Experimental Y. ruckeri Infection in Fish

ERM infection was performed using Y. ruckeri (CSF007-82) virulent isolate that was obtained from the National Centre for Cool and Cold Water Aquaculture, Kearneysville, West Virginia, USA, and stored at −80 °C in the Clinical Division of Fish Medicine, University of Veterinary Medicine, Vienna. Prior use, one colony was inoculated in tryptic soy (TS) broth (Sigma-Aldrich, Austria) and was cultured overnight at 22 °C in an orbital incubator, 144 revolutions per minute (rpm) [31 (link)]. For the experimental challenge, the bacterial suspension was adjusted at a theoretical concentration of 2 × 10⁹ CFU ml⁻¹, which was assessed using a spectrophotometer (Eppendorf BioPhotometer^®, Eppendorf, Hamburg, Germany). Water level was lowered to reach 50 L/tank, then the bacterial challenge was performed by 2 h bath exposure to the bacterial suspension to reach a final concentration of 2 × 10⁶ CFU ml⁻¹ [77 (link)]. Pathogen-free group was exposed to sterile phosphate-buffered saline (PBS) (Sigma-Aldrich, Austria). For spreading the infection, the water flow was temporarily stopped while the airflow was constantly maintained. Then, the water flow was resumed after 2 h of exposure.