Despite the availability of commercial vaccines against several pathogens, infectious diseases continue to cause substantial economic losses in aquaculture. Current vaccine development focuses on exploring antigen delivery systems that enable efficient needle-free, mass vaccination. Bacterial spores offer a promising platform for oral vaccine delivery, as they are highly resistant structures that can act as adjuvants and antigen carriers. This technology has been explored in recent years, mostly using spores from laboratory strains, for which a variety of genetic tools have been optimized. The use of spores of autochthonous probiotic strains for displaying antigens remains to be further explored. In this study, we engineered two fish-gut Bacillus subtilis strains (FI314 and FI442) with probiotic potential to display the immunogenic proteins OmpK or TolC of Vibrio spp. Their immunomodulatory effect was evaluated using in vitro, ex vivo, and in vivo approaches. In RTgutGC cells, both FI314 and FI314-TolC spores induced an up-regulation of innate immune markers, including il1b and il8, while FI314 spores down-regulated casp3a2 expression. These effects were not observed with the probiotic FI442. In European seabass gut explants, FI314-TolC spores induced the expression of il10, while all spores induced the upregulation of ifng after bacterial challenge with V. anguillarum. In vivo, however, feeding European seabass with diets containing FI314, FI314-OmpK, or FI314-TolC spores for 30 days did not elicit a robust adaptive immune response, as indicated by the lack of significant modulation of immune-related genes and unchanged serum IgM levels. RNA-seq analysis of the distal intestine showed that FI314 spores induced a down-regulation of cell proliferation pathways. while OmpK-carrying spores affected innate immunity pathways. The results of this study indicate that the immunomodulatory effects of autochthonous probiotics are strain dependent. FI314 antigen-displaying spores were insufficient to induce an effective adaptive immunity, under the conditions tested. This study emphasizes the importance of optimizing bacterial strain selection, antigen choice, and immunization regimen when designing oral spore-based vaccines for fish.
autochthonous probioticsBacillusfish healthoral vaccinevibriosisFundação para a Ci≖ncia e a Tecnologia10.13039/501100001871The author(s) declared that financial support was received for this work and/or its publication. This research was funded by national funds through FCT - Fundação para a Ciência e a Tecnologia, I.P., and by the European Commission’s Recovery and Resilience Facility, within the scope of UID/04423/2025(https://doi.org/10.54499/UID/04423/2025), UID/PRR/04423/2025 (https://doi.org/10.54499/UID/PRR/04423/2025), and LA/P/0101/2020 (https://doi.org/10.54499/LA/P/0101/2020) and by Project PTDC/CVT-CVT/2477/2021 (https://doi.org/10.54499/PTDC/CVT-CVT/2477/2021). FCT is also greatly acknowledged for the PhD fellowship of GG 2021.07724.BD (https://doi.org/10.54499/2021.07724.BD).section-at-acceptanceMarine Biotechnology and BioproductsIntroduction
The continuous and rapid expansion of aquaculture is crucial for meeting the growing global demand for sustainable, high-quality animal protein sources for the human population (Norman et al., 2019; FAO, 2024). The intensification of aquaculture, however, is accompanied by an increased incidence of infectious diseases caused by bacteria, viruses, and parasites, which impact production efficiency, profitability, and sustainable growth (Rowley et al., 2024; Maezono et al., 2025; FAO, 2024).
Vibriosis is one of the most significant bacterial infections affecting aquaculture (Mohamad et al., 2019; Triga et al., 2023). It is characterized as a hemorrhagic septicemia, and infected fish typically exhibit skin lesions, ulcerations, loss of appetite, and lethargy (Frans et al., 2011; Ina-Salwany et al., 2018). Although Vibrio spp. are ubiquitous in marine environments, with some species even being present in fish intestines (Ofek et al., 2023; Yoshida et al., 2022), several species, such as V. anguillarum (Gao et al., 2025; Kapetanovic et al., 2022; Frans et al., 2011), V. harveyi (Triga et al., 2023; Da Fonseca Ferreira et al., 2025), and V. vulnificus (Janampa-Sarmiento et al., 2024; Carmona-Salido et al., 2021), cause disease outbreaks worldwide (Kapetanovic et al., 2022; Kumarage et al., 2022; Deris et al., 2022). Besides its impact on fish health and welfare, vibriosis is also a public health concern as some Vibrio spp. are considered zoonotic, with increasing reports on human infections (Archer et al., 2023). In particular, V. vulnificus and V. parahaemolyticus are highly pathogenic to humans, as they can be easily transmitted through contact with contaminated water or by consuming undercooked seafood (Carmona-Salido et al., 2021; Naknaen et al., 2024; Martinez-Urtaza et al., 2016).
In Mediterranean aquaculture, European seabass (Dicentrarchus labrax) is one of the most intensively farmed marine species (FAO, 2024; FEAP, 2024). European seabass is highly susceptible to Vibrio infections throughout all production stages. Although commercial vaccines are available for vibriosis, outbreaks still occur frequently and cause significant losses, particularly during the hatchery and juvenile stages (Muniesa et al., 2020). Prevention of disease outbreaks through vaccination is by far the most effective and economically sustainable strategy (Ma et al., 2019). Most vaccines available in the market are injectable, which are labor-intensive, induce handling stress that can increase mortality, and are not suitable for early developmental stages when fish are particularly vulnerable to disease (Tammas et al., 2024; Du et al., 2022).
Efforts are currently focused on developing alternative vaccination strategies, particularly oral vaccines (Radhakrishnan et al., 2023; Embregts and Forlenza, 2016; Angulo et al., 2020; Kumar et al., 2024). Oral vaccination enables large-scale immunization, even in small fish or larvae, minimizes handling stress, and directly targets the mucosal immune system, which serves as the primary line of defense in aquatic organisms (Du et al., 2022). However, two significant challenges still hinder the development of successful oral vaccines: the first is the potential degradation of the antigen when passing through the harsh conditions of the gastrointestinal tract, such as the low pH and digestive enzymes; the second is ensuring a consistent antigen uptake by all fish, which correlates with how much of the oral vaccine is needed to induce a protective immune response (Mutoloki et al., 2015).
Therefore, the development of delivery systems capable of protecting the antigen and eliciting robust, long-lasting protection remains a key aspect of current fish vaccine research. Bacterial spores, particularly those of Bacillus spp., have been explored as promising vaccine delivery platforms in various animal species (Dai et al., 2018; Wang et al., 2019b; Min et al., 2024), including aquatic animals (Liang et al., 2023; Mai et al., 2022; Gonçalves et al., 2022; Docando et al., 2022a; Gao et al., 2021; Sun et al., 2020; De Stefano et al., 2025). Their resistance to physical and chemical stressors (Setlow, 2014) enables them to withstand passage through the gastrointestinal tract without losing structural or functional integrity (Soylemez-Milli et al., 2025; Koopman et al., 2022).
B. subtilis spores can be directly incorporated into animal feeds, bypassing additional protection methods such as encapsulation. Moreover, the ease of large-scale production and stability during long-term storage without refrigeration further enhances their industrial appeal (Payne et al., 2024). B. subtilis spores can be genetically engineered to display heterologous antigens on their surface, acting as both adjuvants and antigen carriers by interaction with the host’s gut-associated lymphoid tissues (GALT) (Saggese et al., 2023; Liang et al., 2023; Mai et al., 2022; Gao et al., 2021; Jiang et al., 2017; Docando et al., 2022b). Additionally, some B. subtilis strains are already used as probiotics in aquaculture (Nayak, 2020), contributing to an overall improved gut health, enhanced growth performance, and stimulation of both innate and adaptive immune responses (Olmos et al., 2020; Kuebutornye et al., 2019; Yu et al., 2025; Neissi et al., 2024).
