Virulence Charicterization Of Streptococcus Suis Serotype 9 Isolated In China

Streptococcus suis(SS) is an important pathogen of human and animal.Thirty-five capsular serotypes of S.suis have been described(types 1/2 and 1 through 34).Serotype 2(SS2) is considered to be the most-virulent serotype and prevalence in worldwide.Strains of Streptococcus suis serotype 2,7,9 seemed to be most commonly isolated from diseased animals.To investigate the population of these type from abundance samples,a multiplex PCR was developed for rapid and sensitive detection of Streptococcus suis serotype 2,7,9.Multiplex PCR used to detected samples from slaughter pigs,sensitivity and specificity of the multiplex PCR were evaluated.Results:3 SS9 strains and 12 SS2 strains were detected from 160 samples,a single PCR used to amplify the undetected samples, found no false-negative samples.The sensitivity of multiplex PCR was 500CFU per reaction.Results showed that multiplex PCR was a highly sensitive and specific tool,ideal for fast diagnosis and surveying epidemics.The virulence-associated gene type of SS9 was not clear,7 virulence-associated genes detected by PCR assay,namely the glutamate dehydrogenase gene(gdh),the capsular polysaccharides gene(cps),the extracellular factor gene(epf),the muramidase-released protein gene(mrp),the suilysin gene(sly),the fibronectin-binding protein gene(fpbs) and the virulence-associated sequence orf2.Results:cps9H,gdh,fpbs were found in all 7 SS9 strains;epf,sly were not found in all 7 stains,mrp existed in GZ0565 and GD0606;orf2 only detected in GZ0565.It suggested that virulence-associated gene type differed between different SS9 strains.Virulence-associated gene type of SS9 differed to the usual virulence-associated gene type(cps2+/gdh+/epf-/mrp+/sly+/fbps+/orf2+) of SS2,GZ0565 isolated from a pig suffered meningitis had low homology of cps9H with other SS9 isolates from pig tonsils,by comparing their 300bp cps9H sequence.Differ the virulence of 7 SS9 strains isolated from China,7 SS9 isolates,SS9 reference strain 22083 and SS2 isolate HA9801 were used to attack ICR mice.LD₅₀ of GD0606 and GZ0565 were tested on mice.Immune mice with inactivated GZ0565 and GD0606 and live avirulent strain GD0627,GZ0565 and GD0606 attacked mice.GZ0565 and GD0606 caused mice death,others including SS2 virulent strain HA9801 did not.GZ0565 had 100%(10/10) protective rate in mice against GZ0565 and GD0606 challenge,GD0606 and GD0627 had 80%(8/10) and 50%(5/10)protective rate in mice against GD0606 challenge, had 60%(6/10) and 30%(3/10) in mice against GZ0565 challenge.It concluded that virulent to mice of SS9 related to their resource;mice could evaluate the virulence of SS9 isolates.SS9 strain GZ0565 and SH040917,SS2 strain HA9801 were used to attack the Guangdong local pigs,each strain attacked 3 pigs.Inactivated former strains were used to immune pigs,each strain immued 3 pigs.GZ0565 immuned group attacked by HA9801, HA9801 immuned group attacked by GZ0565,SH040917 immuned group attacked by GZ0565.Results:All pigs attacked by 5×10⁶CFU HA9801 died in 16h;GZ0565 attacked pigs got arthritis,no pigs died;SH040917 not caused pigs die,and no typical clinical symptom found.GZ0565 immune group protected the HA9801 attacked,but not completely-2/3 protected;SH040917 immune group had low protection against GZ0565-1/3 protected;inactivated HA9801 immune group got apparatus pathological changes,no arthritis.it can be concluded that HA9801 was most virulent,followed by GZ0565,SH040917 was avirulent.Some antigens existed in HA9801 and GZ0565,were the basic elements of vaccine for multi-serotype.GZ0565 and SH040917 differed in virulence and immune protection,though they had same CPS.