High-throughput Sequencing Analysis of the Main Pathogens of Ulcer Diseased Plectropomus leopardus
WANG Lei2, ZHANG Tian-Shi2, LIU Yang2, ZHANG Zi-Wei2, LI Kai-Min2, ZHU Chun-Hua1, CHEN Song-Lin2,*
1 Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang)/Guangdong Research Centre on Reproductive Control and Breeding Technology of Indigenous Valuable Fish Species, Zhanjiang 524088, China; 2 Yellow Sea Fisheries Research Institute/Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs/Qingdao National Laboratory for Marine Science and Technology/Laboratory for Marine Fisheries Science and Food Production Processes, Chinese Academy of Fishery Sciences (CAFS), Qingdao 266071, China
Abstract：Plectropomus leopardus is a kind of newly farmed fish with high economic value. In recent years, skin ulcer disease occurs in both juvenile and adult fish, resulting in a large number of deaths and great economic losses. It is important to identify the main pathogenic bacteria for disease-resistant breeding. In this study, the diseased fish and normal fish were collected from Hainan, and the skin microbial sequencing and analysis were carried out on 3 generation high-throughput sequencing method. The results showed that there were 403 unique operational taxonomic units (OTUs) in the normal group and 72 unique OTUs in the ulcerated group. The number of OTUs shared by the two groups was 74. In the normal group, there were 16 phyla, 30 classes, 63 orders, 96 families, 145 genera and 168 species were identified on average, while 9 phyla, 12 classes, 27 orders, 38 families, 55 genera and 67 species were identified on average in the diseased fish skin. The difference between two groups indicated that pathogenic bacteria inhibited the growth of normal bacteria, resulting in a significant decrease in the diversity of bacteria structure. There were great differences in dominant bacterium composition between normal group and diseased group. Proteobacteria and Firmicutes were dominant phyla in normal group, while Bacteroidetes and Epsilonbacteraeota were dominant phyla in the diseased fish. ANOVA analysis of variance showed that the content of Vibrio in diseased fish skin was significantly increased, and Vibrio harveyi, V. tubiashii and a specie of uncultured Vibrio were annotated in KRONA analysis. Based on above research, a strain of V. Harveyi was isolated and identified from the ulcer skin of P. leopardus, named as V. harveyi stain Dx21. After the artificial infection with the intraperitoneal injection, the result showed that the V. harveyi stain Dx21 could cause surface ulceration and death in juvenile starfish, which was similar to natural infection. The half-lethal dose (LD50) was 1.0×105 cfu/mL for P. leopardus fries weighing 100 g. This study revealed the flora characteristics of the skin of diseased P. leopardus, suggesting that V. harveyi is one of the main pathogens. This study has certain guiding significance for the skin ulcer disease prevention and control of P. leopardus and the development of vaccine.
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