Anti-Virulence Potential of a Chionodracine-Derived Peptide against Multidrug-Resistant Pseudomonas aeruginosa Clinical Isolates from Cystic Fibrosis Patients.

Pseudomonas aeruginosa is an opportunistic pathogen causing several chronic infections resistant to currently available antibiotics. Its pathogenicity is related to the production of different virulence factors such as biofilm and protease secretion. Pseudomonas communities can persist in biofilms that protect bacterial cells from antibiotics. Hence, there is a need for innovative approaches that are able to counteract these virulence factors, which play a pivotal role, especially in chronic infections. In this context, antimicrobial peptides are emerging drugs showing a broad spectrum of antibacterial activity. Here, we tested the anti-virulence activity of a chionodracine-derived peptide (KHS-Cnd) on five P. aeruginosa clinical isolates from cystic fibrosis patients. We demonstrated that KHS-Cnd impaired biofilm development and caused biofilm disaggregation without affecting bacterial viability in nearly all of the tested strains. Ultrastructural morphological analysis showed that the effect of KHS-Cnd on biofilm could be related to a different compactness of the matrix. KHS-Cnd was also able to reduce adhesion to pulmonary cell lines and to impair the invasion of host cells by P. aeruginosa. A cytotoxic effect of KHS-Cnd was observed only at the highest tested concentration. This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against P. aeruginosa clinical strains.

Design and Characterization of Myristoylated and Non-Myristoylated Peptides Effective against Candida spp. Clinical Isolates.

The increasing resistance of fungi to antibiotics is a severe challenge in public health, and newly effective drugs are required. Promising potential medications are lipopeptides, linear antimicrobial peptides (AMPs) conjugated to a lipid tail, usually at the N-terminus. In this paper, we investigated the in vitro and in vivo antifungal activity of three short myristoylated and non-myristoylated peptides derived from a mutant of the AMP Chionodracine. We determined their interaction with anionic and zwitterionic membrane-mimicking vesicles and their structure during this interaction. We then investigated their cytotoxic and hemolytic activity against mammalian cells. Lipidated peptides showed a broad spectrum of activity against a relevant panel of pathogen fungi belonging to Candida spp., including the multidrug-resistant C. auris. The antifungal activity was also observed vs. biofilms of C. albicans, C. tropicalis, and C. auris. Finally, a pilot efficacy study was conducted on the in vivo model consisting of Galleria mellonella larvae. Treatment with the most-promising myristoylated peptide was effective in counteracting the infection from C. auris and C. albicans and the death of the larvae. Therefore, this myristoylated peptide is a potential candidate to develop antifungal agents against human fungal pathogens.

Molecular and cellular characterization of European sea bass CD3ε+ T lymphocytes and their modulation by microalgal feed supplementation.

The CD3 coreceptor is a master T cell surface marker, and genes encoding CD3ζ, γδ, and ε chains have been reported in several teleost fish. Here, a complete cDNA sequence of CD3ɛ chain was identified from a sea bass (Dicentrarchus labrax L.) gill transcriptome. Its basal expression was quantified in both lymphoid and non-lymphoid organs of sea bass juveniles with real-time qPCR analysis. After either in vitro stimulation of head kidney leukocytes with the T-cell mitogen phytohaemagglutinin or in vivo stimulation with an orally administered Vibrio anguillarum vaccine, CD3ε expression levels increased in head kidney leukocytes, confirming that CD3ε T cells may play important roles in fish systemic protection against pathogens. Further, three peptides were designed on the CD3ɛ cytoplasmic tail region and employed as immunogens for antibody production in rabbit. One antiserum so obtained, named RACD3/1, immunostained a band of the expected size in a western blot of a sea bass thymocyte lysate. The distribution of CD3ε+ lymphocyte population in the lymphoid organs and mucosal tissues was addressed in healthy fish by IHC. In decreasing percentage order, CD3ε+ lymphocytes were detected by flow cytometry in thymus, peripheral blood leukocytes, gills, head kidney, gut, and spleen. Finally, a significant in vivo enhancement of CD3ε+ T intestinal lymphocytes was found in fish fed on diets in which 100% fish meal was replaced by the microalgae Nannochloropsis sp. biomass. These results indicate that CD3ε+ T cells are involved in nutritional immune responses.

