Evaluation of a DNA vaccine with self-designed CpG sequences against J genotype IHNV infection in rainbow trout (Oncorhynchus mykiss).

Rainbow trout suffer from infectious hematopoietic necrosis virus (IHNV) outbreaks, which lead to massive mortality and huge economic loss worldwide. The approved commercial vaccine is used for the prevention of IHN in Canada. Given that Chinese domestic J-genotype isolates are different from North American IHNV isolates, the development of an effective DNA vaccine against Chinese J-genotype isolates is urgent. In this study, we developed a DNA vaccine encoding glycoprotein based on our previously isolated IHNV GS21 strain and self-designed CpG sequences were supplemented as molecular adjuvants. The vaccinated rainbow trout were significantly protected against IHNV with approximately a relative percent survival (RPS) of 94.74% compared to the unvaccinated group. Moreover, the specific antibody of IgM and neutralizing antibody (NAb) was significantly provoked after the vaccination. Particularly, the antiviral immune response was rapidly evoked in the early stage of vaccination including the up-regulation of Mx-1, IFN-Ⅰ, and IFN-γ. The IHNV load in vaccinated fish was apparently lower than that in the unvaccinated group. Furthermore, the integration of exogenous genes into the host chromosome and the spread of antimicrobial-resistant genes were not found. These results suggested that our vaccine enhances robust immune responses and evokes considerable protection against IHNV with limited genetically modified risk, which is an effective and promising vaccine candidate for further prevention of IHNV outbreaks.

Genome-wide association analysis of the resistance to infectious hematopoietic necrosis virus in two rainbow trout aquaculture lines confirms oligogenic architecture with several moderate effect quantitative trait loci.

Infectious hematopoietic necrosis (IHN) is a disease of salmonid fish that is caused by the IHN virus (IHNV), which can cause substantial mortality and economic losses in rainbow trout aquaculture and fisheries enhancement hatchery programs. In a previous study on a commercial rainbow trout breeding line that has undergone selection, we found that genetic resistance to IHNV is controlled by the oligogenic inheritance of several moderate and many small effect quantitative trait loci (QTL). Here we used genome wide association analyses in two different commercial aquaculture lines that were naïve to previous exposure to IHNV to determine whether QTL were shared across lines, and to investigate whether there were major effect loci that were still segregating in the naïve lines. A total of 1,859 and 1,768 offspring from two commercial aquaculture strains were phenotyped for resistance to IHNV and genotyped with the rainbow trout Axiom 57K SNP array. Moderate heritability values (0.15-0.25) were estimated. Two statistical methods were used for genome wide association analyses in the two populations. No major QTL were detected despite the naïve status of the two lines. Further, our analyses confirmed an oligogenic architecture for genetic resistance to IHNV in rainbow trout. Overall, 17 QTL with notable effect (≥1.9% of the additive genetic variance) were detected in at least one of the two rainbow trout lines with at least one of the two statistical methods. Five of those QTL were mapped to overlapping or adjacent chromosomal regions in both lines, suggesting that some loci may be shared across commercial lines. Although some of the loci detected in this GWAS merit further investigation to better understand the biological basis of IHNV disease resistance across populations, the overall genetic architecture of IHNV resistance in the two rainbow trout lines suggests that genomic selection may be a more effective strategy for genetic improvement in this trait.

Fulvic acid modulates mucosal immunity in fish skin: Sustainable aquaculture solution or environmental risk factor?

This is the first study determining the effects of bath exposure to fulvic acid, a humic substance, on the skin mucosal immunity of rainbow trout (Oncorhynchus mykiss). Humic substances have recently been gaining attention for their increasing concentrations in aquatic ecosystems and their use as supplements in sustainable aquaculture. This study demonstrated that water exposure to fulvic acid at concentrations of 5 mg C/L and 50 mg C/L increased lysozyme and alkaline phosphatase activities in the mucus by approximately 2-fold and 2.5 to 3.2-fold, respectively. Furthermore, exposure to 50 mg C/L resulted in a 77.0% increase in mucosal immunoglobulin concentrations compared to the other groups. Importantly, all mucus samples demonstrated significant antibacterial activity against Yersinia ruckeri, with control mucus reducing bacterial growth by 44.5% and exposure to fulvic acid increasing this effect to 26.3%. Although these modulations show promise for application in aquaculture, alterations of the beneficial microbiota from long-term exposure in natural waters can be expected. Monitoring the rising concentrations of humic substances in natural water bodies is therefore urgently needed. Overall, this study represents the first investigation revealing the ability of humic substances to modulate skin mucosal immunity and the capacity to combat microorganisms.

