Discovery and Characterization of the ddx41 Gene in Atlantic Salmon: Evolutionary Implications, Structural Functions, and Innate Immune Responses to Piscirickettsia salmonis and Renibacterium salmoninarum Infections.

The innate immune response in Salmo salar, mediated by pattern recognition receptors (PRRs), is crucial for defending against pathogens. This study examined DDX41 protein functions as a cytosolic/nuclear sensor for cyclic dinucleotides, RNA, and DNA from invasive intracellular bacteria. The investigation determined the existence, conservation, and functional expression of the ddx41 gene in S. salar. In silico predictions and experimental validations identified a single ddx41 gene on chromosome 5 in S. salar, showing 83.92% homology with its human counterpart. Transcriptomic analysis in salmon head kidney confirmed gene transcriptional integrity. Proteomic identification through mass spectrometry characterized three unique peptides with 99.99% statistical confidence. Phylogenetic analysis demonstrated significant evolutionary conservation across species. Functional gene expression analysis in SHK-1 cells infected by Piscirickettsia salmonis and Renibacterium salmoninarum indicated significant upregulation of DDX41, correlated with increased proinflammatory cytokine levels and activation of irf3 and interferon signaling pathways. In vivo studies corroborated DDX41 activation in immune responses, particularly when S. salar was challenged with P. salmonis, underscoring its potential in enhancing disease resistance. This is the first study to identify the DDX41 pathway as a key component in S. salar innate immune response to invading pathogens, establishing a basis for future research in salmonid disease resistance.

Salmonid MyD88 is a key adapter protein that activates innate effector mechanisms through the TLR5M/TLR5S signaling pathway and protects against Piscirickettsia salmonis infection.

The membrane-anchored and soluble Toll-like Receptor 5 -TLR5M and TLR5S, respectively-from teleost recognize bacterial flagellin and induce the pro-inflammatory cytokines expression in a MyD88-dependent manner such as the TLR5 mammalian orthologous receptor. However, it has not been demonstrated whether the induced signaling pathway by these receptors activate innate effector mechanisms MyD88-dependent in salmonids. Therefore, in this work we study the MyD88 dependence on the induction of TLR5M/TLR5S signaling pathway mediated by flagellin as ligand on the activation of some innate effector mechanisms. The intracellular and extracellular Reactive Oxygen Species (ROS) production and conditioned supernatants production were evaluated in RTS11 cells, while the challenge with Piscirickettsia salmonis was evaluated in SHK-1 cells. Our results demonstrate that flagellin directly stimulates ROS production and indirectly stimulates it through the production of conditioned supernatants, both in a MyD88-dependent manner. Additionally, flagellin stimulation prevents the cytotoxicity induced by infection with P. salmonis in a MyD88-dependent manner. In conclusion we demonstrate that MyD88 is an essential adapter protein in the activation of the TLR5M/TLR5S signaling pathway mediated by flagellin in salmonids, which leads downstream to the induction of innate effector mechanisms, promoting immuno-protection against a bacterial challenge with P. salmonis.

Temporal Gene Expression Signature of Plasma Extracellular Vesicles-MicroRNAs from Post-Smolt Coho Salmon Challenged with Piscirickettsia salmonis.

Piscirickettsiosis is the most important bacterial disease in the Chilean salmon industry, which has borne major economic losses due to failure to control it. Cells use extracellular vesicles (EVs) as an inter-cellular communicators to deliver several factors (e.g., microRNAs) that may regulate the responses of other cells. However, there is limited knowledge about the identification and characterization of EV-miRNAs in salmonids or the effect of infections on these. In this study, Illumina sequencing technology was used to identify Coho salmon plasma EV-miRNAs upon Piscirickettsia salmonis infection at four different time points. A total of 118 novels and 188 known EV-miRNAs, including key immune teleost miRNAs families (e.g., miR-146, miR-122), were identified. A total of 245 EV-miRNAs were detected as differentially expressed (FDR < 5%) in terms of control, with a clear down-regulation pattern throughout the disease. KEGG enrichment results of EV-miRNAs target genes showed that they were grouped mainly in cellular, stress, inflammation and immune responses. Therefore, it is hypothesized that P. salmonis could potentially benefit from unbalanced modulation response of Coho salmon EV-miRNAs in order to promote a hyper-inflammatory and compromised immune response through the suppression of different key immune host miRNAs during the course of the infection, as indicated by the results of this study.

