| Frequent outbreak of bacterial diseases is a major restraint for the development of marine aquaculture. Nowadays, the use of probiotics and immunostimulants in aquaculture provides good prospect for sustainable development. The first aim of this study is to find out the potential relationship between the microflora and disease outbreaking through comparing the environmental microflora of diseased turbots with that of healthy turbot, in which we can screen out any isolates that have the potential to be probiotics. Secondly, on the basis of probiotics candidates, more tests, such as antagonism and pathogenicity, are performed to determine whether they are suitable to be applied into aquatic animals, followed by identification of the isolates. Finally, a fast detecting method based on PCR technique is established to facilitate the monitoring of probiotics in the environment. Moreover, possible mechanism is speculated through challenging Artemia franciscana protected by DnaK which is overexpressed by YS2 to Vibrio campbellii, which expands the fundamental theory of immunostimulatants.During June 2006 and April 2007, samplings were done for 6 disease outbreaks of turbot, one of which was explicitly studied. The symptoms were gnathoschisis and liver atrophy. Futher works were casted on water quality, total bacterial count, total viable count, total heterotrophic count and total vibrio count. After comparison, both groups had normal water quality, however, the total heterotrophic count of water and intestine in diseased group was lower than that of healthy group, while vibrio count was much higher. PCR-RFLP was used for clustering the microflora, and the results agreed with that of plating method, which showed richer diversity in healthy group than that of diseased group.Probiotics have been well applied in aquaculture. In this study, an antagonistic strain JG1 was screened out by inhibiting test on 234 isolates using V. harveyi SF1 as the indicator strain, which was pathogenic to marine fish. The result of inhibitory spectrum test showed that JG1 was antagonistic to various species, including Vibrio spp., Aeromonas spp., and Photobacteria, especially Vibrio spp. The strongest inhibitory effect showed on V. anguillarum VIB72, Aeromonas salmonicida AS1 and A. hydrophila AHK1 and AHU1. The result of identification and classification of JG1 showed that JG1 was Gram-negative, polar flagellum and golden-yellow pigmented. Combining with 16S rDNA analysis and phenotypic traits, JG1 was finally identified as Pseudoaltermonas flavipulchra. JG1 was isolated from rearing water of healthy turbots. This is the first time to report Pseudoalteromonas flavipulchra that had anagonistic effect to several bacterial pathogens and showed such good prospect of probiotic protential. Later on, pathogenicity of JG1 was tested on zebra fish, scallop, clam and mantis shrimp and showed none pathogenic effect. Challenge test in liquid culture between JG1 and V. campbelliiVC demonstrated that JG1 could inhibit VC. In vivo colonizing ability of JG1 was tested using gnotobiotic Artemia system and gives weak performance in the 7 days run. Therefore, JG1 might work more efficiently by means of adding into water environment. Moreover, a pair of species-specific primers JG153f and JG864r was designed for P. flavipulchra and a fast detecting technique was established based on PCR amplification of gyrB gene.Immunostimulant is another widely-used way for disease control in aquaculture. DnaK is a kind of Hsps, which has immunostimulant effect. The protective effects of YS2, which overexpresses DnaK, against VC in axenic, open and large volume open culture were tested using gnotobiotic Artemia and showed positive results. The variation of environmental microflora of Artemia was studied by comparing DGGE profiles. It turned out to be no significant difference, which implies that the protective effect of DnaK may not be related to the variation of inner microflora of host, but may be related to the enhancement of host immune system.
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