| Streptococcus agalactiae belongs to group B streptococcal(GBS) with broad host range, which can infect humans, mammals, reptiles, amphibians, fish and so on. Since 2009, S.agalactiae outbreaks in tilapia culture zones in south of China largely. The infection rate and mortality rate of tilapia are higher, individual rate by more than 50% and mortality rate over 95%. It causes serious economic losses. The disease has become one of the most serious bacterial diseases affecting tilapia aquaculture in China. But the understanding of its pathogen research is little, which is the interaction among health, pathogenic bacteria and the water quality. Although antibiotics have certain effect, the long-term use can cause drug resistance of bacteria, it is reported that the occurrence of bacterial drug resistance and pathogensis have close relationship with environment. Bacterial quorum sensing system(QS) regulates the expression of certain genes to adapt the change of external environment. Therefore, we studied key enzymes of QS-lux S to provide a theoretical basis for research on pathogenic mechanism of S. agalactiae. Moreover, the long-term use of antibiotics is not the most scientific method, vaccine is still the best way to control the disease. Due to limitations of the fish immune, oral vaccine is necessarily aquatic vaccine research. Biodegradable PLGA nano/micro ball and their derivatives as carriers for vaccine are used in vaccine and gene therapy reportly. Chitosan, PLGA has the characteristics of the biodegradable property and biocompatibility, especially the positively charged characteristic of chitosan can enhance the adsorption of plasmid with negatively charge and the effect of cell infiltration. Therefore, it is thought that chitosan and PLGA have potential advantages as an oral immune carrier. The main research content is as follows.1. The isolation and identification of tilapia S. agalactiae. 47 strains of S. agalactiae from more than 100 samples of tilapia with illness and death were isolated and identified by gram staining, biochemical indentification and PCR technology. The serotypes of strains were serum type Ia through molecular serotype analysis. Evolutionary relationship of S. agalactiae was not restricted by the host through RSCU values analysis. In long-term evolution process, S. agalactiae was an independent replication, not like virus was restricted by the host. The study provides information for epidemiological investigation of S. agalactiae.2. S- Ribosylhomocysteinase(lux S) activity analysis of S. agalactiae. Lux S is key enzyme of QS, the study on antibiotic resistance and virulence affected by lux S gene will provide theoretical basis for pathogenesis of S. agalactiae. Using p SET4 S suicide vector, recombination suicide vector was constructed with homologous arms and chloramphenicol selection label, then the recombination suicide vector was electrotransformed into wild type S. agalactiae. Lux S gene deletion mutant was obtained via homologous recombination and lux S gene replaced by chloramphenicol select tags through PCR identification. Through the determination of bacterial growth curve, growth rate of lux S gene deletion mutant strain had no difference obviously with WT strain. Lux S gene deletion mutant strain lost bioluminescent ability through determination of Vibro harveyi. Lux S deletion mutant reduced susceptibility to norfloxacin and cefradine and reduced virulence to tilapia. And lux S deletion mutant reduced adhesion to epithelial cells and acid resistance. Exogenous 7.4 n M AI-2 is the best concentration to return function of lux S gene mutant.3. Immunogenicity proteins screeing of S. agalactiae. According to the result of bioinformatics prediction, immunogenicity proteins including surface immunogenic protein(sip), capsular polysaccharide glycosyl transferase(cps E) and secreted protein of Ⅶ secretion system(ESAT6) were chose as the research object. The genes encoding three proteins were amplified from S. agalactiae strain(serotype Ia) by PCR technology. According to electrophoresis, the length of sip protein encoding gene was 1305 bp, the length of cps E protein encoding gene was 450 bp, the length of ESAT6 protein encoding gene is 294 bp. The three proteins connected into prokaryotic expression vector p ET32 a plasmid using vitro cloning technology. Using IPTG indued prokaryotic expression, three kinds of proteins can successfully expressed in E. coli. Sip and ESAT6 proteins expressed in the form of soluble protein, cps E protein expressed in the form of inclusion body protein. The purified proteins had good immunogenicity with mouse polyclonal antibody. The study provides foundation for the further research on immune efficacy in animal.4. Chitosan-PLGA packages oral vaccine of S.agalactiae and its immune effect. Eukayotic recombinant expression plasmids including pc DNA-sip 、 pc DNA-cps E 、pc DNA-sip-cps E、pc DNA-IL8-sip-cps E、pc DNA-sip-IL8-cps E were constructed in this study. The best technology of chitosan-PLGA package oral DNA vaccine of S. agalactiae was 2mg/ml PLGA, 0.3 mg/ml chitosan, 2 mg/ml PVA. Its average microsphere diameter was 846.9 nm and its average Z protential is 48.0 m V. Three doses containing 20 μg/tilapia, 50 μg/tilapia and 100 μg/tilapia immuned tilapia, The serum were collected every 7d and ELISA serm titer was determined, the result showed that titer of oral vaccine was higher than DNA vaccine, and the highest serum titer of oral vaccine was in 21 d. The liver, spleen, kidney, gill, heart and intestinal tissue of tilapia were collected every 7d and the expressions of tilapia tissues were determined, the result showed that oral vaccine can effectively express in tilapia tissues. After 30 d of immunity, 2LD50(2×108 cfu/m L) dose challenged immuned tilapia and the relative immune protection rate was determined, the result showed that the RPS of oral vaccine was high than DNA vaccine and the RPS of oral vaccine was between 25-100%.In all, the results showed that sip, cps E and ESAT6 proteins had good immunogenicity and the three proteins can used as new subunit vaccine candidates. Chitosan-PLGA package oral vaccine had strong immune protection to tilapia and had the importance of clinical practiae. |
