Screening And Studies Of Antagonistic Bacillus Against Streptococcus Agalactiae

Streptococcus agalactiae is one of the main pathogenic bacteria of Streptococcus disease in Tilapia, in recent years, it is the main cause of the outbreak of Tilapia Streptococcus disease in main aquaculture areas of Guangdong and Hainan province. Antibiotic drugs were usully used to treat Streptococcus agalactiae, however, the antibiotic therapy tend to exist problems such as drug residues and resistance, it is urgent to explore the biological methods to control the Tilapia Streptococcus disease, this paper is intended to isolate a strain of bacillus with good effect of antagonism Streptococcus, have certain gastrointestinal tolerability and can use safely, besides study the influence of the strain on the growth and serum indexes of Tilapia, and on the protection rate of Streptococcus. The main results are as followed:1.Isolating of antagonistic bacillusIn this study we focused on isolating a strain of bacillus with good effect of antagonism Streptococcus by oxford cup method, then using 16 s rDNA sequences to identificate the candidate strains, then through artificial simulation of gastrointestinal tolerance test and evaluate the resistance of each candidate strain, and the safety test of Tilapia was determined by blood plate and intraperitoneal injection. The results show that:we isolated four strains of bacillus with good effect of antagonism Streptococcus, after tolerability and safety testing, at lase, we determined to use the Bacillus sp. J4 for subsequent experiments, in the artificial gastric fluid when adjust pH to 4,3,2 and through 4 hours cultivate, the relative survival rates was 94.53%, 84.53%,43.24%, in artificial intestinal fluid and in the bile salt concentration of 0.4% through 4 hours cultivate, the relative survival rate of was 97.48% and 91.88%, the Bacillus sp. J4 have a good resistance to the gastrointestinal, at the same time, the security test confirmed that it doesn't produce the hemolysin, and the concentration (up to 1 X 109cfu/mL) of J4 on Tilapia is safety.2.The Bacillus sp. J4 influence on the growth performance and serum indexes of TilapiaIn this study, we selected 360 tails healthy Tilapias and the weight about 20g, randomly divided into four groups, respectively BO, B1, B2, B3 group, each group set up 3 repeat, and each repeat 30 tails fish, BO group were fed with basal diet, group B1, B2 and B3 were fed based diet supplemented the Bacillus sp. J4 with 1×10^6, 1×10^7 and 1×10^8cfu/g diets respectively, test period is 56 days, measure the growth performance of Tilapia, at the same time, take samples of Tilapia serum every 14 days, and determination of blood indexes. The results show that:compared to group BO, B2 and B3 group can significantly improve the Tilapia's WGR and SGR (P<0.05), and significantly reduce FCR of Tilapia (P<0.05), but the effect of HSI and CF was not significant (P>0.05), at the same time,it's not significant of WGR? SGR and FCR in group B1, B2 and B3 (P>0.05), and start from 14 days, the group B2, B3 of Tilapia lysozyme(LSZ) activity were significantly higher than group BO (P<0.05), and at 42 days,28 days respectively to achieve their maximum activity; as superoxide dismutase (SOD) activity in serum, from 14 days to the end of the trial, group B2 and B3 were significantly higher than the control group BO (P<0.05), and at 42 days,28 days respectively to achieve their maximum activity; at the beginning of 28 days, the serum alkaline phosphatase (AKP) activity in the group B2 and the B3 were significantly higher than the control group BO (P<0.05), and in 56 days the activity increased with time; and start from 14 days, the serum levels of malondialdehyde (MDA) in group B2 and B3 were significantly lower than those in control group BO (P<0.05), and all down to the minimum value in 42 days; the serum catalase (CAT) activity of group B2 and B3 were not significantly different from the control group BO (P<0.05); in the serum index, there was no significant difference at all time between the group B2 and B3 (P> 0.05). So the group B2 and B3 all can increased the non-specific immunity and antioxidant ability of Tilapia, and enhanced the ability of disease resistance. At all time points, the group B1's serum LSZ, AKP, SOD and CAT activity, MDA content had no significant difference compared with the control group BO (P> 0.05).3.The Bacillus sp. J4 influence on the protection rate of Tilapia StreptococcusIn this study, after 56 days feed, we moved the group BO, B1, B2 and B3 of Tilapia to the aquarium, each group set up 3 repeat, each repeat 10 tails Tilapia, Then use the optimal concentration of Streptococcus agalactiae for Tilapia intraperitoneal injection and observed 14 days, and calculate the relative protection rate. The results show that:compared with the control group B0, the group B2 and B3 significantly reduced the average mortality (P<0.05), the relative protection rate was 66.67% and 75% respectively, there was no significant difference in the average mortality between the B2 and B3 groups (P>0.05), but Tilapia's death time of group B3 was delayed compared with the B2 group, there was no significant difference between the group B1 and the control group BO (P>0.05), the relative protection rate was only 8.33%.