Author: marina02

Dietary Supplementation with Euglena gracilis Enhances Mucosal Immune Responses in Fish

Introduction: The aquaculture industry is rapidly expanding due to global population growth and increasing consumer acceptance of farmed fish. However, disease outbreaks remain a major constrain, often exacerbated by the aquatic environment. To address and overcome this challenge, the scientific community is seeking sustainable solutions that enhance fish immunity while reducing pressure on wild fish populations.

Microalgae, rich in bioactive compounds, have shown promise in this context. Euglena gracilis, a microalga from the Euglenales family, produces high levels of β-1,3-glucan, omega-3 fatty acids, protein, and vitamins C and E—compounds known to stimulate the immune system. Therefore, dietary supplementation with E. gracilis may support fish immune function.

This study aimed to evaluate the immunomodulatory potential of E. gracilis bioactive compounds using European seabass (Dicentrarchus labrax) as a model species.

Methodology: European seabass were randomly assigned to five dietary groups, each in triplicate (70 fish per tank), and fed one of five diets for 15 days: a commercial control diet, G1, G2, J1, and J2. At the end of the feeding period, T0 samples (3 fish per tank) were collected. The remaining fish were then challenged with Tenacibaculum maritimum through a 1-hour bath in aerated seawater. After 24 hours, tissues (head kidney, gut, and skin) were collected for gene expression analysis. Mortality was monitored for 12 days.

Results: The effect of bioactive compounds from E. gracilis on mucosal immunity was assessed through mRNA expression of immune-related genes. Results from the present study showed a transversal pro-inflammatory response against T. maritimum across all tissues regardless the dietary treatments. Iron metabolism genes were notably affected, with elevated hepcidin expression and corresponding downregulation of the iron exporter ferroportin.

Among dietary treatments, J2 showed the strongest local immunomodulatory effects, marked by the upregulation of several immune-related genes—demonstrating the immune-boosting potential of this particular microalga.

Conclusion: Diets supplemented with compounds extracted from E. gracilis effectively modulated immune responses and delayed disease progression in European seabass. These findings underscore the importance of mucosal immunity in pathogen defence and support the use of microalgae as a sustainable alternative to chemical prophylaxis. Such approaches contribute to a more resilient aquaculture industry and enhanced food security.

This work was funded by EEA Grants through project PT-INNOVATION-0102-MICROBOOST.

1. SARAIVA, MARCIA, CIIMAR, Presenter
2. Silva-Brito, Francisca, CIIMAR, Author
3. Santos, Paulo, CIIMAR, Author
4. Conceição, Luis E. C., Sparos, Author
5. Cunha, Pedro, GreenCoLab, Author
6. Cardoso, Helena, Allmicroalgae, Author
7. Pereira, Helena, Allmicroalgae, Author
8. Gonçalves, Ana T., Sparos, Author
9. Costas, Benjamin, CIIMAR, Author

Development of an mRNA-lipid nanoparticle vaccine against infectious salmon anemia

Introduction: mRNA vaccines demonstrated strong prophylactic efficacy against SARS-CoV-2 infections during the COVID-19 pandemic, which increased the interest for applying this technology to fight other viral diseases. Recently, we tested and confirmed the principle of mRNA vaccination in lipid nanoparticles (LNP) in Atlantic salmon using a reporter gene. This indicates that the vaccine platform has potential to efficiently express antigens and elicit protective immune responses in salmonids. In this study we further aim to develop and evaluate the efficiency of an mRNA vaccine against infectious salmon anemia (ISA) in Atlantic salmon.

Methodology: Two different vaccine inserts were designed, encoding the ISAV hemagglutinin-esterase (HE) and fusion (F) surface proteins. The mRNA was chemically modified with N1-methyl-pseudouridine and encapsulated in LNP prior to intramuscular administration in pre-smolt Atlantic salmon. A cohabitation model, where vaccinated fish were exposed to virus-injected shedder fish 8 weeks post-vaccination, was used to evaluate vaccine efficacy. Two control groups were included: one injected with PBS and another with mock mRNA-LNP. The experiment was performed in two separate tanks, one for monitoring mortality and the other for assessing viral load.

