Screening And Evalution Of Bacillus Spp. Against Vibrio Disease In Shrimp Aquaculture

Part 1: In order to study the flora structure in the intestinal tract and carrying the virus condition of the different aquatic farm shrimp, we used conventional method of bacteria isolation, cultivation, purification and bacteria 16 Sr DNA sequences analysis to analyze the total number of culturable bacteria, the species and numbers of dominant bacteria and we used Nested polymerase chain reaction(Nested PCR) method to detect shrimp virus in Litopenaeus vannamei and Fenneorpenaeus Chinensis cultivated in different farm of Jiangsu, Shandong and Republic of Korea. The results shown that the total number of culturable bacteria of Litopenaeus vannamei and Fenneorpenaeus chinensis is between 105 and 109 CFU/g, and identificated the dominant bacteria isolated from the intestinal tract to genus(species). The results shown that the dominant bacterial is respectively belong to Lactococcus sp, Vibrio sp, Bacillus sp, Photobacterium sp, Shewanella sp, Arthrobacter sp and Microbacterium sp. Litopenaeus vannamei and Fenneorpenaeus chinensis samples are tested positive for WSSV, 6 batches of shrimp samples which is tested positive WSSV, are isolated Vibrio sp from intestinal tract, occupied of culturable bacterial was 33%-93.58%, 2 batches of shrimp samples which is tested positive WSSV, are isolated Shewanella sp, from intestinal tract, occupied of culturable bacterial was 21.67%-34.21%, 4 batches of shrimp samples which is tested positive WSSV, are isolated Photobacterium sp, from intestinal tract, occupied of culturable bacterial was 21.03%-66.83%.Part 2: Adopt the filter method and cocultivation method screened out 4 strains of Bacillus bacteria have obvious antagonism effect to the Vibrio harveyi. The serial number of Bacillus bacteria are 2012070203, 2011120601, 20110910012 and 2011120601, respectively, which were kept and identificated by the our laboratory.The 16 S r DNA of strains sequence analysis, compared with those of relative strains and construct phylogenetic tree. Combined with phenotypic characteristics of bacteria and 16 Sr DNA sequence analysis, strains 2012070203, 2011120601, 20110910012 and 2011120601 were identified as Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium.The 4 strains identified Bacillus bacteria were added to the feed as 4 kinds of experiment feeding.After feeding Litopenaeus vannamei 21 d, the shrimp was injected Vibrio harveyi with 106 cfu/ml and observed 14 d, the accumulative total mortality of the control group was 100%, the experimental group of Bacillus cereus and Bacillus megaterium’s cumulative mortality rates were 91.11% and 95.55% respectively, there is no significant difference compare to control group(P > 0.05). The experimental group of Bacillus licheniformis and Bacillus pumilus’ s the cumulative mortality rate were 77.78% and 84.44% respectively, significantly lower than that of control group(P < 0.05). Part 3: Experiment shrimp and infection are the same as chapter 3. Litopenaeus vannamei was feed two different diets: Control group(commercial fodder) and experimental group(adding the 1.0×108cfu/ml Bacillus licheniformis into the commercial fodder). Shrimps were sampled every 7 days to analysis the intestinal total number of bacteria and vibrio number changes. After injected Vibrio harveyi, at the time of 0 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 72 h and 7 d sampling, analysis the effects of adding Bacillus licheniformis in the feed on intestinal bacterial and non-specific immune gene expression of Litopenaeus vannamei. The result showed that the experimental group can significantly reduce shrimp intestinal vibrio number compared with control group(P<0.05). After affected the V. harveyi, the experimental group’s lysozyme m RNA relative expression quantity in 12 h, 18 h, 24 h, 36 h, 48 h, 72 h of were significantly higher than that of control group(P<0.05). After affected the V. harveyi, the experimental group’s Toll receptor m RNA relative expression quantity in 6h、12h、18h、24h、36h、48h、72h、7d were significantly higher than that of control maximum levels at 18h-24 h. The experimental result indicates: adding probiotics in the feed can effectively improve the ability of Litopenaeus vannamei to resist the V. parahaemolytics’ s infection, the improvement of this ability could be through improve the disease resistance related gene expression. group(P<0.05).Part 4: Litopenaeus vannamei was feed four different diets: Control group(commercial fodder) and experimental group(feed supplemented with Bacillus licheniformis; or with Bacillus subtilis and with mixed diet comprising of Bacillus licheniformis +Bacillus subtilis at the ratio of 1:1), final concentration of probiotics in experimental feeding was 107cfu/g. After feeding Litopenaeus vannamei 21 d, the shrimp was injected V. parahaemolytics with 1×106cfu/ml and observed 14 d, the accumulative total mortality of the control group was 100%, the cumulative mortality rate of Bacillus licheniformis group were 73.34%, the cumulative mortality rate of Bacillus subtilis group were 77.78%, the cumulative mortality rate of Bacillus licheniformis +Bacillus subtilis group were 68.89%, the cumulative mortality of three experiment group all lower than control group(P<0.05). The results showed that adding the probiotics in the feed can improve Litopenaeus vannamei ability to resist V. parahaemolytics infection, and the effect of composite probiotic is superior to single probiotics.Part 5: The experiment shrimp and infection are the same as chapter 5. Litopenaeus vannamei was feed two different diets: Control group(commercial fodder) and experimental group(feed supplemented with mixed diet comprising of Bacillus licheniformis +Bacillus subtilis at the ratio of 1:1). After injected Vibrio parahaemolytics, at the time of 0 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 72 h and 7d sampling, using real time PCR method study the Litopenaeus vannamei non-specific immune gene expression of Bacillus licheniformis + Bacillus subtilis experimental group, which is the highest protection group in the Vibrio parahaemolytics infection. After affected the V. parahaemolytics, the relative expressions of IMD, penaiedin 3a, pro PO, LZM and Crustin were obviously up-regulated in hemolymph of shrimp fed diets supplemented with the compound probiotics(Bacillus licheniformis + Bacillus subtilis) compared to the control during the immunization. In hemolymph, the relative expression of IMD, penaiedin 3a, pro PO, LZM and Crustin were increased to the...