Analysis Of Pathogenic Characteristics Of Non-typeable Strains Of Streptococcus Suis(SS-NT)

Streptococcus suis（SS）with 29 serotypes and a significant number of serovar-nonstreptococcal strains（SS-NT）is the predominant pathogen causing porcine streptococcal disease.In recent years,the detection rate of SS-NT has been increasing in veterinary clinical cases,but its pathogenic characteristics have been poorly reported so far.Therefore,in this study,16 strains of SS-NT isolated and identified in the laboratory were tested to lay the foundation for further effective prevention and control of streptococcal disease in pigs.Methods of study:（1）Morphology,culture and biochemical characteristics,biofilm-forming（BF）ability and drug susceptibility of 16 strains of SS-NT（NT-1～NT-16）were examined using 2 strains of SS2（HF2 and HF3）as reference strains.（2）PCR and multilocus sequence typing（MLST）were performed to determine the virulence genotype and ST type of SS-NT.（3）Intraperitoneal attack screening of strains lethal to mouse and LD₅₀of the mouse determined by cumulative method.（4）The inactivated organisms were prepared in formaldehyde and immunized in rabbits and mice respectively,and the AGP was used to determine the reactivity with rabbit antiserum;the immunogenicity assay included ELISA to detect Ig G antibody and cytokines IL-4,IL-10,IFN-γ,TNF-βand MCP-1 in mouse serum,SBT to detect the bactericidal ability of the serum,and MTT to detect the proliferation index（SI value）of splenic lymphocytes,using flow cytometry to detect the percentage of CD4⁺and CD8⁺T lymphocyte subpopulations,and used peritoneal attack to detect the immune protection rate,tissue charge number and inhibition of tissue damage.Results:（1）according to HF2 and HF3,the morphology and colony characteristics of16 strains of SS-NT microscopically were consistent,some strains differed in hemolysis when anaerobic;the utilization ability of inulin,mycoside,salicin and hesperidin was different;all of them could form BF,among which NT-3,7 and 12 were 3+,NT-14 was1+,12 strains of SS-NT and 2 strains of SS2 were 2+;there were no SS-NT NT 100%sensitive drugs,a total of 8 resistance profiles,NT-2,3,8,12 and 2 SS2 strains with the same resistance profile.（2）Both carry fbps and gapdh genes,no epf genes,sly,orf2 and mrp genes carry 75%,44%and 38%in order,total 6 virulence genotypes,NT-3 and 14are the same as HF2 virulence genotypes;contains 13 ST types,ST3235（NT-4 and 5）and ST3236（NT-13）are new ST types.（3）The LD₅₀of NT-1,2,11 and 15 were 1.13×10⁸,1.88×10⁸,1.95×10⁸and 2.61×10⁹CFU/only in that order.（4）NT-1,2,11 immune group antiserum can react with the corresponding antigen and cross-antigen,serum Ig G antibody titer is 1:6400,1:3200,1:1600,serum sterilization rate is 66.7%,58.9%,59.5%,cytokine level,SI value and CD4⁺,CD8⁺peripheral blood T lymphocyte subset percentage are significantly increased,the immune protection rate against NT-1 is 90%,10%,60%,NT-2 is 30%,80%,20%,NT-11 is 60%,30%,80%,HF3 is 20%,0%,10%,3 SS-NT strains inactivated whole bacteria can effectively inhibit the colonization of their own strains,there is cross-immune protection between strains,except for mice immune to NT-2 after HF3 challenge tissue lesions are more serious,there is no significant difference between tissue load number and the challenge control group,strain NT-1 and NT-11 can effectively inhibit HF3 colonization.Conclusion:the morphological characteristics of SS-NT were stable,but the hemolytic ability and biochemical characteristics were different;the ST types were more diverse and scattered,and the virulence genotypes were complex;the BF formation ability was different among strains,and its strength was not correlated with pathogenicity;there were strong strains of SS-NT with similar virulence to the reference strain,and the most virulent strain had the best antigenicity;SS-NT were all multi-drug resistant strains.The differences in drug resistance among the strains were related to the isolation area,BF formation ability and other factors.