Semiconductivity Transition in Silicon Nanowires by Hole Transport Layer.

The surface of nanowires is a source of interest mainly for electrical prospects. Thus, different surface chemical treatments were carried out to develop recipes to control the surface effect. In this work, we succeed in shifting and tuning the semiconductivity of a Si nanowire-based device from n- to p-type. This was accomplished by generating a hole transport layer at the surface by using an electrochemical reaction-based nonequilibrium position to enhance the impact of the surface charge transfer. This was completed by applying different annealing pulses at low temperature (below 400 °C) to reserve the hydrogen bonds at the surface. After each annealing pulse, the surface was characterized by XPS, Kelvin probe measurements, and conductivity measured by FET based on a single Si NW. The mechanism and conclusion were supported experimentally and theoretically. To this end, this strategy has been demonstrated as an essential tool which could pave a new road for regulating semiconductivity and for other low-dimensional nanomaterials.

Molecular, Cellular and Functional Analysis of TRγ Chain along the European Sea Bass Dicentrarchus labrax Development.

In jawed vertebrates, adaptive immune responses are enabled by T cells. Two lineages were characterized based on their T cell receptor (TcR) heterodimers, namely αß or γδ peptide chains, which display an Ig domain-type sequence that is somatically rearranged. γδ T cells have been less extensively characterized than αß and teleost fish, in particular, suffer from a severe scarcity of data. In this paper, we worked on the well-known model, the European sea bass Dicentrarchus labrax, to broaden the understanding of teleost γδ-T cells. The T cell receptor chain (TR) γ transcript was expressed at a later developmental stage than TRß, suggesting a layered appearance of fish immune cells, and the thymus displayed statistically-significant higher mRNA levels than any other organ or lymphoid tissue investigated. The polyclonal antibody developed against the TRγ allowed the localization of TRγ-expressing cells in lymphoid organs along the ontogeny. Cell positivity was investigated through flow cytometry and the highest percentage was found in peripheral blood leukocytes, followed by thymus, gut, gills, spleen and head kidney. Numerous TRγ-expressing cells were localized in the gut mucosa, and the immunogold labelling revealed ultrastructural features that are typical of T cells. At last, microalgae-based diet formulations significantly modulated the abundance of TRγ+ cells in the posterior intestine, hinting at a putative involvement in nutritional immunity. From a comparative immunological perspective, our results contribute to the comprehension of the diversity and functionalities of γδ T cells during the development of a commercially relevant marine teleost model.

Cold Adaptation in Antarctic Notothenioids: Comparative Transcriptomics Reveals Novel Insights in the Peculiar Role of Gills and Highlights Signatures of Cobalamin Deficiency.

Far from being devoid of life, Antarctic waters are home to Cryonotothenioidea, which represent one of the fascinating cases of evolutionary adaptation to extreme environmental conditions in vertebrates. Thanks to a series of unique morphological and physiological peculiarities, which include the paradigmatic case of loss of hemoglobin in the family Channichthyidae, these fish survive and thrive at sub-zero temperatures. While some of the distinctive features of such adaptations have been known for decades, our knowledge of their genetic and molecular bases is still limited. We generated a reference de novo assembly of the icefish Chionodraco hamatus transcriptome and used this resource for a large-scale comparative analysis among five red-blooded Cryonotothenioidea, the sub-Antarctic notothenioid Eleginops maclovinus and seven temperate teleost species. Our investigation targeted the gills, a tissue of primary importance for gaseous exchange, osmoregulation, ammonia excretion, and its role in fish immunity. One hundred and twenty genes were identified as significantly up-regulated in Antarctic species and surprisingly shared by red- and white-blooded notothenioids, unveiling several previously unreported molecular players that might have contributed to the evolutionary success of Cryonotothenioidea in Antarctica. In particular, we detected cobalamin deficiency signatures and discussed the possible biological implications of this condition concerning hematological alterations and the heavy parasitic loads typically observed in all Cryonotothenioidea.

Identification, molecular characterization and functional analysis of interleukin (IL)-2 and IL-2like (IL-2L) cytokines in sea bass (Dicentrarchus labrax L.).

