Isolation,Identification And Genome-Wide Research Of Vibrio Phages From Shrimp Farm

In recent years,with the rapid transformation of the aquaculture model from coarse to high-density intensification,the aquaculture environment was declined rapidly,resulting occurrence of the diseases frequently,and caused severe damage to the aquaculture industry.Among them,the diseases caused by bacteria such as Vibrio has become a key factor constraining the sustainable development of the aquaculture industry.The farmer's over-reliance and unreasonable use of antibiotics and other chemical drugs caused bacterial resistance.Bacteriophages have received more and more attention because their have potential in replace antibiotics.In this study,the Vibrio which was isolated from shrimp farm as the host bacteria isolated the phages.Identification,biological characteristics and genome-wide research of phages.1.Isolation and identification of host Vibrio and bacteriophagesIn this study,100 shrimp samples and 183 aquaculture samples were collected from 66 Penaeus vannamei farms in Fujian,Guangdong,and Shandong provinces.Vibrio alginolyticus,Vibrio harveyi,and Vibrio parahaemolytic were selectively isolated using molecular biology methods.The isolated vibrio includes 196 yellow and 90 green strains.In this study,the 286 vibrio isolated from shrimp aquaculture tank as a potential host used for phage isolation.The results showed that 14 strains of Vibrio isolated 18 strains of Vibrio alginolyticus phage and 1 Vibrio harveyi bacteriophage.Identification of species of 14 host strains,using morphological characteristics,physiological and biochemical identification and16 S rDNA molecules Identification,Biolog identification,HSP60 gene and other methods.The identification results show that except V149 is V.harveyi,the other 13 strains are Vibrio alginolyticus.Therefore,according to the host identification results,?V039A,?V039B,?V039C,?V067A,?V067B,?V067C,?V067D,?V114,?V130,?V141,?V154,?V170,?V172,?V173,?V177,?V208,?V213,?V214 are the phage of alginolyticus,and ?V149 is Harvey's Vibrio phage.2.Biological characteristics of bacteriophageAccording to phage electron microscopy observations,19 phages belong to Caudovirales.Among them,8 strains belong to Myoviridae(?V039A,?V039B,?V039C,?V067D,?V114,?V170,?V172 and ?V208),9 strains belong to Podoviridae(?V067A,?V067B,?V067C,?V130,?V141,?V154,?V173,?V177 and ?V213),and 2 strains belong to Siphoviridae(?V149 and ?V214).The results of phage digestion showed that except ?V154 and ?V172 may be ss DNA,the others were dsDNA.All phage was insensitive to chloroform and no envelope.The results of cross-cleavage experiments of phage and host indicated that bacteriophage could lyse more than 4 strains of V.alginolyticus,include ?V130,?V154,?V170,?V173,and?V177,and all belonging to broad spectrum bacteriophage.The multiplicity experiment results showed that the optimal multiplicity of infection(MOI)for ?V149 was 0.0001 with high yield.One-step growth curve of bacteriophage showed that phage ?V067A,?V067B,?V170,?V177and ?V213 had strong lysis ability.By studying the thermal stability of bacteriophage ?V170,it was found that its thermal stability is good and it is sensitive to temperatures above 65 �C.3.Phage genome-wide researchThe bacteriophage ?V039C genome was circular dsDNA with a total length of 43405 bp and a GC content of 42.95 %.A total of 77 ORFs were annotated.The bacteriophage ?V141genome was a linear dsDNA with a total length of 43313 bp and a GC content of 48.17%.A total of 49 reading dialogs were annotated.The bacteriophage ?V172 genome was a linear dsDNA with a total length of 22378 bp and a G+C content of 49.52%.39 ORFs were annotated.The phage ?V208 genome was a circular dsDNA with a length of 45521 bp and a G+C content of43.2%.A total of 76 ORFs were annotated.The homology analysis was performed on the four genomes.The results showed that the highest coverage rate was only 2% in the homologous sequences retrieved from phage ?V039C,suggesting that phage ?V039C may be a newly discovered phage of V.alginolyticus.Only 2homologous sequences were found in the phage ?V141 genome.The highest coverage rate was only 3%.This indicates that ?V141 is likely to be a newly discovered lytic phage of V.alginolyticus.Two homologous sequences were found in the phage ?V172 genome,and the coverage with Vibrio vulnificus phage SSP002 was as high as 97%.This indicates that ?V172 is likely to be a phage in which SSP002 is the same strain or closely related to each other.In the homologous sequence retrieved from the phage ?V208 genome,the highest coverage rate was only 3%,indicating that ?V208 is also very likely to be a newly discovered lytic phage of V.alginolyticus.