Phage therapy is a biologically friendly strategy for controlling bacterial pathogens in aquaculture as an alternative to antibiotics and rapid spread of multidrug resistant pathogens. For efficient phage therapy, phage needs to reach target organs and/or tissues of animals in adequate amounts at right time during bacteria infection. Previously, we isolated and characterized multidrug resistant Edwardsiella tarda infecting phage ETP-1 from farm seawater. In this study, we investigated ETP-1 dispersion and persistency efficiency following two modes of ETP-1 administrations, namely (1) intra peritoneal (i.p.) injection and (2) continuous ETP-1 exposure for 12 days to zebrafish at 105,108 and 1011 PFU/mL. In addition, biocontrol efficacy of ETP-1 was tested and transcriptional responses were examined for selected immune genes in kidney and gut tissues at 1, 7, 10 and 12 day post exposure (dpe) to determine safety in concern of phage therapy.
Results showed that ETP-1 disseminated to all the tissues as quickly as day 1 by two modes of phage administration. In i. p. injected tissues, persistency was decreased in different levels with the time, while in bath exposure (1011 PFU/mL), persistency looked consistent throughout the experiment. Moreover, among immune genes (il1-β, tnf-α, il-6, il-10, cxcl-8a and sod-1), no significant differences (P<.05) in the mRNA expression were observed in ETP-1-treated and control groups, except il1-β and sod-1 at 1 dpe in kidney. In the gut, except il1-β and cxcl-8a, other genes were down regulated at day 1, but with the time, levels were became basal or slightly higher (except il1-β) than the controls of respective time points. This indicates, even though, the ETP-1induced some cytokine genes at certain time points, it did not show adverse apparent immune stimulation in host, and use of phage at 1011 PFU/mL was safe. In the zebrafish that bath exposed to ETP-1 for 12 days, and simultaneously challenged with E. tarda (1.08 × 105 CFU fish−1), the survival rate was higher in ETP-1 exposed fish (68%) than the control (18%) until 4 days post challenge. In summary, our data indicate that even the absence of host bacteria as a phage carrier, ETP-1 could transport to the neighboring internal organs. Moreover, results suggest that during systemic bacterial infection stage, phage therapy may work efficiently to kill E. tarda cells, which internalize in those organs.
Keywords: E. tarda; phage ETP-1; immune responses; phage distribution; zebrafish