| Streptococcus suis is an important swine pathogen and 33 S. suis serotypes have been identified based on differences in their capsular antigens. S. suis serotype 2 (S. suis 2 or SS2) is the most frequently isolated serotype worldwide and can cause meningitis, septicemia, arthritis, endocarditis, pneumonia and even acute death in swine. In addition to causing disease in pigs, it is also an important zoonotic agent for humans in contact with diseased pigs or their products. In 1998, SS2 outbreaks in humans in Jiangsu, China infected 25 people of which 14 died. Between July and August 2005, an outbreak of SS2 infection occurred in Sichuan, China, involving 204 cases, of whom 38 died.Due to high economic losses as well as threat to human life, the study of SS2 pathogenesis and the development of new vaccines is crucial for prevention and control of this disease. In this study, a micorarray was used to detect the differences in gene expression of a SS2 two component signal transduction (TCTS) 1660hk/rr gene deletion mutant and the wild type organism. Results show that totally 249 genes had differential expression patterns. The Endothelin-converting enzyme 1 gene (ecel) was found to be the most downregulated. The ecel gene deletion mutan was constructed by a homologous recombination based method. In vitro and in vivo data demonstrates that the ecel deletion mutan was attenuated. In order to find an effective subunit vaccine the 6-phosphogluconate-dehydrogenase (6pgd) was cloned and expressed. Sequencing this gene found that it has high similarity at the nucleotide level with other bacterial, for example the S. pneumoniae. The recombinant 6PGD protein inhibits the SS2 adhesion to host cells. Immunising animals with the recombinant 6PGD protein provided 80% and 50% protection of mice and swines respectively against SS2 high virulence strain infection. The main research contents include:1. Gene different expression of 1660hk/rr deleted mutant strainIn this study we used a microarry which was made by using available the SS2 05ZYH33 strain whole genome sequence. To compare with the wild type SS2 strain SC19 gene expression, the TCTS 1660hk/rr gene deletion mutant shows that 89 genes were down-regulated 2-fold and 41 genes were upregulated 2-fold. Of the 89 genes,25 were downregulated more than 5 fold and 4 were upregulated to the same level. In order to obtain more information, we altered the cut off gene expression change level to 1.5 times. In total 152 genes were down regulated and 99 were upregulated accounting for 11.4% of all the ORFs within the SS2 genome.2. Construction of ecel mutantPrimers were designed to amplify 1034bp of the ecel gene upstream and 1041bp of the downstream sequence based on the SS2 strain 05SSU0153 ecel gene sequence. The two PCR products were purified and cloned into a pMD18T vector resulting in the construction of pMD18T-PS and pMD18T-PX. pMD18T-PS was digested with HindⅢand Sal I, pMD18T-PX digested with Sal I and EcoR I. The suicide vector pSET4s was digested with the same enzymes gradually, after the ligation was completea suicide plasmid, pSET4s-0153 was obtained. The resulting suicide recombinant plasmid pSET4s-0153 was transformed by electroporation into the SS2 wild strain SC19. Through a double feature selection of antibiotics and temperature, the 05SSU0153 gene deletion mutant strain was obtained and namedΔ0153. The gene deletion mutant was confirmed by PCR3. Characterization of the SS2 mutantsHeredity and stability of the SS2 ecel gene deleted mutantΔ0153 was confirmed by continuous passage culture and PCR this confirmed that the mutation was stable. The growth rate of the ece1 deletion mutant was compared to the wild type strain SC19 by measuring OD600 at different time points. The results indicated no difference between parent strain SC19 and mutant strainΔ0153.4. Virulence in pigsIn order to test the virulence capacity of mutant strainΔ0153, pigs were intravenously infected with three different doses 5×105cfu/ml, 1×106cfu/ml and 5×106cfu/ml of wildtype (SC19) and mutant strainΔ0153. Clinical symptoms and survival were observed and recorded daily after the initial infection. The pathological anatomy and pathology were also used to evaluate the virulence of different strains of