequence (VP561-512) was truncated, thereby obtaining five truncated antigen fragments: VP561-210, VP56100-209, VP56210-310, VP56310-500, and VP56410-500. Then, using flagellin C (FlaC) from Aeromonas hydrophila as an adjuvant, recombinant Pichia pastoris expressing VP561-512-FlaC, VP561-210-FlaC, VP56100-209-FlaC, VP56210-310-FlaC, VP56310-500-FlaC and VP56410-500-FlaC were constructed, and recombinant proteins were examined by SDS-PAGE and WB assays. The experimental fish was divided into 8 groups (blank control, P-pPIC3.5K, P-VP561-512-FlaC, P-VP561-210-FlaC, P-VP56100-209-FlaC, P-VP56210-310-FlaC, P-VP56310-500-FlaC, P-VP56410-500-FlaC) to evaluate the protective effect. The survival rate of the P-VP56310-500-FlaC group (54 %) was significantly higher than that of the blank control group (18 %). Oral P-VP56310-500-FlaC vaccine can effectively increase serum enzyme activities (lysozyme, total antioxidant capacity and complement component 3). The mRNA expressions of immune genes (TLR5a, IL-6, IFN1, Mx2, IL-1β, MHCII, IgM, IgT) in the P-VP56310-500-FlaC group were significantly increased. The vaccine P-VP56310-500-FlaC significantly inhibited the replication of GCRV in the spleen, head kidney and midgut, and reduced the degree of damage to the spleen. The above results indicate that oral P-VP56310-500-FlaC vaccine can effectively control GCRV infection, providing an effective strategy for the prevention of viral diseases in aquaculture.

Intestinal oxidative imbalance, inflammatory activation, and microbial dysbiosis are increasingly recognized as major factors affecting the health status of intensively cultured largemouth bass (Micropterus salmoides). Chlorogenic acid (CGA) is a plant-derived polyphenolic compound that is well known for its antioxidant and anti-inflammatory properties. Although its inclusion as a dietary supplement in aquafeeds has garnered increasing interest, its effects on intestinal health in largemouth bass under normal rearing conditions remain largely unknown. Hence, the present investigation was designed to elucidate how incrementally elevated dietary CGA concentrations modulate oxidative capacity, inflammatory gene expression, mucosal histological features, and enteric microbiota structure in juvenile largemouth bass. In this study, largemouth bass were fed diets containing 0, 200, 400, or 600 mg/kg CGA for 70 days, and we evaluated intestinal antioxidant capacity, inflammation-related transcriptional responses, histomorphology, and gut microbiota were assessed. The results demonstrated that CGA supplementation significantly raised intestinal total antioxidant capacity as well as the activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase, while substantially decreasing malondialdehyde levels (p < 0.05). The transcription levels of cat, gsh-px, gst, sod, nrf2, and ucp2 were markedly upregulated, whereas keap1 expression was downregulated (p < 0.05). CGA supplementation also reduced the transcription levels of several proinflammatory genes, including il-1β, il-6, il-8, tnf-α, nf-κb, p50, map3k, jak2, as well as stat3, while significantly elevating il-10 expression, in the 200 and 400 mg/kg groups, tgf-β expression was also lowered (p < 0.05). Histological examination revealed that CGA supplementation influenced intestinal morphology in a dose-dependent manner; the most consistent improvements in villus length, villus width, and muscularis thickness occurred at the 400 mg/kg level, whereas the 600 mg/kg level showed weaker effects in several parameters compared with the control. Furthermore, 16S rRNA sequencing indicated that CGA altered microbial diversity, community structure, and predicted functional profiles in the intestine. In summary, dietary CGA supplementation was associated with enhanced intestinal antioxidant capacity, modified inflammation-related transcriptional responses, changes in intestinal morphology, and shifts in the gut microbial community of largemouth bass. Within the tested dose range, the 400 mg/kg group exhibited relatively favorable responses in several measured indicators under the present experimental conditions.

