Purification of antimicrobial factor from granules of channel catfish peripheral blood leucocytes

The channel catfish ( Ictalurus punctatus) is extensively used in aquaculture in the Southeast US and is susceptible to many bacterial infections acquired from its pond environment. Research is needed to better understand the defensive response and innate immunity of channel catfish against fish pathogens like Edwardsiella ictaluri and Aeromonas hydrophila. The main objectives were purification and characterization of an innate antimicrobial factor isolated from catfish leucocytes that has both bactericidal and antiviral activities. Oxygen-independent mechanisms of innate immunity for killing microorganisms have not been identified in leucocytes of channel catfish. Leucocytes were separated from catfish blood, and granule extracts were obtained by homogenization, centrifugation, and extraction with 10% acetic acid. The granule extracts were further purified by gel filtration chromatography. Bactericidal assays against the two fish pathogens and SDS–PAGE analysis were done on the isolated antimicrobial factor. Determination of antiviral activity of the factor was done by in vitro tissue culture using herpes simplex virus-type 1. Mass spectrometry analyses were done for molecular weight (655 Da), purity, and structural characterization of the innate non-peptide antimicrobial factor.