The importance of the Atlantic salmon peritoneal cavity B cell response: Local IgM secreting cells are predominant upon Piscirickettsia salmonis infection.

The intraperitoneal route is favored for administration of inactivated and attenuated vaccines in Atlantic salmon. Nevertheless, the immune responses in the teleost peritoneal cavity (PerC) are still incompletely defined. In this study, we investigated the B cell responses after intraperitoneal Piscirickettsia salmonis (P. salmonis) challenge of Atlantic salmon, focusing on the local PerC response versus responses in the lymphatic organs: spleen and head kidney. We observed a major increase of leukocytes, total IgM antibody secreting cells (ASC), and P. salmonis-specific ASC in the PerC at 3- and 6-weeks post infection (wpi). The increase in ASC frequency was more prominent in the spleen and PerC compared to the head kidney during the observed 6 wpi. The serum antibody response included P. salmonis-specific antibodies and non-specific antibodies recognizing the non-related bacterial pathogen Yersinia ruckeri and the model antigen TNP-KLH. Finally, we present evidence that supports a putative role for the adipose tissue in the PerC immune response.

Transcriptomic analysis reveals a Piscirickettsia salmonis-induced early inflammatory response in rainbow trout skeletal muscle.

Skeletal muscle is the most abundant tissue in teleosts and is essential for movement and metabolism. Recently, it has been described that skeletal muscle can express and secrete immune-related molecules during pathogen infection. However, the role of this tissue during infection is poorly understood. To determine the immunocompetence of fish skeletal muscle, juvenile rainbow trout (Oncorhynchus mykiss) were challenged with Piscirickettsia salmonis strain LF-89. P. salmonis is the etiological agent of piscirickettsiosis, a severe disease that has caused major economic losses in the aquaculture industry. This gram-negative bacterium produces a chronic systemic infection that involves several organs and tissues in salmonids. Using high-throughput RNA-seq, we found that 60 transcripts were upregulated in skeletal muscle, mostly associated with inflammatory response and positive regulation of interleukin-8 production. Conversely, 141 transcripts were downregulated in association with muscle filament sliding and actin filament-based movement. To validate these results, we performed in vitro experiments using rainbow trout myotubes. In myotubes coincubated with P. salmonis strain LF-89 at an MOI of 50, we found increased expression of the proinflammatory cytokine il1b and the pattern recognition receptor tlr5s 8 and 12 h after infection. These results demonstrated that fish skeletal muscle is an immunologically active organ that can implement an early immunological response against P. salmonis.

Interferon Gamma Induces the Increase of Cell-Surface Markers (CD80/86, CD83 and MHC-II) in Splenocytes From Atlantic Salmon.

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.

Effect of cortisol on the immune-like response of rainbow trout (Oncorhynchus mykiss) myotubes challenged with Piscirickettsia salmonis.

Salmonids are a species of high commercial value in Chilean aquaculture, where muscle is the final product of the industry. Fish can be affected by stress during intensive cultures, increasing susceptibility to infections. Recently, we reported that muscle is an important focus of immune reactions. However, studies have shown the immunosuppressive effect of stress only in lymphoid organs, and few studies have been conducted on muscle and immunity. Hence, we determine the effects of cortisol on the immune-like response of fish myotubes challenged with Piscirickettsia salmonis by three trials. First, rainbow trout primary culture of muscle was cultured and treated with cortisol (100 ng/mL) for 3 and 4 h. Second, myotubes were challenged with P. salmonis (MOI 50) for 4, 6 and 8 h. And third, muscle cell cultures were pretreated with cortisol and then challenged with P. salmonis. The mRNA levels of glucocorticoid pathway and innate immunity were evaluated by qPCR. Cortisol increased the klf15 levels and downregulated the innate immune-related tlr5m gene and antimicrobial peptides. P. salmonis challenge upregulated several immune-related genes. Finally, cortisol pretreatment followed by P. salmonis challenge differentially modulated stress- and immune-related genes. These data suggest that fish muscle cells possess an intrinsic immune response and are differentially regulated by cortisol, which could lead to bacterial outbreaks in muscle under stress conditions.

