| At present, sea cucumber culture has become the most important aquaculture species in the north of China, and production value has achieved 200 billion yuan. However, the diseases cause massive mortality and great economic damage, so its sustainable development was serious constrained. Conventional approaches to control diseases with chemicals include use of antimicrobial drugs, pesticides, and disinfectants. Unfortunately, the abuse of such antimicrobials in disease prevention and growth promotion can lead to the evolution of resistant strains of bacteria. Sea cucumber has no specific immunity, so using bacterin to prevent disease is impossible. Therefore, the research of probiotics for aquatic animals is increasing with the demand for environment-friendly aquaculture. The study mainly includes four contents.1. By the means of morphological, physiological and biochemical tests, and 16S rDNA sequence analysis, three bacterial strains which possess antagonistic effects on common pathogenic bacteria of Apostichopus japonicus were identified. The bacterial strain TYIA-16 was identified as Bacillus subtilis, TYIA-25'was identified as Bacillus cereus, and TYIA-26 was identified as Enterococcus faecium. Among them, Enterococcus faecium could resist low temperature, even grow under 0℃.2. The experiment studied the growth speciality of experimental bacteria, and found the optimal growth condition of 4 bacillus strains. The temperature test suggested that all bacteria could resist high temperature, but not grow below 6. All experimental bacteria could decompose feedstuff of sea cucumber. Bacterial strain TYIA-15'could antagonized other strains mightily. In order to make the probiotics to the bottom of pond effectively, zeolite powder which was safety to sea cucumber was selected as carrier of Bacillus spores, and the results showed its adsorbability rate to experimental bacteria exceed 95% and could carry spores to the bottom of tank effectively. The spores absorbed by zeolite powder could maintain activity for long time after dried under 45℃~55℃, and livability was 70%~80% after half year when original number of bacteria was 1.5×109cfu/g .3. The experiment was conducted to evaluate the effects of single and compound Bacillus preparation on growth, digestive enzyme activities of intestine, immunity and intestinal bacteria of Apostichopus japonicus. Sea cucumbers were fed three different diets: control (nonsupplemented basic diet) or basic diet supplemented with 1.5×107 cfu/g Bacillus subtilis or with 0.5×107 cfu/g Bacillus subtilis and 0.5×107CFU/g Bacillus atrophaeus and 0.5×107 cfu/g Bacillus cereus respectively. The results showed: At first stage (water temperature was 14℃~16℃), sea cucumber fed single or compound Bacillus showed relatively higher growth rate and activity of protease compared with those fed control diet (P<0.05). However, no significant difference about growth rate and activity of protease were observed at second stage (water temperature was 12~14℃). However, diet with supplementation of Bacillus could decrease the activity of amylase in intestine. The experiment suggested that the Bacillus could significantly improve immune indicators of sea cucumber, such as acid phosphatase (ACP) activity and alkaline phosphatase (AKP) activity in coelomic fluid (P<0.05),but no influence was observed on superoxide dismutase activity (SOD). The challenge experiment showed that sea cucumber fed the diet with Bacillus had lower cumulative mortality compared with the control and significant difference was observed between the control and compound Bacillus groups. The experiment also suggested Bacillus subtilis could influence the number of total bacteria counts (TBC) and Vibrio bacteria counts (VBC) in intestine. And, the experiment detected the influence of single Bacillus preparation on sea cucumber culture water quality. The results showed Bacillus had no influence on pH, and dissolved oxygen (DO) decreased in experimental groups on a certain extent. Bacterial strains of TYIA-15'and TYIA-16 could decrease the increment of NH4+-N. The number of Bacillus rised at first, and then descended after achieving zenith in experimental groups, no Bacillus was observed in control group.4. One dominant bacterial strain was isolated from diseased sea cucumbers which suffered'skin ulcer syndrome', and artificial infection test proved that it was the causative pathogen associated with the disease. The bacterium was identified as Bacillus cereus.
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