For analysis of the effect of PoCD9.1 overexpression, pCNPoCD9.1 and pCN3 were transfected into FG cells as described above. After transfection for 24 h, the cells were infected with E. piscicida for 6 h. The bacterial number was determined as described above. For PoCD9.3, the effect of PoCD9.3 overexpression was examined in vivo, and thirty fish were divided into two groups: pCNPoCD9.3 and pCN3 diluted in PBS (200 μg/mL). The fish were injected intramuscularly (i.m.) with 50 μL of pCNPoCD9.3 and pCN3. At 5 days after plasmid administration, spleen and muscle tissues of 5 fish from each group were collected for RNA and DNA extraction. The remaining fish were infected with E. piscicida as described above. Spleens from five fish were taken under aseptic conditions at 24 and 48 hpi. The bacterial number in the tissues was determined by plate count [45]. Briefly, the tissues were homogenized in PBS, and the homogenates were diluted serially. Then, the dilutions were plated on LB agar plates. The plates were incubated at 28 °C for 24 h, and the colonies that appeared on the plates were enumerated. The experiment was performed three times.

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