Challenge test
Bacterial strain
A virulent strain of A. hydrophila was isolated from a naturally diseased cultured O. niloticus during 2010 in a private fish farm at Kafer El-shekh governorate. It was cultured in brain–heart infusion broth (Lab M, USA) at 25 °C for 24 h. The broth culture was centrifuged at 1500g/10 min. The bacterial precipitate was re-suspended in phosphate buffered saline. The bacterial concentration was adjusted to 1.5 × 106 CFU/ml using the plate counting technique [27].
Coating of feed pellets
The fish basal diet was mixed thoroughly with the saline culture (weight equal volume) to obtain 1.5 × 106 cfu/g food. The food loaded with pathogenic bacteria was coated with gelatine. This preparation was performed on the day of challenge. Based on the data for the daily requirements of the fish, the amount of bacteria in the experimental feed was 2.5 ± 0.2 × 106 cfu/fish/day [30].