Recent studies have focused on host-associated (autochthonous) microorganisms as probiotics, capable of efficiently colonizing the gut (Yang et al., 2022; Ji et al., 2023), modulating local immune responses (Van Doan et al., 2021), and enhancing disease resistance (Amoah et al., 2023; Etyemez Buyukdeveci et al., 2023). The use of host-associated bacteria as probiotics is a safer and more ecologically efficient strategy for promoting fish health (Lazado et al., 2015). Host-associated Bacillus spp. isolated from the gut of European seabass and gilthead seabream (Sparus aurata) have shown strong probiotic potential (Santos et al., 2021b). These isolates exhibited antimicrobial activity against relevant pathogens, including Vibrio spp. (Santos et al., 2021a), and produced extracellular compounds capable of modulating leukocyte activity in fish (Santos et al., 2022). Given their host-adapted nature, immunomodulatory properties, and antimicrobial activity, host-derived Bacillus spp. emerge as promising candidates not only as probiotics, but also as biologically compatible platforms for oral vaccine delivery for aquaculture applications.
In a previous study, we demonstrated that an outer membrane protein (OmpK) of Vibrio spp. could be successfully displayed on the surface of spores from the laboratory strain B. subtilis 168, and increased the survival of zebrafish (Danio rerio) larvae and European seabass upon challenge with Vibrio spp (Gonçalves et al., 2022). Outer membrane proteins have been a major focus in vaccine development against Vibrio spp. due to their conserved nature and surface exposure, a structural characteristic that is essential for their immunogenicity (Singh et al., 2024).
The present study aimed to evaluate the immunomodulatory effects of spores derived from B. subtilis strains isolated from fish gut, which possess potential probiotic properties (Santos et al., 2021b). By displaying two Vibrio outer membrane proteins – OmpK and TolC – as antigens, and employing in vitro, ex vivo, and in vivo models, we investigated how stimulation with recombinant antigen-displaying spores influenced the expression of immune-related genes in fish intestinal cells and tissues, and how dietary inclusion of these spores modulated the intestinal transcriptomic profile of the animals. This integrative approach aimed to provide new insights into the role of engineered autochthonous B. subtilis spores as immunostimulants and potential vaccine delivery systems to promote sustainable disease management in aquaculture.
Material and methodsBacterial strains, growth conditions and purification of spores
Bacterial strains used in this work are listed in Table 1. Bacillus subtilis strains (B. subtilis 168, B. subtilis FI314, and B. subtilis FI442) were routinely cultured in Luria-Bertani (LB) medium (Becton Dickinson) at 37°C. V. anguillarum was grown in Brain Heart Infusion (BHI, Becton Dickinson) at 28°C. Cultures were grown aerobically for 24 h, and, when required, the medium was supplemented with 5 µg mL-1 chloramphenicol for selective growth of B. subtilis.
Bacterial strains and plasmids used in this work.
Name
Relevant genotype/phenotype a
Source, Reference or construction b
Bacterial strain
Vibrio anguillarum DSM 21597
Type Strain (ATCC 19264)
DSMZ
B. subtilis 168
trpC2
A.O. Henriques collection
CRS220
ΔamyE::PcotYZ-cotY-6his-ompK, CmR
MBAqua collection (Gonçalves et al., 2022)
CRS278
ΔamyE::PcotYZ-cotY-6his-tolC, CmR
This study, B. subtilis 168 x pGG17
B. subtilis FI314 (FI314)
Fish isolate with probiotic properties
MBAqua collection (Santos et al., 2021b)
CRS281 (FI314O)
ΔamyE::PcotYZ-cotY-6his-ompK, CmR
This study, B. subtilis FI314 x DNA from CRS220
CRS280 (FI314T)
ΔamyE::PcotYZ-cotY-6his-tolC, CmR
This study, B. subtilis FI314 x pGG17
B. subtilis FI442 (FI442)
Fish isolate with probiotic properties
MBAqua collection (Santos et al., 2021b)
CRS291 (FI442O)
ΔamyE::PcotYZ-cotY-6his-ompK, CmR
This study, B. subtilis FI442 x DNA from CRS220
CRS293 (FI442T)
ΔamyE::PcotYZ-cotY-6his-tolC, CmR
This study, B. subtilis FI442 x DNA from CRS278
Plasmid
p1CSV-CotY-C
bla amyE3´ PcotYZ-cotY-rfp cat amyE5´
(Bartels et al., 2018)
pGG17
bla amyE3´ PcotYZ-cotY-6his-tolC cat amyE5´
This study
Antibiotic resistance indicated: Cm – chloramphenicol.
Bacterial strains were obtained from bacterial collections (BCCM/LMG, Belgian Coordinated Collections of Microorganisms, Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences of Ghent University, Ghent, Belgium; DSMZ, DSM Collection, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; from our laboratory stocks (MBAqua collection) or kindly supplied by A. O. Henriques (Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Portugal).
Sporulation of B. subtilis was induced by nutrient exhaustion using Difco Sporulation Media (DSM, Becton Dickinson) supplemented with 1 mM Ca(NO3)2, 0.01 mM MnCl2, and 0.001 mM FeSO4. Spores were produced and purified following a previously established protocol (Tavares et al., 2013) with minor modifications (Rangel et al., 2024). Spore purity and concentration were evaluated by plating serial dilutions in Bott and Wilson buffer (1.24% K2HPO4, 0.76% H2PO4, 0.1% trisodium citrate, 0.6% [NH4]2SO4, pH 6.7) on LB agar plates before and after a 20 min heat treatment at 80°C to eliminate vegetative cells. After an overnight incubation at 37°C, spore titers were calculated as colony-forming units (CFU) per milliliter (CFU mL-1), and spores were stored at -20°C until further use.
Construction of recombinant <italic>Bacillus subtilis</italic> strains carrying OmpK or TolC
Genomic DNA isolated from strain CRS220, a derivative of B. subtilis 168 carrying the fusion CotY-6His-OmpK (Gonçalves et al., 2022), was used to transform B. subtilis strains FI314 and FI442, which were previously isolated from fish and characterized for their use as probiotics (Santos et al., 2021b, 2022, 2021), following previously established protocols (Cutting and Vander Horn, 1990). The chloramphenicol-resistant (CmR) strains were designated CRS281 (FI314O, Table 1) and CRS291 (FI442O, Table 1). The process is schematically represented in Supplementary Figure S1.
A PCR was performed to amplify a 1294 bp fragment of the tolC coding sequence (GenBank accession: CP031492.1), excluding the first 66 nucleotides (encoding a signal peptide) and incorporating an N-terminal 6-histidine tag. The reaction utilized oligonucleotide primers 6His-tolC-F and tolC-SpoVec-R (Table 2), along with genomic DNA from V. anguillarum (Table 1) as the template. A second PCR with oligonucleotide primers 6His-SpoVec-F and tolC-SpoVec-R (Table 2) resulted in a 1333 bp fragment. The amplified DNA was cloned into the plasmid p1CSV-CotY-C, as described by Bartels et al. (2018), resulting in the plasmid pGG17 (Table 1). Following linearization with 10 U of ScaI (Thermo Scientific), pGG17 was used to transform B. subtilis 168 and B. subtilis FI314 competent cells, yielding strains CRS278 and CRS280 (FI314T), respectively (Table 1). Genomic DNA isolated from the strain CRS278 carrying the fusion CotY-6His-OmpK was used for the transformation of B. subtilis FI442 originating strain CRS293 (FI442T; Table 1). The plasmid pGG17 and genomic DNA from all recombinant strains obtained during this work were used as templates for a PCR that amplified the amyE region to confirm the correct integration and sequence assembly.
Oligonucleotides primers used for cloning purposes.
Native or introduced restriction. sites are indicated in different colors (NgoMIV, SpeI).