Characterization of the activity of 2'-C- methylcytidine against infectious pancreatic necrosis virus replication.

Infectious pancreatic necrosis virus (IPNV) is the pathogen of infectious pancreatic necrosis (IPN), which can cause high mortality in salmonids, harm the healthy development of salmon-trout aquaculture, and lead to huge economic losses. However, in China, there is currently neither a commercially available vaccine to prevent IPNV infection nor antiviral drugs to treat IPNV infection. The genome of IPNV consists of two segments of dsRNA named A and B. Segment B encodes the RNA-dependent RNA-polymerase (RdRp) VP1 which is essential for viral RNA replication and is therefore considered an important target for the development of antiviral drugs. In this study, we investigate whether 2'-C-methylcytidine (2CMC), a nucleoside analog which target viral polymerases, has an inhibitory effect on IPNV both in vitro and in vivo. The results show that 2CMC inhibits IPNV infection by inhibiting viral RNA replication rather than viral internalization or attachment. In vivo experiment results showed that 2CMC could inhibit viral RNA replication and reduce viral load in rainbow trout (Oncorhynchus mykiss). In our study, we have revealed that 2CMC has a potent inhibitory effect against IPNV infection. Our data suggest that 2CMC is an attractive anti-IPNV drug candidate which will be highly valuable for the development of potential therapeutics for IPNV.

Dynamic Distribution of Infectious Pancreatic Necrosis Virus (IPNV) Strains of Genogroups 1, 5, and 7 after Intraperitoneal Administration in Rainbow Trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) is the causative agent of rainbow trout (Oncorhynchus mykiss) IPN and causes significant loss of fingerlings. The currently prevalent IPNV genogroups in China are genogroups 1 and 5. However, in this study, we isolated and identified a novel IPNV, IPNV-P202019, which belonged to genogroup 7. Here, a total of 200 specific-pathogen-free rainbow trout (10 g average weight) were divided randomly into four groups to investigate the distribution of different IPNV strains (genogroups 1, 5, and 7) in 9 tissues of rainbow trout by means of intraperitoneal (ip) injection. Fish in each group were monitored after 3-, 7-, 14-, 21- and 28- days post-infection (dpi). The study showed no mortality in all groups. The distribution of IPNV genogroups 1 and 5 was similar in different tissues and had a higher number of viral loads after 3, 7, or 14 dpi. However, the distribution of IPNV genogroup 7 was detected particularly in the spleen, head kidney, and feces and had a lower number of viral loads. The results of this study provide valid data for the distribution of IPNV in rainbow trout tissues and showed that IPNV genogroups 1 and 5 were still the prevalent genogroups of IPNV in China. Although rainbow trout carried IPNV genogroup 7, the viral load was too low to be pathogenic.

Integrative mRNA-miRNA interaction analysis reveals the molecular mechanism of skin color variation between wild-type and yellow mutant rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) is an important economic fish in China. Skin color affects the economic value of trout. However, the molecular mechanism of the skin color variation between wild-type (WR) and yellow mutant rainbow trout (YR) is unclear. We sequenced mRNAs and miRNAs of dorsal skin to identify key color variation-associated mRNAs and miRNAs between WR and YR. Overall, 2060 out of 3625 differentially expressed genes were upregulated in YR, and 196 out of 275 differentially expressed miRNAs were downregulated in WR. We identified three key YR-upregulated genes related to the formation of xanthophores (GCH1, SLC2A11, and SOX10). Interestingly, several genes related to melanogenesis (TYR, TYRP1, TYRP2, MC1R, MITF, PMEL, SLC45A2, and OCA2) were downregulated in WR. Integrated analysis identified five miRNAs that target at least two skin color-related genes (miR-495-y, miR-543-y, miR-665-z, miR-433-y, and miR-382-x). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of target genes identified noncoding RNA metabolic process as the most significantly enriched GO term, and several metabolic pathways associated with skin color were enriched significantly, such as tyrosine metabolism, histidine metabolism, and vitamin B6 metabolism. Quantitative real-time PCR of selected mRNAs and miRNAs validated the reliability of the integrated analysis. This study provides in-depth insights into the molecular mechanism of skin color variation between WR and YR, which will accelerate the genetic selection and breeding of rainbow trout with consumer-favored traits.

