Identification And Distribution Of Putative Virulent Genes Of Streptococcus Equi Ssp. Zooepidemicus

Streptococcus equi ssp.zooepidemicus is a gram's positive group C Streptococcus, which infects a wide range of animals. It can cause meningitis, septicemania, arthritis, endocarditis and sudden death in pigs, which made substantial economic losses in pig industry and became a substantial threat to human health especially those involved in pig industry. Though several virulent factors such as M-like protein, FBPS, IgG-binding protein have been identified, the pathogenic mechanism of SEZ is still unclear. In this study, suppression subtractive hybridization (SSH) was used to analyse the genomic differences between strain ATCC35246 and ST171. Fifteen fragments of putative virulent genes were found by this method and the distribution of six fragments in other streptococcus strains were also detected.SSH was carried out between virulent strain ATCC35246 and avirulent strain ST171 in order to find the fragments of genes unique to the virulent strains. The tester (ATCC35246) and driver (ST171) genomic DNAs were digested with RsaI after extracted from the bacteria. The tester DNA was then divided into two portions, each of which was ligated with a different adaptor. The tester-specific sequences were amplified after two hybridizations and two PCRs. The PCR amplification product was cloned into PMD-18T simple vetor and transformed into E. coli TOP10 competent cells.A subtractive library including 800 clones was constructed and 296 clones were picked randomly to perform the colony PCR ,the result of which showed that these clones contained 100-2000bp inserted fragments.Fix the 296 positive amplifies PCR products onto the nylon membranes, which were then hybridized with the tester DNA and diver DNA labeled with DIG.. 20 genomic regions were absent in ST171 and the DNA sequence of these regions in ATCC35246 were determined. These DNA fragments containing putative virulent genes encoded 15 proteins, which were homology to proteins involved in some aspects of cell surface structure, molecule synthesis, energy metabolism, regulation, transport systems and some unknown functions, which were not reported before.PCR primers were designed based on six representative fragments, whose distribution in other Streptococcus strains were performed by PCR detection. The validity of PCR was proved that the result showed these six fragments could be all detected in ATCC35246 while only one fragment present in ST171. The fragments were widely distributed in other S.zooepidemicus strains and could be detected in some other Streptococcus strains. But they were not detected in Streptococcus suis type 2 strains.This study find some putative virulent genetic fragments, the results of which is helpful to the research of pathogenic mechanism of S.zooepidemicus and PCR detection of virulent strains.