Using Bacteriophages To Control Skin Ulceration Syndrome Of The Sea Cucumber (Apostichopus Japonicus) Caused By Vibrio Spp.

Skin ulceration syndrome is one of the most epidemic and serious disease during sea cucumber cultivation and hampers the consistent and healthy development of sea cucumber cultivation industry. At present, antibiotics and sanitizers were extensively used to prevent and cure skin ulceration disease of the sea cucumber. However, some important findings indicate that this strategy has limited success:drug residues, development of multi-drug resistance in bacteria, ecological and public health impacts. This study intends to apply lytic phage to control skin ulceralion of sea cucumbers, developing a new way of healthy aquaculture.By using plate screening techniques, bacterial strains were isolated from the body wall of sea cucumbers suffering from skin ulceration syndrome in Dalian. Induced infection assays were performed to test whether these isolated strains were pathogens. Bacterial strains were identified by morphological observation, physiological and biochemical indexes test and sequence similarity analysis of16S rDNA genes. Two bacterial strains were isolated named as VDL-1and VDL-2. Induced infection experiments showed that two bacterial isolates were virulent since they induced the same signs as natural infection. VDL-2had higher virulence than that of VDL-1. Both VDL-1and VDL-2were rod-shaped with polar flagella and had no capsule. The physiological and biochemical indexes between VDL-1and VDL-2had some different. VDL-1was identified as Vibrio cyclitrophicus and VDL-2was identified as Vibrio tasmaniensis.Isolated strains were used as host to isolate and purify bacteriophages via double-layer plate method. Morphology of the isolated phage was observed by transmission electron microscope. One-step growth curve, the sensitive to chloroform and the host range were performed to characterize bacteriophages. Bacterial challenge assay were carried out by measuring the development of co-cultures’optical density at600nm. Two bacteriophages (PVDL-1and PVDL-2) were isolated from seawater using VDL-1and VDL-2as host strains, respectively. PVDL-1was classified as Siphoviridae and it had a latent period of35min and an estimated burst size of218PFU/cell. PVDL-2was classified as Myoviridae and it had a latent period of30min and an estimated burst size of110PFU/cell. Both of PVDL-1and PVDL-2owned host specificity and were insensitive to chloroform. PVDL-1showed high lytic activity in vitro against VDL-1. While VDL-2evolved to PVDL-2resistant bacteria after incubating for6h.Phage PVDL-1and PVDL-2were delivered by immersion way during phage therapy experiment. The efficiencies of phage therapy were assessed by comparing the survival rate of sea cucumbers among negative group, positive group, phage preventive group and phage therapeutic group. PVDL-1treatment groups could improve the survival rate from45%to90%. PVDL-2preventive group and therapeutic group could improve the survival rale from0%to more95%and55%respectively.Results from the vitro and therapy experiments demonstrated that PVDL-1and PVDL-2could be used as phage biological agents to replace antibiotics for treating diseases of cultured sea cucumber.