In mammals, interleukin (IL)-2, initially known as a T-cell grow factor, is an immunomodulatory cytokine involved in the proliferation of T cells upon antigen activation. In bony fish, some IL-2 orthologs have been identified, but, recently, an additional IL-2like (IL-2L) gene has been found. In this paper, we report the presence of these two divergent IL-2 isoforms in sea bass (Dicentrarchus labrax L.). Genomic analyses revealed that they originated from a gene duplication event, as happened in most percomorphs. These two IL-2 paralogs show differences in the amino acid sequence and in the exon 4 size, and these features could be an indication that they bind preferentially to different specific IL-2 receptors. Sea bass IL-2 paralogs are highly expressed in gut and spleen, which are tissues and organs involved in fish T cell immune functions, and the two cytokines could be up-regulated by both PHA stimulation and vaccination with a bacterial vaccine, with IL-2L being more inducible. To investigate the functional activities of sea bass IL-2 and IL-2L we produced the corresponding recombinant molecules in E. coli and used them to in vitro stimulate HK and spleen leukocytes. IL-2L is able to up-regulate the expression of markers related to different T cell subsets (Th1, Th2 and Th17) and to Treg cells in HK, whereas it has little effect in spleen. IL-2 is not active on these markers in HK, but shows an effect on Th1 markers in spleen. Finally, the stimulation with recombinant IL-2 and IL-2L is also able to induce in vitro proliferation of HK- and spleen-derived leukocytes. In conclusion, we have demonstrated that sea bass possess two IL-2 paralogs that likely have an important role in regulating T cell development in this species and that show distinct bioactivities.

Identification of an IgD/IgT chimera in the European sea bass (Dicentrarchus labrax L.).

Three classes of immunoglobulins have been identified in Teleosts: IgM, IgT/Z and IgD. They are fundamental for fish immune responses and, therefore, their functional activities are heavily investigated. In this paper, we describe the identification of a new IgD/IgT chimera in sea bass (Dicentrarchus labrax) from a gills transcriptome. This transcript joined the first six constant domains of the IgD chain with the two terminal constant domains of IgT, generating a long in-frame coding sequence with a junction between the canonical δ6 exon splicing donor site and the τ3 exon splicing acceptor site. Studies performed on genomic DNA confirmed the presence of the sequence and identifies and intronic region of 656 bp within this joining region. The basal expression of the IgD/IgT chimera was investigated both in silico and in vivo: high level of expression was found in gills, gut and head kidney. Moreover, IgD/IgT transcripts were up-regulated after in vitro stimulation of sea bass HK leukocytes with LPS. The IgD/IgT chimera was found also in two congener species, Morone saxatilis and Morone chrysops. It is not possible to have a precise idea on the evolutionary scenario that lead to the appearance of this sequence due to the lack of genomic information, but we could speculate that an ancestral duplication of the entire IgH locus was followed by the chimerization of Cδ/Cτ in one of the two loci. Finally, the IgD/IgT high basal expression in tissues and organs fundamental for sea bass immune response and its modulation after LPS stimulation provide a very preliminary indication that this unusual Ig variant could have a functional activity.

Structural Analysis and Design of Chionodracine-Derived Peptides Using Circular Dichroism and Molecular Dynamics Simulations.

Antimicrobial peptides have been identified as one of the alternatives to the extensive use of common antibiotics as they show a broad spectrum of activity against human pathogens. Among these is Chionodracine (Cnd), a host-defense peptide isolated from the Antarctic icefish Chionodraco hamatus, which belongs to the family of Piscidins. Previously, we demonstrated that Cnd and its analogs display high antimicrobial activity against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species). Herein, we investigate the interactions with lipid membranes of Cnd and two analogs, Cnd-m3 and Cnd-m3a, showing enhanced potency. Using a combination of Circular Dichroism, fluorescence spectroscopy, and all-atom Molecular Dynamics (MD) simulations, we determined the structural basis for the different activity among these peptides. We show that all peptides are predominantly unstructured in water and fold, preferentially as α-helices, in the presence of lipid vesicles of various compositions. Through a series of MD simulations of 400 ns time scale, we show the effect of mutations on the structure and lipid interactions of Cnd and its analogs. By explaining the structural basis for the activity of these analogs, our findings provide structural templates to design minimalistic peptides for therapeutics.