Western blot analysis
Spores’ suspensions of the probiotic engineered strains carrying OmpK (FI314O and FI442O) or TolC (FI314T and FI442T), as well as the parental strains (FI314 and FI442) were prepared (section 2.1), quantified at 580 nm, mixed with 2x Loading Buffer (10% glycerol, 10% 2-mercaptoethanol, 100 mM dithiothreitol (DTT), 4% SDS, 0.05% bromophenol blue, 0.125 M Tris) and boiled for 8 min. Proteins from the spore’s coat were resolved by SDS-PAGE in a 15% acrylamide gel. The Western blot analysis was carried out as described by Gonçalves et al. (2022) using a 6His-Tag monoclonal antibody (1:1000; HIS6.H8; Invitrogen).
<italic>In vitro</italic> stimulation of RTgutGC cells
The rainbow trout (Oncorhynchus mykiss) intestinal epithelial cell line RTgutGC (Kawano et al., 2011) was used to assess the transcriptional response upon exposure to B. subtilis spores. Cells were cultured as described elsewhere (Docando et al., 2022b), distributed into 24-well plates at approximately 1×106 cells per well, and incubated at 19°C for 24 h. Cells were then stimulated with the parental probiotic spores (FI314 and FI442), OmpK-spores (FI314O and FI442O), or TolC-spores (FI314T and FI442T) at a final concentration of 1×108 CFU mL-1, or left untreated as negative controls. After 24 h of incubation, cells were harvested and stored in TRI® reagent (Zymo Research) at -80°C until RNA extraction.
<italic>Ex vivo</italic> stimulation of European seabass gut
Seven European seabass of approximately 200 g were fasted for 48 h and euthanized by immersion in an overdose of 2-phenoxyethanol (1 mg L-1). The intestines were collected and processed as described by Docando et al. (2022b) with minor modifications. Briefly, each intestine was collected in 20 mL of ice-cold Leibovitz medium (L-15, Thermo Fisher) containing 1% penicillin-streptomycin (P/S, Gibco™) and 2% fetal bovine serum (FBS, Gibco™). Tissues were then transferred into new media, and pyloric caeca, fat, and gut contents were removed. The intestines were opened longitudinally, cut into fragments of approximately 1 cm2, and transferred to 24-well plates. To ensure experimental homogeneity, one fragment from the anterior portion and one from the distal portion of the intestine were used for each treatment. Gut explants were either stimulated with 1×108 CFU mL-1 spores or left untreated as negative controls and incubated at 24°C. After 24 h, one representative sample was collected, and the remaining tissue was challenged with 1 ×107 CFU mL-1 of V. anguillarum (Fontinha et al., 2024) for 2 h. Explants were collected in RNAlater and stored at -80°C for further RNA extraction.
To confirm the viability of gut explants, an intestine was processed as described to determine the activity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) following 24 h of stimulation as described elsewhere (Erickson-DiRenzo et al., 2015; Fontinha et al., 2024). The formation of formazan confirmed tissue viability, and the viability index was calculated as formazan absorbance (abs)/tissue weight (mg) (Supplementary Figure S2).
European seabass <italic>in vivo</italic> trialFish immunization, experimental design and sampling
European seabass juveniles were quarantined for 2 weeks and then transferred to an experimental recirculating aquaculture system consisting of 24 fiberglass tanks with a water capacity of 100 L, thermoregulated to 22.0 ± 1.0°C. Tanks were supplied with a continuous flow of filtered seawater with controlled parameters (salinity: 33.0 ± 1.0 g L-1; O2 7.5 mg L-1; NH4+ and NO2- ≤ 0.05 mg L-1). After two weeks of acclimation to the experimental system, 25 fish with an initial mean body weight of 17.00 ± 0.01 g were randomly distributed to each tank in triplicate groups for each experimental diet. Fish were hand-fed twice a day until apparent satiation, six days a week for 30 days.
Four experimental diets were formulated to be isoproteic (44%) and isolipidic (17%): a control diet (CTR, spore free), without addition of B. subtilis spores, and 3 other CTR-like diets supplemented with 5×109 CFU Kg-1 spores from the parental B. subtilis FI314 without antigens (FI314 diet), spores displaying the OmpK antigen (FI314O diet), or spores displaying the TolC antigen (FI314T diet). All diets were identical in composition, differing only by the absence or presence of spores. All ingredients were well mixed and dry-pelleted in a laboratory pellet mill (California Pellet Mill, CPM, Crawfordsville, IN, USA) through a 2 mm die. The pellets were dried in an oven at 40 °C for 24 h and stored at 4°C until use. Spore quantification and viability in each diet were measured by homogenizing 100 mg of the diet in B&W salts and proceeding as described above (section 2.1).
At the end of the trial, fish were fasted for 24 h. Blood was collected from the caudal vein of four fish from each tank, after which fish were euthanized and sampled for head kidney and distal intestine. Blood was allowed to clot for 1 h at room temperature, and then stored overnight at 4°C. The next day, blood was centrifuged at 10000 x g for 10 min at 4°C, followed by serum collection and storage at -80°C until further use. Head kidney and distal intestine samples, used for gene expression analysis, were collected in RNA later, placed at 4°C overnight and stored at -80°C. Distal intestine samples were collected from three other fish, pooled per tank, and snap-frozen in liquid nitrogen before storage at -80°C for the RNAseq analysis.
Indirect ELISA for detection of <italic>Vibrio anguillarum</italic> antibodies
An indirect ELISA was carried out to detect antibodies in the serum of fish fed the experimental diets. A 96-well flat-bottom microtiter plate (Nunc Flat Bottom MaxiSorp™) was coated with a freshly prepared V. anguillarum cell suspension at approximately 2 × 107 CFU mL−¹ per well and then incubated at room temperature. After 4 h, wells were washed and blocked with 1x PBS supplemented with 10% low-fat milk for 2 h. Subsequently, the wells were rewashed and incubated overnight at 4°C with fish serum diluted 1:6 in 1x PBS or BSA (20 µg mL−¹). Wells coated with 1x PBS served as blanks. The next day, wells were washed and incubated with 100 µL of anti-European seabass IgM monoclonal antibody (Aquatic Diagnostics Ltd.) diluted 1:33 in 1x PBS for 1 h at room temperature. After washing, wells were incubated with a goat anti-mouse IgG (H+L) HRP-conjugated secondary antibody (Invitrogen), diluted 1:10000 for 1 h. Detection was performed by adding 100 µl of TMB (3,3’,5,5’-tetramethylbenzidine) substrate for 20 minutes. The reaction was stopped by adding 100 µL of 2 M H2SO4, and absorbance was measured immediately at 450 nm.
RNAseq analysis
Intestinal tissue samples (each sample consists of 3 pooled intestinal fragments per tank) were homogenized in TRIzol™ Reagent (Zymo Research), and total RNA was extracted according to the manufacturer’s protocol. RNA concentration, purity, and integrity were assessed using the 5400 Fragment Analyzer (Agilent Technologies). Subsequently, sequencing libraries were prepared by Novogene Co., Ltd. (Cambridge, UK) using the Illumina NovaSeq X Plus Series (PE150). Each sample consisted of pooled distal intestines from three fish, from the same tank (n = 3 per treatment). The raw reads were processed using Illumina Casava 1.8 software, and clean data were obtained after removing reads containing adapters, poly-N regions, and low-quality reads. The Q20, Q30, and GC content of the clean data were calculated (Supplementary Table S1). Paired-end clean reads were aligned to the reference genome (dlabrax2021, GenBank: GCA_905237075.1) using HISAT2 software (2.2.1). Read numbers were counted using the featureCounts software (2.0.6), and the expression level of each gene was expressed as the number of Fragments Per Kilobase Million (FPKM). Differential expression analysis for comparing two groups was performed using the DESeq2 R package (version 1.42.0), with the resulting p-value adjusted using Benjamini and Hochberg’s method (p-adj). Differentially expressed genes (DEGs) were considered when p-adj ≤ 0.05 and |log2(foldchange)| ≥ 1. For the analysis of DEGs, transcriptomes of the probiotic-treated group (FI314) were compared to the control group (CTR). DEGs between fish fed antigen-displaying spores (FI314O or FI314T) and fish fed the parental spores (FI314) were also performed. Enrichment analyses were performed using clusterProfiler (version 4.8.1) with Gene Ontology (GO) and the KEGG pathways database. Since D. labrax is not represented in the KEGG database, gene annotation was performed using Danio rerio Ensembl orthologs.
