Screening And Studying The Mechanism Of Action Of The Disease-resistant Microorganisms Of Shrimp

Probiotics are recognized as safe and effective shrimp immune-enhancingsubstances, and are one of the main strategies to control shrimp diseases, In current,the lack of screening and applied research on new and efficient functional strains. Inthe study, isolated several strains of predominant bacteria from healthy shrimpdigestive tract and the culture environment, to examine the anti-WSSV infectioneffect on the basis of further analysis of the Bacillus firums PC024/Vibrioalginolyticus LV463added to the shrimp feed and study farmed shrimp immuneactivity, the expression levels of immune-related molecules, as well as theproliferation rate of WSSV in the shrimp body. This research has made Bacillusfirums/Vibrio alginolyticus with the use can significantly improve the ability ofanti-WSSV infection Litopenaeus vannamei; disease-resistant strains by increasingthe level of shrimp immunity and disease resistance gene expression to slow down thevirus in shrimp body proliferation, which play important results of the anti-virusprotection role. Strains screened in this study is expected to feed additives used inshrimp aquaculture production, and become one of the effective way of preventionand treatment of shrimp white spot disease ecology. The main research content andresults of the following aspects:1. Comparison and apply of the main identification methods of marinebacteria.To develop more accurate, efficient and convenient method of bacterialidentification, This experiment utilize16S rRNA sequence analysis, bacterial fattyacids by gas chromatography, ATB microbial identification system, Biolog systematicidentification of four methods for identification of16strains of marine bacteria.Results showed16S rRNA sequence analysis and bacterial fatty acids by gaschromatography which is fewer identification steps and cost less than ATB and Biologsystematic identification. Compared with16S rRNA sequence analysis and ATBsystem, bacterial fatty acids and Biolog systematic identification is much accurate inidentifying species. In practice, According to their own experimental requirements and equipment conditions to select the appropriate method for bacteria identification,and sometimes in order to get more accurate results, we need require the identificationof several methods used in combination.2. Identification and biological characteristics analysis of a marine Bacillus.A strain PC024was isolated from intestinal of healthy Fenneropenaeus chinensis.Morphology characteristics and staining property shows the strain was Gram-positive,side spores and had flagella. Cultured for24h on2216E solid medium, the colony wasround, slightly raised middle, a diameter of about2mm, the cell was rod-shaped. Itshowed highest identity to Bacillus firmus by using fatty acid analysis and Biologsystematic identification. Based on16S rRNA sequence analysis the strain is100%identical to several species in Bacillus genus and the phylogenetic tree revealed that itis most close to Bacillus firmus. Taken together of16S rRNA sequence analysis, ATBMicrobial identification system, BIOLOG Systematic Identification, and bacterialfatty acids by gas chromatography, four methods of identification results, the isolatewas identified as Bacillus firmus.3. The effect of four shrimp symbiotic single or multiple bacteria onLitopenaeus vannamei resistance to WSSV infection. Bacillus firmus PC024,Bacillus cereus PC465, Vibrio alginolyticus LV463and PC773, Added single orcombination bacteria (including live/inactivated) and wrapped in the feed.Shrimps were challenged with WSSV after20days of feeding these experimentaldiets. The cumulative mortality of shrimp was recorded during the infectionexperimental period. Challenge experiment shows that B.firmus/V.alginolyticuscomposite group (B/V group) of cumulative mortality rate was significantly lowerthan the other experimental group and control group (P <0.05). Based on these results,it is sugggested that feeding shrimp intestine probiotics significantly improve theability of antiviral infection of L.vannamei, Especially feeding with B.firmus andV.alginolyticus.4. A feeding experiment of35days was conducted to investigate the effects ofadding probiotics in the feed on non-specofic immune enzyme activites ofLitopenaeus vannamei.Two experimental diets (Bacillus firmus alone, Bacillusfirmus/inactivated Vibrio alginolyticus) and the basal diet. Six shrimps were sampled every5days and some humoral immune parameters of serum and the hepatopancreasof shrimp were evaluated. Results showed that: During the immunization andinfection WSSV，The activity of acid phosphatase (ACP), superoxide dismutase(SOD), lysozyme (LZM), catalase (POD) and phenol oxidase (PO) and proteincontent of serum and the hepatopancreas in each experimental group than the controlgroup increased significantly (P <0.05). The results indicate: Treatment with addingprobiotic can enhance the shrimp immune function, thereby enhancing the ability ofthe shrimp anti-WSSV infection.5. Effects of adding Bacillus firums/Vibrio alginolyticus in the feed onnon-specific immune factors of hemolymph of Litopenaeus vannamei. The mRNArelative expression level of prophenoloxidase (proPO), superoxide dismutase (SOD),lysozyme(LZM), Heat shock protein70(Hsp70), LPS-β-1,3-glucan bindingprotein(LGBP), Signal transduction and activator of transcription(STAT) andβ-1,3-glucan binding protein(βGBP) of hemolymph of L.vannamei were detected.Theresults indicated that the relative expression of seven genes were significantly higherthan the control group during the immunatizion (P<0.05); However, during theinfection WSSV, the relative expression of seven immune factors demonstrated thatfirst up-regulation to maximum and downward. The results suggest that treatmentwith adding probiotic can activate the expression of immune factors of hemolymph,thereby enhancing the ability of the shrimp anti-WSSV infection.6. Effects of adding Bacillus firums/Vibrio alginolyticus in the feed onnon-specific immune factors expression of lymphoid of Litopenaeus vannamei.Analyzed changes in relative expression levels of seven immune factors during theimmunization and infection WSSV. The mRNA relative expression level ofprophenoloxidase (proPO), superoxide dismutase (SOD), lysozyme (LZM), Heatshock protein70(Hsp70), LPS-β-1,3-glucan binding protein(LGBP), Signaltransduction and activator of transcription(STAT) and β-1,3-glucan bindingprotein(βGBP) of hemolymph of L.vannamei were detected. The results indicated thatthe relative expression of seven immune factors were significantly higher than thecontrol group during the immunization (P<0.05); However, During the infectionWSSV, the relative expression of seven immune factors demonstrated that first up-regulation to maximum and downward. Results suggest that probiotic supplementcan activate the expression of the shrimp hemolymph of immune-related molecules,thereby enhancing the ability of the shrimp anti-WSSV infection.7. Effects of adding probiotics in the feed on WSSV virus proliferation rateon Litopenaeus vannamei infected with WSSV. Preparation of feed and shrimpfarming with the same Chapter3, WSSV virus copy number in muscle and gill tissuesof shrimps at different times were detected by quantitative PCR. The results showedthat WSSV viral copy number in treatment with probiotics at each sampling timepoint were significantly lower than the control group (P <0.05). The results suggestthat probiotic supplement can reduce the shrimp infected with WSSV virus in vivoproliferation rates, thereby reducing the number of deaths of the shrimp.