DEVELOPMENT OF THE FIRST STANDARDIZED BIOTIC CHALLENGE MODEL FOR DOVER SOLE Solea solea  

The growing importance of the aquaculture sector during recent decades renders a diversification of fish species and products imperative. Flatfish species constitute important and credible new aquaculture candidates as exemplified by Dover sole Solea solea (Linnaeus 1758) exhibiting high flesh quality and important market value, and being the subject of an increasing consumer's demand. As for other advanced marine teleost species, production has been severely hampered by difficulties in larval rearing which is reflected in low and unpredictable survival rates. Mortality largely may be induced by bacterial diseases (caused by e.g. Vibrio sp.) thereby decreasing survival and influencing the development of the fish embryo and larvae. Because of the emergence of acquired antimicrobial resistance, there is a great need for alternative measures to overcome disease. In this respect, the use of probionts in aquaculture is popular but scientifically poorly documented. Furthermore, the increasing interest of researchers in the interactions between bacteria and their host, either to unravel the mode of action of probiotics or to investigate how pathogens elicit disease and mortality, underscores the need for reliable experimental models. Nevertheless, only a handful of studies focused on the development of such models for fish larvae and for Dover sole, no such model is available yet. The present study describes a standardized biotic challenge model for Dover sole larvae adopting Vibrio spp. as potential pathogens.

Our research group pinpointed a housing system for Dover sole larvae whereby they are kept individually in 24-well plates. This ensures a standardised and reliable experimental set-up in which the possible death of one larva has no effect on the other larvae, rendering experiments adopting such a system more reproducible. In three separate experiments, different pathogenic candidates in varying concentrations were tested. Five pathogenic strains were adopted: the wild-type Vibrio anguillarum HI610 strain, V. anguillarum Fr and V. harveyi Fr that were both procured from a disease outbreak in sea bass larvae in a French hatchery, V. tapetis isolated from skin blisters and liver of Dover sole and V. tapetis 2 originating from skin ulcers of wild dab (Limanda limanda). One of these strains was added to the well water of larvae at 10 days after hatching in a final concentration of 10^5, 10^6 or 10^7 colony forming units (CFU)/ml. No significant differences in survival were noticed between the larvae inoculated with V. anguillarum wild-type, V. harveyi Fr, V. tapetis, V. tapetis 2 and the control group at any of the  concentrations tested. Vibrio anguillarum Fr administered at a final concentration of 10^6 CFU/ml well water elicited a larval mortality of 36%, while the control group displayed 5% mortality. When this strain was supplied to the larvae at a concentration of 10^7 CFU/ml, 48% mortality was observed, compared with 5% of the unchallenged larvae.

The model as described above is to be regarded as a powerful tool for investigating the pathogenesis of V. anguillarum infections in Dover sole larvae and for evaluating curative or preventive treatments against vibriosis.