Pike-Perch (Sander lucioperca) and Rainbow Trout (Oncorhynchus mykiss) Fed with an Alternative Microorganism Mix for Reducing Fish Meal and Oil-Fishes' Growth Performances and Quality Traits.

In the last decades, several plant-based materials were used for the substitution of fish meal and oil in aquaculture. The present study evaluated the fish quality and the sensory differences of rainbow trout (Oncorhynchus mykiss) and pike-perch (Sander lucioperca) from three different feeding groups, which were fed a commercially available industrial (standard) diet, a control diet, and a special microorganism-based feed mix. This feed mainly consisted of a mix made of Rhodotorula glutinis, Crypthecodinium cohnii, and Arthrospira sp. and had 50% less fish meal and fish oil compared to typical control diets. At the beginning, the pike-perch population was six months old, and the rainbow trout population was 15 months old. The feeding study duration was 16 weeks and every four weeks the growth performance and several morphometric parameters were recorded. Afterwards, sensory evaluation took place to identify possible trends. Sensory evaluation revealed that the rainbow trout groups did not show any significant differences to the standard and control fish fillets with regard to odor, texture, and taste. The effects on rainbow trout growth performances and carcass parameters were similar to the standard group. The feed mix was not optimal for pike-perch farming, which was also reflected by significantly adversely affected growth performance and carcass parameters. The sensorial evaluation showed an opposite trend: here, only small differences in the fillets from the feed mix and standard/control diet were observed.

Effects of elderflower extract enriched with polyphenols on antioxidant defense of salmon leukocytes

BACKGROUND: In fish farming, the plant extracts containing antioxidant compounds have been added to the diet for enhancing pathogen resistance. In vitro studies evaluating the antioxidant effect of herbal extracts on fish cell models have focused on ROS production and the respiratory burst mechanism. However, the effects on enzymatic antioxidant defense on salmon leukocytes have not been evaluated. This study aims to evaluate the enzymatic antioxidant defense and ROS-induced cell damage in Salmon Head Kidney-1 (SHK-1) cell line exposed to polyphenol-enriched extract from Sambucus nigra flowers. RESULTS: Firstly, the Total Reactive Antioxidant Power (TRAP) assay of elderflower polyphenol (EP) was evaluated, showing 459 and 489 times more active than gallic acid and butyl hydroxy toluene (BHT), respectively. The toxic effect of EP on salmon cells was not significant at concentrations below 120 mg/ mL and no hemolysis activity was observed between 20 and 400 mg/mL. The treatment of SHK-1 cell line with EP decreased both the lipid peroxidation and protein oxidation induced by H2O2, which could be associated with decreasing oxidative stress in the SHK-1 cells since the GSH/GSSG ratio increased when only EP was added. CONCLUSIONS: These results suggest that plant extracts enriched with polyphenols could improve the enzymatic antioxidant defense of salmon leukocytes and protect the cells against ROS-induced cell damage

A ribonucleoprotein transfection strategy for CRISPR/Cas9-mediated gene editing and single cell cloning in rainbow trout cells.

BACKGROUND: The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example. RESULTS: Ribonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively. CONCLUSIONS: The designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function.

Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l-1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l-1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l-1 and 10 mg l-1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l-1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l-1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l-1 though values were significantly similar with concentration from 16 to 128 mg l-1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l-1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

Negligible risk of zoonotic anisakid nematodes in farmed fish from European mariculture, 2016 to 2018.

