The in vitro immunomodulatory effect of extracellular products (ECPs) of Vagococcus fluvialis L21 on European sea bass (Dicentrarchus labrax) leucocytes

doi:10.1016/j.fsi.2014.11.037

Fish & Shellfish Immunology

Volume 42, Issue 2, February 2015, Pages 517-521

https://doi.org/10.1016/j.fsi.2014.11.037 Get rights and content

Highlights

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Evaluation of the levels of immune genes in sea bass HK leucocytes after incubation with the ECPs of V. fluvialis L21.
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We used real-time PCR to investigate in vitro the dynamic of expression of immune-related.
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ECPs of Vagococcus fluvialis L21 induce an early inflammatory response in HK leucocytes.
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In vivo studies are necessary to better understand the interaction between ECPs and the immune response in European Sea bass.

Abstract

The immune associated genes, interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin 10 (IL-10), tumor necrosis factor-α (TNF-α), ciclo-oxigenase-2 (COX-2), and Mx gene were studied by real-time PCR in head-kidney leucocytes of sea bass after incubation with the extracellular products (ECPs) of the probiotic strain Vagococcus fluvialis L21 and polyinosinic:polycytidylic acid (POLY I:C), at different times (T1.5, T6, T12, T24, T48 and T72). In general, we can observe how pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and COX-2 studied displayed a strong peak after stimulation with 1.5 h of ECPs of V. fluvialis L21, significant differences (P < 0.05) exist with other periods and with the POLY I: C at the same time. Similarly to the case of IL-10 also produced a statistically significant (P < 0.05) peak of expression on leukocytes that were stimulated with the ECPs of V. fluvialis L21. In the case of Mx gene expression, we note that in almost all sampling times there is an up-regulation of the Mx gene in leucocytes incubated with ECPs and POLY I:C compared to the control and Mx expression was higher in leucocytes that were stimulated with the ECPs of V. fluvialis for all times, except in T24. With these results we can consider that the ECPs of V. fluvialis L21 have a great power of stimulating the in vitro expression of immune-related genes and may even be useful as adjuvants for vaccine in aquaculture.

Introduction

Infectious diseases are one of the most limiting factors for the successful development of aquaculture industry. Among other control strategies, probiotics are considered an alternative tool for disease control, which consequently can prevent the use of antibiotics [1], [2], [3], [4]. Today, not only alive bacterial strains are used as probiotics, but also inactivated ones, which have beneficial effects on the host, both in vitro and in vivo [5], [6], [7]. In addition, recent researches shows that purified components of the bacterial strains may have beneficial effect on the health of the hosts. Abbass et al. [8] showed that the cell wall proteins, outer membrane proteins (OMP) and lipopolysaccharides (LPS) of the probiotic strains Aeromonas sobria GC2 and Bacillus subtilis JB-1 conferred protection against Yersinia ruckeri in salmonids. Moreover, the probiotic supplementation has begun to be used as an adjuvant to increase the immunogenicity of vaccines in humans and animals [9], [10].

In order to better understand the mechanism of action of the Vagococcus fluvialis L21 strain, identified and characterized as probiotic strain with protective effect against vibriosis in sea bass by Vibrio anguillaum by Sorroza et al. [11], and considering that the this strain has a clear immunomodulatory activity in vitro, as well as the ability to induce cytokine production related to the immune response [7], [12], the aim of this study was to determine, in vitro, the effect of extracellular products (ECPs) of V. fluvialis L21 on the dynamics of expression of genes (IL-1β, IL-6, IL-10, TNF-α, COX-2, Mx) involved in the immune response in sea bass leucocytes.

Section snippets

Fish

Thirty-six sea bass with an average weight of 150 g were provides by the Canary Institute of Marine Sciences (ICCM) to study the dynamics of expression of cytokines related to the immune response. The fish were kept in tanks of 1000 l of capacity with open water circulation, constant aeration and natural photoperiod of 12 h. Fish were fed daily with a commercial diet Skretting (Burgos, Spain). After checking the sanitary status, fish were slaughtered by overdose of 2-phenoxyethanol (Panreac),

Immune-related gene expression

Fig. 1 show the immune-related gene expression in sea bass leucocytes incubated with the ECPs of V. fluvialis L21 and the POLY I: C. We can observe how pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and COX-2 studied displayed a strong peak after stimulation with 1.5 h of ECPs of V. fluvialis L21, significant differences (P < 0.05) exist with other periods and with the POLY I: C at the same time. The peak expression markedly diminished in the case of COX-2 or more is maintained prolonged as is

Discussion

Although the use of probiotic strains is an essential tool in aquaculture practice, sometimes there are certain risks caused by the use of alive probiotic strains since their administration may interfere with the ecosystems [5], [17]. On the other hand, there is some concern in the scientific community about the possible development of virulence by some fish probiotic strains, due to the possible horizontal transfer of resistance plasmids [18]. Although so far there has been no notification,

Conclusion

Overall, taking into account previous results obtained with strain V. fluvialis L21 in which it was demonstrated the protective effect of this strain in sea bass against experimental infection with V. anguillarum as well as stimulation of non-specific cellular immune response and the effect on gene expression, we can consider that the ECPs of V. fluvialis L21 have a great power of stimulating in vitro the expression of immune-related genes and may even be useful as adjuvants for vaccine in

Acknowledgments

Lorena Román was recipient of PhD Grant by Cabildo Insular de Gran Canaria. The authors wish to thank CANEXMAR SL and Instituto Canario de Ciencias Marinas (ICCM) for providing the fish for this research. This research was supported by a grant from the Canary Islands Government, Spain (PI2007/047).

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Full length articleThe in vitro immunomodulatory effect of extracellular products (ECPs) of Vagococcus fluvialis L21 on European sea bass (Dicentrarchus labrax) leucocytes

Highlights

Abstract

Introduction

Section snippets

Fish

Immune-related gene expression

Discussion

Conclusion

Acknowledgments

Fish Shellfish Immunol

Fish Shellfish Immunol

Vaccine

Vaccine

Vet Microbiol

Vet Microbiol

Vet Immunol Immuno

Prev Vet Med

Dev Comp Immunol

Fish Shellfish Immunol

Mol Immunol

Fish Shellfish Immunol

Fish Shellfish Immunol

Fish Shellfish Immunol

Fish Shellfish Immunol

Fish Shellfish Immunol

Fish Shellfish Immunol

Fish Shellfish Immunol

Immunol Lett

Dev Com Immunol

Full length article
The in vitro immunomodulatory effect of extracellular products (ECPs) of Vagococcus fluvialis L21 on European sea bass (Dicentrarchus labrax) leucocytes