Isolation And Identification Of Pathogenic Bacteria In Abalone, And Analysis Of The Relationship Between Dynamics Of Bacterial Community And Abalone Diseases

Abalone is an important economic shellfish species in China. Since1960s, abalone producers had developed methods of abalone aquaculture. However, along with the enlargement of abalone farming scale, which strengthened the level of intensive culturing, abalone aquaculture encountered frequent disease outbreaks. This situation had became the bottleneck of the abalone industry development. Bacterial infection was considered to be one of the most important factors that led to abalone deaths. In this paper, we combined traditional cultivated method, the method of identification of pathogenic bacteria, molecular ecology methods, and high-throughput sequencing method, to investigate the dominant species and its characteristic that led to abalone disease outbreaks, to study and analysis the relationship of the composition and dynamics in microbial community and the process of abalone disease outbreaks. The main results are as follows:1, Dominant species AP37was isolated from moribund abalones during disease outbreaks from2009to2011, and the isolate AP37exhibited the strongest virulence. After immersion challenge with106CFU/mL of AP37, abalone mortalities of0,53and67%were induced at water temperatures of20℃,24℃, and28℃, respectively. Following intramuscular injection, AP37showed a low LD50(median lethal concentration) value of2.9×102CFU/g (colony forming units per gram abalone wet body weight). For further analysis of virulence, AP37was screened for the production of extracellular factors. The results showed that various factors including presence of flagella and production of extracellular enzymes, such as lipase, phospholipase and haemolysin, could be responsible for pathogenesis. Multi-locus sequence analysis (MLSA) of concatenated sequences, including the rpoD, rctB, gyrB, toxR and pyrH genes, identified strain AP37as V. harveyi. In antibiotic susceptibility tests, strain AP37exhibited susceptibility to7antibiotics and resistance to13.2, To understand the relationship between microorganisms and the disease of farmed juvenile abalones(Haliotis discus hannai), we analyzed the composition of the microbial flora associated with juvenile abalones using both a traditional culturing method and terminal restriction fragment length polymorphism (T-RFLP) analysis. The cultivation results showed that the number of bacterial colony forming units (CFUs) in healthy juvenile abalones from a healthy pond (HA) was higher than in the other samples, and Nesterenkonia sandarakina was the dominant bacteria in the HA samples; in samples from unhealthy ponds, moribund abalones (DM) exhibited a higher bacterial diversity, and the Vibrio genus comprised up to93.8%of total bacterial CFUs; Conversely, the healthy juveniles in the diseased ponds, which were still attached to the bricks, demonstrated the lowest numbers of CFUs. Vibrio atlanticus, which was isolated from the HM (moribund abalones in healthy ponds), DA (healthy abalones in unhealthy ponds) and DM samples, comprised a large proportion of the bacterial composition of these samples. The result of T-RFLP analysis on bacterial diversity of samples was consistent with the cuLtivation result. A terminal restriction fragment length of504bp was obtained from the HM, DA and DM samples, and occupied a large proportion. This result implied that TRF of504bp is an important indicator fragment. The strain which was identified as V. atlanticus also produced TRF of504bp, so it may be associated with this abalone disease.3, Using high-throughput sequencing method, we compared the microbial structure and variation in the gut and hepatopancreas Haliotis diverscolor in different heathy status, and also in the surrounding water which held the abalone samples. Results showed that Mycoplasma, which belongs to phylum Tenericutes, distributed widely in abalone gut and hepatopancreas, and in the aquaculture water. Besides, it was the most dominant bacterial component in gut of all abalone samples, and the propotion of Mycoplasma in cDN A extracted from healthy samples was less than it in cDNA from unhealthy samples. It implied that Mycoplasma presented steadily in abalone, and the aquaculture water, and it may be the potential pathogenic bacteria. Psychrilyobacter tended to be more prevalent in heathy abalone gut samples (AH, ah) than in less-active abalones and moribound abalones (AD, ad, AM). Besides, the difference was more obvious in the corresponding cDNA samples. Prolixibacter possessed the largest proportion in AM sample than other samples. Interesting, genus Vibrio had lager proportion in heathy abalones than in less-active and moribound abalones regardless in DNA or cDNA libaraies. On the other hand, microbial communities from hepatopancreas showed distinct composition. Among them,δ-Proteobacteria was the dominant bacteria group, and one unidentified marine bacteria named SAR324had the greastes percentage in each status.