Molecular Epidemiology Study Of Tilapia Streptococcosis And The Immune Function Explatation Of THSP70in This Disease

Streptococcicosis is one of the most severe diseases that infect the farmed tilapia in the world. It is now globally epidemic and can cause significant economic losses for the tilapia industry. As a tilapia production and exportation country, in China the streptococcicosis caused about160millions US dollars in the tilapia production during2009to2012. The epidemic situations of such severe disease has been enormously changed in recent years. For now, there is no effective preventive and control measure for this disease. In present study, the prevelent strains of streptococcicosis in the tilapia raising areas in China, including Guangdong, Guangxi, Fujian, Hainan and Yunnan provinces, during the past7years (2006-2012) were isolated and their serotypes, genotypes, species shift reasons and the immmunogenicities of the prevelent strains were studied to investigate the epidemiology of this pathogen in China. Meanwhile, the tilapia heat shock protein70(tHsp70) were expressed in yeast expression system and its immunogical functions were explored from the gene, cell and organism aspects in current study.The tilapia streptococcus pathogen library covering the largest source, amount and most detailed information of the major streptococcus strains in China has been built in current study. This library collected and organized totally210clinical strains of tilapia streptococcus from Guangdong, Guangxi, Hainan, Fujian and Yunnan provinces during2006to2012. The identification results exhibited that the prevelent strains of tilapia streptococcus has been shifted from previous Streptococcus iniae (91.30%, before2008) to current S. agalactiae (97.86%, from2009to2012). Three serotypes, la, Ib and Ⅲ, were found in these prevelent strains of S.agalactiae and the predominant serotype was la which accounted for94.67%of all S.agalactiae strains. The serotypes and PFGE types of these strains varied with geographical and temporal factors. Moreover, the immunogenicities of the prevelent strains of S.agalactiae were associalted with their serotypes and PFGE types, and it is difficult to obtain one vaccine candidate strain that can be used to protect all other genetic diverse strains from the same serotype. The reason why the prevelent species of tilapia streptococcus shifted was also explored from both aspects of epidemiological investigation and laboratory experiment, which turned out the2008extreme cold weather that lasted for more than30days in the south of China and the virulence diversity of the S. iniae and S. agalactiae may contribute to such species shift.Another important study was the secretory expression of tHsp70in the yeast expression system and subsequent biological and immunological researches on this purified protein. The gene of tHsp70was first cloned and used to construct the efficient and stable pichia pastoris secretory expression system, KM71/pPIC9K/tHSP70. The concentrition of expressed tHsp70was up to30mg/L. The purified tHsp70was ATPase and peptide-binding positive and could significantly enhance the growth rate, the nitric oxide (NO) release and the phagocytic activity of tilapia peritoneal macrophages (P<0.01). Additionally, the antigen phagocytic activity for the S. iniae thallus was also enhanced by this protein. Morover, the tilapia lymphocyte proliferations induced by the S. iniae thallus were significanlty increased by stimulation of tHsp70(10μg/mL)(P<0.01), whereas the inhibitions of the S. iniae extracellular products (ECP) to the lymphocyte proliferation were decreased. The gene expression analysis demonstrated that the levels of IL-8, Hsp70, CXCR4and PGRN in tilapia peritoneal macrophages and the levels of ICER, PGK, MMP9and MHCII in peripheral blood lymphocytes were significantly increased by treatment of tHsp70(P<0.01). Based on above results, a tHsp70-peptide microparticle oral vaccine against tilapia S. iniae was obtained by using particle package techniques and the immunological results showed the protection rate was76.78%and69.09%on10and20days post immunization, which was significantly higher than controls, respectively (P<0.01).Current study has been acquired the comprehensive data about the tilapia streptococcicosis occurrence and development regularity and the pathogen distribution and changing characteristics. Furthermore, the direct reason for the tilapia streptococcus species shift has also been figured out in this study. This not only provides direct consults for the scientific prevention and control, and further vaccine development, but also lay a substantial foundation for the novel fish oral vaccine explortion based on the tHsp70biological and immunological characteristics.