Recombinant DnaK orally administered protects axenic European sea bass against vibriosis

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture and is a hindering factor for successful sustainable aquaculture of this commercially valuable species. Priming of the innate immune system through administration of immunostimulants has become an important approach to control disease outbreaks in marine fish larviculture. This study was conducted to evaluate immunostimulation by Escherichia coli HSP70 (DnaK) in axenic European sea bass larvae in order to protect the larvae against vibriosis. DnaK stimulates the immune response in crustaceans and juvenile fish against bacterial infections. The use of axenic fish larvae allows to study immunostimulation in the absence of an interfering microbial community. At 7 days post-hatching, larvae received a single dose of alginate encapsulated recombinant DnaK. Two non-treated control groups in which animals either received empty alginate microparticles (C1) or no alginante microparticles (C2 and C3) were included in the study. Eighteen hours later, all larvae, except the ones from group C3 (non-infected control) were challenged with V. anguillarum (10(5) CFU, bath infection). Mortality was daily recorded until 120 h post infection and at 18, 24, and 36 h post infection, larvae were sampled for expression of immune related genes. Results showed that V. anguillarum induced an immune response in axenic sea bass larvae but that the innate immune response was incapable to protect the larvae against deadly septicaemic disease. In addition, we showed that administration of alginate encapsulated DnaK to axenic European sea bass larvae at DAH7 resulted in a significant, DnaK dose dependent, upreglation of immune sensor, regulatory and effector genes. Significant upregulation of cxcr4, cas1 and especially of hep and dic was correlated with significant higher survival rates in V. anguillarum infected larvae. In the future recombinant DnaK might perhaps be used as a novel immunostimulant in sea bass larviculture

Drinking activity and microparticle size selection in early post-hatching axenic European sea bass (Dicentrarchus labrax L.) larvae

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Preparation and characterization of alginate microparticles containing a model protein for oral administration in gnotobiotic European sea bass (Dicentrarchus labrax) larvae

Aquaculture is the fastest growing animal production sector. However, the production of marine fish is still hampered by the high mortality rate in the first few weeks after hatching. Mortality in larvae is often caused by microbial infections. Today, the incorporation of immunostimulants into microparticles provides us new tools to enhance disease resistance in marine larviculture. In this study, we prepared alginate microparticles loaded with the model antigen fluorescein isothiocyanate conjugated-bovine serum albumin. Optimum concentrations of alginate and CaCl2, the correct alginate viscosity and the appropriate preparatory conditions led to the creation of desirable microparticles with the correct size for oral feeding in gnotobiotic European sea bass larvae. The prepared alginate microparticles were stable in sea water and were successfully ingested by gnotobiotic sea bass larvae at day after hatching 7 without causing any negative effects. Results suggest the suitability of this drug delivery system for targeting the innate immune system of fish larvae in order to enhance disease resistance and thus reduce mortality in larviculture

Recombinant ferritin-H induces immunosuppression in European sea bass larvae (Dicentrarchus labrax) rather than immunostimulation and protection against a Vibrio anguillarum infection

Vibrio anguillarum causes high mortality in European sea bass (Dicentrarchus labrax) larviculture. In this study, we evaluated if the recombinant sea bass ferritin-H could stimulate the innate immune system of gnotobiotic European sea bass larvae resulting in protection against a V. anguillarum challenge. We also evaluated the effect of a V. anguillarum infection on the transcription of immune-related genes in gnotobiotic European sea bass larvae. Recombinant sea bass ferritin-H was produced, encapsulated in calcium alginate microparticles and orally delivered to sea bass larvae at seven days after hatching. Our results showed V. anguillarum caused an acute infection, resulting in high mortality. The infection significantly upregulated the expression of tlr3, tlr5, casl, il1 beta, tnf alpha, mif, il10, cc1, cxcl8 at 18, 24 and 36 h post infection, but not of the chemokine receptor genes cxcr4 and ccr9. There was no protective effect of ferritin-H. Remarkably, ferritin-H caused significantly higher transcript levels for cxcr4 and ccr9. Sea bass ferritin-H was more likely involved in immune-suppression and results point in the direction of a negative regulation of CXCR4 resulting in inhibition of cell proliferation, differentiation and migration which is detrimental to innate immunity and might explain the non-protective effect of ferritin-H in fish larvae

The road to an immune-priming strategy: a case of gnotobiotic European sea bass (Dicentrarchus labrax) larvae

The search for alternative disease control in aquaculture practice is becoming more important to overcome the increasing occurrence of antibiotic resistance. Gnotobiotic animal models to study immune priming with prophylactic agents are an ideal platform to observe host-responses. Our study presents a 70 kDa recombinant bacterial heat shock protein, known as DnaK, to prime the innate immunity of gnotobiotic European sea bass larvae model that was developed at the Laboratory of Aquaculture and Artemia Reference Center (Dierckens et al., 2009). Several studies have suggested that heat shock protein can mediate both innate and adaptive immune responses (Robert, 2003). Previously, DnaK significantly improved survival of Artemia franciscana against a Vibrio campbellii infection (Sung et al., 2009). In the present study, two treatment groups of sea bass larvae were fed once with a high dose (1mg) and a low dose (0.5mg) of DnaK encapsulated in alginate microparticles at day 7 after hatching. After 18h, larvae were challenged with a pathogenic Vibrio anguillarum strain HI-610 at a density of 105 cfu ml-1. The efficacy of DnaK to protect sea bass larvae against infection was monitored by counting the survival of the larvae after 18, 24 and 36h. A qPCR was conducted to observe the expression of nine innate immune-related genes. Our results showed that the survival of challenged larvae from both treatment groups (high and low dose) were not significantly different (p<0.05) compared to the alginate control group. However, gene expression analysis showed a significant up-regulation of the innate immune-related genes in the larvae fed with a high dose (1mg) of DnaK-alginate microparticles compared to the control group after 18 and 24h of the V. anguillarum challenge. The significant up-regulation includes: pro-inflammatory genes (interleukin-1ß, interleukin-8 and tumor necrosis factor-α), the anti-inflammatory gene (interleukin-10) chemotactic cytokines receptor genes (CXCR4, CCR1 and CCR9) and the macrophage migration inhibition factor gene, MIF. Furthermore, the inflammatory cytokine-converting enzyme Caspase-1 was significantly increased for both dosages. Most of these genes were not significantly different 36h after challenge. Although no protection against V. anguillarum infection was observed in the survival, the recombinant DnaK protein did show a strong modulation on the innate immune responses on the gene expression level of the gnotobiotic European sea bass larvae. In conclusion, the use of DnaK as immunostimulants through non-diet feeding suggests to have a beneficial effect on disease resistance. Future studies focusing on the dose-response relationship are needed