Protein-Based Vaccine Protect Against Piscirickettsia salmonis in Atlantic Salmon (Salmo salar).

An effective and economical vaccine against the Piscirickettsia salmonis pathogen is needed for sustainable salmon farming and to reduce disease-related economic losses. Consequently, the aquaculture industry urgently needs to investigate efficient prophylactic measures. Three protein-based vaccine prototypes against Piscirickettsia salmonis were prepared from a highly pathogenic Chilean isolate. Only one vaccine effectively protected Atlantic salmon (Salmo salar), in correlation with the induction of Piscirickettsia-specific IgM antibodies and a high induction of transcripts encoding pro-inflammatory cytokines (i.e., Il-1ß and TNF-α). In addition, we studied the proteome fraction protein of P. salmonis strain Austral-005 using multidimensional protein identification technology. The analyzes identified 87 proteins of different subcellular origins, such as the cytoplasmic and membrane compartment, where many of them have virulence functions. The other two prototypes activated only the innate immune responses, but did not protect Salmo salar against P. salmonis. These results suggest that the knowledge of the formulation of vaccines based on P. salmonis proteins is useful as an effective therapy, this demonstrates the importance of the different research tools to improve the study of the different immune responses, resistance to diseases in the Atlantic salmon. We suggest that this vaccine can help prevent widespread infection by P. salmonis, in addition to being able to be used as a booster after a primary vaccine to maintain high levels of circulating protective antibodies, greatly helping to reduce the economic losses caused by the pathogen.

Characterization and expression analysis of Nod-like receptor 3 (NLRC3) against infection with Piscirickettsia salmonis in Atlantic salmon.

The nucleotide binding oligomerization domain like receptors, or NOD like receptors (NLRs), are intracellular receptors responsible for recognizing pathogens in vertebrates. Several NLR mammalian models have been characterized and analyzed but few studies have been performed with teleost species. In this study, we analyzed the nucleotide sequence of six mRNA variants of NLRC3 in Atlantic salmon (SsNLRC3), and we deduced the amino acid sequence coding for two different isoforms with a total length of 1135 amino acids and 1093 amino acids. We analyzed the phylogeny of all variants, including a Piscirickettsia salmonis infection in Atlantic salmon. All variants and their expression pattern during infection were analyzed using real-time qPCR. One of the analyzed variants was over-expressed during the early stages of Piscirickettsia salmonis infection, and we were able to identify two different SsNLRC3 isoforms. Lastly, we observed that an alteration in the amino acid sequence of one of the isoforms can directly affect the pathogen recognition function.

Transcriptome Profiling of Atlantic Salmon (Salmo salar) Parr With Higher and Lower Pathogen Loads Following Piscirickettsia salmonis Infection.

Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis, is one of the most devastating diseases of salmonids. However, the transcriptomic responses of Atlantic salmon (Salmon salar) in freshwater to an EM-90-like isolate have not been explored. Here, we infected Atlantic salmon parr with an EM-90-like isolate and conducted time-course qPCR analyses of pathogen load and four biomarkers (campb, hampa, il8a, tlr5a) of innate immunity on the head kidney samples. Transcript expression of three of these genes (except hampa), as well as pathogen level, peaked at 21 days post-injection (DPI). Multivariate analyses of infected individuals at 21 DPI revealed two infection phenotypes [lower (L-SRS) and higher (H-SRS) infection level]. Five fish from each group (Control, L-SRS, and H-SRS) were selected for transcriptome profiling using a 44K salmonid microarray platform. We identified 1,636 and 3,076 differentially expressed probes (DEPs) in the L-SRS and H-SRS groups compared with the control group, respectively (FDR = 1%). Gene ontology term enrichment analyses of SRS-responsive genes revealed the activation of a large number of innate (e.g. "phagocytosis", "defense response to bacterium", "inflammatory response") and adaptive (e.g. "regulation of T cell activation", "antigen processing and presentation of exogenous antigen") immune processes, while a small number of general physiological processes (e.g. "apoptotic process", development and metabolism relevant) was enriched. Transcriptome results were confirmed by qPCR analyses of 42 microarray-identified transcripts. Furthermore, the comparison of individuals with differing levels of infection (H-SRS vs. L-SRS) generated insights into the biological processes possibly involved in disease resistance or susceptibility. This study demonstrated a low mortality (~30%) EM-90-like infection model and broadened the current understanding of molecular pathways underlying P. salmonis-triggered responses of Atlantic salmon, identifying biomarkers that may assist to diagnose and combat this pathogen.