BackgroundThe increasing demand for raw or undercooked fish products, supplied by both aquaculture and fisheries, raises concerns about the transmission risk to humans of zoonotic fish parasites. This has led to the current European Union (EU) Regulation No 1276/2011 amending Annex III of Regulation (EC) No 853/2004 and mandating a freezing treatment of such products. Zoonotic parasites, particularly anisakid larvae, have been well documented in wild fish. Data on their presence in European aquaculture products, however, are still scarce, except for Atlantic salmon (Salmo salar), where the zoonotic risk was assessed as negligible, exempting it from freezing treatment.AimTo evaluate the zoonotic Anisakidae parasite risk in European farmed marine fish other than Atlantic salmon.MethodsFrom 2016 to 2018 an observational parasitological survey was undertaken on 6,549 farmed fish including 2,753 gilthead seabream (Sparus aurata), 2,761 European seabass (Dicentrarchus labrax) and 1,035 turbot (Scophthalmus maximus) from 14 farms in Italy, Spain and Greece. Furthermore, 200 rainbow trout (Oncorhynchus mykiss) sea-caged in Denmark, as well as 352 seabream and 290 seabass imported in Italy and Spain from other countries were examined. Fish were subjected to visual inspection and candling. Fresh visceral organs/fillet samples were artificially digested or UV pressed and visually examined for zoonotic anisakid larvae.ResultsNo zoonotic parasites were found in any of the fish investigated.ConclusionsThe risk linked to zoonotic Anisakidae in the examined fish species from European mariculture appears negligible. This study laid the groundwork for considerations to amend the current EU regulation.

Glyphosate disrupts sperm quality and induced DNA damage of rainbow trout (Oncorhynchus mykiss) sperm.

As a widespread pollutant, glyphosate (GLY) adversely affects the aquatic environment and can impair the reproductive ability and functions of fish. The purpose of the current study was to assess in vitro effect of GLY on rainbow trout (Oncorhynchus mykiss) sperm cells. The sperm cells were exposed to different GLY concentrations (2.5, 5, 10 mg/L). Sperm motility parameters were analyzed with computer assisted sperm analysis. DNA fragmentation (%) was measured by the comet assay using fluorescence microscopy. With increased GLY concentration, sperm motility and duration decreased after exposure. DNA fragmentation (% DNA in tail) in sperm cells was higher in treatments containing GLY than control (p < 0.05). Consequently, sperm cells are sensitive to low doses of GLY, and this can negatively affect natural populations.

GFAP expression in the optic nerve and increased H2S generation in the integration centers of the rainbow trout (Oncorhynchus mykiss) brain after unilateral eye injury.

Hydrogen sulfide (H2S) is considered as a protective factor against cardiovascular disorders. However, there are few reports on the effects of H2S in the central nervous system during stress or injury. Previous studies on goldfish have shown that astrocytic response occurs in the damaged and contralateral optic nerves. Glial fibrillary acidic protein (GFAP) concentration in the optic nerves of rainbow trout has not been measured previously. This study further characterized the astrocytic response in the optic nerve and the brain of a rainbow trout (Oncorhynchus mykiss) after unilateral eye injury and estimated the amount of H2S-producing enzyme cystathionine ß-synthase (CBS) in the brain of the rainbow trout. Within 1 week after unilateral eye injury, a protein band corresponding to a molecular weight of 50 kDa was identified in the ipsi- and contralateral optic nerves of the rainbow trout. The concentration of GFAP in the injured optic nerve increased compared to the protein concentration on the contralateral side. The results of a quantitative analysis of GFAP+ cell distribution in the contralateral optic nerve showed the largest number of GFAP+ cells and fibers in the optic nerve head. In the damaged optic nerve, patterns of GFAP+ cell migration and large GFAP+ bipolar activated astrocytes were detected at 1 week after unilateral eye injury. The study of H2S-producing system after unilateral eye injury in the rainbow trout was conducted using enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemistry of polyclonal antibodies against CBS in the integrative centers of the brain: telencephalon, optic tectum, and cerebellum. Enzyme-linked immunosorbent assay results showed a 1.7-fold increase in CBS expression in the rainbow trout brain at 1 week after unilateral eye injury compared with that in intact animals. In the ventricular and subventricular regions of the rainbow trout telencephalon, CBS+ radial glia and neuroepithelial cells were identified. After unilateral eye injury, the number of CBS+ neuroepithelial cells in the pallial and subpallial periventricular regions of the telencephalon increased. In the optic tectum, unilateral eye injury led to an increase in CBS expression in radial glial cells; simultaneously, the number of CBS+ neuroepithelial cells decreased in intact animals. In the cerebellum of the rainbow trout, neuroglial interrelationships were revealed, where H2S was released, apparently, from astrocyte-like cells. The organization of H2S-producing cell complexes suggests that, the amount of glutamate produced in the rainbow trout cerebellum and its reuptake was controlled by astrocyte-like cells, reducing its excitotoxicity. In the dorsal matrix zone and granular eminences of the rainbow trout cerebellum, CBS was expressed in neuroepithelial cells. After unilateral eye injury, the level of CBS activity increased in all parts of the cerebellum. An increase in the number of H2S-producing cells was a response to oxidative stress after unilateral eye injury, and the overproduction of H2S in the cerebellum occurred to neutralize reactive oxygen species, providing the cells of the rainbow trout cerebellum with a protective effect. A structural reorganization in the dorsal matrix zone, associated with the appearance of an additional CBS+ apical zone, and a decrease in the enzyme activity in the dorsal matrix zone, was revealed in the zones of constitutive neurogenesis. All experiments were approved by the Commission on Biomedical Ethics, A.V. Zhirmunsky National Scientific Center of Marine Biology (NSCMB), Far Eastern Branch, Russian Academy of Science (FEB RAS) (approval No. 1) on July 31, 2019.

