Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM+ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

L-methionine supplementation modulates IgM+ B cell responses in rainbow trout.

The interest in dietary amino acids (AAs) as potential immunomodulators has been growing the recent years, since specific AAs are known to regulate key metabolic pathways of the immune response or increase the synthesis of some immune-related proteins. Methionine, tryptophan and lysine are among the ten essential AAs for fish, meaning that they cannot be produced endogenously and must be provided through the diet. To date, although dietary supplementation of fish with some of these AAs has been shown to have positive effects on some innate immune parameters and disease resistance, the effects that these AAs provoke on cells of the adaptive immune system remained unexplored. Hence, in the current study, we have investigated the effects of these three AAs on the functionality of rainbow trout (Oncorhynchus mykiss) IgM+ B cells. For this, splenic leukocytes were isolated from untreated adult rainbow trout and incubated in culture media additionally supplemented with different doses of methionine, tryptophan or lysine in the presence or absence of the model antigen TNP-LPS (2,4,6-trinitrophenyl hapten conjugated to lipopolysaccharide). The survival, IgM secreting capacity and proliferation of IgM+ B cells was then studied. In the case of methionine, the phagocytic capacity of IgM+ B cells was also determined. Our results demonstrate that methionine supplementation significantly increases the proliferative effects provoked by TNP-LPS and also up-regulates the number of cells secreting IgM, whereas tryptophan or lysine have either minor or even negative effects on rainbow trout IgM+ B cells. This increase in the number of IgM-secreting cells in response to methionine surplus was further verified in a feeding experiment, in which the beneficial effects of methionine on the specific response to anal immunization were also confirmed. The results presented demonstrate the beneficial effects of dietary supplementation with methionine on the adaptive immune responses of fish.

Transcriptomic and targeted immune transcript analyses confirm localized skin immune responses in Atlantic salmon towards the salmon louse.

Atlantic salmon (Salmo salar) are highly susceptible to infestations with the ectoparasite Lepeophtheirus salmonis, the salmon louse. Infestations elicit an immune response in the fish, but the response does not lead to parasite clearance, nor does it protect against subsequent infestations. It is, however, not known why the immune response is not adequate, possibly because the local response directly underneath the louse has been poorly evaluated. The present study describes the transcriptomic response by RNA sequencing of skin at the site of copepodid attachment. Analysing differentially expressed genes, 2864 were higher and 1357 were lower expressed at the louse attachment site compared to uninfested sites in the louse infested fish, while gene expression at uninfested sites were similar to uninfested control fish. The transcriptional patterns of selected immune genes were further detailed in three skin compartments/types: Whole skin, scales only and fin tissue. The elevation of pro-inflammatory cytokines and immune cell marker transcripts observed in whole skin and scale samples were not induced in fin, and a higher cytokine transcript level in scale samples suggest it can be used as a nonlethal sampling method to enhance selective breeding trials. Furthermore, the immune response was followed in both skin and anterior kidney as the infestation developed. Here, newly moulted preadult 1 stage lice induced a higher immune response than chalimi and adult lice. Overall, infestation with salmon louse induce a modest but early immune response with an elevation of mainly innate immune transcripts, with the response primarily localized to the site of attachment.

Immunostimulant properties of full-length and truncated Marinobacter algicola flagellins, and their effects against viral hemorrhagic septicemia virus (VHSV) in trout.

