Intestinal Colonization and Diarrhea Induction by Vibrio cholerae in Adult Zebrafish

Place adult zebrafish in sterile infection water and add a pathogenic Vibrio cholerae strain at the desired density.

Cover the beaker with a perforated lid to prevent the fish from jumping out and incubate.

The zebrafish ingest the bacteria, which reach the intestine, propel through the mucus layer using their flagella, and adhere to the epithelium.

They colonize the epithelium and secrete toxins that disrupt intestinal integrity, leading to leakage of ions, fluids, and proteins.

The toxins also trigger epithelial mucus secretion, promoting bacterial persistence.

The accumulation of fluid, mucus, and intestinal contents results in watery diarrhea, detectable by increased water turbidity.

Post-incubation, collect the fish and discard the infected water into a disinfectant.

Transfer the fish to sterile water to remove surface bacteria.

Collect and transfer the fish again to sterile water for further analysis.

After preparing the V.cholerae cultures, inoculate the zebrafish by immersion. First, place four or five zebrafish into a 400 milliliter beaker containing 200 milliliters of sterile infection water. Then add 1 milliliter of V.cholerae culture for the desired infection concentration.

Then, cover the beaker with a perforated lid. After preparing all the beakers, transfer them to a glass front incubator set at 28 degrees Celsius. For a transitory exposure, after about six hours, pour out the beaker water through a fish net to collect the fish. The infected water must be disposed of in bleach to kill the bacteria.

Then, place the fish into 200 milliliters of sterile infection water for five minutes. After five minutes, run this water through a fishnet, disposing of the water in bleach. Now, the fish can go into a new clean beaker with 200 milliliters of sterile infection water.