Mortality was recorded daily during the challenge period. Viral RNA load at 11-, 12-. 13-, and 14-weeks post vaccination was quantified using qPCR.

Additionally, to screen for the presence of neutralizing antibodies in vaccinated fish a neutralization assay and will be performed with samples prior to and after viral challenge.

Results and conclusion: The study is ongoing, and the results will be presented at the meeting.

Keywords: Atlantic salmon, mRNA vaccines, infectious salmon anemia

Funding: FHF – Norwegian Seafood Research Fund, # 901746.

1. Dahl, Lars Ole, Norwegian University of Life Sciences, Presenter
2. Braaen, Stine, Norwegian University of Life Sciences, Author
3. Wessel, Øystein, Norwegian University of Life Sciences, Author
4. Hak, Sjoerd, SINTEF Industry, Department of Biotechnology and Nanomedicine, Author
5. Borgos, Sven Even, SINTEF Industry, Department of Biotechnology and Nanomedicine, Author
6. Molska, Alicja, SINTEF Industry, Department of Biotechnology and Nanomedicine, Author
7. Hernandez, Fernando de Carlos, SINTEF Industry, Department of Biotechnology and Nanomedicine, Author
8. Parot, Jérémie, SINTEF Industry, Department of Biotechnology and Nanomedicine, Author
9. Fosse, Johanne Hol, Norwegian Veterinary Institute, Author
10. Rimstad, Espen, Norwegian University of Life Sciences, Author

Extracellular vesicles secreted by Pediococcus pentosaceus exhibited the resistance to megalocytivirus in barramundi kidney cell line

Barramundi (Lates calcarifer), an economically important aquaculture species in Taiwan, is prone to infectious disease outbreaks under high-density farming conditions. Among these, red sea bream iridovirus (RSIV) is one of the most severe pathogens causing high mortality in both farmed and wild fish populations worldwide. RSIV is a member of the Megalocytivirus genus, belonging to Iridoviridae family. Studies have shown that barramundi infected with RSIV at 80 days post-hatching can experience up to 100% cumulative mortality within 14 days. Recent studies have indicated that extracellular vesicles (EVs) also participate in antiviral mechanisms. EVs are membrane-bound particles released by cells, including bacteria, carrying specific DNA, RNA, proteins, and lipids, and serve as mediators of intercellular communication that regulate physiological and pathological processes. Probiotics are widely used in aquaculture to enhance fish health. Previous studies have shown that Pediococcus pentosaceus strain 4012 (LAB4012) can prevent bacterial infections and viral diseases such as nervous necrosis virus (NNV) and grouper iridovirus (GIV) in fish. However, it remains unclear whether the extracellular vesicles (EV4012) secreted by LAB4012 are the primary factor responsible for its antiviral effects and what the underlying antiviral mechanisms are. Therefore, this study aims to purified EV4012 and to assess whether EV4012 can resist RSIV infection.Using tangential flow filtration (TFF), the EV4012 was purified from LAB4012 culture supernatant. The characteristics of EV4012, including structures, particle sizes, and particle amounts, were analyzed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Moreover, we investigated the antiviral activity of EV4012 against RSIV in barramundi kidney cell line. The results showed that EV4012 could neutralize RSIV. Therefore, EV4012 shows promise as a preventive agent against RSIV in barramundi aquaculture.

