Influencing Factors on Up-stream Process of Edwardsiella ictaluri Bacteriophage Preparation

Abstract

Catfish filet (Pangasianodon hypophthalmus (Sauvage) is an important export product of East Asian countries. Aquaculture of these fish has often been threatened by a serious disease, bacillary necrosis of pangasius (BNP) that is caused by Edwarsiella ictaluri bacteria. Bacteriophage therapy is considered an alternative reagent to antibiotics for controlling the disease. In this study, a batch process was used to prepare vB_EiA_PVN09 bacteriophage product toward its application in aquaculture of catfish in Vietnam. The influencing factors in the upstream process investigated here included medium type, pH, divalent cations, infection time point, MOI value, aeration, and temperature. The stability of phage product (raw lysate) during storage was also assessed. The results showed that E. ictaluri medium (EIM) broth was the best for formation of phage progenies (PFU mL-1) but did not indicate phage product stability during storage. Tryptic Soy Broth (TSB) broth was selected to investigate the influencing factors. It was found that the most sensitive factor was medium pH, which, when lower than 6.0, would completely suppress the process. The second sensitive factor was phage infection time point. Adding phage later than 4 h reduced 1 – 2.5 log PFU mL-1. Mg2+ or Ca2+ could enhanced phage yields by 1.5 - or 3-fold. MOI (0.01 - 1.0) and temperature (24 - 30 °C) did not cause significant difference in the yield while aeration (0 - 150 rpm) had slightly effects. The preparation made under conditions of pH 6.0 containing 0.6 mM Ca2+, 150 rpm, at 28 °C, phage infection point at 3 h with MOI = 0.1 and further incubation for 5 h was able to produce 9.72 ± 0.40 log PFU mL-1 (n=5) phage progenies. Phage product had a half-life of about 30 d at room temperature. Our study has pre-established an upstream process for E. ictaluri bacteriophage preparation in a 40 mL batch with verified factors.