Vaccine Preparation And Characterization Of Extracellular Products From S.iniae Isolated From Tilapia

Streptococcus iniae is known to be a major bacterial disease in mariculture animal systems. Disease outbreaks attributed to S.iniae has been reported in some mariculture aquatic animal farm production. In recent years , mass mortalities of many cultured kinds of maricultured fishes,such as Paralichthys olivaceus, tilapia and Sciaenops ocellatus have occurred frequently infected by S.iniae in china .In this study, we isolated one strain from the diseased tilapia collected from the coast of the South China Sea, and identified it as S.iniae by biology system and 16SrRNA PCR. The pathogenicity of S.iniae to tilapia was also tested and its 50% lethal dose (LD50) value in tilapia was 6.81×10⁷cfu/mL. In addition, S.iniae was more senstive to Tetracycline,Benzylpenicillin G,Ciprofloxacin,Novobiocin,Cefuroxime,Piperacillin,Doxycycline et al, but resistant to Neomycin,Kanamycin.S.iniae has high nutritional requirement and we have to make a preparation for the development of vaccine , so we optimized its culture conditions. There have some factors that effect the growth of S iniae, for example, pH, gaseous environment, temperature and growth factors in the fluid culture medium were studied to optimize its growth parameters. The results indicated that the initial pH 7 - 8 and the 30 degree's temperature were optimal for the S.iniae, while whether there was dissolved oxygen or not had small effect on its growth. The maximal bacteria's concentration could be achieved by supplying 10 mg/mL yeast powder and 2 mg/mL glucose in the BHI. And we also got its growth curve : 0-4h is growth delay phase, 4-24h is logarithmic phase, 24-48h stable phase, and then is decline phase.The biologic activities of S.iniae extracellular products were studied. The results showed that S.iniae ECP has amylase, protease, lecitinase, gelatinase and lipase activities, but no urease activity. It could dissolve the blood cell of tilapia , which can get the haemolysis potency of 1:64. The ECP had good stablity , and the optimal temperature was 55℃. Some chemical agents had also many effects on the ECP, whose results showed that the EDTA,PMSF,DTT could reduce the activities of ECPase to 72.4%,77.6%,72.4% respectivly, and Cu²⁺,Ca²⁺,K⁺,Mg²⁺ could reduce the activities of ECPase, while Fe³⁺, Co²⁺, Mn²⁺ could activate its activities. The pathogenicity of ECP to tilapia was tested, which showed S.iniae ECP has toxicity to tilapia and the mortality will raise with the increase of ECP concentration. We analyzed the main protein of ECP by SDS-PAGE and two dimensional electrophoresis. The SDS-PAGE analysis exhibited that the main protein of the ECP were arranged between 26 kDa and 95kDa, while the two dimensional electrophoresis showed 120 spots. In addition, we studied the ECP of western-blot with rat anti S.iniae ,which showed four proteins ,whose molecular weight were 26kDa,37 kDa,95 kDa,97 kDa respectively.The formalin killed S.iniae vaccine and white oil adjuvant vaccine were prepared and used to vaccinate the tilapia for eight weeks. After immunization the fish were challenged by intraperitioneal injection of S.iniae. The tilapia immunized with vaccine showed that there were significantly higher serum bacteriolytic activities, lysozyme activities ,SOD,and antibody activity than those fish in the control group. And the formalin killed S.iniae vaccine and white oil adjuvant vaccine showed high immunoprotection with RPS of 65.43%, 88.89% respectively.