Differential Effects of Dietary Supplementation of Krill Meal, Soybean Meal, Butyrate, and Bactocell® on the Gene Expression of Atlantic Salmon Head Kidney.

The head kidney is a key organ that plays a fundamental role in the regulation of the fish immune response and in the maintenance of endocrine homeostasis. Previous studies indicate that the supplementation of exogenous dietary components, such as krill meal (KM), soybean meal (SM), Bactocell® (BA), and butyrate (BU), can have a significant effect on the immune function of the head kidney. The aim of this study was to investigate the differential effect of these four dietary ingredients on the transcriptional profiles of the head kidney of the Atlantic salmon. This study revealed that just a small number of genes were responsive to the feeding regime after a long-term (12 weeks) treatment, and evidenced that the most significant alterations, both in terms of the number of affected genes and magnitude of changes in gene expression, were detectable in the BU- and KM-fed groups compared with controls, while the SM diet had a nearly negligible effect, and BA had no significant effects at all. Most of the differentially expressed genes were involved in the immune response and, in line with data previously obtained from pyloric caeca, major components of the complement system were significantly affected. These alterations were accompanied by an increase in the density of melanomacrophage centers in the KM- and SM-fed group and their reduction in the BU-fed group. While three types of dietary supplements (BU, KM, and SM) were able to produce a significant modulation of some molecular players of the immune system, the butyrate-rich diet was revealed as the one with the most relevant immune-stimulating properties in the head kidney. These preliminary results suggest that further investigations should be aimed towards the elucidation of the potential beneficial effects of butyrate and krill meal supplementation on farmed salmon health and growth performance.

Lack of in vivo cross-protection of two different betanodavirus species RGNNV and SJNNV in European sea bass Dicentrachus labrax.

Viral encephalopathy and retinopathy (VER) is a severe infective disease characterized by neuropathological changes in several fish species associated with high mortality. The etiological agent is a virus belonging to the Nodaviridae family, genus Betanodavirus. To date, four different betanodavirus species have been officially recognized by International Committee on Taxonomy of Viruses (ICTV), namely the red-spotted grouper- (RGNNV), the striped jack- (SJNNV), the barfin flounder- (BFNNV) and the tiger puffer nervous necrosis virus (TPNNV). Moreover, two reassortants RGNNV/SJNNV and SJNNV/RGNNV have been described. Betanodaviruses can be classified into three different serotypes (A, B and C) that are antigenically different, so none (between serotype A and C) or partial (between serotype B and C) cross-immunoreactivity has been detected in vitro. In this study we investigated the in vivo cross-protection of the two main betanodavirus species (RGNNV and SJNNV), which belong to distinct serotype, by immunizing intraperitoneally (IP) juvenile sea bass with formalin inactivated RGNNV and SJNNV vaccines, followed by a challenge with RGNNV. Fish IP vaccinated with inactivated RGNNV showed a high protection value (85%). Serological analyses highlighted a great specific anti-NNV immunoglobulin M (IgM) production against the homologous virus, while a good seroconversion with low neutralization property was highlighted against the heterologous virus. In fish IP vaccinated with inactivated SJNNV the protection recorded was equal to 25%, significantly lower respect to the one provided by RGNNV IP vaccine. ELISA test detected good IgM production against the homologous virus, and a lower, but still detectable IgM production against the heterologous one. By contrast, serum neutralization test highlighted a poorly detectable antibody production unable to neutralize either the homologous or the heterologous virus. These results confirm that the two serotypes are not cross-protective in vivo. According to these findings, the production of multivalent formulation, or at least the provision of different types of vaccines based on both fish and virus species requirement, should be recommended in order to broaden the range of protection.

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.

The African coelacanth genome provides insights into tetrapod evolution.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Immuno-related gene transcription and antibody response in nodavirus (RGNNV and SJNNV)-infected European sea bass (Dicentrarchus labrax L.).