Gene expression analysis by qPCR
Total RNA was extracted from RTgutGC cells, gut explants, head-kidney, and distal intestine using the Direct-zol™ RNA Miniprep kit (Zymo Research) following the manufacturer’s instructions. RNA was quantified using the μDrop™ Plate (Thermo Scientific) in a Multiskan Go Plate Reader (Thermo Scientific) and converted to cDNA using the NZY First-Strand cDNA Synthesis Kit (NZYTech). Gene transcription levels were determined using real-time quantitative PCR (qPCR) on a CFX Connect Real-Time System (Bio-Rad). Reactions were carried out using 0.5 μL of two-fold diluted cDNA, 0.5 μL of each set of primers at 10 μM (Supplementary Table S2), 5 μL of SsoAdvanced Universal SYBR® Green Supermix (BioRad), and 3.5 μL DNAse/RNase/Protease-free water in a final volume of 10 μL. Primer efficiency (between 90% and 110%) was validated using two-fold dilutions of cDNA and calculated using the slope obtained from the regression line of the quantification cycle (Ct) and the relative cDNA concentration, according to the equation E = 10(-1/slope) (Pfaffl, 2001). PCR reactions were incubated at 95°C for 30 s, followed by 40 cycles of 15 s at 95°C and annealing for 30 s at the appropriate temperature (Supplementary Table S2). Each cDNA sample was run in duplicate, and a negative control was included for each experiment. Relative gene expression was calculated using Pfaffl (2001) methodology. Data were normalized using reference genes: β-actin (actb) and ribosomal protein S20 (rps20) were used for rainbow trout, and small ribosomal subunit 40s (40s) and elongation factor 1α (ef1a) for European seabass. All values were expressed as mean normalized values ± standard error of the mean.
Statistical analysis
Data analysis was done using GraphPad Prism 10 software. All data were tested for normality using the Shapiro-Wilk test and for homogeneity using the Levene test. Data from gene expression levels of RTgutGC cells, European seabass head kidney, and distal intestine were analyzed by one-way ANOVA, and a p < 0.05 was considered significant. A two-way ANOVA with challenge and treatment as factors was performed on data from gene expression in gut explants. Tukey’s test was performed for comparisons between groups.
ResultsConstruction of recombinant <italic>B. subtilis</italic> strains for spore display of OmpK and TolC
A truncated fragment of the outer membrane protein K (OmpK) was previously displayed in B. subtilis spores fused to CotY (Gonçalves et al., 2022), a protein abundant in the spore’s crust (Bartels et al., 2019). Genomic DNA from the strain carrying the fusion CotY-6His-OmpK (CRS220) was used in this study to transform the putative probiotic strains FI314 and FI442, generating strains CRS281 and CRS291 (Table 1; Supplementary Figure S1). To assess whether displaying a different antigen on the surface of the spores would influence their immunomodulatory properties, the TolC protein was also selected for display. The tolC sequence was inserted into the genomes of FI314 and FI442 by transformation with plasmid pGG17 or genomic DNA (Table 2; Supplementary Figure S1), originating strains CRS280 and CRS293 (Table 2). Sequencing of the amyE region from plasmid pGG17 and from the genomic DNA of strains CRS281, CRS291, CRS280, and CRS293 confirmed the correct sequence assembly (data not shown).
Since all recombinant strains carried a 6-histidine tag fused to the target antigen, a commercial anti-His Tag antibody was used to detect OmpK and TolC in recombinant spores by Western blot. Spores from the parental strains FI314 and FI442 were included as negative controls. Spore coat proteins were resolved in a 15% SDS-PAGE (Figure 1A), and similar profiles were found for the congenic derivatives when compared to each parental strain, with only minor differences. In FI314 spores, one band below 37 kDa and another around 20 kDa (Figure 1A, bands 1 and 2) were faint in the recombinant spores. In CotY-OmpK spores, a low molecular weight band (Figure 1A, band 3) was more intense, and in the case of the CotY-TolC spores, the same happens with a band below 37 kDa (Figure 1A, band 4). In the FI442 spores, congenic spores showed a stronger band between 10 and 15 kDa (Figure 1A, bands 5 and 6).
Detection of the OmpK and TolC proteins at the surface of recombinant probiotic B. subtilis spores. (A) Spores from recombinant B. subtilis probiotic strains carrying the CotY-OmpK or CotY-TolC fusions were produced, and proteins were separated on a 15% acrylamide gel. Strains within the same parental background (FI314 or FI442) showed similar overall protein profiles. Variations in band intensity are highlighted and numbered (1–6). (B) Western blot analysis using an anti-HisTag antibody (1:1000) did not reveal unique bands at the expected molecular weights. Faint bands of approximately 46 kDa, corresponding to the sum of CotY (18 kDa) and 6His-OmpK (28 kDa), were observed in recombinant strains CRS281 (FI314O) and CRS291 (FI442O) (indicated by *). Faint bands of approximately 66 kDa (indicated by **), consistent with the sum of CotY and 6His-TolC (48 kDa), were detected in CRS280 (FI314T) and CRS293 (FI442T) spores. As expected, no specific bands were detected in spores from the parental strains FI314 and FI442.
Gel images display protein bands separated by molecular weight. Panel A shows Coomassie-stained protein bands for FI314 and FI442 strains with different constructs. Panel B is a corresponding immunoblot with marked bands, indicating protein expression or interaction differences under each condition. Molecular weights are indicated on the left side in kilodaltons.
The Western blot, using an anti-His Tag antibody, detected non-specific bands in all strains, including the negative controls. Nevertheless, a band of approximately 46 kDa, consistent with the sum of CotY (18 kDa) and OmpK (28 kDa), was detected in all spores carrying the CotY-OmpK fusion (Figure 1B indicated as *). This band was absent in spores from the parental strains, supporting the possibility that it corresponds to the fusion protein. The same was observed in spores carrying the CotY-TolC fusion (CotY – 18 kDa and TolC – 48 kDa), where a band of approximately 66 kDa was observed in spores of the derivative strains carrying the CotY-TolC fusion (Figure 1B, indicated as **), but not in parental spores.
Immune modulation of RTgutGC cells stimulated <italic>in vitro</italic> with antigen-displaying <italic>B. subtilis</italic> spores
RTgutGC cells were exposed to spores from parental strains (FI314 and FI442), OmpK-displaying spores (FI314O and FI442O), and TolC-displaying spores (FI314T and FI442T). RNA from the exposed cells was used to quantify the expression of genes coding for two pro-inflammatory cytokines, tumor necrosis factor-α (tnfa) and interleukin-1β (il1b), a pro-inflammatory chemokine, interleukin-8 (il8), the stress-related heat shock protein 70 (hsp70), cyclooxygenase-2a (cox2a), involved in the inflammatory process, and caspase 3 (casp3a2), involved in apoptosis.
Stimulation of RTgutGC cells with FI1314 spores in the absence of an antigen resulted in an increased expression of il1b and il8 (Figure 2). The expression of il1b was also significantly upregulated in response to FI1314 spores displaying the TolC antigen. In contrast, exposure to FI314O spores did not affect the expression levels of the analyzed genes when compared to other treatments. Stimulation with any FI442 spores did not result in any significant changes in gene expression. A decrease in casp3a2 expression was in cells exposed to spores from the parental FI314 strain (Figure 2).