1. GUO, YI-TING, INSTITUTE OF FISHERIES SCIENCE, NATIONAL TAIWAN UNIVERSITY, Presenter
2. Wu, Yu-Chi, INSTITUTE OF FISHERIES SCIENCE, NATIONAL TAIWAN UNIVERSITY, Author

development of oral vaccines for aquaculture using spore-display technology

Oral vaccines are highly demanded by the aquaculture sector to allow mass delivery of antigens. Despite this, most commercially available vaccines are injectable, being labor-intensive, expensive, and require individual handling of fish.One possible strategy to create injection-free vaccine delivery vehicles is the use of bacterial spores,extremely resistant structures with wide biotechnological applications.Bacterialspores, particularly those ofBacillus subtilis, are promising delivery vehicles of different molecules through surface display technology. Theextreme resistance of B. subtilisspores, which guarantees passagethrough the harsh gut environmentwithout losing characteristics, is the basis for their oral applications.Spores’ direct incorporation into animal feed, circumventing further protection processes, such as encapsulation, together with their simple production andlong shelf-lifewithout the need for refrigeration, are also attractive characteristics from the industrial point of view. Adding to this “needle-free” and “refrigeration-free” potential,B. subtilisspores adjuvant propertiesand contribution to GALT development,increase their potential as oral delivery systems of antigens.In this work,we displayed at the surface ofB. subtilisspores, antigens from different problematic aquaculture pathogens (e.g.,Vibriospp.,Photobacterium damselae, Edwrdsiella tarda,Aeromonas hydrophila).The developed Sporovaccines were first tested by immersion in zebrafish larvae (Danio rerio). When previously treated with the antigens-carrying spores,zebrafish survival upon a bacterial challenge with each pathogen, increased up to 90% depending on the pathogen targeted.Further, when added to commercial feed and used to orally vaccinate European seabass juveniles, Sporovaccines increased fish survival from 60 to 86%. We then evaluated the effecton the expression of immune-related genes by using an epithelial gut cell line from rainbow trout (Oncorhynchus mykiss)(RTgutGC cell line). Transcription effects were evaluated by real-time quantitative PCR analysis of immune-related genes (e.g., TNF-α,IL-1β,IL-8, COX-2, Hsp70, Casp3a2). Sporovaccines significantly induced the transcription of the pro-inflammatory cytokine IL-1βand pro-inflammatory chemokine IL-8.Our results indicate thatB. subtilisspores can effectively be used as carriers for massive delivery of antigens in fish. This work was funded byFEDER, POCI, PT2020, and FCT/MCTES through project PTDC/CVT-CVT/2477/2021, PhD fellowship 2021.07724.BD (GG), CIIMAR Strategic Funding UIDB/04423/2020 and UIDP/04423/2020.

1. SERRA, CLAUDIA, CIIMAR-UP, Presenter
2. Gonçalves, Gabriela, CIIMAR-UP, Author
3. Santos, Rafaela, CIIMAR-UP, Author
4. Carvalho, Antonio Paulo, CIIMAR-UP, Author
5. Enes, Paula, CIIMAR-UP, Author
6. Díaz-Rosales, Patricia, CIIMAR-UP, Author
7. Couto, Ana, CIIMAR-UP, Author
8. Oliva-Teles, Aires, CIIMAR-UP, Author

Effects of Torenia concolor on immune regulation and resistance to megalocytivirus in barramundi (Lates calcarifer)

Red seabream iridovirus (RSIV) is a highly pathogenic virus that affects farmed barramundi in Asia, leading to high mortality and significant economic losses. Torenia concolor (TC) has bioactive compounds with anti-inflammatory and antiviral properties. In this study, we examined the in vitro mechanisms by which boiled water extracts of TC (TCE) exert anti-RSIV activity. The results indicated that TCE could inhibit RSIV infection by blocking the RSIV receptor and suppressing viral replication, thereby exerting antiviral effects. To evaluate the effects of TC on immune regulation and anti-RSIV activity in vivo, barramundi were fed diets containing different concentrations of TC, including 0 g/kg (TC0), 5 g/kg (TC5), or 10 g/kg (TC10) for 28 days. The results revealed that the supplementation of TC had no significant effect on barramundi growth performance. In addition, immune gene expression (IL-1β, TNF-α, IgM, Mx, SOD, CAT, C3, and lysozyme) was analyzed in the head kidney and spleen of barramundi fed with TC-containing diets for 1, 2, 3, and 4 weeks. The results showed that the gene expression of IL-1β, Mx and C3 was significantly increased in TC5 and TC10 groups. Furthermore, after feeding with TC for 4 weeks and challenging with RSIV, the supplementation with TC could lower the mortality in RSIV-challenged barramundi. In conclusion, TC regulated the immune response and enhanced resistance to RSIV in barramundi, highlighting its potential as an antiviral functional feed additive in aquaculture.

