Study On Biological Characteristics Of Streptococcus Suis Serotype 4 Isolates And Screening Of Strains For Inactivated Vaccine

Streptococcus suis(SS)was the most main pathogen causing Swine Streptococci.According to the difference of capsular polysaccharide,it could been divided into 35 serotypes,of which serotype 1,2,7 and 9 were the main ones.In recent years,the isolation rate of Streptococcus suis serotype 4(SS4)increased gradually,which caused morbidity and mortality,harming the breeding industry and public health.Inactivated vaccine was commonly used to prevent and control Swine Streptococci,because it was convenient for research and development,usement and preservation.Besides,it could been made into multi-valent or multi-combination vaccine.Up to date,more attention were paid to the biological characteristics of Streptococcus suis serotype 2(SS2)rather than SS4.Moreover,there was a lack of cross-protection between different serotypes of SS,and the existing commercial inactivated vaccines were mainly aimed at SS2 and SS7,which couldn't produce immune protection against SS4.Therefore,the research on SS4 vaccine was necessary.In this research,19 SS4 strains isolated from clinical cases were used as test strains,and the following two aspects were studyed.First,the study on the biological characteristics of SS4.19 test strains of SS4 and 2 reference strains of SS2 were used for cultural and morphological observation,biochemical test,mouse pathogenicity test,virulence gene detection,biofilm formation ability determination and multi-locus sequence typing.The results displayed that,compared with SS2,SS4 had a larger colony diameter,the same hemolytic characteristics and microscopy morphology;most strains of SS4(68.4%)had same biochemical characteristics with SS2,minority of strains(31.6%)had incompletely same reaction to lactose,raffinose,synanthrin and mushroom sugar;All strains of SS4 caused meningitis and death in mice,and the LD₅₀of the 7 most virulent strains of SS4(AHshou18-1,AHhuai18-8,AHhuai18-10,JSxu18-4,HBgu18-3,JSxu18-10,HBgu18-4)and 2 strains of SS2 were in the order of 10⁷?10⁸CFU;there were 4 to 8 virulence genes and 5 virulence genotypes in SS4,and gdh+cps4+epf+mrp+sly+gapdh+fbps+orf2+was the dominant virulence genotype(36.8%);all the strains of SS4 had the ability of biofilm formation,weak(1+)mainly(89.5%),and the reticular biofilm structure was observed under scanning electron microscope;there were 3 ST types in SS4 strains,of which ST94 were prevalent(89.5%),while ST1158 and ST1224 were new found.The results indicated that,SS4 strains had identical cultural and morphological characteristics,diverse biochemical characteristics,high carrying-rate and various types of virulence genes,prevalent biofilm formation ability,strong pathogenicity to mice and genetic diversity.Second,the screening of SS4 vaccine strain.Six SS4 strains with the strongest virulence (AHshou18-1,AHhuai18-8,AHhuai18-10,JSxu18-4,HBgu18-3,and HBgu18-4)were screened out through the mouse pathogenicity test.Then,mice were immunized with six kinds of inactivated bacteria combined with ISA 201 mineral oil adjuvant,Ig G titer in serum deteriminated by ELISA,immune protection rate determinated by poison protection test,and the histopathological changes of mouse organs observed.The results displayed that,the serum Ig G titers of AHshou18-1?AHhuai18-8?AHhuai18-10?JSxu18-4?HBgu18-3?HBgu18-4 immune group were 1:1600,1:1600,1:3200,1:6400,1:3200,the immune protection rates were 30%,30%,50%,70%,60%,80%,and the pathological changes of JSxu18-4,HBgu18-3,HBgu18-4 immune groups were slighter than the other immune groups.The results indicated that JSxu18-4 and HBgu18-4 had better antigenicity.After passing in vitro to 30 generations,the LD₅₀ of JSxu18-4 increased to 3.81×10⁸CFU,the Ig G titer decreased to 1:1500,and the immune protection rate decreased to 40%,while the LD₅₀ of HBgu18-4 increased to 2.49×10⁸CFU,the Ig G titer and the immune protection rate remained stable at 1:6400 and 80%.And the pathological changes of HBgu18-4 immune group were slighter compared with JSxu18-4 immune group.The results showed that HBgu18-4 had better genetic stability.In conclusion,HBgu18-4 had strong pathogenicity and good antigenicity,and it's genetic traits were uniform and stable,so it could been used as a candidate strain for SS4 vaccine.