Development of phage delivery by bioencapsulation of artemia nauplii with Edwardsiella tarda phage (ETP-1).

This study proposed that phage-enriched artemia could be a useful tool for transferring phage into the cultured fish (larvae or adult) as a feed, and introduce mode of phage administration and its safety in concern of tissue adaptation for efficient phage therapy in aquatic animals. First, whether Edwardsiella tarda phage (ETP-1) could attach or ingest by the artemia and optimum time period for the ETP-1 enrichment with artemia were investigated. ETP-1 dispersion, abundance and persistency, and zebrafish immune transcriptional responses and histopathology were evaluated after feeding the fish with ETP-1-enriched artemia. Hatched artemia nauplii (36 h) were enriched with 1.90 × 10 ¹¹ PFUmL ^-1 of ETP-1, and maintained at 25 °C. The highest enrichment level was obtained after 4 h (3.00 × 10 ⁹ PFUmL ^-1 ), and artemia were alive and active similar to control for 8 h. ETP-1 disseminated dose dependently to all the tissues rapidly (12 h). However, when feeding discontinued, it drastically decreased at day 3 with high abundance and persistency in the spleen (1.02 × 10 ³ ) followed by the kidney (4.00 × 10 ¹ ) and the gut (1 × 10 ¹ PFUmL ^-1 ) for highest ETP-1-enriched artemia dose. In contrast, during continuous delivery of ETP-1-enriched artemia, ETP-1 detected in all the tissues (at day 10: gut; 1.90 × 10 ⁷ , kidney; 3.33 × 10 ⁶ , spleen; 5.52 × 10 ⁵ , liver; 6.20 × 10 ⁴ PFUmL ^-1 mg ^-1 tissues). Though the phage abundance varied, results indicated that oral fed ETP-1-enriched artemia disperse to the neighboring organs, even the absence of host as phage carrier. Non-significant differences of immune transcriptional and histopathology analysis between ETP-1-enriched artemia fed and controls suggest that no adverse apparent immune stimulation in host occurred, and use of ETP-1 at 10 ¹¹ PFUmL ^-1 was safe. With further supportive studies, live artemia-mediated phage delivery method could be used as a promising tool during phage therapy against pathogenic bacteria to control aquatic diseases.