The Screen And Evaluation Of The Probiotics Of Resistance To Shrimp White Spot Syndrome Virus

Since 1960s, shrimp culture has shown to be the fastest growing section in aquaculture industry. While shrimp culture experienced a sharp drop in 1993, with the government, researchers and farmers's help, the shrimp culture of China once again on the world shrimp farming superpower position. However, in the brilliant achievements behind, there have been many problems:Serious degradation of quality such as shrimp, low immunity, poor disease resistance, frequency of shrimp disease. Although, such as improved germplasm can optimize the way of farming technology to improve the survival rate of shrimp, but shrimp enhanced the immune function is one of the most effective solution. Probiotics can enhance immune function of animals, promote animal health, enhance disease resistance of animals. The experiment was divided into four parts:The study of shrimp immune activity and disease resistance of Photobacterium sp. and Bacillus sp.:The Photobacterium sp. and Bacillus sp.:in the basic diets were isolated from the intestine of healthy shrimp (bacteria content of not less than 1011 CFU/g) of viable bacteria and ultrasonication the 1g/kg. Bacteria content was prepared not less than 10 CFU/g of four immune Feed as the immune experimental group.In this experiment, dietary Bacillus sp. of shrimp acid phosphatase (ACP), alkaline phosphatase (AKP) activity was significantly higher than the other experimental group and control group (P<0.05); 5d, 10d the dietary Bacillus sp. of shrimp immune serum catalase (CAT) activity was significantly higher (P<0.05), other groups was not significant; all the immune group vannamei's superoxide dismutase (SOD) activity was significantly higher (P<0.05); Bacillus sp. immune group shrimp lysozyme was significantly higher (P<0.05). In this experiment, Photobacterium sp. ultrasonication group of anti-WSSV immune survival (0.49±0.15) was significantly higher (P<0.05). Bacillus sp. of the immune experiment anti-WSSV survival rate (0.53±0.12) was significantly higher survival rate.The study of gastrointestinal symbiotic bacteria on shrimp anti-WSSV infection ability:With commercial diet as the control group,O. anthropi and Ensifer sp. (CFU≥1011 CFU/g) were isolated from healthy shrimp gut separation of bacteria and ultrasonication lg/kg, bacteria content was prepared not less than 108 CFU/g of the immune feed as the immune experimental group. After the immune 10d,25d and 35d, conduct the challenge test to the immune group and control group, analysis of probiotics on the different time of the shrimp anti-Disease impact force, and then provide a theoretical basis of the best action time for the probiotics by Taqman real time quantitative PCR, study the effect of the probiotic on WSSV in shrimp.In this experiment, each experimental group in the first 0-4 days after infection has no significant difference in survival (P>0.05); The immune protection of WSSV infection rate of Ensifer sp. up to 41%±2.5% in immune 35days, which is significantly higher than control group (P<0.05). The results showed that the diet containing Ensifer sp. for 35d immune can raise shrimp in a certain degree of resistance to disease.After 10d and 25d immune experiment, WSSV were detected using TaqMan MGB probe. WSSV copy number changes showed an increasing trend in the control group, increased from 103 to 1011. In other immune groups,WSSV copy number of the experimental group were in the 102-107 range.The optimization of culture conditions of Photobacterium sp. and Bacillus sp.:In this study, the optimization of culture conditions from different carbon sources, nitrogen sources of different ratio of carbon, inorganic salt component and the initial medium pH, provide the basis for theory of Photobacterium sp. and Bacillus sp. large-scale industrial production. The experimental results show Photobacterium sp. best growth conditions: initial medium pH value of 7.6; The best carbon source is glucose; By orthogonal test, the optimal culture conditions of Photobacterium sp. was initially identified as: glucose 0.5%(W/V), peptone 1.0%(W/V), beef extract 0.2%(W/V), yeast extract 0.5%(W/V); sodium citrate 1.0‰(W/V), K2HPO4 0.6‰(W/V), KH2PO4 0.1‰(W/V), MgSO4 0.8‰(W/V). Bacillus sp. best growth conditions: initial medium pH value of 6.0; The best carbon source is sucrose; By orthogonal test, the optimal culture conditions of Bacillus sp. was initially identified as:sucrose 0.5%(W/V), peptone 1.0%(W/V), beef extract 0.5%(W/V), yeast extract 0.1% (W/V) sodium citrate 0.7%o (W/V), K2HPO4 1.0‰(W/V), KH2PO4 0.4‰(W/ V),MgSO4 0.2‰(W/V).The effect of Photobacterium sp. and Bacillus sp. colonization of the gut in Fenneropenaeus chinensis:The Photobacterium sp. and Bacillus sp.:in the basic diets were isolated from the intestine of healthy shrimp (bacteria content of not less than 1011 CFU/g) of 1g/kg. Bacteria content was prepared not less than 10s CFU/g of four immune Feed as the immune experimental group.After 10d immune experiment, isolate and cultured the intestinal bacteria, and for 16S rRNA sequencing, identification the different strains of probiotics within the intestinal colonization of shrimp case.In this experiment, after 10d, the intestinal bacterium of Photobacterium sp. immune group identification were Pseudomonas sp.(1.5×108 cfu/g), compared with Pseudomonas sp.(GenBank Accession No. GU396287), identity of 100%, accounting for 80% of the total number of intestinal bacteria. The intestinal bacterium of Bacillus sp. immune group identification were Bacillus sp.(2.67 x108 cfu/g), compared with Bacillus sp. (GenBank Accession No. GU826165), identity of 100%, accounting for the total number of intestinal bacteria 76.2%. The intestinal bacterium of control group identification were Vibrio sp.(4 x108 cfu/g), compared with Vibrio sp. (GenBank Accession No. GQ406759), accounting for 70.6% of the total number of intestinal bacteria.