In normal physiological conditions, germinal center (GC) in secondary lymphoid organs (SLOs) serve as the central sites for the generation of immune memory. How such spatially organized humoral immunity operates in early vertebrates lacking canonical lymphoid architecture remains unclear. Previously, it was thought that this anatomically defined structure was absent in the kidneys of bony fish (which make up half of all vertebrates). This left a fundamental gap in our understanding of the evolution of kidney immunity. Here, we show that teleost fish can initiate and sustain GC-like antiviral humoral responses within the head kidney (HK), a unique lympho-hematopoietic organ. We identified induced HK-associated lymphoid aggregates with GC-response characteristics. These structures revealed polarized cxcr5+ and cxcr4+ B cell regions, robust B cell proliferation, apoptosis, follicular helper T cell (Tfh) assembly, and the initiation of a transcriptional program remarkably similar to that of mammalian GCs. We term these structures HK germinal center–like (HK-GCL) niches. Our findings demonstrate that functional GC programs can be dynamically assembled outside canonical secondary lymphoid organs, revealing an ancestral strategy of humoral immunity in vertebrates and supporting the principle that immune function precedes anatomical form.

Spring viremia of carp virus (SVCV) poses a major threat to carp aquaculture due to the lack of effective antiviral strategies. This study investigates the antiviral potential of baicalin in epithelioma papulosum cyprini (EPC) cells and zebrafish. Baicalin exhibits antiviral activity during both pretreatment and post-infection treatment and shows direct virucidal activity during virus–compound co-incubation. Moreover, baicalin alleviated oxidative stress, preserved mitochondrial function, and reduced apoptosis in infected cells. Proteomic analyses revealed that baicalin suppressed SVCV replication by modulating the AMPK/mTOR signaling pathway, as evidenced by decreased AMPK phosphorylation, increased mTOR phosphorylation, and subsequent inhibition of autophagy. These findings demonstrate that baicalin significantly suppresses SVCV replication and is associated with modulation of the AMPK/mTOR-mediated autophagy pathway. These findings identify baicalin as a promising antiviral candidate against SVCV and provide a foundation for further evaluation in economically important aquaculture species.

Largemouth bass virus (LMBV) is a major pathogen threatening the aquaculture of largemouth bass, causing significant economic losses. Oral vaccination offers a practical and efficient strategy for disease prevention in farmed fish. In this study, a fusion CotC-MCP-3 gene was introduced into Bacillus subtilis 168 (Bs168) to enable surface display of the MCP-3 protein on bacterial spores through the anchoring protein CotC, thereby generating a candidate oral vaccine. At 28 days post-vaccination (dpv), qPCR analysis revealed pronounced upregulation of associated innate and adaptive immunity genes in the gut and head kidney of largemouth bass immunized with the MCP-3 vaccine. Vaccination markedly increased the abundance of IgM+ B cells in both the gut and head kidney, as well as total IgM levels in serum and gut mucus. Concomitantly, LMBV-specific IgM titers and neutralizing activities in both serum and gut mucus were significantly enhanced. Following the experimental LMBV challenge, fish in the MCP-3 vaccine group achieved a relative percent survival (RPS) of 68.7%, accompanied by substantially lower viral loads in the head kidney, gut, and spleen, as well as notably attenuated histopathological lesions compared with the other groups. The Bs168-based MCP-3 oral vaccine provides strong protection against LMBV infection in largemouth bass, as demonstrated by these results, highlighting its potential as a promising strategy for managing viral diseases in aquaculture.

In teleost fish, the gut functions not only as a primary site for nutrient digestion and absorption but also as a critical mucosal immune organ. Although the functional heterogeneity of nutrient absorption among different intestinal segments has been well characterized, the segment-specific features of intestinal immune responses remain poorly understood, particularly with regard to mucosal IgM responses. In this study, we established a virus infection model via intraperitoneal injection and demonstrated that the virus triggers robust innate and adaptive immune responses in the gut of largemouth bass (Micropterus salmoides), accompanied by significant upregulation of multiple immune-related genes. The secondary infection induced a stronger IgM response compared to primary infection, characterized by a significant increase in IgM+ B cell proportions and markedly elevated LMBV-specific IgM titers. In addition, the rise in LMBV-IgM titers may be closely associated with their specific neutralizing activity against the virus. Notably, intestinal IgM responses exhibited clear segmental heterogeneity, with the hindgut displaying substantially stronger IgM responses than the foregut and midgut. Collectively, this study highlights the pivotal role of the hindgut in antiviral intestinal immunity and provides a theoretical foundation for the development of mucosal vaccines in teleost fish.