Assessing the impacts of skin mucus from Salmo salar and Oncorhynchus mykiss on the growth and in vitro infectivity of the fish pathogen Piscirickettsia salmonis.

Piscirickettsiosis is a fish disease caused by the facultative intracellular bacterium, Piscirickettsia salmonis. Even though entry routes of P. salmonis in fish are not fully clear yet, the skin seems to be the main portal in some salmonid species. Despite the importance of fish mucous skin barrier in fighting waterborne pathogens, the interaction between salmonid skin mucus and the bacterium is unknown. This study seeks to determine the in vitro changes in the growth of two Chilean P. salmonis strains (LF-89-like and EM-90-like genotypes) and the type strain LF-89T under exposures to skin mucus from Salmo salar and Oncorhynchus mykiss, as well as changes in the cytotoxic effect of P. salmonis on the SHK-1 cells following exposures. The results suggest that the growth of three P. salmonis strains was not significantly negatively affected under exposures to skin mucus (adjusted at 100 µg total protein ml-1 ) of O. mykiss (69 ± 18 U lysozyme ml-1 ) and S. salar (48 ± 33 U lysozyme ml-1 ) over time. However, the cytotoxic effect of P. salmonis, pre-exposed to salmonid skin mucus, on the SHK-1 cell line was reliably identified only towards the end of the incubation period, suggesting that the mucus had a delaying effect on the cytotoxic response of the cell line to the bacterium. These results represent a baseline knowledge to open new avenues of research intended to understand how P. salmonis faces the fish mucous skin barrier.

Non-Specific Antibodies Induce Lysosomal Activation in Atlantic Salmon Macrophages Infected by Piscirickettsia salmonis.

Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.

Pharmacological iron-chelation as an assisted nutritional immunity strategy against Piscirickettsia salmonis infection.

Salmonid Rickettsial Septicaemia (SRS), caused by Piscirickettsia salmonis, is a severe bacterial disease in the Chilean salmon farming industry. Vaccines and antibiotics are the current strategies to fight SRS; however, the high frequency of new epizootic events confirms the need to develop new strategies to combat this disease. An innovative opportunity is perturbing the host pathways used by the microorganisms to replicate inside host cells through host-directed antimicrobial drugs (HDAD). Iron is a critical nutrient for P. salmonis infection; hence, the use of iron-chelators becomes an excellent alternative to be used as HDAD. The aim of this work was to use the iron chelator Deferiprone (DFP) as HDAD to treat SRS. Here, we describe the protective effect of the iron chelator DFP over P. salmonis infections at non-antibiotic concentrations, in bacterial challenges both in vitro and in vivo. At the cellular level, our results indicate that DFP reduced the intracellular iron content by 33.1% and P. salmonis relative load during bacterial infections by 78%. These findings were recapitulated in fish, where DFP reduced the mortality of rainbow trout challenged with P. salmonis in 34.9% compared to the non-treated group. This is the first report of the protective capacity of an iron chelator against infection in fish, becoming a potential effective host-directed therapy for SRS and other animals against ferrophilic pathogens.

Host genetic variation explains reduced protection of commercial vaccines against Piscirickettsia salmonis in Atlantic salmon.

Vaccination is a widely used control strategy to prevent Piscirickettsia salmonis causing disease in salmon farming. However, it is not known why all the currently available commercial vaccines generally fail to protect against this pathogenic bacteria. Here, we report, from two different populations, that between-family variation is a strong intrinsic factor that determines vaccine protection for this disease. While in some full-sib families, the protection added by vaccination increased the survival time in 13 days in comparison with their unvaccinated siblings; in other families, there was no added protection by vaccination or even it was slightly negative. Resistance to P. salmonis, measured as days to death, was higher in vaccinated than unvaccinated fish, but only a moderate positive genetic correlation was obtained between these traits. This disputes a previous hypothesis, that stated that both traits were fully controlled by the same genes, and challenges the use of unvaccinated fish as gold standard for evaluating and selecting fish resistant to P. salmonis, particularly if the offspring will be vaccinated. More studies are necessary to evaluate if variation in the host immune response to vaccination could explain the between-family differences in resistance observed in vaccinated fish.