Transcriptional responses of RTgutGC cells to antigen-displaying spores from different probiotic B. subtilis strains. Cells were stimulated with 1 × 108 CFU mL-1 spores from two probiotic strains (FI314 or FI442) and respective recombinant spores displaying OmpK (FI314O or FI442O) or TolC (FI314T or FI442T) antigens, for 24 h at 19°C. The control (CTR) corresponds to unstimulated cells. Relative gene expression levels of immune-related genes tnfa, il1b, il8, hsp70, cox2a and casp3a2 were measured by qPCR and normalized to the mean expression of reference genes actb and rps20. Data shown is the mean ± SEM of 4 replicates. Differences between groups were evaluated by ANOVA, followed by Tukey post-hoc test. Different lowercase letters indicate statistically significant differences between groups (p < 0.05).
Bar graphs showing normalized mRNA expression levels for tnfa, il1b, il8, cox2a, hsp70, and casp3a2 across different treatments: CTR, FI314, FI314O, FI314T, FI442, FI442O, and FI442T. Varied expression is indicated, with significant differences marked by letters a and b.Immune modulation of fish gut explants stimulated <italic>ex vivo</italic> with antigen-displaying <italic>B. subtilis</italic> spores upon <italic>V. anguillarum</italic> challenge
To further explore the results obtained in the RTgutGC cell line, European seabass gut explants were stimulated with spores from the parental probiotic strains (FI314 or FI442), as well as with recombinant spores displaying OmpK (FI314O or FI442O) or TolC (FI314T or FI442T), followed by a bacterial challenge with V. anguillarum. The effect of treatment, challenge, and their interaction on the expression of several immune-related genes was evaluated.
Independent of the bacterial challenge, treatment with spores significantly affected the expression of tnfa, il10, igm, cd4, ifng, and mhci. The bacterial challenge with V. anguillarum affected the expression of tnfa, il10, cd4, ifng, and mhci across treatments (Figure 3).
Transcriptional responses of fish gut explants stimulated with antigen-displaying spores from different probiotic B. subtilis strains upon challenge with Vibrio anguillarum. Explants were stimulated with 1 × 108 CFU mL−¹ spores from two probiotic strains (FI314 or FI442) and respective recombinant spores displaying OmpK (FI314O or FI442O) or TolC (FI314T or FI442T), for 24 h at 24°C. Unstimulated explants served as controls (CTR). Following spore stimulation, explants were challenged with 1 × 107 CFU mL−¹ V. anguillarum for 2 h. Relative expression of the immune-related genes tnfa, il1b, il10, igm, cd4, ifng, mhci and mhcii was determined by qPCR and normalized to the mean expression of the reference genes 40s and ef1a. Data are presented as mean ± SEM of 6 biological replicates. Data was analyzed by two-way ANOVA with Treatment and Challenge as variables. When p < 0.05 in the Treatment variable, Tukey’s post-hoc test was performed to analyze global differences between treatments, which are represented in the “Non-challenged” panel of each graph (to simplify interpretation) by different uppercase letters. When a significant interaction was detected (p < 0.05), differences between treatments were analyzed by a Tukey post-hoc test separately within the challenged and non-challenged groups, and different lowercase letters indicate statistically significant differences between treatments (p < 0.05). Differences between the same treatment before and after the challenge are represented by asterisks, in the “Challenged” panel of each graph (*p < 0.05, **p < 0.01, ***p < 0.001).
Bar charts showing normalized mRNA expression levels for genes (tnfa, il1b, il10, igm, cd4, ifng, mhci, mhcii) under different treatments and challenges. Each chart compares expression across conditions like “non-challenged” and “challenged.” Statistical significance is indicated by different letters above bars and asterisks. P-values are provided for treatment, challenge, and their interaction.
An interaction between treatment and challenge was observed in the expression of il10 and ifng. Stimulation with TolC-displaying FI314 (FI314T) and FI442 (FI442T) spores upregulated the expression of il10 in challenged gut explants, compared with non-challenged, although only treatment with FI314T induced a significant increase, compared with the other treatments. In contrast, treatment with spores regardless of the strain and presence of antigen, increased the expression of ifng after bacterial challenge, compared to the control (Figure 3).
Overall, stimulation with probiotic spores increased the expression of igm, cd4, and mhci compared to the control. Expression of tnfa was higher in explants treated with all FI314-derived spores and with antigen-displaying FI442 spores (FI442O and FI442T), although it was only significantly different from the control in FI314T treatment. The expression of il1b was affected only by the bacterial challenge, and no significant differences were detected in mhcii expression across treatments or challenge conditions (Figure 3).
Immune response of European seabass upon oral immunization with antigen-displaying <italic>B. subtilis</italic> spores
Since FI314 and its derivatives elicited more pronounced immune responses in both RTgutGC cells and gut explants, the in vivo trial only tested FI314 spores and their recombinant derivatives displaying OmpK (FI314O) or TolC (FI314T). FI314 derivatives were selected not only due to these similar effects, but also because this strain is well characterized for its probiotic potential, with previous in vivo studies demonstrating its capacity to modulate leukocyte responses upon bacterial infection (Santos et al., 2022, 2021).
To evaluate the immune response induced by the experimental diets, three complementary approaches were used: (i) an indirect ELISA to detect serum antibodies raised against V. anguillarum, (ii) gene expression analysis in the head kidney and distal intestine to assess systemic and mucosal immune modulation, and (iii) RNA-seq transcriptomic analysis of the distal intestine of fish to complete a comprehensive overview of transcriptional changes.
ELISA detection of anti-<italic>V. anguillarum</italic> antibodies
After the immunization period, serum from fish fed experimental diets was used to indirectly determine anti-OmpK and anti-TolC antibody production by ELISA, using V. anguillarum cell extracts and an anti-seabass antibody (Figure 4). Analysis of serum of fish fed probiotic spores carrying OmpK or TolC antigens showed no significant differences in antibody levels compared with fish fed the parental spores (FI314) or the control diet (CTR).
Antibody titer in the serum of European seabass fed antigen-displaying B. subtilis spores for 30 days. Fish were fed daily for 30 days, a control (CTR) diet without spores, a diet containing probiotic strain FI314, or diets supplemented with FI314-derivatives displaying OmpK (FI314O) or TolC (FI314T). Detection of anti -V. anguillarum antibodies was done by indirect ELISA in the sera of individual fish (n=12) using V. anguillarum cell-extracts and anti- European seabass antibody. Each blot represents the OD450nm of individual samples. Bars represent the mean ± SEM. Data were analyzed by one-way ANOVA, and no significant differences were detected.
Bar graph showing optical density (OD450 nm) for four groups: CTR, FI314, FI314O, and FI314T. Each bar represents mean values with error bars and individual data points. The axis ranges from 0.00 to 2.50.Gene expression in head kidney and distal intestine
In the head kidney, il1b expression was significantly upregulated in fish fed FI314O spores compared to the control and FI314T groups. No significant differences were detected in tnfa, il10, cd4, mhci, or igm expression (Figure 5). Nonetheless, a trend toward increased tnfa expression was observed in fish fed antigen-carrying spores (FI314O and FI314T), as well as for cd4 expression in the FI314O group (Figure 5).In the distal intestine, no significant differences among groups were found in the expression of tnfa, il1b, il10, cd4, and mhci (Figure 6). However, a tendency for higher il1b, il10, and cd4 expression was observed in the FI314O group.
Transcriptional responses in the head kidney of European seabass fed antigen-displaying B. subtilis spores for 30 days. Fish were fed daily for 30 days with a diet containing spores from the probiotic FI314 or spores containing the OmpK (FI314O) or TolC (FI314T) antigens. The control (CTR) corresponds to samples from fish fed a diet without spores. Relative gene expression levels of immune-related genes tnfa, il1b, il10, cd4, mhci and igm were measured by qPCR and normalized to the mean expression of reference genes 40s and ef1a. Data shown is the mean ± SEM of 9 biological replicates. Differences between groups were evaluated by ANOVA, followed by Tukey-test. Different lowercase letters indicate statistically significant differences between groups (p < 0.05).
