Introduction
Bacterial diseases represent a significant issue in Mediterranean aquaculture. The number of new pathogens identified has increased over time, and one of the most common causes of the emergence of infectious diseases in marine fish is the Aeromonas species (Tansel Tanrikul & Dinçtürk , 2021). Over the past decade, Aeromona veronii has become a major problem in European sea bass (Dicentrarchus labrax) farming in some Mediterranean countries, with outbreaks occurring when water temperatures reach their highest values (Smyrli et al., 2019). Due to factors such as climate change, the duration of periods with higher water temperatures is increasing, which not only leads to a higher incidence of outbreaks but also allows them to persist over time. Additionally, the rise in water temperatures contributes to the proliferation of other emerging pathogens (Combe et al., 2023). 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 required (Yilmaz et al., 2022). To assess their effectiveness, the development of new standardized fish health models is necessary. In response to this challenge, we are working on the development of infection models for A. veronii , with plans to expand them to other emerging pathogens such as Lactococcus garvieae. To date , we have standardized two infection models for A. veronii , using intraperitoneal (IP) injection and bath immersion. These models have been tested under different conditions to evaluate their reproducibility. Preliminary results suggest that pathogenicity varies significantly with temperature, but it is also influenced by factors such as fish size, fish batch, and culture density, among others. Our next steps involve developing and validating a cohabitation infection model for A. veronii , as well as extending this approach to other emerging pathogens.
Materials and methods
B acterial isolates from internal organs of a sick fish (real outbreak in a Turkish farm) were streaked on tryptic soy agar (TSA) plates supplemented with 2% NaCl . The TSA plates were incubated at 25°C for 24-48 h and colony morphology was observed. The pure colonies were streaked again on TSA plates to determinate the specie by proteomic analyses (MALDI-TOF). O nce confirmed, the A. veronii strain was cultured for cryopreservation and added to the CTAQUA strain collection (CT0068) . A stock of different vials was generated from a liquid culture in tryptic soy broth (TSB) supplemented with 2% NaCl, with bacteria grown for 24 hours at 25 °C under agitation (150 rpm). Subsequently, bacteria were centrifuged to remove the culture medium and resuspended in a cryopreservation medium (50 % glycerol in Phosphate Buffer Saline (PBS)) at a final concentration of 1×10⁹ Colony Forming Units (CFU)/mL. Vials were stored at -80 °C. After an initial virulence test in which 10 European sea bass were inoculated by IP injection to confirm that the strain caused mortality, dose-response trials were conducted. I nfection by IP injection was carried out by inoculating 0.1 ml of bacterial suspension per fish. This suspension was prepared by diluting a cryovial with an initial concentration of 1×10⁹ CFU/mL in PBS to obtain the concentrations to be tested. Bath infection was carried out by scaling up the culture in TSB supplemented with 2% NaCl at 25°C under agitation (150 rpm) from one of the cryovials (1 mL) until obtaining a sufficient amount of culture at a concentration of 1×10⁹ CFU/mL to prepare the dilutions to be tested in 10-liter buckets with seawater. Then, fish (20 gr) were placed in the buckets for 1 hour with aeration. The handling of the animals was performed under deep anesthesia induced by an anesthetic (benzocaine). In all the tests conducted, a control group was included, in which the fish were handled in the same way as the experimental fish but without exposure to the pathogen. Control fish were inoculated with PBS in the case of IP injection or placed only in seawater in the bucket for bath immersion. All concentrations were validated in triplicate (10 fish/tank) and after pathogen exposure, the fish were transferred to tanks in a Recirculating Aquaculture System (RAS), where all culture parameters were monitored throughout the trial. IP injection tests were carried out at 22°C, while the bath tests were conducted at 22°C and 25°C to evaluate the dose-response at different temperatures.
Results
Graph 1 shows the cumulative mortality results for the different bacterial concentrations of A. veronii according to the tested route of administration and temperature.
In sight of the results, it can be concluded that there is a good dose-response relationship for the CT0068 isolate when administered to pre-ongrowing sea bass both by IP injection and by bath, making these models suitable for validating preventive strategies and treatments against A. veronii.
References
Combe, M.; Reverter , M.; Caruso, D.; Pepey , E. & Gozlan, R. E. (2023). Impact of Global Warming on the Severity of Viral Diseases: A Potentially Alarming Threat to Sustainable Aquaculture Worldwide. Microorganisms, 11 (4) – 1049: 1-14.
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.
Tansel TanrikuL , T. & Dinçtürk , E. (2021). A New Outbreak in Sea Bass Farming in Turkey: Aeromonas veronii. Journal of the Hellenic Veterinary Medical Society, 72(3): 3051–3058.
Smyrli M.; Triga, A.; Dourala , N.; Varvarigos, P.; Pavlidis, M.; Ha Quoc, V. & Pantelis, K. (2019) . Comparative Study on A Novel Pathogen of European Seabass. Diversity of Aeromonas veronii in the Aegean Sea. Microorganisms, 7 (504): 1-24.