Luminal uptake of Vibrio (Listonella) anguillarum by shed enterocytes--a novel early defence strategy in larval fish.

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.

Bacterial host interaction of GFP-labelled Vibrio anguillarum HI-610 with gnotobiotic sea bass, Dicentrarchus labrax (L.), larvae.

The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ-free sea bass larvae were orally exposed to a V. anguillarum HI-610 pathogen labelled with the green fluorescent protein (GFP-HI-610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post-exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid- and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.

Development of a bacterial challenge test for gnotobiotic sea bass (Dicentrarchus labrax) larvae.

The use of probiotic microorganisms in aquaculture is gaining a lot of interest. Gnotobiotic model systems are required in order to fully understand the effects and modes-of-action of these microorganisms, as the native microbial communities present in non-sterile animals can lead to false conclusions. In this study, a gnotobiotic sea bass larvae (Dicentrarchus labrax) test system was developed. In order to obtain bacteria-free animals, the eggs were disinfected with glutaraldehyde and subsequently incubated in a solution of rifampicin and ampicillin. Axenity was confirmed using culture-dependent and -independent techniques. The gnotobiotic larvae were fed axenic Artemia sp. from 7 days after hatching onwards. In the challenge test, one of the three opportunistic pathogens, Aeromonas hydrophila, Listonella anguillarum serovar O1 and O2a, was added to the model system via the water and encapsulated in Artemia sp. Only serovar O2a led to increased mortality in the sea bass larvae. The presented gnotobiotic model can be used for research on, among others, reciprocal metabolic effects between microorganisms and the host (e.g. as measured by gene expression), immunostimulants, pharmacological research and the histological development of the gastrointestinal tract and growth of larvae.