Bar charts showing normalized mRNA expression levels for six genes: tnfa, il1b, il10, cd4, mhci, and igm, across four groups labeled CTR, FI314, FI314O, and FI314T. Each chart compares expression levels, with variations in bar heights indicating differences among groups. Some charts have annotations (a, b) signifying statistically significant differences.
Transcriptional responses in the distal intestine of European seabass fed antigen-displaying B. subtilis spores for 30 days. Fish were fed daily for 30 days with a diet containing spores from the probiotic FI314 or spores containing the OmpK (FI314O) or TolC (FI314T) antigens for 30 days. The control (CTR) corresponds to samples from fish fed a diet without spores. Relative gene expression levels of immune-related genes tnfa, il1b, il10, cd4 and mhci were measured by qPCR and normalized to the mean expression of reference genes 40s and ef1a. Data shown is the mean ± SEM of 9 biological replicates. No significant differences were observed between groups.
Bar graphs showing normalized mRNA expression levels for tnfa, il1b, il10, cd4, and mhci across four groups: CTR, FI314, FI314O, and FI314T. Each graph displays data with error bars representing variability.Transcriptional analysis by RNA-seq
Twelve distal intestine pools (3 pools per each treatment: CTR, FI314, FI314O and FI314T) were successfully sequenced, producing an average of 55,084,016 ± 20,406,331 raw reads per pool and an average of 54,378,398 ± 19,829,408 clean reads with an average Q30 of 96.2 ± 0.4%. Each sample had an average of 91.00 ± 0.03% mapped reads, and each treatment group had more than 49 M reads in total (data compiled in Supplementary Table S1).
Principal component analysis (PCA) did not separate FI314 and CTR groups along PC1 (explaining 20% of the variance) or PC2 (explaining 14% of the variance). In contrast, FI314O and FI314T groups formed distinct clusters in the PCA plot (Figure 7).
Principal component analysis (PCA) of transcriptomic profiles from the distal intestine of European seabass fed antigen-displaying B. subtilis spores for 30 days. Different colors represent the experimental diets: blue, control (CTR); dark blue, diet containing B. subtilis FI314 spores (FI314); and green and dark green, diets containing spores displaying an antigen (FI314O and FI314T, respectively).
Scatter plot of Principal Component Analysis (PCA) showing data points distributed along two axes: PC1 with twenty percent variance and PC2 with fourteen percent variance. Points are color-coded by sample name: CTR (light blue), FI314 (blue), FI314O (light green), and FI314T (green).
Differentially expressed genes (DEGs) were identified from the RNA-seq profile to determine the transcriptional differences between fish fed the CTR diet and those fed with B. subtilis FI314 spores (Figure 8). DEGs analysis showed that the dietary inclusion of spores downregulated 505 genes and upregulated 267 genes (DESeq2, p-adj < 0.05).
Volcano plots of differentially expressed genes (DEGs) in the distal intestine of European seabass fed antigen-displaying B. subtilis spores for 30 days. DGEs were evaluated between (A) fish fed the control diet (CTR) and those fed the diet with B. subtilis FI314, (B) fish fed the diet with B. subtilis FI314 and those fed the diet containing spores displaying OmpK antigen or (C) TolC antigen.
Three volcano plots labeled A, B, and C show gene expression data. The x-axis indicates log2FoldChange, and the y-axis shows -log10(p-value). Red dots represent upregulated genes, blue dots for downregulated, and gray for no change. A displays 267 up and 505 down, B shows 147 up and 152 down, while C has 292 up and 276 downregulated genes. Dotted lines denote significance thresholds.
To further explore how the oral delivery of antigen-displaying spores influenced the host intestinal transcriptome, additional comparisons were performed between the recombinant spore groups and the parental one (FI314O vs. FI314 and FI314T vs. FI314). The DEG analysis showed that the incorporation of the OmpK antigen resulted in the modulation of 299 genes (147 upregulated and 152 downregulated). In contrast, the inclusion of the TolC antigen affected 568 genes (292 upregulated and 276 downregulated).
Functional enrichment analysis using the Gene Ontology (GO) database was conducted to identify biological processes affected by the dietary inclusion of FI314 spores and recombinant antigen-displaying derivatives. Feeding fish with the diet FI314 significantly downregulated GO categories related to DNA replication and cellular proliferation (Supplementary Figure S3), in comparison with fish fed the CTR diet. In contrast, inclusion of OmpK-displaying spores compared to FI314 spores modulated biological processes associated with the immune system, as reflected by the upregulation of genes encoding chemokine 3-like and IL-6 subfamily cytokine M17, as well as unclassified genes, and the downregulation of genes related to vitronectin A, vitronectin B, and the uncharacterized gene LOC127367333 (Table 3). The dietary inclusion of TolC-displaying spores, compared to the parental spores, resulted in the upregulation of genes associated with DNA replication and metabolic processes, as indicated by GO categories (Supplementary Figure S3).
Differentially expressed immunity-related genes in the distal intestine of European seabass fed with OmpK-displaying B. subtilis FI314 spores compared to the parental FI314.
Functional enrichment analysis using the GO database identified (p adj < 0.05) immune-related genes up- and downregulated in the distal intestine of spores fed the OmpK-carrying spores in comparison with fish fed the parental FI314 spores.
Discussion
The use of Bacillus subtilis as a probiotic in aquaculture has been extensively explored. Dietary supplementation with B. subtilis, either as vegetative cells or spores, has been shown to i) promote fish growth, ii) modulate the gut microbiota, iii) stimulate both innate and adaptive immune responses, and iv) improve disease resistance in several fish species (Etyemez Buyukdeveci et al., 2023; Touraki et al., 2012; Neissi et al., 2024; Docando et al., 2022b; Amoah et al., 2023; Raida et al., 2003; Yu et al., 2025). In addition to their probiotic traits, B. subtilis spores are intrinsically robust (Setlow, 2014) and stable, making them highly suitable for biotechnological applications (Payne et al., 2024). In aquaculture, this has led to growing interest in using B. subtilis not only as a probiotic but also as a mucosal vaccine delivery platform (Bahrulolum and Ahmadian, 2025).
Several studies have developed oral vaccines by displaying heterologous antigens on the surface of B. subtilis spores targeting major fish pathogens, including bacteria, viruses, and parasites (Zhang et al., 2025; Hou et al., 2025; Yao et al., 2019; Docando et al., 2022a; Mai et al., 2022). Laboratory strains of B. subtilis, such as B. subtilis 168, B. subtilis WB600, or B. subtilis PY79, which are well-characterized and easily manipulated due to the availability of sophisticated genetic tools, have served as the primary choice of chassis for this technology (Zhang et al., 2025; Hou et al., 2025; Pham et al., 2017).
In our previous work, using spore-surface display technology, we developed an oral vaccine against vibriosis that enhanced zebrafish survival against two Vibrio spp., as well as European seabass survival against V. anguillarum (Gonçalves et al., 2022). We chose CotY protein as an anchor (Bartels et al., 2018) and displayed a truncated version of OmpK (antigen from Vibrio spp.) on the surface of spores from the laboratory strain B. subtilis 168 (Earl et al., 2008). Building up on those findings, the current study sought to display the same antigen (OmpK) in the spores of two putative probiotic B. subtilis strains, FI314 and FI442, previously isolated from the gut of European seabass and gilthead seabream (Santos et al., 2021b), combining the advantages of a fish-associated probiotic (Santos et al., 2021a, 2022) with the platform for antigen delivery. These strains were selected based on previous in vitro observations of quorum-quenching, anti-biofilm, and anti-growth activities against fish pathogens, including Vibrio spp (Santos et al., 2021b).
