Isolation And Characterization Of Pathogenic Bacteria And Lytic Bacteriophages From Skin Ulcer Syndrome Sea Cucumber (Apostichopus Japonicus)

Skin ulcer syndrome can cause severe mortality and morbidity among sea cucumber aquaculture, with the whole industry suffering great economic loss currently. It has been proved that vibrio is a main pathogen causing sea cucumber ulceration. Nowadays, antibiotic abuse and resistance have drawn public tremendous attention. It is urgent for us to search alternative method for antimicrobial drugs.Vibrio was isolated from skin ulcer syndrome sea cucumber (Apostichopus japonicus), and artificial challenges experiment were used to prove its pathogenicity, further, physiological, biochemical test and molecular identification were simultaneously carried out to taxonomy the bacterium. Additionally, lytic bacteriophages were isolated basing on the enrichment method, finally, lytic bacteriophages cocktail will be applied for phage therapy. Artificial challenges including intraperitoneal injection and immersion bath were further to confirm the bacterium pathogenicity. In order to identify and classify the pathogen, morphologic observation by scan electronic microscopy, physiological index and 16S rDNA PCR were performed individually. Sewage from aquafarm was centrifuged, and then suspension was collected, filtrating through membrane. The filtration was inoculated into the broth media. To obtain the high titer phage stock, the bacterium culture mixture was then centrifuged and filtrated through membrane. Double-layer agar method was used to purify bacteriophages with different plaque sizes. Transmission electronic microscopy was applied to observe the bacteriophages morphology.Intraperitoneal injection could cause the ejection of digest system with the number of bacteria approximately 5×103CFU; however, skin ulcer syndrome was outbreak under the immersion challenge with the bacteria concentration of 1×103 CFU/ml in 3 days. Further identification showed that the strain belongs to V. harveyi. Bacteriophage VP-1 and VP-2 were obtained by the enrichment method, belonging to Siphoviridae. The phage genome was digested by some restriction enzyme, in some way; obviously the genomes of VP-1 and VP-2 belonged to double-strand DNA. The bacterium growth was inhibited by bacteriophages cocktail in the In vitro bacteriostasis experiment. Bacteriophage VP-2 one step growth curve was drew, and the lytic cycle was approximate 40 min, moreover, about 100 progenies was released per bacterium. The pathogen also developed resistance to both phage VP-1 and VP-2.