Cold-Resistant Heterotrophic Ammonium and Nitrite-Removing Bacteria Improve Aquaculture Conditions of Rainbow Trout (Oncorhynchus mykiss).

The aim of this study was isolation and characterization of heterotrophic bacteria capable of ammonium and nitrite removal at 15 °C (optimal temperature for growing rainbow trout Oncorhynchus mykiss). Environmental isolates were grown in liquid media containing ammonium or nitrite, and best strains in terms of growth and ammonium or nitrite removal were identified via 16S rRNA sequencing. Dyadobacter sp. (no. 68) and Janthinobacterium sp. (no. 100) were selected for optimal adaptation to growth at 15 °C and best ammonium and nitrite removal (P < 0.05), respectively. A heterotrophic ammonium and nitrite removal (HAN) microbial complex, containing selected strains, was prepared and applied in a trout culture system. After 10 days, the effect of microbial HAN complex was investigated in terms of ammonium and nitrite removal, as well as stress and immune indices present in the plasma of cultivated trout. Compared to a standard cultivation setup, addition of the HAN complex had a clear beneficial effect on keeping the un-ionized ammonia and nitrite level below prescribed standards (P < 0.05). This resulted in reduction of stress and immune reactions of cultivated fish (P < 0.05), leading to an augmentation of final weight and survival. Application of the selected microbial complex resulted in a significant improvement of the aquaculture ecosystem.

Phenotypic and Genetic Predictors of Pathogenicity and Virulence in Flavobacterium psychrophilum.

Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in farmed rainbow trout (Oncorhynchus mykiss), with the multilocus sequence typing (MLST) clonal complex (CC) CC-ST10 accounting for the majority of outbreaks globally. The development of alternative strategies to antibiotic treatment of BCWD using bacteriophage-based control of F. psychrophilum, or virulence factors as targets for therapy, requires knowledge of the phage-sensitivity of outbreak strains and of universal traits contributing to their pathogenicity. To examine the association between virulence and both genetic (MLST sequence type (ST) and PCR-serotype) and phenotypic characteristics (adherence, antibiotic resistance, colony spreading motility, hemolytic and proteolytic activity), the median lethal dose (LD50) of 26 geographically disparate F. psychrophilum isolates was determined in rainbow trout. Furthermore, the in vitro sensitivity of the isolates against five bacteriophages was determined by the efficiency of plating (EOP). The tested F. psychrophilum isolates were mainly represented by CC-ST10 genotypes (22 out of 26) and showed up to 3-log differences in LD50 (8.9 × 103 to 3.1 × 106 CFU). No association between MLST ST and virulence was found because of a high variation in LD50 within STs. All identified serotypes (0, 1, and 2) were pathogenic, but ten most virulent isolates belonged to serotype 1 or 2. Isolates of high (LD50 < 105 CFU), moderate (LD50 = 105-106 CFU), and weak (LD50 > 106 CFU) virulence were similar in phenotypic characteristics in vitro. However, the only non-virulent CC-ST10 isolate was deficient in spreading motility and proteolytic activity, indicating that the characteristics are required for pathogenicity in F. psychrophilum. Univariate correlation studies found only non-significant associations between LD50 and the measured phenotypic characteristics, and the multivariable analysis did neither reveal any significant predictors of virulence. The majority of isolates (16 out of 26) were sensitive to at least four bacteriophages, with up to a 6-log variation in the EOP. Most CC-ST10 isolates (16 out of 22) were sensitive to the examined phages, including 5 out of the 7 most virulent isolates represented by prevalent and antibiotic-resistant STs. Our findings suggest that control of BCWD using lytic phages or interventions targeting shared characteristics of pathogenic F. psychrophilum strains should be further explored.

Distinct response of immune gene expression in peripheral blood leucocytes modulated by bacterin vaccine candidates in rainbow trout Oncorhynchus mykiss: A potential in vitro screening and batch testing system for vaccine development in aquaculture.

Fish aquaculture is the world's fastest growing food production industry and infectious diseases are a major limiting factor. Vaccination is the most appropriate method for controlling infectious diseases and a key reason for the success of salmonid cultivation and has reduced the use of antibiotics. The development of fish vaccines requires the use of a great number of experimental animals that are challenged with virulent pathogens. In vitro cell culture systems have the potential to replace in vivo pathogen exposure for initial screening and testing of novel vaccine candidates/preparations, and for batch potency and safety tests. PBL contain major immune cells that enable the detection of both innate and adaptive immune responses in vitro. Fish PBL can be easily prepared using a hypotonic method and is the only way to obtain large numbers of immune cells non-lethally. Distinct gene expression profiles of innate and adaptive immunity have been observed between bacterins prepared from different bacterial species, as well as from different strains or culturing conditions of the same bacterial species. Distinct immune pathways are activated by pathogens or vaccines in vivo that can be detected in PBL in vitro. Immune gene expression in PBL after stimulation with vaccine candidates may shed light on the immune pathways involved that lead to vaccine-mediated protection. This study suggests that PBL are a suitable platform for initial screening of vaccine candidates, for evaluation of vaccine-induced immune responses, and a cheap alternative for potency testing to reduce animal use in aquaculture vaccine development.

Improving a fish intestinal barrier model by combining two rainbow trout cell lines: epithelial RTgutGC and fibroblastic RTgutF.

An in vitro model of the fish intestine is of interest for research and application in diverse fields such as fish physiology, aquaculture and chemical risk assessment. The recently developed epithelial barrier model of the fish intestine relies on the RTgutGC cell line from rainbow trout (Oncorhynchus mykiss), cultured in inserts on permeable membranes. Our aim was to extend the current system by introducing intestinal fibroblasts as supportive layer in order to reconstruct the epithelial-mesenchymal interface as found in vivo. We therefore initiated and characterized the first fibroblast cell line from the intestine of rainbow trout, which has been termed RTgutF. Co-culture studies of RTgutGC and RTgutF were performed on commercially available electric cell substrate for impedance sensing (ECIS) and on newly developed ultrathin, highly porous alumina membranes to imitate the cellular interaction with the basement membrane. Cellular events were examined with non-invasive impedance spectroscopy to distinguish between barrier tightness and cell density in the ECIS system and to determine transepithelial electrical resistance for cells cultured on the alumina membranes. We highlight the relevance of the piscine intestinal fibroblasts for an advanced intestinal barrier model, particularly on ultrathin alumina membranes. These membranes enable rapid crosstalk of cells cultured on opposite sides, which led to increased barrier tightening in the fish cell line-based epithelial-mesenchymal model.

Immunomodulatory effects of orally administrated florfenicol in rainbow trout (Oncorhynchus mykiss) following experimental challenge with streptococcosis/lactococcosis.

