Development And Preliminary Application Of Molecular Diagnostic Technique Of Streptococcus Suis Serotype2

Swine streptococcosis was one of the most important bacterial infectious diseases,causing meningitis, arthritis, septicaemia, endocarditis and sudden death of pigs, and etc.Streptococcus suis is one of the important agents of Swine streptococcosis. So far,35capsularserotypes have been identified, serotype1,2,7and9were the most prevalent and mostpathogenic serotype, which lead to great economic loss in the swine industry. Meanwhile, Swinestreptococcosis was one of the important amphixenosises, cauing great attention in public health.The study was carried out in order to establish rapid, simple and specific detection methods ofimmunology and molecular biology, and to investigate the carrier rate and serotype distribution ofstreptococcus suis in Henan province, which may lay the foundation of rapid diagnosis and controlof the disease.1. Identification of virulence genes and sequence analysis of cps2J gene ofStreptococcus suis serotype2isolated from Henan ProvinceTo investigate virulence genes and mutation of cps2J gene of eight Streptococcus suisserotype2(SS2) strains isolated from Henan province, PCR methods for detecting seven virulencegenes of SS2were developed, based on the specific primers for virulence genes of gdh, cps2J, mrp,ef, sly, orf2and fbps. Complete cps2J gene was amplified, sequenced and analyzed. The resultswere as follows: the detection rate of gene gdh, cps2J, mrp, ef, sly, orf2and fbps were100%(8/8),100%(8/8),62.5%(5/8),75%(6/8),87.5(7/8),100%(8/8) and100%(8/8), respectively. Thesequences of cps2J genes of eight SS2isolates achieved above98%nucleotide homology with thatof SS2strains isolated from other areas, amino acid sequence homology achieved to above97%.Sequence analysis indicated homology between the eight SS2strains and reference strains at homeand abroad was high and genetic relationship was close.2. Expression and immunogenicity analysis of cps2H gene of streptococcus suisIn order to analysis the immunogenicity of cps2H gene of streptococcus suis, The cps2Hgene was amplified by PCR form ZMDSC-2. The cps2H gene comprised1371bp encoding457amino acids.The cps2H gene was cloned into a prokaryotic expression vector pET32a(+) and expressed in E.coli by IPTG induction. The expressed products were analyzed by SDS-PAGE andwestern blotting. The results showed that the fusion protein was66.2KDa in size and reactedstrongly with antiserum of streptococcus suis. The protein was used to immunize mice andantibody against the recombinant cps2H antigen was tested by ELISA. Antibody could be testedon day7post-immunization, and reached peak on day28, indicating that the fusion protein hadstrong immunogenicity. These results showed that the recombinant cps2H antigen has goodimmunogenicity, and it might allow to develop effective vaccination strategies againststreptococcus suis infection.3. Rapid detection of Streptococcus suis serotype2by loop-mediated isothermalamplificationA loop-mediated isothermal amplification (LAMP) assay was developed for rapid, sensitiveand specific detection of SS2. A set of four primers were designed based on capsularpolysaccharide （cps2H） gene of SS2. LAMP was conducted under optimized conditions.Ladder-like DNA fragments were observed on agarose gel electrophoresis for amplified productsfrom SS2positive samples. The sensitivity the developed LAMP was1000times higher than thatof conventional PCR, with detection limit of0.186fg/ul. The whole detection process takes onlyone hour. The LAMP described in this study was proved to be a sensitive, specific and rapid wayfor the detection of SS2. It has a potential application for both laboratorial and field detection ofSS2.4. Development and application of multiplex PCR assay for rapid detection ofStreptococcus suis species and its main pathogenic serotypes1,2,7and9Five pairs of primers were designed in this multiplex PCR assay, which was based on thesequences of the species-specific gene coding for glutamate hydrogenase(gdh) of streptococcussuis and serotypes-specific genes of cps1I, cps2H, cps7H and cps9G coding for the capsule ofstreptococcus suis serotypes1(and14),2(and1/2),7and9, respectively. By optimizing the singleand multiplex PCR conditions and primers concentrations, a stable multiplex PCR assay wasestablished. The PCR assay was rapid, specific and sensitive. The multiplex PCR assay was thenapplied to the detection of61tonsils. The results indicted that the61tonsils included47tonsils ofgdh+(77.0%),9tonsils of serotype2(14.8%),3tonsils of serotype7(4.9%) and20tonsils ofserotype9(32.8%). The result proved that the multiplex PCR could be used for rapid disgnosisand epidemiological investigation of streptococcus suis.5. Development and application of an indirect hemagglutination assay fordetection of antibodies against streptococcus suis serotype2To establish an indirect hemagglutination assay (IHA) for detection of antibodies against SS2, the glutaraldehyde-fixed chicken red blood cells (CRBC) were sensitized by Capsularpolysaccharide of SS2.The best antigen concentration is2.25μg/mL. The serum is positive ifantibody≥1:16. The positive sera against Escherichia Coli, haemophilus parasuis, Enterococcus,SS1, SS7, SS9, staphylococcus aureus, Salmonella typhimurium, Pasteurella multocida showednegative reaction to the CPS antigen of SS2used in IHA. Examination of1044serum samplesrevealed that the positive ratio of antibody against SS2was61.69%. These data indicated that theIHA established is sensitive, specific for detecting antibody against SS2.