Epidemiology Of Streptococcus Suis In Chongqing And Cloning And Sequence Analysis Of GDH Gene

1 The establishment of Multiplex PCR of serotype 1, 2, 7, 9 of S.suisFive pairs of primers were designed, in which 4 pairs of them were based on capsular polysaccharide(cps) biosynthsis genes specific for serotype 1, 2, 7 and 9, the other pairs of them were based on the glutamate dehydrogenase (gdh) gene specific for S.suis and mutiplex PCR assays of S.suis serotype 1, 2, 7 and 9 were developed to amplify gdh and cps1J,gdh and cps2J,gdh and cps7H,gdh and cps9G. After serial tests, multiplex PCR of serotype 1, 2, 7, 9 were established successfully. In the serial tests, the Specific assays of mutiplex PCR was demonstrated by detecting the 35 serotypes of S.suis and one strain of Streptococcus group B and one strain of Streptococcus group C, and by sequencing the products of mutiplex PCR; in the sensitive assays, mutiplex PCR of S.suis serotype 1, 2, 7and 9 could be detected definitely when the template contained as few as 100 cfu; mutiplex PCR of serotype 2 was used to detect 12 strains of serotype 2 known, its coincidence rate is 100%; mutiplex PCR of serotype 1, 7, 9 were used to identify 10 strains of S.suis suspected, The results showed that 2 strains were serotype 7, 2 strains were serotype 9; in the same time , mutiplex PCR of serotype 1, 2, 7, 9 were tested periodic in 1 year, the results showed that mutiplex PCR of serotype 1, 2, 7, 9 were stability.2 The detection and identification of S.suis in Chongqing1360 palatine tonsil samples of clinical healthy pigs and 116 tissue samples of diseased pigs were collected from 20 different district of Chongqing in china. By means of multiplex PCR of S.suis serotype 1, 2, 7 and 9 assays and plate agglutination test, 224 strains of S.suis , in which 5 strains belonged to serotype 2(from 3 different district) ,4 strains belonged to serotype 9(from 2 different district), 4 strains belonged to serotype 7 (from 4 different district), were detected and identified from the 1360 palatine tonsils. the detection rate of S.suis hit 16.5%. 8 strains of S.suis(not serotype 1, 2 ,1/2, 7,9, 14) were detected and identified from the 116 tissue samples.1 strain of serotype 1/2 was isolated in the detection and it was the first report in china.3 The detection, sequencing and new findings of mrp and epf of S.suis isolated from ChongqingMuramidase released protein(MRP, mrp) and Extracellular protein factor(EF, epf) were the two important virulence factors of S.suis. Different molecular weight of MRP and EF, such as MRP , MRP~S, EF~*, was correlated with the difference of virulence and pathogenicity of S.suis. 26 strains of S.suis serotype 2(including strains isolated fromChongqing), in which 25 strains isolated from pigs, 1 strain islated fom diseased human, were identified with mrp and epf. The results showed that most strains isolated from the diseased pigs(including diseased human) were mrp~+epf~+, except strain SH0401was mrp~+ epf~*, and most strains isolated from the palatine tonsil of clinical healthy pigs were mrp~-epf~+, except strain CQTN63 was mrp~+epf~+. The epf of 10 strains of S.suis serotype 2 were sequenced ,its identities hit 99%.15 strains of S.suis serotype 1, 1/2, 7, 9 (including strains isolated from Chongqing) were identified with mrp and epf. It was found that 3 strains of serotype 7 and 1 strain of serotype 1/2 were mrp~s(474bp), and the mrp~s in this test was never reported even now. 1 strain BJ09 was mrp*(about 2000bp). All mrp~s and mrp~* were sequenced. the results showed that the 464bp of mrp~s were same with mrp of serotype 2(1148bp), but the mrp of serotype 2 have more 670bp than mrp~s of serotyoe 7 and 1/2. The 800bp of mrp~* of strain BJ09 were same with mrp of serotype 2, but the other of mrp of strain BJ09 were different completely with mrp of serotype 2. 15 strains of S.suis serotype 1, 1/2, 7,9 were epf~-.4 The cloning and sequence analysis of gene encoding glutamate dehydrogenase (GDH) of S.suisResearch indicated that glutamate dehydrogenase(GDH) of S.suis is a protective antigen, and can be used as a diagnostic antigen. Compared to other protein, GDH have low point mutation rate, and have high conservatism. Therefore GDH have more wider and wider prospect.GDH gene whole open reading fram (ORE, 1347bp) of 44 strains of S.suis, in which 43 belonged to serotype 1, 2, 1/2, 7, 9, 4, 19, 27, 30, 31, the other belonged to unknown serotype, were sequenced and sequence analysis. The results showed that the homology of gdh of all strains of serotype 2 hit above 99%, the homology among these serotypes most of could reached to 94% above. Compared to serotype 2, the nucleotide of gdh of other serotype have genesised mutation in the site 162, 279, 534, 898,916, 978, 981,991,1011, 1023,1068, 1077,1083, 1098,1104, 1107, 1137, 1158 and 1224 all, the results showed that the transform of this nucleotide have stronger correlation on the serotype changed of S.suis, this also demonstration that this sites were important mutation site. At the same, two regions (978~1023 site and 1060～1120 site) were hypevariable regions, and the mutations of nucleotide in the two regions also have stronger correlation on the transform of serotype.The sequences of amino acids which deductioned analysis indicate, the homology of amino acids sequences which 44 strains have reached to 97% above, and also have typical conservation sequences that the family of GDH 1 has. At the same we find that besides serotype 2, the amino acids have transformed at the follow three sites which its 299 site (from S to A), 305 site (from K to E),330 site (from K to E), demonstration that the transform of amino acids at these sites have largly correlation on the transform of serotype.