SS2. The results showed that compared with wildtype strain SC19, mutant strainΔ0153 post-infection showed mild clinical symptoms of disease and the death rate decreased significantly. All these results suggested the mutant strain A0153 significantly attenuated when compared to the wildtype strain SC19.5. Expression 6PGD protein and the bacterial inhibition testThe 6pgd gene of SS2 was amplified by PCR withprimers designed according to the published Genbank SS2 6pgd gene sequence and cloned to the expression vector pET-28. The plasmid was transformed into E. coli expression strain BL21 (DE3) and induced by IPTG. Recombinant 6PGD protein was isolated using histidine purification reagents and confirmed by western blot. The ability of the 6PGD protein to inhibit SS2 virulence by monitoring adhesion to host Hep2 and HeLa cells was tested. The results showed that after adding the 6PGD protein, SS2 adhesion to Hep2 and HeLa cells was decreased by about 72% and 66%. This may indicate that 6PGD protein may play as an adhesion factor in SS2. 6. 6PGD protein protection in miceTwenty 6-week-old Balb/c female mice were randomly divided into two experimental groups of ten to evaluate the efficacy of the 6PGD protein as a subunit vaccine. Group one was immunized subcutaneously with 20 mg of purified r6PGD mixed with 100 mL complete Freund's Adjuvant on day 0 (primary immunization). At days 14 and 28, the mice were immunized with 20 mg of r6PGD protein emulsified with 100 mL incomplete Freund's Adjuvant according to the manufacturer's protocol. The other group was immunized subcutaneously with adjuvant as a control. Ten days after the third vaccination, the animals from both groups were challenged intraperitoneally with 2.5×108 CFU per mouse of log-phase SS2 virulence strain SC19 in 0.2 mL TSB. Mice were bled at days 0,14,28 and 38, serum samples were detected by 6PGD ELISA. Clinical symptoms and survival were monitored daily after infection. The results showed that anti-6PGDantibody can be detected at day 14 and that on day 38 the specific antibody reaches a higher level. After infection, the adjuvant control mice showed significant clinical symptoms and all died in 48h. Anatomicallymultiple organ bleeding was noted and SS2 could beisolated from various organs. Some of the 6PGD protein immunized group mice showed clinical symptoms of disease but finally 80% were protected. Afternatomical examination of the surviving mice, no significant changes could be observed and the challenge organismcould not be isolated from organs.7. 6PGD protein protection in pigsTwenty 4-week-old pigs were taken from a herd that was SS2 free and were randomly assigned to two groups of 10 pigs each. Group one were immunized intramuscularly with 500 mg of purified 6PGD mixed with 500 mL complete Freund's adjuvant on day 0. At day 14, the pigs were immunized with 500 mg 6PGD emulsified with 500 mL incomplete Freund's adjuvant, according to the manufacturer's protocol. The other group was immunized with only the adjuvant as the control group. Two weeks after the booster immunization, all twenty pigs in both immunizedcontrolgroups were intravenously challenged with 1×106 CFU per pig of a log-phase culture of SS2 virulence strain SC19 in 1 mL TSB. On days 0,14, and 28, blood samples were obtained from each pig by precaval vein bleeding and serum samples were detected by 6PGD ELISA. Clinical symptoms and survival were monitored daily after infection. After 7 days observation, all the surviving pigs were killed to observe the pathological changes in the organs and also take the tissues for pathology studies. The results showed that the anti-6PGD protein specific antibody could be detected on day 14 and that on day 28 the antibody reaches a higher level. After infection, the adjuvant control pigs showed significant clinical symptoms and all died at 48h. Anatomically the animals were foundto have multiple organs with hyperemia, hemorrhage and the challenge organism could be isolated from various organs. In the 6PGD protein immunized group, some pigs had significant clinical symptoms and 20% died within 48h. Finally,50% of the immunized pigs were protected. After anatomical examination of the surviving pigs, most of the organs did not contain culturable SS2. |
