and encodes ampicillin resistance. The transformed E. ictaluri were confirmed by PCR using E. ictaluri-specific primers (Russo et al., 2009). Twenty-four 15-mL tubes were filled with theront solution at 8 mL per tube. Edwardsiella ictaluri was added to theronts as follows: (1)

0 CFU mL−1 (no bacteria); (2) 4 × 103 CFU mL−1; (3) 4 × 105 CFU mL−1; and (4) 4 × 107 CFU mL−1. Theronts in 12 tubes were exposed to E. ictaluri for 1 h and the remaining 12 tubes for 4 h. Triplicate tubes were used for each combination of E. ictaluri concentration and exposure time. At the end of each sampling time, formalin was Selleckchem Sorafenib added to each tube to fix theronts at 1% for 30 min. Theronts were washed with sterile water three times and harvested by centrifugation at 240 g for 3 min.

The supernatant was discarded, and theronts were suspended in 0.5 mL sterile water in a flow cytometer tube. The number of theronts carrying fluorescent E. ictaluri was counted for each sample using the Coulter Epics flow cytometer (Beckman Coulter, Inc.) equipped with a 15 mW argon ion laser operating at 488 nm. Theronts without E. ictaluri exposure were included as negative controls. The percentage of theronts fluorescing was determined from ~ 1000 theronts in each sample. Fish infected Dabrafenib manufacturer with maturing tomonts were anesthetized with 150mgL1 tricaine methanesulfonate (MS-222) and rinsed in tank water, and the skin was gently scraped to dislodge the parasites. Four six-well plates were filled with 300 tomonts well−1. Each plate had three

treatments with two wells per treatment in all plates. Edwardsiella ictaluri was added to wells in each plate as follows: (1) 0 CFU mL−1; (2) 4 × 105 CFU mL−1; and (3) 4 × 107 CFU mL−1. Tomonts were exposed to E. ictaluri at room temperature 4-Aminobutyrate aminotransferase for 2 h. Then, the bacterial suspension and unattached tomonts were removed from each well. Fresh tank water was added to each well to wash (three times) the attached tomonts and remove suspended bacteria. After washing, 30 mL fresh tank water was added to each well and incubated at 22 ± 2 °C. One plate was sampled at 2, 4, 8, or 24 h postexposure to E. ictaluri. At the end of each sampling time, the attached tomonts (2–8 h) or theronts (24 h) were harvested and fixed with 1% formalin. After washing three times with clean water, one drop of tomont or theront sample and one drop of Gel/Mount™ aqueous mounting medium (Sigma) were placed on a slide and covered with a cover slip. The slides were viewed with an Olympus BX41 fluorescence microscope and photographed with an Olympus DP70 digital microscope camera. The distribution of E. ictaluri on the parasite (tomont specimens) was examined using a Zeiss Axioplan 2 microscope (Göttingen, Germany) fitted with a Bio-Rad Radiance 2000 confocal scan head. Laser scanning was controlled using Lasersharp 2000 software (Bio-Rad).