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.

Probiotics are considered promising feed additives for enhancing fish health and production performance in aquaculture. This study evaluated the effects of dietary supplementation with Sporolactobacillus laevolacticus on growth performance, feed utilization, intestinal health, and physiological responses in juvenile coho salmon (Oncorhynchus kisutch). Fish were fed a control diet or diets supplemented with S. laevolacticus at 0.89 × 107, 0.90 × 109, or 0.87 × 1011 CFU/g for 10 weeks. Compared with the control, S. laevolacticus supplementation significantly increased final body weight, weight gain rate, specific growth rate, and protein efficiency ratio, while decreasing the feed conversion ratio (p < 0.05). It also significantly enhanced intestinal protease and α-amylase activities, improved serum biochemical and immune-related parameters, and promoted better intestinal morphology (p < 0.05). Additionally, S. laevolacticus supplementation led to elevated expression of antioxidant-related genes, reduced expression of pro-inflammatory genes, and altered gut microbial composition, characterized by a decrease in Proteobacteria and increases in Firmicutes and Lactobacillales. Among the tested dosages, 0.90 × 109 CFU/g produced the most consistent improvements in growth performance, digestive function, intestinal health, antioxidant and immune responses, and gut microbial composition, and was therefore identified as the optimal supplementation level. Collectively, dietary S. laevolacticus at 0.90 × 109 CFU/g improved growth performance and intestinal health in juvenile coho salmon, highlighting its potential as a probiotic candidate for coho salmon aquaculture.

Grass carp hemorrhagic disease (GCHD) caused by GCRV-II poses a severe threat to aquaculture, yet mechanistic studies are hampered by the lack of appropriate in vitro models. Here, we established GCB75, a novel astrocyte-like cell line derived from grass carp brain, stably passaged over 66 times with a diploid karyotype (2n = 48) and glial fibrillary acidic protein (GFAP) expression. Key findings show GCB75 exhibits high susceptibility to GCRV-II, supporting robust replication (at least peak titer 1.38 × 108 pfu/mL) with characteristic cytopathic effects (CPE), viral inclusion bodies (VIBs), and autophagosome-mediated release. Importantly, GCRV-II propagated in GCB75 retains full virulence, inducing 100% mortality in grass carp with classic GCHD symptoms. This first brain-derived astrocyte model for GCRV-II fills a critical gap, enabling studies on neurotropism and host-virus interactions. Its potential spans pathogenesis research, antiviral drug screening, and vaccine development, addressing urgent needs in aquaculture disease control.

Sex-specific differences in disease resistance are well-documented in vertebrates, with immune responses often influenced by sex hormones and other physiological factors. However, the relationship between gut microbiota and immunity in driving these differences is not fully understood, particularly in non-mammalian vertebrates such as teleost fish. This study explores the role of sexual dimorphism in gut immunity and microbiota dynamics in juvenile common carp (Cyprinus carpio) following infection with the spring viremia of carp virus (SVCV). Using transcriptomic analysis and 16S rRNA sequencing, we assessed gut microbiota and immune responses in male, female, and all-female carps. Females exhibited heightened antiviral and inflammatory responses, which were associated with significant bacterial translocation and microbial dysbiosis. In contrast, males showed greater disease resistance and maintained a more stable gut microbiota. These results emphasize a significant correlation between changes in gut microbiota and sex-specific immune responses and provide valuable insights into the mechanisms underlying immunological dimorphism in teleost fish. These findings have important implications for improving aquaculture management and developing targeted disease control strategies.