Expression of DC-SIGN-like C-Type Lectin Receptors in Salmo salar.

C-Type Lectin Receptors (CTLR) are involved in the activation of innate and adaptative immune responses. Among these receptors, the Dendritic Cell-Specific ICAM-3-Grabbing nonintegrin (DC-SIGN/CD209) has become a hot topic due to its ability to bind and facilitate the infections processes of several pathogens. Although well characterized in mammals, little documentation exists about the receptor in salmonid fishes. Here, we report the sequence and expression analysis of eight DC-SIGN-like genes in Salmo salar. Each receptor displays structural similarities to DC-SIGN molecules described in mammals, including internalization motifs, a neck region with heptad repeats, and a Ca+2-dependent carbohydrate recognition domain. The receptors are expressed in multiple tissues of fish, and fish cell lines, with differential expression upon infection with viral and bacterial pathogens. The identification of DC-SIGN-like receptors in Salmo salar provides new information regarding the structure of the immune system of salmon, potential markers for cell subsets, as well as insights into DC-SIGN conservation across species.

Infection and microbial molecular motifs modulate transcription of the interferon-inducible gene ifit5 in a teleost fish.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are involved in antiviral defense. Members of this protein family contain distinctive multiple structural motifs comprising tetratricopeptides that are tandemly arrayed or dispersed along the polypeptide. IFIT-encoding genes are upregulated by type I interferons (IFNs) and other stimuli. IFIT proteins inhibit virus replication by binding to and regulating the functions of cellular and viral RNA and proteins. In teleost fish, knowledge about genes and functions of IFITs is currently limited. In the present work, we describe an IFIT5 orthologue in Atlantic salmon (SsaIFIT5) with characteristic tetratricopeptide repeat motifs. We show here that the gene encoding SsaIFIT5 (SsaIfit5) was ubiquitously expressed in various salmon tissues, while bacterial and viral challenge of live fish and in vitro stimulation of cells with recombinant IFNs and pathogen mimics triggered its transcription. The profound expression in response to various immune stimulation could be ascribed to the identified IFN response elements and binding sites for various immune-relevant transcription factors in the putative promoter of the SsaIfit5 gene. Our results establish SsaIfit5 as an IFN-stimulated gene in A. salmon and strongly suggest a phylogenetically conserved role of the IFIT5 protein in antimicrobial responses in vertebrates.

Non-lysosomal Activation in Macrophages of Atlantic Salmon (Salmo salar) After Infection With Piscirickettsia salmonis.

Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis is able to survive in host macrophages, localized within a vacuole like-compartment which prevents lysosomal degradation. However, the relevant aspects of the pathogenesis of P. salmonis as the host modulation that allow its intracellular survival have been poorly characterized. In this study, we evaluated the role of lysosomes in the response to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A gentamicin protection assay was performed to recover intracellular bacteria and the 16S rDNA copy number was quantified through quantitative polymerase chain reaction in order to determine the replication of P. salmonis within macrophages. Lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon macrophage-enriched cell cultures, accompanied by an increase in the detection of the 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and replication appears to be favored by a perturbation of the lysosomal degradation system. We observed a modulation in the total number of lysosomes and lysosomal acidification following infection with P. salmonis. Collectively, the results of this study showed that infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal response which may be associated with host immune evasion mechanisms of P. salmonis that have not been previously reported.

Temperature modulates the immunological response of the sub-antarctic notothenioid fish Eleginops maclovinus injected with Piscirickettsia salmonis.