1. LIN, ZHE-LI, INSTITUTE OF FISHERIES SCIENCE, NATIONAL TAIWAN UNIVERSITY, TAIPEI 10617, TAIWAN, Presenter
2. WU, YU-CHI, INSTITUTE OF FISHERIES SCIENCE, NATIONAL TAIWAN UNIVERSITY, TAIPEI 10617, TAIWAN, Author

A Method a pH-Responsive Chitosan–Alginate Encapsulated Oral Vaccine Against Streptococcus iniae in Barramundi (Lates calcarifer)

Introduction
Streptococcus iniae is a significant pathogen in aquaculture, associated with high mortality in juvenile barramundi (Lates calcarifer). While injectable vaccines using formalin-inactivated antigens with oil adjuvants confer strong protection, their use is constrained by handling requirements and limited feasibility for booster application. Oral vaccination offers a practical alternative, especially for smaller fish, but remains hindered by antigen degradation in the gastrointestinal tract and limited mucosal uptake. This study investigates a pH-responsive oral vaccine using chitosan-alginate encapsulation to improve antigen protection and delivery.

Methodology
A layer-by-layer encapsulation method was explored to formulate microparticles containing whole-cell formalin-inactivated S. iniae, using chitosan, TPP, and alginate. Fourteen formulations were screened based on particle size, zeta potential (depicting surface charge), and structural stability in simulated gastric and intestinal fluids. A lead candidate was selected for a fully blinded in vivo trial in barramundi juveniles (~10 g) with to oral vaccine, injectable vaccine, and control groups. Blood, skin, and gut mucus were sampled at 7, 14, 21, and 28 days post-vaccination. Antibody responses were measured by ELISA, and fish were challenged with S. iniae at 2 and 4 weeks post-vaccination. The trial is ongoing from April–July 2025.

Results

Zeta potential shifted from –10.40 ± 0.85 mV (pre-chitosan) to +17.19 ± 1.20 to +26.68 ± 2.64 mV after chitosan coating, with further variation following alginate encapsulation. Microparticles formed via chitosan–TPP–alginate layering had median sizes ranging from 3.56 ± 0.18 to 43.63 ± 4.41 µm. These surface charge changes, supported by SEM imaging, confirmed successful formation of a polymer matrix entrapping whole-cell antigens. Incubation in simulated gastric fluid (pH 2-4, pepsin) and simulated intestinal fluid (pH 7-9, trypsin) allows selection of optimised formulations with optimal gastric stability and properties suitable for mucosal delivery at intestinal conditions.

Conclusions

This study evaluates the potential of pH-responsive chitosan–alginate encapsulation for effective oral delivery of S. iniae antigens in barramundi. By protecting antigens during gastric transit and enabling controlled release in the intestine, this approach addresses key limitations of conventional oral vaccines. The platform offers a scalable, less invasive alternative to injectable delivery and may enable broader vaccine access across aquaculture systems.