Postbiotics are functional metabolites derived from bacteria that have been shown to promote growth, alleviate stress in aquatic organisms, and enhance the host immune system. They offer unique advantages over traditional probiotics in terms of safety and gastrointestinal stability, highlighting their tremendous potential in aquaculture. However, the variety of postbiotics formulations available for aquaculture and the research on their effects on host growth and disease resistance remain limited. In our study, we utilized a potentially applicable aquaculture strain of Bacillus velezensis to produce postbiotics and evaluate their modulatory effects on growth and immune regulation in largemouth bass. Our results demonstrate that supplementation with an appropriate concentration of postbiotics significantly promotes growth and gut health in largemouth bass, with the most pronounced effects observed in the 50 mL/kg group. Moreover, postbiotics feeding induced the expression of innate antiviral genes (dhx58, trim25, mx1, and ifnγ) and adaptive immune-related genes (igm, igt, cd8α, and cd4) in the gut and head kidney and markedly enhanced the activities of serum innate immune enzymes, including lysozyme, superoxide dismutase, and acid phosphatase. Importantly, postbiotics supplementation increased the proportion of IgM+ B cells and IgM secretion, thereby activating humoral immune responses and mediating enhanced antiviral defense in largemouth bass. In conclusion, this study systematically investigated the optimal effective concentration of Bacillus velezensis-fermented postbiotics for largemouth bass and demonstrated its important role in promoting host growth and enhancing disease resistance. These findings provide a theoretical foundation and strategic support for the application of postbiotics in antiviral control in aquaculture.

In vertebrates, the development of intestinal immunity is closely associated with dynamic changes in the gut microbiota. However, stage-associated differences in intestinal immunity and gut microbial communities remain poorly characterized in teleost fish. In this study, transcriptomic analysis combined with 16S rRNA gene sequencing was employed to characterize intestinal immunity and gut microbial communities in juvenile and adult common carp (Cyprinus carpio). Transcriptomic profiling revealed marked developmental differences in intestinal immune function. Juvenile carp exhibited a predominantly innate immune phenotype, characterized by elevated expression of pro-inflammatory cytokines, antimicrobial peptides, and lysozyme-related genes. This immune profile was accompanied by enhanced mucosal barrier function and a relatively pro-inflammatory intestinal environment. In contrast, adult carp displayed increased expression of genes associated with adaptive immunity, suggesting that adult common carp exhibit relatively stronger adaptive immune characteristics than juvenile fish. Gut microbiota analysis demonstrated significant stage-dependent differences in microbial diversity and community composition. Juvenile fish were enriched with bacterial taxa potentially associated with innate immune activation, whereas adult fish harbored distinct microbial communities linked to intestinal homeostasis and barrier maintenance. Furthermore, correlation analyses identified significant associations between specific microbial taxa and innate immune-related gene expression, suggesting a close association between gut microbiota composition and intestinal immune characteristics in juvenile and adult common carp. Collectively, these findings reveal stage-associated differences in intestinal immunity and gut microbial communities between juvenile and adult common carp, thereby providing insights into intestinal immune characteristics at different developmental stages in teleost fish.

Aquaculture faces disease outbreaks that cause significant economic losses. Yeast (3- glucan, a bioactive component of yeast cell walls, enhances intestinal immune function, though its underlying mechanism remains unclear. Herein, this study used coho salmon (20.02 +/- 0.15 g), with yeast (3- glucan added to the basal diet at concentrations of 0, 125, 250, 500, and 1000 mg/kg. Each treatment group included three replicates, with 20 fish per replicate, and the cultivation period lasted for 8 weeks. This study demonstrated that the dietary inclusion of yeast (3- glucan significantly enhanced the growth performance of coho salmon and markedly improved intestinal physical barrier function, as evidenced by the upregulation of zo1, zo2, occludin, claudin15, and claudin19 gene expression. Additionally, it significantly strengthened the intestinal immune barrier, as reflected by the upregulated expression of IgD, IgM, IgT, il-15, and tgf-beta 1. Notably, our findings also suggest that the immunomodulatory role of yeast (3- glucan lies in its regulation of intestinal probiotics ( Clostridium sensu stricto 1 , Romboutsia, Bacillus, and Turicibacter), which mediate lipid metabolism, leading to the production of unsaturated fatty acids and antimicrobial peptides, thereby strengthening the host's ability to resist pathogenic invasions. Furthermore, in vitro experiments demonstrated that the intestinal mucus from coho salmon fed yeast (3- glucan significantly inhibited viral invasion. Our findings demonstrate that yeast (3- glucan enhances host immune function by improving the composition of the gut microbiota and its metabolic products, thereby alleviating virus-induced intestinal damage.

