| In order to reduce utility of antibiotics for control of Streptococcus agalactiate, which causes streptococcosis of tilapia and results in great economic losses, the rapid detection, the rapid detection(indirect competetive ELISA) was established for diagnosis of the pathogen in early stage of the bacterial infection. Subsequentily, the Chinese herbal medicines compound was used in time to treat the streptococcosis in early stage.The S. agalactiate suspension was used as the antigen to immunize rabbit, then the anti-S. agalactiate polyclonal antibody was prepared, the antibody titer determined by indirect ELISA was 2.048×106 cfu·mL-1. The indirect competitive enzyme-linked immunosorbent assay(ELISA) was established for detection of S. agalactiate from Oreochromis niloticus. The optimal concentration of the coating antigen was 10 μg·mL-1, the working concentration of anti-S. agalactiate antibody was 1: 3200, then the gression equation was y=-17.138x+162.3, R2=0.9979, and the most suitable detection range was 9.6567×103~6.25×107 cfu·m L-1. The variation coeffieients of intra-assay and inter-assay were 5.02% and 7.38%, respectively, and less than 10%. This indicated that the indirect competetive ELISA can be used for detection of S. agalactiate at early stage of the bacterial infection in tilapia.The 7 species among the 230 Chinese herbal medicines showed well antibacterial efficacy, that is, Gall chinensis, Chebulae immaturus, Terminalia chebula, Spatholobi caulis, Salvia miltiorrhiza, Cleistocalyx operculatus, Punica granatum. Among the 7 species, G. chinensis and C. immaturus were the best anti-bacterial medicines with the minimal inhibitory concentration(MIC) and the minimal bactericidal concentration(MBC) of 12.5 mg·mL-1, T. chebula was the second with MIC and MBC of 50 mg·mL-1, S. Caulis, S. miltiorrhiza, C. operculatus, and P. granatum ranked the third with MIC and MBC of 100 mg·m L-1. Several Chinese herbal medicines had synergistic effect when they were combined together to inhibit S. agalactiate. Three compounds showed significantly antibacterial efficacy against S. agalactiate and others pathogens of tilapia, such as S. iniae, Aeromonas sobria, A. hydrophila, Vibrio vulnificus, Edwardsiella tarda, A. veronii. The three Chinese herbal medicine compounds were as follows: compound 4 was consisted of G. chinensis: C. immaturus: P. granatum: S. Caulis: C. operculatus = 2: 4: 2: 1: 1; Compound 12 was consisted of G. chinensis: T. chebula: P. granatum: S. Caulis: C. operculatus = 1: 4: 3: 1: 1; and Compound 16 was consisted of G. chinensis: C. immaturus: T. chebula: P. granatum = 2: 4: 2: 2.The LC50 values at 24, 48 and 96 h of compound 4 were 927.668, 854.577 and 626.512 mg·m L-1. The safe concentration(SC) value of compound 4 was 217.56 mg·mL-1. The LC50 values at 24, 48 and 96 h of compound 12 were 2451.15, 1750.71 and 1164.57 mg·mL-1. The safe concentration(SC) value of compound 12 was 267.93 mg·m L-1. The LC50 values at 24, 48 and 96 h of compound 16 were 1062.25, 913.00 and 676.21 mg·m L-1. The safe concentration(SC) value of compound 12 was 158.483 mg·mL-1.The challenge tests with S. agalactiae showed that survival rate of infected tilapia was 80% in the treatment diet containing 4% compound 4, 73.3% in the diet containing 2% compound 4, and 66.7% in the diet containing 1% compound 4, while the survival rate was 0 in control. In the same challenge tests the survival rates of infected tilapia treated with diets containing compound 12 at 4%, 2%, and 1% were 93.3%, 73.3%, and 70%, respectively, but the survival rate was 0 in control. Meanwhile, the survival rates of infected tilapia treated with diets containing compound 16 at 4%, 2%, and 1% were 80%, 60%, and 40%, respectively, when the survival rate was 0 in control.The Chinese herbal compound 12 in tilapia diet could not only promote the growth of trial tilapia,but also stimulate proliferation of blood cells(especially immune-relative blood cells) as well as increase the activity of SOD and LSZ enzymes of trial tilapia. |
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