Additionally, to evaluate the effect of different antigens and possibly enhance vaccine efficiency, we also selected TolC, an outer membrane protein from Vibrio spp., which has been previously assessed as a potential target for vaccine development (Wang et al., 2020; Zhu et al., 2019). Both OmpK and TolC are outer membrane proteins recognized as common immunogenic motifs among Vibrio spp. They have been associated with protective immune responses in several fish species (Lee et al., 2021; Silvaraj et al., 2020; Zhang et al., 2007; De Stefano et al., 2025; Gonçalves et al., 2022; Zhu et al., 2019) and have attracted attention for the development of experimental vaccines against vibriosis (Li et al., 2014).
Proper surface localization of the antigen is essential for efficient recognition by gut immune cells (Mutoloki et al., 2015; Embregts and Forlenza, 2016; Salinas and Parra, 2015). It is also important to ensure that antigen display does not compromise the spore structure and intrinsic resistance. The correct genomic integration of each fusion was confirmed by sequencing the amyE region of all recombinant strains, where the double-crossover event occurs. Subsequent Western blot analysis also detected the fusions CotY-OmpK and CotY-TolC. However, the signal intensity was not very strong among recombinant spores, and non-specific bands were observed, which reflects the highly complex and resilient architecture of the spore coat and crust, composed of multiple concentric layers of highly cross-linked proteins (Plomp et al., 2014). CotY participates in stable complexes with itself and other morphogenetic proteins (Liu et al., 2015; Bartels et al., 2019), which difficult the complete denaturation of fusion proteins and affect electrophoretic behavior. Additionally, the His-Tag epitope can be partially masked within the spore shell. Also, the detection of the 6-histidine tail is not always consistent (Debeljak et al., 2006), which results in weak and unspecific signal detection. Besides structural limitations, spore-surface display studies indicate that recombinant fusion proteins can be present at relatively low copy numbers compared to non-recombinant proteins (Saggese et al., 2023), resulting in reduced Western blot sensitivity but does not necessarily indicate a failed surface display. Western blot analysis using specific antibodies to detect the anchor protein from the spore and the target antigen should be considered in the future to enhance the detection of recombinant proteins.
After confirming the successful fusion integration, we evaluated the immunostimulatory potential of all recombinant and parental spores by analyzing their transcriptional effect on a rainbow trout intestinal epithelial (RTgutGC) cell line, as well as on European seabass intestinal explants. Both approaches have been utilized in multiple studies to evaluate the effects of mucosal adjuvants (Martin-Martin et al., 2020), probiotics (Docando et al., 2022b), and immunostimulants (Fosse et al., 2025; Wang et al., 2019a), and also contribute to the refinement of animal use. Importantly, intestinal epithelial cells constitute one of the first host barriers, sensing microbial-associated molecular patterns through pattern recognition receptors and initiating inflammatory and immune responses (Salinas and Parra, 2015).
In the present study, spores of the probiotic FI314 upregulated the expression of il1b and il8 in intestinal epithelial cells. IL-1β is a key cytokine that triggers inflammation upon activation of pattern-recognition receptors in the host (Zou and Secombes, 2016). IL-8 is a potent chemokine that recruits immune cells and triggers an oxidative burst in neutrophils, thereby amplifying local inflammatory responses (Zhao et al., 2022). Our findings are consistent with previous studies demonstrating the immunostimulatory potential of B. subtilis spores. For instance, two B. subtilis strains isolated from European seabass were shown to induce the upregulation of il1b, il8, tnfa and il10 in intestinal epithelial cells and gut explants of rainbow trout in a strain- and dose-dependent manner (Docando et al., 2022b). Similarly, dietary supplementation with B. subtilis spores increased the expression of il1b and tnfa in Nile tilapia (Oreochromis niloticus), in a strain-dependent manner (Galagarza et al., 2018). In contrast, a downregulation of il1b, il8 and tnfa was observed at low dietary levels of B. subtilis spores in largemouth bass (Micropterus salmoides), but no significant effect was observed at higher levels (Du et al., 2021). Together, these observations suggest that probiotic spores stimulate innate immunity, reinforcing that transcriptional responses to B. subtilis are highly dependent on strain characteristics and dosage. As with FI314 spores, FI314-TolC spores upregulated il1b, whereas FI314-OmpK spores did not induce significant changes in pro-inflammatory gene expression. These responses suggest that antigen engineering may modulate the interaction with intestinal epithelial receptors, as previously reported for engineered spores tested in mice, where antigen fusion affected immune recognition (Piekarska et al., 2017). A down-regulation of casp3a2 in intestinal epithelial cells exposed to FI314 spores suggests a potential anti-apoptotic effect. Caspase-3 is a crucial executioner caspase involved in programmed cell death in both fish and mammals (Song et al., 2022). In line with our results, oral administration of Bacillus spp. probiotics also reduced caspase-3 levels under oxidative stress conditions in rats, supporting an anti-apoptotic and mucosal-protective effect (Wu et al., 2019). In the hybrid grouper (Epinephelus fuscoguttatus ♀× Epinephelus lanceolatus ♂), dietary supplementation with different B. subtilis strains also downregulated casp3a2 in the liver, while improving intestinal integrity and stress resilience in a strain-dependent manner (Han et al., 2024).
An ex vivo model provides a more physiologically relevant context than an in vitro assay, as it preserves the native intestinal architecture and contains different cell types that contribute to immune signaling (Coccia et al., 2019; Salinas and Parra, 2015). Furthermore, a bacterial challenge with V. anguillarum was also conducted ex vivo to evaluate how this pathogen modulates the expression of immune-related genes in the gut of European seabass. In the present study, different treatments modulated the expression of il10 and ifng after V. anguillarum challenge. Expression of il10 was generally higher in spore-treated explants following the challenge, although this effect was only significant with FI314T. This anti-inflammatory mediator suppresses the immune response and inhibits excessive inflammation following bacterial infections (Fajardo et al., 2022; Zhu et al., 2023). Further, upon challenge with V. anguillarum, the transcription levels of ifng were significantly upregulated in explants that had been previously treated with spores. However, spore treatment alone also did not modulate ifng expression; instead, it acted as a priming stimulus, enabling a stronger ifng response upon pathogen exposure. In fish, IFN-γ promotes macrophage activation and Th1-like cell-mediated immune responses (Zou and Secombes, 2016; Pereiro et al., 2019). IFN-γ contributes to defense against intracellular pathogens in fish, such as Aeromonas salmonicida (Hu et al., 2021) and Edwardsiella tarda (Velazquez et al., 2017). Although V. anguillarum is not typically considered an intracellular pathogen, an upregulation of ifng has already been observed in European seabass challenged with V. anguillarum (Saleh et al., 2024). Additionally, an up-regulation of ifng was also observed in the hindgut of grass carps orally immunized with antigen-displaying B. subtilis spores against Clonorchis sinensis (Jiang et al., 2017). Taken together, the results from the in vitro and ex vivo screening indicate an upregulation of immune-related genes in fish exposed to FI314, FI314-OmpK, and FI314-TolC. This, however, was not observed with FI442.
