Molecular characterization and immunohistochemical localization of tilapia piscidin 3 in response toAeromonas hydrophilainfection in Nile tilapia

The antimicrobial activity of tilapia piscidin 3 (TP3) was determined in vitro against a locally isolatedAeromonas hydrophila. A 388bpfragment was amplified from the TP3 cDNA and sequenced. The coding sequence (CDS) of TP3 was estimated to be 231bpcodes for 76 amino acids long and stop codon. In silico analysis was performed to detect both the signal peptide and the prodomain cleavage sites to follow the amino acids number 22 and 70, respectively. Based on this, a peptide 23 amino acids long with a remarkably high computed antimicrobial probability was synthesized and used in the subsequent experiments. The antimicrobial activity of TP3 was determined with minimum inhibitory concentration (MIC) and minim um bactericidal concentration (MBC) methods. TP3 exhibited relatively weak antimicrobial activities against the tested bacteria. A challenge experiment was then performed in Nile tilapia with low and high doses ofA. hydrophila, followed by timely recognition; after 3, 6, 24 h, and 7 days of the specific TP3 gene expression, immunohistochemical localization was also performed. Histopathological examination revealed provoked inflammatory responses and congestion in the same organs of TP3 expression. Immunohistochemical localization showed thatA. hydrophilainduced tilapia fish to express TP3 after 24 h within the gills, intestine, hepatopancreas, spleen, and posterior kidney. In quantitative real time (RT)-polymerase chain reaction analysis, the high dose showed higher mRNA expression levels than the low dose, and its expression levels increased in theA. hydrophila-infected fish. It was therefore concluded that TP3 plays an essential role in fish immunity.