Adjuvants that would help optimize fish vaccines against bacterial and viral pathogens are highly demanded by the aquaculture sector. Flagellin has been proposed as an immunostimulant and an adjuvant for more than a decade. However, the adjuvant ability of flagellins with hypervariable region deleted is still unclear in fish. In this study, we evaluated the immune-stimulating capacity of two recombinant flagellins, the wild-type flagellin F from Marinobacter algicola and a version with the hypervariable region deleted (FredV2), to induce the transcription of a wide range of immune genes using two rainbow trout cell lines: a monocyte/macrophage-cell line (RTS-11) and an epithelial cell line from intestine (RTgutGC). Additionally, we studied the capacity of both flagellins to limit the replication of viral hemorrhagic septicemia virus (VHSV) on the RTgutGC cell line. Our results demonstrated that both recombinant flagellins can significantly increase the transcription of IL-1ß1, IL-6, and IL-8 in both cell lines. However, other cytokines such as IFNγ1, and TNFα or antimicrobial peptides such as hepcidin were induced by both flagellins in RTgutGC but not in RTS-11 cells. Furthermore, both flagellins were capable of reducing the replication of VHSV in RTgutGC cells. Although the immunostimulatory and the antiviral capacities exerted by F were slightly more potent than those obtained with FredV2, the effects were retained after losing the hypervariable region. Our results provide new information on the immunostimulating and antiviral capacities of flagellins that point to their potential as suitable adjuvants for the future optimization of vaccines in aquaculture.

Bacillus subtilis Expressing the Infectious Pancreatic Necrosis Virus VP2 Protein Retains Its Immunostimulatory Properties and Induces a Specific Antibody Response.

Bacillus subtilis has been documented in the past years as an effective probiotic for different aquacultured species, with recognized beneficial effects on water quality, fish growth and immune status. Furthermore, its potential as a vaccine adjuvant has also been explored in different species. In the current work, we have used B. subtilis spores as delivery vehicles for the presentation of the VP2 protein from infectious pancreatic necrosis virus (IPNV). For this, the VP2 gene was amplified and translationally fused to the crust protein CotY. The successful expression of VP2 on the spores was confirmed by Western blot. We then compared the immunostimulatory potential of this VP2-expressing strain (CRS208) to that of the original B. subtilis strain (168) on rainbow trout (Oncorhynchus mykiss) leukocytes obtained from spleen, head kidney and the peritoneal cavity. Our results demonstrated that both strains significantly increased the percentage of IgM+ B cells and the number of IgM-secreting cells in all leukocyte cultures. Both strains also induced the transcription of a wide range of immune genes in these cultures, with small differences between them. Importantly, specific anti-IPNV antibodies were detected in fish intraperitoneally or orally vaccinated with the CRS208 strain. Altogether, our results demonstrate B. subtilis spores expressing foreign viral proteins retain their immunomodulatory potential while inducing a significant antibody response, thus constituting a promising vaccination strategy.

Mechanisms Used by Probiotics to Confer Pathogen Resistance to Teleost Fish.

Probiotics have been defined as live microorganisms that when administered in adequate amounts confer health benefits to the host. The use of probiotics in aquaculture is an attractive bio-friendly method to decrease the impact of infectious diseases, but is still not an extended practice. Although many studies have investigated the systemic and mucosal immunological effects of probiotics, not all of them have established whether they were actually capable of increasing resistance to different types of pathogens, being this the outmost desired goal. In this sense, in the current paper, we have summarized those experiments in which probiotics were shown to provide increased resistance against bacterial, viral or parasitic pathogens. Additionally, we have reviewed what is known for fish probiotics regarding the mechanisms through which they exert positive effects on pathogen resistance, including direct actions on the pathogen, as well as positive effects on the host.

Vaccination and immune responses of European sea bass (Dicentrarchus labrax L.) against betanodavirus.

This review summarizes the available knowledge on the immune defences of European sea bass against antigenic preparations derived from the viral encephalopathy and retinopathy virus (betanodavirus), which represents a major threat to the health of this fish species. The nodavirus is widely present and differentiates into several strains that infect invertebrates (in insects, alphanodavirus) and teleost fish, and thus may represent a great problem for farmed fish species. Many efforts have been directed to discovering new immunizations to induce protection in sea bass, especially at young stages, and these efforts have included employing diverse betanodavirus strains, antigen preparation, vaccination routes, and the addition of adjuvants and/or immunostimulants. The obtained results showed that inactivated preparations of betanodavirus that were administered intraperitoneally may induce both immune recognition and protection. Attempts at performing mucosal immunization by immersion and/or oral administration, which is a vaccination route that is highly preferred for sea bass, have shown intriguing results, and more studies are necessary for its improvement. Overall, the objective of identifying a reliable vaccine that also cross-protects against different genotypes or reassortant viruses for use in European sea bass against betanodavirus appears to be an attainable goal in the near future.

