Isolation And Identification Of Streptococcus Suis In Henan Province And Prokaryotic Expression Of Virulence-associated Genes

Swine streptococcosis was one of the most important bacterial infectious diseases, causing meningitis, arthritis, septicaemia, endocarditis and sudden death of pigs, and etc. S. suis is one of the important agents of Swine streptococcosis. So far, 35 capsular serotypes have been identified, with types 1(and 14), 2(and 1/2), 7 and 9 were the most prevalent and most pathogenic serotypes, which lead to great economic loss in the swine industry. Meanwhile, Swine streptococcosis was one of the important amphixenosises, causing meningitis, septicaemia, permanent hearing loss of human, causing great attention in public health. The study was carried out in order to establish rapid, simple and specific detection methods of immunology and molecular biology, and to investigate the carrier rate and serotype distribution of S. suis in Henan province, which may lay the foundation for rapid diagnosis and control of the diseases.1. Establishment of PCR assays for rapid identification of S. suisFive pairs of primers were designed, which are based on the sequences of the species-specific gdh gene coding for glutamate hydrogenase of S. suis and serotypes-specific genes of cps1I, cps2H, cps7H and cps9G coding for the capsules of S. suis serotypes 1(and 14), 2(and 1/2), 7, and 9, respectively. The applicons were 689bp, 441bp, 542bp, 232bp and 330bp, respectively. By optimizing the PCR conditions of annealing temperature and Mg2+, dNTP and primers concentrations, a stable tetramerous PCR assay and a single PCR assay were established, respectively. To evaluate the specificity, 12 strains of other bacterial species related to S. suis or isolated from pigs were analyzed. The PCR assay was then applied to the detection of 39 tonsils, and several PCR products were sequenced to confirm the PCR assay. The PCR assay is rapid, exact, specific and sensitive. When genomic DNA of S. suis serotype 2 was used as template for PCR, the detection threshold of the tetramerous PCR assay was 2.52 CFU/assay. The results showed that the PCR assay can be applied in rapid detection of S. suis and its main pathogenic serotypes from tonsils.2. Isolation, identification and prevalence of S. suis in diseased pig herds in Henan Eighty-three palatine tonsils of clinical diseased pigs from 33 pig farms in 14 regions of Henan province were detected by the PCR assay established, and S. suis-like strains were isolated from the tonsils at the same time and identified by morphology observation, Gram staining, PCR assay, and sequencing analysis, and PCR amplicons of partial were gel retrieved for sequencing as a confirmation. A total of 77 tonsils were detected as S. suis positive, and the rate was 92.8%. Among these samples, 15 tonsils were type 2 positive, account for 18.1%; 4 tonsils were type 7 positive, account for 4.8%; type 9 positive was 26, account for 31.3%; and type 1 wasn't detected. A total of 77 strains of S. suis were isolated, and among these strains, 7 strains are serotype 2, account for 9.2%; 3 strains are serotype 7, account for 4.0%; 4 strains are serotype 9, account for 5.3%; and serotype 1 wasn't isolated. The morphous of S. suis strains isolated was needlepoint-like, off-white or gray, small colonies ofα,βorγhaemolysis, the bacteria are positive cocci, often arranged in pairs or short chain, long chain by chance in the Gram-stained smears. The results demonstrated that high infection rate of S. suis was found in Henan, with type 9 the most prevalent serotype, then type 2, and the last type 7, no S. suis type 1 was found in Henan. S. suis of pathogenic serotypes often simultaneously or secondary infected with HCV and PCV2.3. Cloning and sequence analysis of the gene coding for glutamate hydrogenaseBy using PCR assay, the gdh genes were amplified from the genomic DNA of S. suis of five strains containing serotypes 1(and 14), 2(and 1/2), 7, 9 and serotype unknown, and cloned into pMD-18T vector, and after transformation into E. coli JM109 competent cells, the recombinant plasmids were extracted, identified, sequenced, analysed and compared with another 16 gdh nucleotide and deduced amino acid sequences of published strains from GenBank. Five gdh genes were successfully cloned and sequenced. Sequence analysis showed that the five sequences had close relationship with the gdh genes of S. suis published in GenBank. The homology of the 21 nucleotide sequences was 96.4%～100%, which was 98.4%～100% for the deduced amino acid sequences, with only 5 amino acid difference at most. Within 15 strains of serotype 2, the homology of nucleotide sequences and deduced amino acid sequences were 99.9 %～100 % and 99.6 %～100 % , respectively. Among those, the homology of deduced amino acid sequences from 13 isolates out of 15 serotype 2 strains were 100%, another 2 were only one amino acid difference. By analyzing all the 21 amino acid sequences, 6 sequences other than 15 serotype 2 were found with the same three amino acid differences compared with 15 serotype 2, which were Ser→Ala at the position of 299aa, and Lys→Glu at the position of 305aa and 330aa, respectively. Apart from this, there were only one or two amino acid differences in other position of few sequences. Predicting antigenic epitopes of the GDH proteins of the five strains shows 21 or 22 epitopes, in which 13 or 14 epitopes were S.suis specific. The results indicated that the gdh genes cloned in this study were very conservative, thus it can be expressed in prokaryocyte or eukaryocyte and its product could be used in immunological research.4. Prokaryotic expression of the gdh and cps9G whole genes of S. suisTwo pairs of primers were designed and the gdh and cps9G whole genes were amplified by using PCR from genomic DNAs of SS2 PDSWG-1 and SS9 PY-2 isolated in Henan province, respectively. The genes were digested with BamHⅠand Xho I, and cloned into pET32a(+) vector, two recombinant expression plasmids of pET32a-gdh and pET32a-cps9G were constructed. Then the plasmids were transformed into E. coli BL21(DE3) competent cells induced by IPTG for expression and the expression products were analyzed by SDS-PAGE and Western-blotting. SDS-PAGE showed a fusion protein band of about 68.8KDa was successfully expressed by E.coli BL21 containing the recombinant plasmid pET32a-gdh induced by 0.8 mmol/L IPTG for 3h. Another fusion protein band of about 50.7KDa was successfully expressed by E.coli BL21 containing the recombinant plasmid pET32a-cps9G induced by 1.2 mmol/L IPTG for 4h. Both the two fusion protein products have particular biologic activity by Western blotting analysis, which may provide good antigens for immunological test methods and producing specific monoclonal antibody(McAbs) and may lay the foundation for preparing subunit vaccine and test kits.