The Effects Of Dietary Supplement Of Probiotics On The Disease-resistance And Intestinal Microbial Composition Of Litopenaeus Vannamei

Part1: In order to detect rapidly Bacillus cereus and Vibrio alginolyticus of the strain fromshrimp, the antisera were produced from the specific pathogen free (SPF) of New Zealand whiterabbits which were immunized with V. alginolyticus and B. cereus, and the titer of the polyclonalantibody were higher than1:2000. The antisera were used as primary antibody and the goatanti-rabbit IgG-HRP was used as secondary antibody in indirect ELISA which was applied forrapid detection of V. alginolyticus and B. cereus. The optimum diluted concentrations of theantiserum were determined to be1:10000in indirect ELISA. The optimum coated concentrationof the antigen was determined to be106CFU/ml. The goat anti-rabbit IgG-HRP was determined tobe1:1000. The sensitivity of the serum was tested, and the lowest concentration of two strains ofmarine bacterium could be detected was106CFU/ml. The anti-Bacillus cereus serum has crossreaction only with B. thuringiensis. Cross reaction of the anti-Vibrio alginolyticus serum with thestrains of other bacterial species in the genus of Vibrio were detected, and all the results werenegative. Our laboratory have purificated109strains of marine bacteria from9sample batches,which include Penaeus monodon, Fenneropenaeus chinensis and Penaeus vanmamei. With theestablished indirect ELISA method, the department of marine bacteria were been detected rapidly.Six strains of Vibrio were positive, and all of Bacillus were negative.Part2: In order to research the effect of Photobacterium damselae, Bacillus cereus, Vibrioalginolyticus and Bacillus firmus, on anti-WSSV infection and intestinal microflora ofLitopenaeus vannamei, Probiotics were added to basic feed respectively which concentration ofviable bacteria was108CFU/g. The test was divided into six group(A-F group), which theexperimental group (A-F group) was fed of the immune feed everyday while the challenge controlgroup (G group)and the blank control group(H group) were fed of basic feed. Shrimps werechallenged with white spot syndrome virus (WSSV) after three weeks of feeding experiment. Thewhole experiment was end after35days. Result of challenge experiment showed that shrimps ofexperimental group were all died, while immune protective rate of the experimental group were46.4%,21.74%,35.29%,19.71%,14.08%,21.25%, and survival rate of the blank control groupwas91.67%.The immune protective rate of A group was highest. The test showed thatPhotobacterium damselae could improve significantly shrimps’ disease resistance of theexperimental group. We should study the reason of the improvement of disease resistance further.Part3: In order to research the effect of Photobacterium damselae on anti-WSSV infectionand intestinal microflora of Litopenaeus vannamei, Photobacterium damselae (number PC463)was added to basic feed which concentration of viable bacteria was108CFU/g. Shrimps weredivided into three groups, which the experimental group was fed of the immune feed everyday while the challenge control group and the blank control group were fed of basic feed. Shrimpswere challenged with white spot syndrome virus (WSSV) after three weeks of feeding experiment.The whole experiment was end after35days. Result of challenge experiment showed that shrimpsof experimental group were all died, while cumulative mortality of the experimental group was53.56%and survival rate of the blank control group was91.67%. Sampled at Day0,10,20,28,35,the method of high throughput sequencing of16S rDNA gene’s V4region was used to determinediversity and composition of bacterial communities in each of these samples. Experiment showed:(1)The intestinal microflora of Litopenaeus vannamei were mainly with Proteobacteria andBacteroidetes, while Verrucomicrobia and Planctomycetes were at lower rate. Proteobacteria wasmainly with Vibrio, Photobacterium and Pseudoalteromonas. Bacteroidete was mainly withFlavobacterium. The proportion of Proteobacteria was becoming lower with the growth of shrimp,while Bacteroidetes was becoming higher.(2) The proportion of Proteobacteria was becominghigher in the intestinal microflora when shrimp was challenged with WSSV, and the proportion ofVibrio was becoming lower significantly.(3)In intestinal microflora of the experimental group’shrimps, the proportion of Proteobacteria was higher than the control group, while Bacteroideteswas lower than the control group. This result showed Photobacterium damselae could reduce therate of Vibrio in the intestinal microflora.(4)The proportion of Proteobacteria was0.8%inintestinal microflora of the experimental group’ shrimps, while rate of the control group’ was0.2%. The range of Proteobacteria was small in the experimental group’ shrimps.(5)Photobacterium damselae couldn’t protect shrimp from infection with WSSV, while could play aprotective immune effects and immune protective rate was46.4%. The survival was not detectedwith WSSV. Through this experiment, we could realize that intestinal microflora of healthyshrimps, the effect for shrimps challenged with WSSV and shrimps of the experimental group.Photobacterium damselae could improve significantly shrimps’ disease resistance of theexperimental group. With analysis the reason of the improvement of disease resistance, we couldrealize fishery probiotics and use it exactly.