Three 1-L beakers were filled with 200 mL of theront solution each at a concentration of 6800 theronts mL−1. Edwardsiella ictaluri was added to each beaker as follows:

(1) 0 CFU mL−1; (2) 4 × 105 CFU mL−1; and (3) 4 × 107 CFU mL−1. After exposure to E. ictaluri for 1 h, theronts were harvested by centrifugation in 50-mL tubes at 240 g PI3K Inhibitor Library for 3 min and the supernatant discarded. Theronts were then washed (three times) with fresh tank water and centrifuged, and the supernatant was discarded to remove nonadherent bacteria. After washing, theronts were suspended in 100 mL tank water and enumerated with a Sedgwick-Rafter cell (Xu et al., 2000). Six 2-L beakers were used with 1 L water and 30 channel catfish fingerlings distributed in each container. The fish (3.3 ± 0.5 cm in length and 0.3 ± 0.1 g in weight) were acclimated to laboratory conditions 3 days prior to the trial. Water in each beaker was reduced to 0.5 L. The theronts exposed to various concentrations of E. ictaluri were added to each beaker at 1000 theronts fish−1 (two beakers for each treatment). Five fish were sampled from each beaker at 4 h, 1 day, and 2 days post-theront exposure. The remaining 15 fish in each beaker were monitored for mortality. Each sampled fish was put in a 1.5-mL microcentrifuge tube, labeled, and washed with sterile water three times. Each fish was homogenized after adding 0.5 mL sterile water to a clean microcentrifuge

tube using a 1.5-mL pellet pestle. Half of the fish tissue from each sample was transferred to a 15-mL tube with 5 mL brain heart infusion Target Selective Inhibitor Library research buy (BHI) broth containing 100 μg mL−1 ampicillin and incubated at 28 °C for 24 h with shaking. The pZsGreen-transformed Dolichyl-phosphate-mannose-protein mannosyltransferase E. ictaluri was able to grow in BHI with ampicillin, but other autochthonous bacteria were inhibited. The presence of E. ictaluri was examined by florescence microscopy at 24 h postculture. The remaining fish tissue was frozen at −20 °C for DNA extraction and used for qPCR. The tissues preserved at −20 °C were used to extract DNA and quantitate E. ictaluri with qPCR. Total genomic DNA of E. ictaluri

in fish tissues was extracted by the DNeasy Tissue kit and eluted into 200 μL water according to the manufacturer’s instructions. DNA yield and purity were determined using a Nanodrop ND-1000. The gDNA was stored at −20 °C until use. One-step qPCR was performed as described by Bilodeau et al. (2003) using E. ictaluri-specific primers (forward 5′-ACTTATCGCCCTCGCAACTC-3′ and reverse 5′-CCTCTGATAAGTGGTTCTCG-3′) and a dual-labeled probe (5′-CCTCACATATTGCTTCAGCGTCGAC-3′). Reactions were completed using an Applied Biosystems 7500 with the following conditions: 50 °C for 2 min, 95 °C for 2 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Extracted DNA from fish tissue (1 μL) was used as template in qPCR, and the DNA concentration in fish tissue was determined via the standard curve [threshold cycle (Ct) values vs. DNA concentration of E. ictaluri].