engleski

Expression of immune markers in in vitro stimulated peripheral blood leukocytes of the European sea bass (Dicentrarchus labrax) by autochthonous intestinal Alteromonas sp. and Pseudoalteromonas sp.

The European sea bass (Dicentrarchus labrax) is known as the most important commercial fish reared in the Mediterranean, reaching over 260.000 tonnes in 2013. Its rearing systems varies from more extensive (coastal lagoons), to the intensive, highly producing inland recirculation systems or net cages. Rearing conditions of each of those systems, always represent a trade-off between the ultimate health status of the fish and its growth rate. Probiotics are beneficial and easy to use feed additives composed of live microorganisms that have an antimicrobial effect through modifying the intestinal microbiota, secreting antibacterial substances, competing with pathogens to prevent their adhesion to the intestine, or for nutrients necessary for pathogen survival, eliciting an antitoxin effect, and modulating the host immune system. We have isolated autochthonous bacteria from intestine of farmed sea bass and chosen two strains based on their biochemical properties ; Alteromonas sp. and Pseudoalteromonas sp. Bacteria were cultured, PCR-identified and assessed through public repositories GenBank and RBD, and tested for pathogenicity in fish at two concentrations (low 105 CFU/ ml and high 108 CFU/ ml). For in vitro assay, sea bass peripheral blood leukocytes (PBL) were prepared using 51% iso-osmotic Percoll solution, seeded in 6-well plates with L-15 medium supplemented with 15% FCS and 1% penicillin/streptomycin. After 2 h of PBL settlement in wells, bacterial isolates were added in culture media at 108 CFU/ ml, except in control wells and left for 1 h, 3 h, 5 h and 12 h. RNA was extracted by TriReagent, quantified and transcribed in cDNA, and sea bass specific primers for target genes ; lysozyme g (lys), Mx protein (Mx), caspase 3 (casp3), inteleukine 10 (IL-10), tumour necrosis factor α (TNF-α), and reference genes 18s rRNA, β-actin (β-act) and elongation factor (ef) were used for real-time PCR in a LightCycler 480 System. We demonstrated that both bacteria induced congruent immune response in PBL, showing very similar kinetics when compared to control PBL. Target genes were differentially expressed already 1 h post- stimulation, mostly maintaining such kinetics till the termination of the experiment, supporting immunomodulatory properties of both isolates.

autochthonous bacteria; peripheral blood leukocytes; probiotic; sea bass

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