Isolation, identification and biological properties of 3 lytic bacterio phages against AHPND-associated Vibrio parahaemolyticus

To isolate and identify lytic bacteriophages against AHPND-associated Vibrio parahaemolyticus and analyze their biological properties, lytic bacteriophages were isolated from Fujian and Mexico seawater samples by double-layer agar culture method, and one strain of V. parahaemolyticus strain associated with AHPND was used as the host bacterial strain for phage isolation and amplification. The phages were classified and identified by plaque shapes, restriction endonucleases, whole genome sequence, phylogenetic tree building and transmission electron microscopic technique. The lysis range and biological properties including optimal multiplicity of infection (MOI), thermal stability, pH stability, stability to peracetic acid and survival stability at different temperature were determined. The results showed that 3 lytic bacteriophages against AHPND-related V. parahaemolyticus were isolated, named P46, P48 and VP7. Their plaques were translucent circle. The plaque diameter of P46 was 4-5 mm. The plaque diameter of P48 and VP7 were 5-6 mm. The nucleotides of 3 bacteriophages were all dsDNA. P46, P48 and VP7 all had an icosahedral head with about 60 nm diameter. Their tails were about 18-20 nm in length and 8 nm in width. 3 phages belonged to the familiy Podoviridae. The cocktail of P46, P48 and VP7 lysed 91.5% of AHPND-associated V. parahaemolyticus strains and non-VP_AHPND 92.3% respectively. And they all could not identify non-V. parahaemolyticus bacteria. The optimal MOI of P46, P48 and VP7 were 0.001, 0.1 and 0.001 respectively. The optimal pH of P46 and VP7 were 6-8 and P48`s was 6-9. Three bacteriophages were stable at 60 °C and had higher survival rate at 4 °C for 50 weeks. P46, P48 and VP7 also tolerated peracetic acid with universal bactericidal concentration. Alignment with the sequences of the conserved protein sequences of NCBI showed that P46, P48 and VP7 had low homology with other bacteriophages. Bacteriophages P46, P48 and VP7 might be 3 novel Podoviridae bacteriophages. This study is the first report of lytic Podoviridae bacteriophages against AHPND-associated V. parahaemolyticus in China. The results may enrich the resources of bacteriophages against AHPND-associated V. parahaemolyticus and provide potential application of phages as a biocontrol agent.