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 × 1011 PFUmL-1 of ETP-1, and maintained at 25 °C. The highest enrichment level was obtained after 4 h (3.00 × 109 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 × 103) followed by the kidney (4.00 × 101) and the gut (1 × 101 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 × 107, kidney; 3.33 × 106, spleen; 5.52 × 105, liver; 6.20 × 104 PFUmL-1mg-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 1011 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.

Complete genome analysis of the novel Edwardsiella tarda phage vB_EtaM_ET-ABTNL-9.

This work describes the characterization and genome annotation of a new lytic phage, vB_EtaM_ET-ABTNL-9 (referred to as PETp9), isolated from waste water samples collected in Dalian, China, that can kill bacteria of the species Edwardsiella tarda. The genome of phage PETp9 is a circular double-stranded DNA molecule that is 89,762 bp in length with a G+C content of 37.26%, contains 132 ORFs, and encodes one tRNA. Phylogenetic analysis indicated that phage PETp9 should be considered a novel phage.

Isolation and characterization of phage (ETP-1) specific to multidrug resistant pathogenic Edwardsiella tarda and its in vivo biocontrol efficacy in zebrafish (Danio rerio).

Edwardsiella tarda phage (ETP-1) was isolated from marine fish farm water to characterize its effect against pathogenic multidrug-resistant E. tarda. According to transmission electron microscopy results, ETP-1 is classified as a member of the family Podoviridae. ETP-1 showed MOI dependent E. tarda growth inhibition, a latent period of 60 min, and burst size of 100 PFU per infected cells. In host range tests, five out of eight E. tarda strains were sensitive to ETP-1 which had efficiency of plating index in the range 1-1.28. ETP-1 was stable over a broad range of pH and temperature. The size of the ETP-1 genome was predicted to be approximately 40 kb. Zebrafish exposed to ETP-1 showed no adverse gene responses to the inflammatory mediator cytokines, il1-ß, tnf-α, il-6, and il-10, the chemokine, cxcl-8a, and reactive oxygen species, sod-1. When zebrafish were 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 phage exposed fish (68%) compared to that of the control (18%) until 4 days post challenge. Our results suggest that ETP-1 can be used as a potential bio-therapeutic candidate to control multi-drug resistant E. tarda infection in aquaculture.

Genomic characterization of bacteriophage pEt-SU, a novel phiKZ-related virus infecting Edwardsiella tarda.

A bacteriophage infecting Edwardsiella tarda (named pEt-SU) was isolated from freshwater collected in Chung-ju, South Korea. The whole genome of pEt-SU was 276,734 bp in length, representing the first giant phage infecting Edwardsiella reported to date. A total of 284 putative open reading frames were predicted and annotated. Morphology and genome analyses verified that pEt-SU may be distantly related to the phiKZ-like phages, a well-known giant myovirus. The findings in this study provide new insights into the phages infecting E. tarda ads well as fundamental data for the study of giant phages.

Full-genome sequence of a novel myovirus, GF-2, infecting Edwardsiella tarda: comparison with other Edwardsiella myoviral genomes.

Edwardsiellosis, which is caused by Edwardsiella tarda, a Gram-negative bacterium, is one of the most serious infectious diseases in both marine and freshwater fish farms worldwide. Previously, we reported the complete genome sequences of three E. tarda-lytic bacteriophages (two podoviruses and a myovirus), which were isolated from fish tissues and fish-rearing seawater. Further genomic information regarding E. tarda phages is important for understanding phage-host interactions as well as for applications of the phages for the control of disease. Here, we report the complete genome sequence of a novel E. tarda phage (GF-2) of myovirus morphology (family Myoviridae), isolated from tissue homogenates of a cultured Japanese flounder (Paralichthys olivaceus) that succumbed to edwardsiellosis in Japan. The size of the entire genome was 43,129 bp, with a GC content of 51.3 % and containing 82 open reading frames (ORFs). The GF-2 genome possesses lysogeny-related genes that have not been found in the reported Edwardsiella phage genomes. Comparative genomics of Edwardsiella myophages suggest that the C-terminal domains of the tail fiber proteins have relevance to their host specificity. Thus, GF-2 genome information provides a novel resource for our understanding of the molecular mechanisms involved in their host specificity and for detection of E. tarda in aquaculture environments.

Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infection.

Bacterial ghosts may be generated by the controlled expression of the phiX174 lysis gene E in Gram-negative bacteria and they are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. The Edwardsiella tarda ghost (ETG) vaccine was prepared using this technology and tested in vaccination trials. Control groups included mice immunized with formalin-killed E. tarda (FKC) or mice treated with phosphate-buffered saline (PBS), respectively. The results showed that serum IgA and IgG antibody titers were significantly higher in the ETG-vaccinated group compared to the other groups. In addition, CD8+ T cell counts in peripheral blood were elevated in the ETG groups. Most important, ETG-immunized mice were significantly protected against E. tarda challenge (86.7% survival) compared to 73.3 and 33.3% survival in the FKC-immunized and PBS-treated control, respectively, suggesting that an ETG oral vaccine could confer protection against infection in a mouse model of disease.