1. TRAN, LAM, School of the Environments, The University of Queensland, Presenter
2. Baldelli, Alberto, School of Agriculture and Food Sustainability, The University of Queensland, Author
3. Saming, Francisca, Sydney School of Veterinary Science, The University of Sydney, Author
4. Grondahl, Lisbeth, School of Chemistry and Molecular Biosciences, The University of Queensland, Author
5. Barnes, Andrew, School of the Environments, The University of Queensland, Author

Dietary administration of Shewanella putrefaciens Pdp11 extracts modulates gene expression of RGNNV-infected European sea bass

Introduction

Viral encephalopathy and retinopathy is a neurological disease, caused by the nervous necrosis virus, that provokes high mortalities in cultured sea bass (Dicentrarchus labrax). As a prophylactic measure, our previous studies explored the use of food supplemented with sonicated extracts of Shewanella putrefaciens Pdp11 (SpPdp11), resulting in a significantly increased survival rates of RGNNV-challenged sea bass (Moreno et al., 2023). The current study is a step forward, looking into the effects of this postbiotic on the transcriptomic response of RGNNV-infected sea bass.

Materials and Methods

Two groups of sea bass were established: control feed fish, and fish receiving feed supplemented with SpPdp11 extracts. After 30 days of feeding, fish were intramuscularly injected with RGNNV, and RNA was extracted from brains sampled at 72 h post-infection. RNAseq was performed to obtain 30 million 150-bp reads per sample using Illumina HiSeq 2000. The SPETO-RNA workflow (Delgado et al., 2023) was applied for data analysis. A database of KO terms associated with sea bass genes was generated with Blast Koala and functional enrichment analysis performed through KE pathways.

Results and Discussion

The analysis of the gene expression following the diet supplemented with the postbiotic revealed increased expression of pathways related to the nervous system. Similarly, activation of the adipocytokine signaling pathway, where interferon plays a key role, was observed, supporting the hypothesis of an immunomodulatory effect of this diet prior to infection.

The analysis of RGNNV-infected fish showed deregulation of 6,819 genes (29.5% of the annotated genes), primarily involved in responses to infections and cellular damage, and activation of pathways related to neurological damage, consistent with the altered behavior typical of RGNNV infections. In addition, we recorded inhibition in pathways associated with nerve stimuli. These results provide a deeper understanding of how RGNNV affects the nervous system.

Finally, the analysis of infected fish after feeding with the postbiotic showed significant decrease in the number of genes inhibited by the viral infection in sea bass receiving SpPdp11. These are genes involved in synaptic signaling and neuronal development pathways, suggesting a modulating effect of the diet supplemented with SpPdp11 extracts on pathways leading to nervous system degeneration in sea bass RGNNV infections.

Funding: Regional Project (Junta de Andalucía), ref: P18-RT-1067 and Spanish National Project, ref. PID2020-115954RB-I00.

1. GARCIA-ROSADO, ESTHER, UNIVERSITY OF MALAGA, Presenter
2. Moreno, Patricia, UNIVERSITY OF MALAGA, Author
3. Diaz-Martínez, Luis, UNIVERSITY OF MALAGA, Author
4. Bautista, Rocio, UNIVERSITY OF MALAGA, Author
5. Moriñigo, Miguel A., UNIVERSITY OF MALAGA, Author
6. Balebona, M. Carmen, UNIVERSITY OF MALAGA, Author
7. Béjar, Julia, UNIVERSITY OF MALAGA, Author
8. Alonso, M. Carmen, UNIVERSITY OF MALAGA, Author

Subunit vaccine development for prevention of Tenacibaculosis in Atlantic salmon

Introduction: The development of ulcers due to bacterial infections in farmed Atlantic salmon (Salmo salar) poses a significant and ongoing challenge for the Norwegian aquaculture industry. Outbreaks of tenacibaculosis, caused by Tenacibaculum finnmarkense, present severe issues for newly transferred smolt, manifesting as ulcer formation and acute mortality. Currently, there is no effective vaccine available to mitigate this disease. This study aims to evaluate whether immunization with recombinant expressed antigens derived from T. finnmarkense, could serve as a strategy to provide protection against tenacibaculosis.