An in vivo trial was conducted to validate the results obtained from the in vitro and ex vivo approaches, as inter-organ signaling, microbiota interactions, and neuroendocrine regulation (Cortez and Guedes, 2023) are essential to assess the immune response in fish. Based on the in vitro and ex vivo findings, only FI314 spores were selected for the in vivo evaluation. Feeding European seabass with FI314, FI314-OmpK, or FI314-TolC spores for 30 days did not elicit a robust adaptive response. In the head kidney, the expression of tnfa, il10, cd4, mhci and igm remained unchanged, while il1b was significantly upregulated in fish fed FI314-OmpK spores. In the distal intestine, no significant modulation of immune markers was detected. This result contrasts with the in vitro findings, where FI314-TolC induced a stronger upregulation of immune-related genes, highlighting the greater complexity of the whole organism when receiving the oral vaccine (Yu et al., 2020). Similarly, the serum IgM titer remained unchanged across treatments, which was measured by ELISA using a cell suspension of V. anguillarum. Future studies should consider including serum from Vibrio-infected or vaccinated fish to further validate a lack of humoral activation. Nonetheless, this result is consistent with the lack of igm transcriptional modulation in the head kidney. Several factors may explain this outcome, including insufficient antigen dose, limited antigen uptake, or prolonged antigen exposure, which may trigger immune tolerance mechanisms, as observed in previous studies. For instance, feeding grouper (Epinephelus spp.) with B. subtilis displaying the VP19 protein induced the expression of igm, ifng, cd4, tnfa and il6 in the hindgut, along with increased specific serum IgM levels at 14 and 21 days post-vaccination (dpv). However, at 28 dpv, no differences with the control group were observed (Liang et al., 2023). Similarly, B. subtilis spores displaying the MCP antigen triggered the up-regulation of igm, il1b and mhci at 7, 14, and 21 dpv in the head kidney of grouper (Epinephelus coioides) (Mai et al., 2022). Spores expressing the Sip protein upregulated mhci and mhcii at 21 dpv, whereas il1b, tnfa, cd4, and igm remained unaltered, consistent with unchanged serum IgM levels, in tilapia (Yao et al., 2019).
Collectively, these studies indicate that spore-based oral vaccines elicit an early activation response followed by immune regulation, at later stages of oral immunization. In fish, prolonged oral stimulation can trigger mucosal tolerance mechanisms aimed at preserving intestinal homeostasis, which are mediated by anti-inflammatory cytokines and other regulatory pathways, such as the activation of regulatory T cells (Lee et al., 2021). Further studies, including additional earlier sampling points, would provide a more comprehensive analysis of the immune response’s evolution to validate this interpretation.
An RNA-seq analysis was performed on distal intestinal samples to obtain a comprehensive view of gene expression changes induced by oral immunization with antigen-displaying B. subtilis spores, which might have been overlooked in targeted qPCR analysis. Samples from the FI314-OmpK and FI314-TolC groups exhibited clustering of replicates, which reduces the statistical power of the analysis and can mask subtle differences in gene expression (Love et al., 2014).
The highest number of differentially expressed genes (DEGs) was observed in fish fed spores lacking the antigen, with changes primarily associated with downregulation of DNA proliferation and regulatory pathways. This was unexpected, as dietary supplementation with probiotics, specifically B. subtilis, is typically associated with histological changes in the gut, such as increased epithelial thickness, goblet cell number, and villus size (Du et al., 2021; Etyemez Buyukdeveci et al., 2023). Nonetheless, the observed down-regulation of cellular proliferation pathways aligns with the reduced casp3a2 expression detected in vitro. This modulation may reflect an anti-apoptotic effect that mitigates epithelial damage under conditions of oxidative stress, inflammation, or infection (Song et al., 2022; Wu et al., 2019; Han et al., 2024).
When samples of fish fed the OmpK-displaying spores were compared with the parental spores FI314, the RNAseq showed an effect related to the immune system. Specifically, the upregulation of genes encoding chemokine 3-like and a fish-specific IL-6 member, M17, a subfamily cytokine M17, and the downregulation of genes related to vitronectin A and vitronectin B. IL-6 has both pro- and anti-inflammatory roles (Bird et al., 2005), while chemokine 3 serves as a key regulator of immune responses, guiding the activation and migration of neutrophils toward areas affected by infection or inflammation (Jayasinghe et al., 2025). However, qPCR analysis of intestine and kidney samples did not show significant differences in the expression of common immune-related genes. This suggests that OmpK-displaying spores effects are not strong enough to be reflected in the expression of canonical immune markers. These findings, together with the transcriptomic responses observed for the FI314 strain, suggest a localized modulation of innate immune signaling by OmpK-displaying spores, rather than a strong systemic activation. Further studies modifying the spore dosage and immunization periods will be necessary to confirm this hypothesis. The dietary inclusion of TolC-displaying spores, compared to the FI314 spores, resulted in the upregulation of genes associated with DNA replication and metabolic processes in European seabass, as indicated by GO biological process categories. This finding is consistent with the in vitro results, where casp3a2 expression was higher in cells exposed to TolC-displaying spores compared to those exposed to the parental FI314 spores. However, no results were related to immune system activation. As discussed above, factors such as immunization scheme, dosage, and the specific antigen displayed affect the responses to oral spore-based vaccines. The mucosal immune response to these antigen-carriers is inherently complex, involving both host-specific mechanisms and the intrinsic characteristics of the probiotic strain, the antigen, and their combined immunogenicity.
Overall, the results of this study indicate that B. subtilis FI314 spores act as mild modulators rather than strong inducers of mucosal inflammation. Spores could trigger intestinal tissues to adopt a responsive yet controlled immune state. In contrast, FI442 spores elicited minimal immunological effects under the same experimental conditions. The different immunomodulatory responses elicited by FI314 and FI442, despite being two B. subtilis strains, highlight the importance of strain-specific traits. In previous studies, these strains exhibited different phenotypes (e.g., differences in antimicrobial activities) and were isolated from distinct host species. Such differences in genetic background, phenotypic characteristics, and host adaptation contribute to the divergent responses observed in this study. Antigen display did not substantially alter the intrinsic probiotic profile but may have refined the immune response, leading to a subtle activation of innate pathways. The differential responses observed between OmpK and TolC-carrying spores highlight the importance of antigen selection and display efficiency in oral spore-based vaccination. Nonetheless, neither antigen was efficient at inducing a robust adaptive immune response measurable on serum IgM levels. However, antibody responses in the intestinal mucus (including IgT) were not assessed in this study and could provide more information regarding adaptive immune activation. This study focused on two autochthonous probiotic B. subtilis strains, and it is plausible that endogenous bacterial strains interact with the host in a homeostatic manner, rather than eliciting a strong activation of innate immunity mechanisms. Strong responses may be easily elicited by “foreign” (allochthonous) spores, such as those isolated from soil or the gut of other animal species.
Future studies should compare the mucosal immune responses induced by autochthonous and allochthonous Bacillus strains, assess earlier sampling points to capture immune-activation phases, evaluate antigen persistence and degradation on the spore surface, and integrate histological and microbiota analyses to gain a comprehensive understanding of the immune response. Such analysis will be essential to fully elucidate the mechanisms behind the local and systemic immune responses to spore-based oral vaccines in fish. Once vaccine development is optimized, an in vivo challenge will be necessary to demonstrate protective efficacy, together with later sampling points of specific antibodies in fish serum, to assess the duration of protection.
Data availability statement
The datasets used in this study (RNA-seq analysis) are deposited in the NCBI database under the archives numbers SRR36723510 to SRR36723521.
Ethics statement
The animal study was approved by Animal Welfare Committee of the Interdisciplinary Centre of Marine and Environmental Research (CIIMAR, ORBEA_CIIMAR27_2019). The study was conducted in accordance with the local legislation and institutional requirements. No potentially identifiable images or data are presented in this study.
RS and CS are guest editors of the special issue “Bacillus spp. for sustainable aquaculture: probiotics, postbiotics, secondary metabolites and biotechnological solutions” of Frontiers in Marine Science.
The remaining author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors CT, AO-T, AC, PD-R and CS declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.
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Edited by: Santhiyagu Prakash, Tamil Nadu Fisheries University, India
Reviewed by: María Inés Becker, Science and Technology for Development Foundation (Fucited), Chile
Manikandan Gurusamy, Tshwane University of Technology, South Africa
Sreeja Narayanan, Cochin University of Science and Technology, India