Background/Objectives: Nocardia seriolae and Aeromonas veronii are two important pathogens that can affect a wide range of fish species and cause substantial economic losses. However, a vaccine that simultaneously protects fish from these two bacterial infections is not yet available. Methods: Three formalin-inactivated whole-cell vaccines prepared from N. seriolae and A. veronii (Monovalent Av, Monovalent Ns and Bivalent Av-Ns) were generated, and their efficacy was evaluated through a range of tests. The immune-related gene expression in the spleen and head kidney, enzyme activity, and specific antibody levels in serum were also detected. Results: All groups of vaccinated fish exhibited increased serum enzymatic activity compared with control fish, which peaked at week 3 after vaccination; in particular, that of the Bivalent Av-Ns group increased remarkably. The expression of immune-related genes in the spleen, head, and kidneys increased after immunization and were significantly enhanced (p < 0.05) in the bivalent vaccine group. Specific antibodies were produced at the 1st wpv, peaked at the 4th to the 5th wpv, and then decreased at the 6th wpv in all vaccinated groups. The Monovalent Av and Monovalent Ns against A. veronii and N. seriolae showed 56.67% and 22.22% RPS, respectively. Moreover, Bivalent Av-Ns offered 33.33% and 76.67% RPS for single infection with N. seriolae or A. veronii, as well as providing 44.44% RPS for dual infection with combined N. seriolae and A. veronii. Conclusions: Our findings indicate that the administration of the A. veronii and N. seriolae bivalent vaccine can protect largemouth bass from both bacterial infections.

Precise identification of protective antigenic epitopes is critical not only for eliciting robust and specific immune responses but also for providing a vital foundation for vaccine development and monoclonal antibody engineering. In this study, we cloned six constructs, five truncated major capsid protein (MCP) of largemouth bass virus (LMBV) fragments (MCP-1, MCP-2, MCP-3, MCP-4, MCP-1&3) and the full-length MCP, into pET-32a expressed them in a prokaryotic system, and evaluated their antigenicity. Furthermore, the antigen protein was administered to fish in a four-week immunization trial. This study demonstrated that MCP-3 markedly enhanced both innate and adaptive immune responses. We also observed a pronounced expansion of IgM+ and IgT+ B cell populations. Notably, MCP-3 enhanced the titers of both serum and mucosal antigen-specific IgM and conferred the strongest neutralizing activity against LMBV. Challenge experiments further confirmed that the MCP-3 group achieved the highest relative percent survival (RPS = 88.89 %) and the lowest post-infection tissue viral loads. In summary, these results indicate that we have identified MCP-3 as a key protective antigen of LMBV, which may serve as a promising target for vaccine development to prevent and control LMBV infection.

The mucosal immune system plays a critical role in defending the body against external pathogens and preserving homeostasis. The polymeric immunoglobulin receptor (PIGR) is a critical component of this system, responsible for facilitating the transport and secretion of soluble polymeric immunoglobulins across epithelial cells, thereby contributing to immune defense. In zebrafish, a pIgR-like (pIgRL) family exists, among which pIgRL4.2 is highly expressed in multiple immune organs, suggesting its potential role in immunity. In this study, spring viremia of carp virus (SVCV) infection significantly downregulates pIgRL4.2 expression. Conversely, overexpression of pIgRL4.2 in EPC cells markedly delays SVCV-induced cytopathic effects and effectively suppresses SVCV replication. Additionally, overexpression of pIgRL4.2 enhances IFN activation induced by both poly(I:C) treatment and SVCV infection. Furthermore, CRISPR/Cas9 was used to generate pIgRL4.2-null zebrafish, and disruption of pIgRL4.2 in zebrafish has been demonstrated to result in a reduction in survival rates following SVCV challenge. This is accompanied by a consistent downregulation of antiviral responsive genes, concomitant with an increase in SVCV replication in pIgRL4.2-deficient zebrafish. Therefore, this study demonstrates that pIgRL4.2 inhibits viral replication by positively regulating the IFN signaling pathway.