Immunoglobulin T from sea bass (Dicentrarchus labrax L.): molecular characterization, tissue localization and expression after nodavirus infection.

BACKGROUND: Immunoglobulins (Igs) are fundamental components of the adaptive immune system of vertebrates, with the IgT/IgZ isotype specific of Teleosts. In this paper we describe the identification of an IgT heavy chain from the European sea bass (Dicentrarchus labrax L.), its molecular characterization and tissue mRNA localization by in situ hybridization. RESULTS: Sea bass IgT consists of 552 aa (Accession Number KM410929) and it contains a putative 19 amino acids long signal peptide and one potential N-glycosylation site. The C-region consists of four CH domains; each contains the cysteine and tryptophan residues required for their correct folding. Based on the recent sequencing of sea bass genome, we have identified five different genomic contigs bearing exons unequivocally pertaining to IgT (CH2, CH3 and CH4), but none corresponded to a complete IgH locus as IgT sequences were found in the highly fragmented assembled genomic regions which could not be assigned to any major scaffold. The 3D structure of sea bass IgT has been modelled using the crystal structure of a mouse Ig gamma as a template, thus showing that the amino acid sequence is suitable for the expected topology referred to an immunoglobulin-like architecture. The basal expression of sea bass IgT and IgM in different organs has been analysed: gut and gills, important mucosal organs, showed high IgT transcripts levels and this was the first indication of the possible involvement of sea bass IgT in mucosal immune responses. Moreover, sea bass IgT expression increased in gills and spleen after infection with nodavirus, highlighting the importance of IgT in sea bass immune responses. In situ hybridization confirmed the presence of IgT transcripts in the gut and it revealed a differential expression along the intestinal tract, with a major expression in the posterior intestine, suggesting the hindgut as a site for the recruitment of IgT+ cells in this species. IgT transcripts were also found in gill filaments and parallel lamellae and, for the first time, we identified scattered IgT positive cells in the liver, with a strong signal in the hepatic parenchyma. CONCLUSIONS: In conclusion, we performed a full molecular characterization of IgT in sea bass that points out its possible involvement in mucosal immune responses of this species.

A formalin-inactivated immunogen against viral encephalopathy and retinopathy (VER) disease in European sea bass (Dicentrarchus labrax): immunological and protection effects.

The European sea bass (Dicentrarchus labrax) is an important farmed fish species in the Mediterranean area, very sensitive to the infection by encephalopathy and retinopathy virus (VERv), or Betanodavirus, which causes massive mortalities. Effective vaccines to fight the pathology are not yet available and in this work we describe a promising intraperitoneal immunization route against VERv of sea bass juveniles. We performed intraperitoneal and immersion immunization trials with a VERv (isolate 283.2009 RGNNV) inactivated by formalin, ß-propiolactone and heat treatment. Interestingly, the intraperitoneal immunization with formalin-inactivated VERv induced a significant antigen-specific IgM production, differently from other inactivation protocols. However, the same formalin-inactivated antigen resulted in very low IgM antibodies when administered by immersion. Following the intraperitoneal injection with formalin-inactivated virus, the quantitative expression of the antiviral MxA gene showed a modulation of transcripts in the gut after 48 h and on head kidney after 24 h, whereas ISG12 gene was significantly up-regulated after 48 h on both tissues. In immersion immunization with formalin-inactivated VERv, a modulation of MxA and ISG12 genes after 24 h post-treatment was detected in the gills. An effective uptake of VERv particles in the gills was confirmed by immunohistochemistry using anti-VERv antibodies. Lastly, in challenge experiments using live VERv after intraperitoneal immunization with formalin-inactivated VERv, we observed a significant increase (81.9%) in relative survival percentage with respect to non-immunized fish, whereas immersion immunization resulted in no protection. Our results suggest that intraperitoneal immunization with formalin-inactivated VERv could be a safe and effective strategy to fight Betanodavirus infection in European sea bass.