Due to current restrictions on the use of chemicals or antibiotics in aquaculture production , effective and sustainable preventive treatments against systemic infections in cultured species are requiered (Yilmaz et al. 2022). To evaluate its effective ness, the development of new standardized fish health models is needed. In this scenario, a bath infection model for European sea bass (Dicentrarchus labrax ) against Tenacibaculum maritimum has been developed in this study . This bacterium, represents one of the most worrying pathogens for Mediterranean aquaculture species. It is a gram-negative filamentous bacterium which, on top of producing high mortality rates, increases fish susceptibility to other pathogens in a variety of marine aquaculture species. Specifically in this study, this model has been used to test a probiotic ( Vibrio lentus ) characterized , in vitro analyzed and cultured by AZTI . The use of probiotics as a method for prophylaxis and control of disease outbreaks in aquaculture has been proposed in recent years (Wanka et al. 2018). These compounds included in functional diets have a huge potential optimizing growth and feed conversion and boosting the immune responses, increasing the resistance of fish to diseases. Therefore, i n this study, fish performance parameters and innate immune indicators have been evaluated as well as fish disease resistance through a new standardized fish health model consisting in exposing fish to T. maritimum and comparing relative percentage of survival (RPS) against a control group (non-probiotic diet) . Complementing these results, a cohabitation infection model with the same bacterial strain is under development. Cohabitation models simulate more accurately disease outbreaks in production farms. However, their development is generally more complex as they are more susceptible to other variables such as fish density or ratio naive/infected fish. Preliminary results are very hopeful so a European sea bass against T. maritimum cohabitation model is expected to be standardized in forthcoming studies.
Materials and methods
European sea bass were fed during 6 weeks with a diet including probiotic V. lentus at 0,5% (probiotic group) or a control diet without probiotic (control group). At the end of the treatment phase, survival, weight gain, specific growth rate and feed conversion factor were calculated. In addition, kidney, plasma and mucus samples were collected to evaluate innate immune indicators using microscopy and absorbance and/or fluorescence techniques. Specifically, phagocytic activity, lysozyme activity, protease activity, anti-protease activity and total proteins have been analyzed . After the treatment phase, fish were exposed to different concentrations of T. maritimum to evaluate the RPS. The strain used in this challenge pertains to CTAQUA strains collection (CT0013 T. maritimum strain) and prior to fish exposure, it has been cultured in FMM (Flexibacter maritimum medium ) at 22 °C and 250 rpm during 24 hours (Pazos et al., 1996). The infection by bath has been carried out in 10-litre buckets, in which fish have remained for 1 hour with aeration exposed to different concentrations of the pathogen (7.50x104, 5.00x10 5 and 1.00x10 6 CFU/ml, in triplicates) . Each concentration has been obtained by diluting the original bacterial culture in seawater with a starting concentration of 1.00x10 9 CFU/mL. After exposure to the pathogen, fish were transferred the tanks of the RAS (Recirculation Aquaculture System) holding system, controlling all culture parameters throughout the test. For the cohabitation tests, infected a non-infected fish were allocated in the same tanks in triplicate in a 1:1 ratio (fish density 10 Kg/m3). I nfected fish acted as infection vectors and were previously exposed to the pathogen strain following the methodology described above for the bath model . In this case, after infection (1.00x108, 1.00x10 7 and 1.00x10 6 CFU/ml) , fish were transferred to the system tanks together with no-infected fish). In order to differentiate the infected/naive populations, naive fish were marked with visible implant elastomer (VIE).
Results did not show differences in growth and feed conversion neither in activity of immune system indicators evaluated between probiotic group and control group but there were differences in the percentage of survival after exposure to the pathogen between groups (table 1). For the development of cohabitation model, we have observed a relation between pathogen concentration and mortality percentage (figure 1).
In sight of the results, it can be concluded that bath infection model with T. maritimum has allowed the validation of the probiotic, which has a potential effect as a preventive treatment. Blue line: infected. Orange line: naive.
Yilmaz, S.; Yilmaz, E.; Dawood, M. A. O.; Ringø, E.; Ahmadifar , E. & Abdel-Latif, H. M. R. (2022). Probiotics, prebiotics, and synbiotics used to control vibriosis in fish: A review. Aquaculture, 547.
Wanka, K. M.; Damerau , T.; Costas, B.; Krueger, A.; Schulz, C. & Wuertz, S. (2018). Isolation and characterization of native probiotics for fish farming. BMC Microbiology, 18 (119).
Pazos F., Y. Santos, A. R. Macias, S. Nuñez & A. E. Toranzo. (1996) Evaluation of media for the successful culture of Flexibacter maritimus . Journal of Fish Disease, 19:193–197.
This research was funded by the FISHEALTH project (CER-20211010) from the Centre for the Development of Industrial Technology (CDTI) and framed in the call for aid aimed at “CERVERA” Technological Centers of Excellence Industrial.