Eleginops maclovinus is a eurythermic fish that under natural conditions lives in environments with temperatures ranging from 4 to 18 °C and can be usually captured near salmon farming areas. The aim of this study was to evaluate the effect of temperature over the innate and adaptive immune response of E. maclovinus challenged with Piscirickettsia salmonis following different treatments: C (control injection with culture medium at 12 °C), C+ (bacterial injection at 12 °C), 18 °C c/A + B (injection with culture medium in acclimation at 18 °C), 18 °C c/A + B (bacterial injection in acclimation at 18 °C), 18 °C s/A + M (injection with culture medium without acclimation at 18 °C) and 18 °C s/A + B (bacterial injection without acclimation at 18 °C). Each injection had 100 µL of culture medium or with 100 µL at a concentration 1 × 108 of live bacteria, sampling six fish per group at 4, 8, 12, 16 and 20 days post-injection (dpi). Expression of the mRNA related with the innate immune response gene (TLR1, TLR5, TLR8, NLRC3, NLRC5, MyD88 and IL-1ß) as well as the adaptive immune response gene (MHCI, MHCII, IgMs and IgD) were measured in spleen and head kidney. Gene expression profiles were treatment-type and time dependent. Levels of Immunoglobulin M (IgM) increased in challenged groups with P. salmonis from day 8-20 post challenge, which suggest activation of B cells IgM + through P. salmonis epitope detection. Additionally, a rise in temperature from 12 °C (C+) to 18 °C (with/without acclimation) also resulted in antibody increment detected in serum with significant differences between "18 °C c/A + B" and "18 °C s/A + B" groups. This is the first study that evaluates the effect of temperature changes and mRNA expression related with immune system gene over time on E. maclovinus, a native wild life fish that cohabits in the salmon farming environment.

The Use of Chitosan-Coated Membrane Vesicles for Immunization Against Salmonid Rickettsial Septicemia in an Adult Zebrafish Model.

The introduction of fish vaccination has had a tremendous impact on the aquaculture industry by providing an important measurement in regard to disease control. Infectious diseases caused by intracellular pathogens do, however, remain an unsolved problem for the industry. This is in many cases directly connected to the inability of vaccines to evoke a cellular immunity needed for long-term protection. Thus, there is a need for new and improved vaccines and adjuvants able to induce a strong humoral and cellular immune response. We have previously shown that membrane vesicles (MVs) from the intracellular fish pathogen Piscirickettsia salmonis are able to induce a protective response in adult zebrafish, but the incorporation of an adjuvant has not been evaluated. In this study, we report the use of chitosan as an adjuvant in combination with the P. salmonis-derived MVs for improved immunization against P. salmonis. Both free chitosan and chitosan-coated MVs (cMVs) were injected into adult zebrafish and their efficacy evaluated. The cMVs provided a significant protection (p < 0.05), while a small but nonsignificant reduction in mortalities was registered for fish injected with free chitosan. Both free chitosan and the cMVs were shown to induce an increased immune gene expression of CD 4, CD 8, MHC I, Mpeg1.1, TNFα, IL-1ß, IL-10, and IL-6, but to a higher degree in the cMV group. Taken together, the results indicate a potential use of chitosan-coated MVs for vaccination, and that zebrafish is a promising model for aquaculture-relevant studies.

Immunological response of the Sub-Antarctic Notothenioid fish Eleginops maclovinus injected with two strains of Piscirickettsia salmonis.

Eleginops maclovinus is an endemic fish to Chile that lives in proximity to salmonid culture centers, feeding off of uneaten pellet and salmonid feces. Occurring in the natural environment, this interaction between native and farmed fish could result in the horizontal transmission of pathogens affecting the aquaculture industry. The aim of this study was to evaluate the innate and adaptive immune responses of E. maclovinus challenged with P. salmonis. Treatment injections (in duplicate) were as follows: control (100 µL of culture medium), wild type LF-89 strain (100 µL, 1 × 108 live bacteria), and antibiotic resistant strain Austral-005 (100 µL, 1 × 108 live bacteria). The fish were sampled at various time-points during the 35-day experimental period. The gene expression of TLRs (1, 5, and 8), NLRCs (3 and 5), C3, IL-1ß, MHCII, and IgMs were significantly modulated during the experimental period in both the spleen and gut (excepting TLR1 and TLR8 spleen expressions), with tissue-specific expression profiles and punctual differences between the injected strains. Anti-P. salmonis antibodies increased in E. maclovinus serum from day 14-28 for the LF-89 strain and from day 14-35 for the Austral-005 strain. These results suggest temporal activation of the innate and adaptive immune responses in E. maclovinus tissues when injected by distinct P. salmonis strains. The Austral-005 strain did not always cause the greatest increases/decreases in the number of transcripts, so the magnitude of the observed immune response (mRNA) may not be related to antibiotic resistance. This is the first immunological study to relate a pathogen widely studied in salmonids with a native fish.