The purpose of the present work was to determine whether florfenicol (FFC) as the prominent broad-spectrum antibiotic could affect serum biochemical and immunological parameters, as well as immune-related genes expression in rainbow trout (55 ±â€¯7.6 g) challenged with the Lactococcus garvieae and Streptococcus iniae. In the first trial, the doses of the pathogens for challenge test were determined based on LD50. The therapeutic dosage of the drug (15 mg.kg-1 BW for 10 consecutive days) was administrated as medicated feed. After anesthesia, blood and kidney samples were collected from individual fish and were kept in deep freezing mode until the beginning of the measurements. Serum biochemical and enzymatic indices were measured using commercial kits. Immune parameters including total immunoglobulin level, lysozyme, ACH50, respiratory burst (RB), and phagocytic activities (PA) and the expression of immune genes namely TNF-α, IL-1ß, IL-8, and IgM was evaluated. The levels of lysozyme and RB activities, as well as the expression of TNF-α and IL-1ß genes, showed a significant increase in the FFC treated/infected fish compared to untreated diseased fish (P < 0.05). In contrast, serum total immunoglobulin and IgM-related genes expression were suppressed following drug administration represented by a significant reduction in untreated streptococcal infected fish compared to other treatments (P < 0.05). However, no significant effect of FFC was observed on serum ACH50 activity, PA values and IL-8-related gene expression (P > 0.05). These results demonstrated that FFC treatment could improve some physiological status including stress resistance and some liver function parameters, and much innate immunity was invigorated, but at the same time, the suppressive effects of FFC on acquainted immunity cannot be ignored.

The effect of Spirulina platensis meal on antioxidant gene expression, total antioxidant capacity, and lipid peroxidation of rainbow trout (Oncorhynchus mykiss).

The study was conducted to investigate the effect of diets containing 0, 2.5, 5, 7.5, and 10% S. platensis meal on total antioxidant capacity (TAC) and lipid peroxidation, as well as the expression of two antioxidant enzyme genes (SOD and CAT) in rainbow trout (Oncorhynchus mykiss). One hundred and eighty fish, with an average initial weight of 101 ± 8 g, were cultured for 10 weeks. At the end of experiment, lipid peroxidation significantly decreased in serum of fish fed with S. platensis and fish fed with 5, 7.5, and 10% microalgae showed a significantly lower value compared to control and 2.5%. Inclusion of 7.5 and 10% S. platensis in diet also decreased lipid peroxidation in liver. TAC significantly increased with increasing level of S. platensis. Expression level of superoxide dismutase (SOD) and catalase (CAT) genes significantly increased in the fish liver after administration of microalgae and fish fed with 10% S. platensis in diet showed the highest level compared to the other treatments. The present study reveals that inclusion of 10% S. platensis in diet can decrease oxidative stress in rainbow trout. Therefore, S. platensis can be used as potential antioxidant for fish farming.

Viral-Infected Change of the Digestive Tract Microbiota Associated With Mucosal Immunity in Teleost Fish.

The digestive tract is a unique series of organs that is inhabited by a range of commensal microbes while also exposed to an overwhelming load of dietary antigens. It is widely known that mammals have evolved complex and efficient immune strategies to protect the mucosa of the digestive tract. However, in the early vertebrates, the roles of mucosal immune defense and microbial communities in the different segments of the digestive tract are not well-understood. Here, we constructed a bath infection model with infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). Importantly, following viral infection, we found that the IHNV distribution and the reactions of immune-related genes had similar trends that decreased across the digestive tract. Hematoxylin and eosin (H & E) and alcian blue (A & B) staining of the trout digestive tract showed that the pathological changes only occurred in the buccal and pharyngeal mucosal tissues. Moreover, the increased diversity of the microbial community was only detected in the buccal mucosa through 16S rRNA gene sequencing, suggesting that the magnitude of the immune response and microbial community changes are related to the IHNV load and the original microbial diversity. In addition, the loss of digestive tract dominant species and increased colonization of opportunistic bacteria were discovered in the buccal mucosal surface indicating that a secondary bacterial infection occurred in this mucosal tissue.