Analysis Of Bacterial Communities From Turbot(Scophthalmus Maximus) Aquaculture System And Construction Of DNA Microarray Detection System For Aquaculture Pathogenic Bacteria

In order to construct robust surveillance and alert system, the distribution of pathogenic bacteria in marine environment and the bacterial communities in the gastrointestinal tract of larval turbot and aquaculture system was studied based on culture-dependent method and molecular biological technology, such as 16 S r RNA gene meta-genome sequencing and phylogenic gene marker screening. Beside, with the development of intensivism aquaculture mode and prevalence of aquatic disease, a DNA microarray-based detection system whose main features include high specificity, parallelize sample processing and cost-effective was optimized and utilized to explore the feasibility for field practice.In this study, bacterial pathogens which responded to diseased larval turbot with syndrome of abdominal distension were surveyed from September 2013 to December 2014. As a result, multiple dominant strains were isolated from three batches of diseased turbot larvae. Only one strain caused the same syndrome with the diseased samples by the injection challenge test and it was identified as Pseudomonas aeruginosa by blast analysis of similarity of 16 S r RNA gene and gyr B gene sequence. Even through with the relative high LD50 concentration of 3.6×108 CFU·m L-1, it is the first reported strain which could infect larval turbot.The distribution of potential pathogens in marine aquaculture was analyzed with the isolation of hemolytic vibrios. 19 strains of hemolytic vibrios were isolated from the coastal environment of Shandong peninsula. Among it, vibrios belong to Splendidus clade(11/19) and Harveyi clade(6/19) were most frequently isolated. Many species of reported aquatic pathogenic vibrios, like V. harveyi, V. alginolyticus and V. owensii was found exist in the aquatic samples. Through infection test, two strains of V. owensii and one strain of V. harveyi showed virulence to larval turbot. And all of the three strains contained the homologic hemolysin encoded gene vhh B of V. harveyi. The result indicated that the existed risk of potential pathogen to aquaculture. In addition, ferric uptake regulation gene(fur) showed high resolution in identification of Vibrionaceae species in contrast to 16 S r RNA gene.The taxonomic profiling of gastrointestinal tract of larval turbot and aquaculture system was acquired based on 16 S r RNA gene meta-genome sequencing technology. The diversity of bacterial communities was higher in water than in fish and Pseudoalteromonas, unclassified Alteromonadales, Pseudomonas, Vibrio and Aliivibrio shared the similar proportion in waters. While Vibrio was the most abundant genus in larvae and Pseudomonas was the sub-dominant genus.Depend on upon results and related data, a DNA microarray platform which based on multiplex-PCR technology was optimized and constructed to detect aquaculture pathogenic bacteria. The finalized microarray included 31 kind of probes which could target 9 species of pathogenic bacteria and no cross hybridization was observed. The constructed microarray also showed high sensitivity in detecting bacteria from diseased turbot sample in contrast to PCR method. And optimized system contained the use of 1 μmol·L-1 oligo probe in a mixed spotting buffer of 0.1 mol·L-1 Na2HPO4 and 25% DMSO, 150 pmol Cy3 labelled random primer performed better through hybridizition for 2 hours at 60 ℃. The detection limit of optimized DNA microarray was 10 fg of genome DNA of V. alginolyticus and it was 2 logs higher than PCR method.