Characterisation of Vibrio anguillarum for the development of vaccine in cod (Gadus morhua).

Abstract

Atlantic cod (Gadus morhua L.) is one of the most promising new fish species introduced to cold water aquaculture due to the large established market in Europe and the USA and the decline in wild stock. So far, the production of farmed cod has been relatively low, with the main hindrance due to diseases. Vibrio anguillarum has been recognised as the biggest disease problem of farmed cod and has slowed the development of a successful cod aquaculture industry. When the first incidences of V. anguillarum occurred in cod aquaculture, vaccines designed for vibriosis in Atlantic salmon (Salmo salar L.) were used in an attempt to combat the disease. However, these vaccines did not provide sufficient protection, possibly because they lacked serotype O2b, which is known to affect cod and to a lesser extent salmonids. Recently, vibriosis vaccines specifically designed to protect Atlantic cod have been formulated, but outbreaks of vibriosis in vaccinated fish are still being reported, suggesting that these formulations are inadequate. The aim of this project was to develop a whole cell inactivated vaccine formulation specifically tailored to protect Atlantic cod against Vibrio anguillarum. The serological classification of V. anguillarum was first investigated by producing a set of monoclonal antibodies (mAbs). Using lipopolysaccharides (LPS) extracted with butan-1-ol, 4 mAbs were selected and shown to react specifically with V. anguillarum serotypes O1, O2a and O2b. A collection of over 150 V. anguillarum isolates were screened using these, which revealed that most of the isolates had been previously correctly classified. A new sub-serotype of V. anguillarum O2 was identified from isolates recovered from outbreaks of vibriosis in Norway as well as Scotland. This new sub-serotype was referred to as O2d since the subserotype O2c has been recently identified in vibriosis cases from Atlantic cod. However, it was shown that the O2c sub-serotype might not belong to the O2 serotype, but in fact belongs to another serotype. To protect Atlantic cod against all the V. anguillarum serotypes (and subserotypes) which they are susceptible to, it is recommended that isolates from serotypes O1, O2a, O2b, O2c and O2d should all be included in a bacterin vaccine for cod. In order to determine which isolates from each of the serotypes to include in the vaccine, a variety of virulence factors of V. anguillarum were investigated in vitro. The interaction of some candidate isolates from O1, O2a and O2b serotypes (O2c and O2d were not identified at the time this part of the study took place) with cod phagocytic cells were studied using flow cytometry. Phagocytosis and respiratory burst of cod macrophages and neutrophils as well as cod serum killing of V. anguillarum were quantified. It was found that isolates within the same serotype displayed varying degrees of resistance to phagocytosis and the subsequent respiratory burst activity as well as that all the V. anguillarum strains tested were resistant to Atlantic cod serum killing. These in vitro assays were found to be very useful in assessing the virulence of V. anguillarum. The isolate within each serotype eliciting the highest percentage of positive phagocytic cells was selected in order to increase the antigen presentation pathway, thus theoretically enhancing the protection elicited by the vaccine. A multivalent formalin-inactivated non-adjuvanted vaccine was prepared which included all the serotypes previously described and was injected intraperitoneally into Atlantic cod. A bath challenge was performed on vaccinated and mock-vaccinated fish, 6 weeks post immunisation, using V. anguillarum isolates from the serotypes O2b, O2c and O2d that were not included in the vaccine. An excellent level of protection was obtained against O2b and O2d (relative percentage survival 100% and 96.4%, respectively), but the challenge with the sub-serotype O2c isolate did not produce any mortality in the control group and needs to be repeated. The vaccine formulation was very efficient at protecting Atlantic cod against vibriosis but further challenges need to be performed with other serotypes included in the vaccine (O1 and O2a), as well as with more isolates from the O2b, O2c and O2d sub-serotype. To conclude, Atlantic cod is a species which will certainly have a major influence in marine aquaculture, but many areas have to be improved. The development of an effective and broad range vaccine to protect cod against Vibrio anguillarum offers another advance which should help Atlantic cod aquaculture to reach its full potential.