Methodology: Genome analysis, coupled with qPCR, was employed to identify genes potentially involved in interactions with the extracellular matrix. Two recombinantly expressed proteins were utilized in preliminary trials to demonstrate that intraperitoneal injection, as opposed to nasal or bath exposure, significantly enhanced serum antibody responses in Atlantic salmon. Subsequently, a new cohort of fish was intraperitoneally vaccinated prior to a challenge experiment with T. finnmarkense to assess the antibody responses and mortality rates in the immunized fish.

Results and Conclusions: Infected fish exhibited characteristic symptoms of tenacibaculosis, including histopathological features consistent with previous descriptions of Tenacibaculum infections. Ulcers were predominantly populated by filamentous rod-shaped bacteria, with significant bacterial presence in collagen-rich tissues. Verified antibody responses in vaccinated groups confirmed the protective effect of the immunization, evidenced by reduced mortality rates in immunized fish. These promising results support the continued development of a recombinant protein-based vaccine against tenacibaculosis in Atlantic salmon.

1. Karslen, Christian, Nofima AS, Norway, Presenter
2. Sveen, Lene, Nofima AS, Norway, Author
3. Småge, Sverre B., Cermaq Group AS, Norway, Author
4. Karlsen, Marius, PHARMAQ part of Zoetis, Norway, Author
5. Duesund, Henrik, Cermaq Group AS, Norway, Author
6. Sandtrø, Ane, PHARMAQ part of Zoetis, Norway, Author

Dietary administration of the host defense peptide piscidin 1 modulates immunity and improve fillet quality in gilthead seabream (Sparus aurata)

Host-defense peptides (HDPs) represent promising candidates for functional and therapeutic applications. Nevertheless, their oral administration is constrained by issues of instability and undesirable organoleptic properties [1]. In this study, we encapsulated piscidin 1 (P1) within carrageenan and pectin microparticles to develop an oral delivery system aimed at overcoming these challenges. P1, an HDP of gilthead seabream (Sparus aurata), comprises 25 amino acids. Piscidins, a family of HDPs found exclusively in teleost fish, are noted for their ability to form an amphipathic α-helix upon membrane binding and their potent antimicrobial activity [2]. Gilthead seabream were fed for 17 days a commercial diet (Skretting) supplemented with 0%, 0.05%, or 0.1% encapsulated P1. Mucus, gut, skin and brain were sampled on days 4 and 17 for humoral immune response (peroxidase, anti-protease, and lysozyme activities) and gene expression analysis (oxidative stress: sod, cat, gpx1, gpx4; cellular stress: hsp-70, hsp-90; anti-inflammatory response: il-10, tgf; adaptive immunity: mhc-II, ifn, il-7; pattern recognition receptors: tlr-5, tlr-7, tlr-8). Additionally, skin, eye, and muscle samples were collected on day 17 for colorimetric analysis. Results indicated a significant increase in anti-protease activity in fish fed with encapsulated P1. Gene expression analysis revealed that sod was the only gene consistently expressed across the gut–skin–brain axis, although the three organs exhibited distinct patterns of up- or down-regulation for the other genes studied. Colorimetric analysis demonstrated that treatment with encapsulated P1 enhanced fillet brightness. Overall, these findings suggest that encapsulated P1 possesses immunomodulatory properties and can improve fillet appearance, underscoring its potential as a therapeutic agent in aquaculture.

Acknowledgements

Claudia Marín-Parra (PRE2021-098414) has PhD grant. This study was funded by the Proyecto de investigación PID2020-113637RB-C21 financiado por MCIN/ AEI /10.13039/501100011033 and forms part of the ThinkInAzul programme supported by MCIN with funding from European Union Next Generation EU(PRTR-C17.I1) and by the Comunidad Autónoma de la Región de Murcia-Fundación Séneca (Spain).

