Bacterial skin pathogens (e.g., Moritella viscosa and Tenacibaculum spp) are affecting fish health and welfare in farmed salmon during seawater stage (Sommerset et al., 2022) . Both bacteria can be the primary cause of an infection, or establish a “consortium of pathogens” together with others such as Aliivibrio wodanis (Karlsen et al., 2014), which increases fish mortality. To control this situation, fish-specific pathogen vaccines are used in salmon farming. However, the effectiveness of some of these immunological strategies still do not meet industry expectations , especially those associated with long-term protection (Figueroa et al., 2022) . In addition, there is a lack of vaccines against emerging bacteria . Interestingly, Trained Immunity-based Vaccines (TIbVs) have been reported in higher vertebrates during recent years (Sánchez-Ramón et al., 2018) . This approach focuses on the innate response and how the vaccines can provide immunological protection against challenges that are not related to the vaccine antigens (Sánchez-Ramón et al., 2018) . Considering this, TIbVs could be relevant to explore in fish, since there are currently no specific vaccines available against Tenacibaculum spp .
In the Resilient Salmon project (RCN 294821), in coordination with Foods of Norway, a Centre for Research- based Innovation at the Norwegian University of Life Sciences (NMBU) , we are exploring nutritional programming with novel aquafeeds containing fucoidan isolated from sugar kelp (Saccharina latissima) to modulate trained immunity-related responses against Tenacibaculum maritimum in vaccinated Atlantic salmon (Salmo salar) .
Materials and methods
Atlantic salmon pre-smolts were maintained at the NMBU Center for Sustainable Aquaculture. B efore the feeding trial, fish were vaccinated with ALPHA JECT micro 6 vaccine (PHARMAQ) which contained ant igens from Aeromonas salmonicida , Listonella anguillarum O1 and O2α, Vibrio salmonicida , M. viscosa and infectious pancreatic necrosis virus. Later, fish were randomly distributed into six 300 L tanks (45 fish per tank) supplied with recirculated fresh water. Each tank was assigned one of three diets (CD: commercial-like diet; FD : CD + 0.2% fucoidan extract from sugar kelp; MG : CD + 0.2% MacroGard®) for four weeks.
After this period, 35 fish per tank were transferred to seawater at the Norwegian Institute for Water Research (Solbergstrand, Norway) and fed the CD for 4 weeks before being intraperitoneal ly injected with inactivated T. maritimum. S amples ( e.g., distal intestine: DI and head kidney: HK) were taken from six fish per tank at different time points: at the end of freshwater stage, after four weeks in seawater, one day post-stimulation and seven days post-stimulation . Total RNA was extracted from the tissue samples, and qPCR was performed to determine the gene expression of specific immune-related biomarkers.
Results and Discussion
I n the different dietary groups, a n immunological profile that could be associated with trained immunity (up-regulation of type-1 immune biomarkers such as il-1b , il-8 and tnf-a at the end of the first stimulus, a decrease in their expression during rest time, and an enhanced response after the secondary stimulus) was detected in HK and DI of vaccinated Atlantic salmon. In higher vertebrates , there are reports showing similar results (Sánchez-Ramón et al., 2018), which have demonstrated the ability of vaccines to modulate the innate immunity against bacterial challenges that are not related to the vaccine antigens. This is interesting to explore in fish, since that there are no specific vaccines against T. maritimum , and because this bacterium is causing a higher number of natural outbreaks in salmon farms in recent years (Sommerset et al., 2022) . Furthermore, the inclusion of fucoidan in novel aquafeeds as a bioactive additive showed up- regulation (one day post-stimulation with T. maritimum ) of genes related to effector molecules such as antimicrobial peptides (e.g., hepcidin) , which are crucial in the innate humoral response . Antimicrobial peptides are part of the first line of defence against a wide spectrum of pathogens and may contribute to a decrease in the use of antibiotics (Valero et al., 2020).
Vaccines could induce the modulation of biomarkers a ssociated with t rained i mmunity against T. maritimum in HK and DI of Atlantic salmon. Moreover, nutritional programm ing using fucoidan may enhance the innate humoral response (e.g., antimicrobial peptides) related to TIbVs in immunological organs . This data supports the proposal to use fucoidan as a feed additive in novel aquafeeds for Atlantic salmon to improve fish health and welfare and to strengthen the sustainability of the aquaculture industry.
Karlsen C, et al. 2014. Co-infection of Atlantic salmon (Salmo salar ), by Moritella viscosa and Aliivibrio wodanis , development of disease and host colonization. Vet. Microbiol. 171(1–2): 112-121.
Figueroa C, et al. 2022. Commercial Vaccines Do Not Confer Protection against Two Genogroups of Piscirickettsia salmonis , LF-89 and EM-90, in Atlantic Salmon . Biology , 11(7): 993.
Sánchez-Ramón S, et al. 2018. Trained Immunity-Based Vaccines: A New Paradigm for the Development of Broad-Spectrum Anti-infectious Formulations. Front. Immunol. 9: 2936.
Sommerset I, et al. 2022. Norwegian Fish Health Report 2021, Norwegian Veterinary Institute Report, series #2a/2022.
Valero Y, et al. 2020. Antimicrobial peptides from fish: beyond the fight against pathogens. Rev Aquacult 12: 224-253