The immune response of European sea bass to RGNNV and SJNNV infections has been evaluated by quantifying the transcription of some genes involved in the IFN I system, as well as in the inflammatory and adaptive immune mechanisms. The transcription of IFN-I, ISG-12, ISG-15 and MxA genes has been analyzed in brain and head kidney, showing that RGNNV genotype induces a more intense response of the IFN I system than SJNNV in both organs. In addition, the results obtained indicate the importance of the inflammatory response in nodavirus pathogenesis, with the transcription of IL-8 and TNF-α significantly higher in brain than in head kidney, being RGNNV the strongest inductor. An important difference between the immune response induced by both genotypes refers to the IgM titre in sera, which was higher in SJNNV-inoculated fish. The acquired response is also important locally, since TR-γ transcription is higher in brain than in head kidney (especially in the RGNNV-inoculated group). To our knowledge, this is the first study addressing the sea bass anti-SJNNV immune response.

Electrochemical synthesis of self-organized TiO2 crystalline nanotubes without annealing.

This work demonstrates that upon anodic polarization in an aqueous fluoride-containing electrolyte, TiO2 nanotube array films can be formed with a well-defined crystalline phase, rather than an amorphous one. The crystalline phase was obtained avoiding any high temperature annealing. We studied the formation of nanotubes in an HF/H2O medium and the development of crystalline grains on the nanotube wall, and we found a facile way to achieve crystalline TiO2 nanotube arrays through a one-step anodization. The crystallinity of the film was influenced by the synthesis parameters, and the optimization of the electrolyte composition and anodization conditions (applied voltage and time) were carried out. For comparison purposes, crystalline anatase TiO2 nanotubes were also prepared by thermal treatment of amorphous nanotubes grown in an organic bath (ethylene glycol/NH4F/H2O). The morphology and the crystallinity of the nanotubes were studied by field emission gun-scanning electron microscopy (FEG-SEM) and Raman spectroscopy, whereas the electrochemical and semiconducting properties were analyzed by means of linear sweep voltammetry, impedance spectroscopy, and Mott-Schottky plots. X-ray photoelectron spectroscopy (XPS) and ultraviolet photoelectron spectroscopy (UPS) allowed us to determine the surface composition and the electronic structure of the samples and to correlate them with the electrochemical data. The optimal conditions to achieve a crystalline phase with high donor concentration are defined.

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.

An ab initio study of hydroxylated graphane.

Graphene-based derivatives with covalent functionalization and well-defined stoichiometry are highly desirable in view of their application as functional surfaces. Here, we have evaluated by ab initio calculations the energy of formation and the phase diagram of hydroxylated graphane structures, i.e., fully functionalized graphene derivatives coordinated with -H and -OH groups. We compared these structures to different hydrogenated and non-hydrogenated graphene oxide derivatives, with high level of epoxide and hydroxyl groups functionalization. Based on our calculations, stable phases of hydroxylated graphane with low and high contents of hydrogen are demonstrated for high oxygen and hydrogen partial pressure, respectively. Stable phases of graphene oxide with a mixed carbon hybridization are also found. Notably, the synthesis of hydroxylated graphane has been recently reported in the literature.

Structure and membrane interactions of chionodracine, a piscidin-like antimicrobial peptide from the icefish Chionodraco hamatus.

Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.

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.

Immune response of the Antarctic teleost Trematomus bernacchii to immunization with Psychrobacter sp. (TAD1).

Adult Trematomus bernacchii have been immunized intraperitoneally with heat-killed cells of the Antarctic marine bacterium Psychrobacter sp. (TAD1) up to 60 days. After immunizations and sampling at various times, fish sera were tested for specific IgM by ELISA, and different tissues (head kidney and spleen) were investigated for transcription of master genes of the acquired immune response (IgM, IgT, TRß, TRγ). Results from ELISA assays showed a time-dependent induction of specific serum anti-TAD1 IgM, and western blot analysis of TAD1 lysates probed with fish sera revealed enhanced immunoreactivity in immunized animals compared to controls. Quantitative PCR analysis of transcripts coding for IgM, IgT, TRß, TRγ was performed in T. bernacchii tissues to assess basal expression, and then on cDNAs of cells from head kidney and spleen of fish injected for 8, 24, and 72 h with inactivated TAD1. The results showed a differential basal expression of transcripts in the examined tissues, and a time-dependent strong up-regulation of IgT, TRß, TRγ genes upon in vivo stimulation with TAD1. These results represent a first in vivo study on the mounting of a specific immune response in an Antarctic teleost species.