|
To investigate how the Strepstococcus invasive fishes variously, 9 isolates are employed in this research, including 5 Strepstococcus iniae and 4 Strepstococcus dysgalactiae. The research focuses on the extracellular products produced by these isolates, analyzing the characteristics from several aspects and using the products as a kind of immunogen in order to check out its immunoeffects on Japanese flounder（Paralichthys olivaceus）.First, physiological and biochemical characteristic analysis and comparison of 9 isolates are carried out, respectively, by means of Rapid STREP 32 E and Columbia sheep blood agar. The results indicate that physiological and biochemical characteristics of 9 isolates vary, which infers different composition of enzymes secreated. Moreover,α,βandγ, three haemolytic types all exist. Then the pathogenicity is detected by challenging flounders. The LD50 of Strepstococcus iniae, NUF849, NUF812, NUF701, NUF693 and NUF633 is 3.7×105 CFU·mL-1, 1.0×106 CFU·mL-1, 4.6×107 CFU·mL-1, 1.4×107 CFU·mL-1 and 2.2×107 CFU·mL-1, respectively. However, low mortality is found when flounders are challenged by large quantities of Strepstococcus dysgalactiae, SD, L2,WZ and HT. NUF849 is the most virulent isolates. Therefore, the 9 isolates are suitable and chosen for the following reseach.Taken NUF849 as an example, preparation of extracellular products is optimized from aspects of culture time and methods of concentration. The final method performs as follow: activated isolates are incubated in BHI broth for 24 h at 28℃, and then a aliquot of suspension is spread on BHI agar overlaid with a sterilized cellophane sheet to be incubated for another 60 h. Bacteria and ECPs are harvested by washing the cellophane sheet with PBS then centrifuged（8000 g,20 min,4℃）. The supernatants are filtered with a 0.22μm filter and dialyzed with diluted water at 4℃, followed by lyopilization and resuspension . Extracellular products of 9 isolates are prepared with the optimized method above, respectively, which are analyzed by SDS-PAGE and Western-blot to clarify the composition of protein and their antigenicity. The results suggest that extracellular products vary among isolates relating to species, sources and virulence, in the presence of MW and abundance. And they can be recognized by the anti-serum from survival flounders, indicating their antigenicity. 9 isolates share a few bands, with MW of 14, 25, 27, 37, 45, 50, 60, 92 kDa and etc. The avirulunt isolates, NUF693 and NUF633, can strongly act with negative serum at MW 60 kDa.Under several conditions, NUF849 is incubated for growth curve and extracellular products. The results of the comparison showed that temperature, salinity, air condition, concentration of glucose and iron can give signals to bacteria to regulate the composition of extracellular products. The changes bands with MW of 25, 27, 37, 42, 45 and 60 kDa, which are sensitive to environmental factors are also antigens. These proteins can be candidate for vaccine. The growth condition alters too, under the environmental changes. We also found the suitable condition for growth of NUF849. NUF849 hold the best growth condition in the broth consisited of 5‰NaCl and glucose at 28℃,with appropriate concentration of serum from flounder.Flounders are vaccinated by FKC, ECPs and FKC+ECPs, 3 kinds of immunogen of NUF849, and then challenged by NUF849 or NUF812. Serum is collected every 7 days and antibody titres, phagocytic activities and SOD, POD and ACP activities are detected. Meantime, FKC, ECPs and FKC+ECPs induced the relative survival of 60%, 40% and 90% （NUF849） and 50%, 30% and 80% （NUF812） after challenge, respectively. The highest immune ability was observed in the flounders treated by FKC+ECPs, for they showed significantly higher antibody titres, phagocytic activities, distinct enzyme activities and the highest relative percent survival.
|