Bacterial sepsis caused by Aeromonas hydrophila (A. hydrophila) and infectious spleen and kidney necrosis virus disease (ISKNVD) caused by infectious spleen and kidney necrosis virus (ISKNV) frequently result in significant mortality among Chinese perch (Siniperca chuatsi). Co-infection of mandarin fish with A. hydrophila and ISKNV occurs from time to time. In this study, a visual detection method for ISKNV and A. hydrophila was developed, using loop-mediated isothermal amplification (LAMP) and pre-addition of hydroxynaphthol blue. Primers for amplifying LAMP in the same system were designed based on the conserved regions of the MCP gene of infectious spleen and kidney necrosis virus, as well as the hlyA gene of A. hydrophila. The results showed that this method amplified bright trapezoidal bands in the presence of only A. hydrophila or ISKNV and both, with sky blue for positive amplification and violet for negative amplification. There was no cross-reactivity with other pathogens, and fragments of 182 bp, 171 bp and 163 bp appeared after digestion of the A. hydrophila LAMP product and 136 bp, 117 bp and 96 bp appeared after digestion of the ISKNV LAMP product. This holds true even when both positive products are present simultaneously. The minimum detection limit of this method was 100 fg for A. hydrophila and 100 fg for ISKNV, and the minimum detection limit for the mixed template was 1 pg. Overall, this method has high sensitivity and specificity to rapidly detect and distinguish between the two pathogens.

The severe damage caused by public health emergencies in recent years indicates that epitope identification and vaccine development remain potential strategies to stop viral epidemics. Despite challenges such as comprehensive virus analysis, computer simulations and antibody-based reverse vaccinology can rapidly localize antigenic epitopes without the need to resolve protein functions, thereby supporting vaccine design. In previous studies, we developed a strategy for peptide vaccine design based on simulated epitopes for tilapia lake virus (TiLV). To further improve vaccine immunogenicity and capitalize on the significance of antibodies for epitope screening, this study presents a structural modification strategy based on antigen-antibody docking for reverse vaccinology in conjunction with single amino acid mutagenesis inducing a robust immune response in tilapia. A high-affinity scFv1 was obtained through screening from the constructed single-chain antibody fragment (scFv) library targeting TiLV. Subsequently, the key dominant epitope 385GYQLASEIRGTIPLSS400 was identified through alanine scanning and antibody binding assays based on the docking. Based on the epitope and scFv1 structure, an enhanced epitope vaccine, S390F, was obtained through single amino acid random mutation modification. Boost immunizations of tilapia elicited significant responses in IgM levels and non-specific immune enzyme activities, upregulated the expression of immune-related genes, and improved RPS of TiLV infection by approximately 25%, effectively preventing viral attack. Meanwhile, S390F activated the expression of the co-stimulatory molecule CD40, suggesting a response from immune cells. The development of peptide-based vaccines through computer-aided design effectively highlights the importance of antibodies and offers valuable insights for the control of emerging diseases. IMPORTANCE:Public health emergencies pose significant threats to both human and environmental health, and the rapid control of emerging diseases often remains challenging due to their unknown characteristics. In this context, vaccines have historically been instrumental in the fight against major diseases, with epitope vaccines emerging as a preferred approach due to their precision and efficacy. In previous studies, we developed a strategy for peptide vaccine design based on simulated epitopes for tilapia lake virus (TiLV). To further improve vaccine immunogenicity and capitalize on the significance of antibodies for epitope screening, this study presents a structural modification strategy based on antigen-antibody docking for reverse vaccinology in conjunction with single amino acid mutagenesis inducing a robust immune response in tilapia. We believe that our findings are highly relevant to the field and contribute to the ongoing efforts to combat public health threats.