| Recently,there are more and more cases has been reported that people developed severe clinical symptoms such as septicemia,meningitis,toxic shock syndrome and even death by ingestion of Streptococcus suis contaminated pork or other related food,indicating the foodborne infection of Streptococcus suis is becoming a serious threat to food safety and human health.The state key special project of research and development of key technologies for food safety pinpoints that the most important task is to solve the severe pollution problem of food origin.As Streptococcus suis normally inhabits in upper respiratory tract of pigs and the bacteria carrier state of a herd is almost 100%,it would be of great importance in food safety and health care if an effective biological control strategy such as immunization could be used to eliminate Streptococcus suis inhabiting in mucosa of the host to control the transmission of the pathogen in the first step of food production.That is why we are focusing on exploration of the live vaccine against Streptococcus suis in this study.Considering the sudden death caused by Streptococcus suis infection involves its ability to escape from innate immune response,which induces severe inflammatory storm,we selected five genes related to innate immune escape named sly,scp A,ssn A,fhb,ssads and constructed the gene-deletion plasmids to delete these five genes in Streptococcus suis strain SC19 to obtain an isogenic five-gene deletion mutant named 2015033.Based on our study,the basic biological characters such as growth character and capsule generation were not affected by deletion of five genes.In vitro experiments demonstrated that the hemolytic activity to sheep red cells,cytotoxicity to five different cells or cell lines（HBMEC,A549,Caco-2,HEp-2,He La）and the growth ability in human blood of 2015033 were significantly attenuated compared with SC19.In vivo experiments demonstrated that 2015033 was found to have diminished virulence in mice model and to be eliminated more easily in host tissue.In addition,detection of six different inflammatory factors（IL-6,IL-10,IL-12p70,TNF-α,IFN-γ,MCP-1）of mice serum showed that 2015033 induced weaker inflammatory response compared with SC19.Immunization with 2015033 via the subcutaneous way induced BALB/c mice to generate high level of Ig G,Ig G1 and Ig G2a antibodies.The Ig G1 titer was higher than the Ig G2a titer which indicated immunization with 2015033 induced both Th2 and Th1 immune response and the former was predominant.Opsonic killing assay showed SC19（ST7）,P1/7（ST1）and LSM102（ST658）strains pretreated with nonimmune mice serum grew well in nonimmune mice blood,but the growth of these strains pretreated with 2015033-immune mice serum was significantly reduced,which indicated that immunization with 2015033induced important immune response against Streptococcus suis.Challenge mice with these different sequence types of Streptococcus suis strains after immunization showed the survival rate of immune mice was much higher than nonimmune mice,indicating immunization with2015033 provided cross-protection against infection with different sequence types of Streptococcus suis strains.Detection of six different inflammatory factors showed that immunization with 2015033 suppressed proinflammatory response caused by SC19 strain infection.As 2015033 still possessed growth ability in human and pig blood,we then deleted other five genes named iga,nud P,ide Ssuis,end Asuis,mrp in 2015033 to generate an isogenic ten-gene deletion mutant named 2015800.The basic biological characters of 2015800 such as growth character and capsule generation were not changed.In vitro experiments demonstrated that the hemolytic activity to sheep red cells and the cytotoxicity to three different cells or cell lines（HBMEC,A549,Caco-2）of 2015800 were significantly attenuated compared with SC19.It is worth mentioning that the growth of 2015800 in both human and pig blood was completely suppressed.In vivo experiments demonstrated that the virulence of 2015800 was significantly reduced in mice model and detection of six different inflammatory factors showed that 2015800 induced weaker inflammatory response compared with SC19. |
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