Bibliography:

[1] A. Mohan, S.R.C.K. Rajendran, Q.S. He, L. Bazinet, C.C. Udenigwe, Encapsulation of food protein hydrolysates and peptides: a review, RSC Advances 5(97) (2015) 79270-79278.

[2] H. Cetuk, J. Maramba, M. Britt, A.J. Scott, R.K. Ernst, M. Mihailescu, M.L. Cotten, S. Sukharev, Differential Interactions of Piscidins with Phospholipids and Lipopolysaccharides at Membrane Interfaces, Langmuir 36(18) (2020) 5065-5077.

1. Marin-Parra, Claudia, University of Murcia, Presenter
2. Reboredo-González, Alba, Miguel Hernández University, Author
3. Valero-Cases, Estefanía, Miguel Hernández University, Author
4. Espinosa-Ruiz, Cristóbal, Institute of Agricultural and Environmental Research and Development of Murcia, Author
5. Esteban, María Ángeles, University of Murcia, Author

Bioengineered outer membrane vesicles as a platform for producing multivalent fish vaccines

Vaccination is crucial for controlling infectious outbreaks and reducing antibiotic use in aquaculture. Existing vaccines focus on bacterial and viral diseases, with limited coverage for parasites and fungi, and often provide incomplete protection. As a result, infectious diseases continue to cause major economic losses, highlighting the need for innovative and improved vaccination strategies. Outer membrane vesicles (OMVs) are nano-sized particles released by Gram-negative bacteria. They are promising vaccine candidates due to their immunostimulatory content, stability, and intrinsic adjuvanticity. OMVs can induce immune responses after oral administration and can be bioengineered to remove toxic elements and incorporate antigens from multiple pathogens, enabling multivalent vaccine design. Although OMVs are already used in licensed human vaccines (e.g., against Neisseria meningitidis), their application in fish vaccination remains largely unexplored. Extending OMV-based vaccine technology to aquaculture, particularly for oral delivery, would represent a major advance, offering easier administration, reduced costs, and minimal animal handling stress.

Recently, we showed that Photobacterium damselae subsp. piscicida (Phdp) releases high numbers of OMVs. Analysis of OMVs from the virulent strain MT1451 revealed the presence of several virulence factors, including the plasmid-encoded AIP56 toxin, making them lethal to European sea bass (Dicentrarchus labrax), and unsuitable for vaccination. However, we generated a plasmid-cured strain and showed that the AIP56-negative OMVs produced by this strain, administered i.p. with no adjuvant to sea bass, provided significant protection (35–38% RPS) against experimental Phdp infection.

In this work, we further enhanced the protective efficacy of these OMVs by deleting an additional toxin that targets macrophages and developed methodologies for incorporating heterologous antigens into the detoxified OMVs. For efficient incorporation, mature antigens were expressed in the detoxified Phdp strain fused to a Sec signal peptide for periplasmic targeting, under control of a strong promoter to maximize expression.

Using this approach, we successfully incorporated antigens from a hyper-virulent Phdp strain, Tenacibaculum maritimum, nervous necrosis virus (NNV), and Vibrio harveyi. I.p. immunization of sea bass with OMVs loaded with antigens from the hyper-virulent Phdp strain elicited antibody responses against the incorporated antigens, demonstrating the feasibility of using these OMVs as a platform for developing multivalent fish vaccines. Future studies should investigate their potential for oral immunization. Creating effective, orally administered, multivalent fish vaccines would enable large-scale low-stress vaccination, ultimately improving fish health and promoting more sustainable aquaculture practices.

1. LOUREIRO, INÊS, I3S – INSTITUTE FOR RESEARCH AND INNOVATION IN HEALTH, Presenter
2. dos Santos, Nuno MS, I3S – INSTITUTE FOR RESEARCH AND INNOVATION IN HEALTH, Author
3. do Vale, Ana, I3S – INSTITUTE FOR RESEARCH AND INNOVATION IN HEALTH, Author