EFFECT OF A Vibrio anguillarum INFECTION IN THE INTESTINAL MICROBIOTA COMPOSITION OF THE ZEBRAFISH Danio rerio MODEL
Zebrafish, a widely used research model, has been used to study the interaction between the gut microbiota and the fish host. These studies revealed that gut microbiota participate in the education of the immune system, maturation of the gut, and promotion of nutrient metabolism in the host. Zebrafish has also been suggested to study the interaction between the host and pathogenic microorganisms, since methodologies for rearing germ-free or gnotobiotic fish have been standardized. In our lab, we have standardized an infection model in zebrafish larvae with the fish pathogen V. anguillarum, an invasive motile bacterium, causing hemorrhagic septicemia. We have observed that the gut represents the principal entry way of the pathogen, as previously described in rainbow trout. In germ-free conditions, V. anguillarum reached higher concentrations in larvae and provoked significantly more mortality than in conventional conditions revealing, the protective role of the host microbiota against this pathogen. Due to the importance of gut microbiota in larval health, the present study investigated how a V. anguillarum infection affects the gut microbiota composition of zebrafish larvae.
We inoculated, by immersion, 5 days post-fertilization (dpf) zebrafish larvae (n=150) with V. aguillarum (107 bacteria/mL). Control larvae (n=150) were inoculated with sterile larval media (E3). Larvae were maintained at 28°C in E3. From 5 to 9 dpf we determine the larval survival, V. anguillarum count (CFU/larvae) and total cultivable bacterial count (CFU/larvae) in trypticase soy agar (TSA) medium. Each bacterial morphotypes from TSA plates were counted, purified and Gram stained.
At days 9 dpf, 75% larvae survived the challenged with V. anguillarum, compared to 97% in control group (p<0.05). Concentration of the pathogen ranged from 3.7 to 4.5 (Log10 CFU/mL) in challenged larvae (Table 1). Total bacterial counts were not affected with V. anguillarum challenge. However, from 5 to 8 dpf V. anguillarum modified gut microbiota because new bacterial morphotypes were detected. Each colony morphotypes is being identified by sequencing their 16S rRNA gene
