Molecular Biologic Study Of Streptococcus Suis Potential Virulence Factors Secreted Nuclease A And Arg-aminopeptidas

The thrity-five different scrotypes(type1/2 and 1 through34)of S.suis have been described. Of the 35 scrotypes, type 2 is the most frequently isolated serotypes from pigs with disease, which can cause many different disease such as: meningitis, septicemia arthritis, endocarditis and sudden death in pigs or human. At pesent, a number of potential virulence factors have been identified and partially characterized, including Extracellular factor, Murimidase-released protein, Suilysin, Immunoglobulin G-binding protein, Capsular Polysaccharide, Bacterial Adherence and Fibronectin-binding protein. Now there were some potential virulence factors has been found they include S.suis Secreted Nuclease A, orf2, 38-Kilodalton Immunogenic and Protective Antigen, Glutamate Dehydrogenase, Arg-aminopeptidas, Casinse, Chymotrypsin-like, DPP IVand so on.S.suis secreted nuclease A (SsnA) activity detection showed that clinical isolates from surface (nasal mucosa or palatine tonsil) or internal (joint, brain or other internal organ) locations revealed a significant relationship between expression of nuclease and isolation from an internal site.Proteases identified as critical virulence factors in numerou microbial pathogens, which are hydrolytic enzymes catalyzing the cleavage of peptide bonds in proteins and peptides, may be a kind of virulence factors of S. suis. Indeed, these enzymes may act on a variety of host proteins including serumand tissue components thus contributing to neutralization of the immune defense system and tissue destruction. It showed that Arg-aminopeptidas(ADS) may be a larvaceous virulence factors.To establish a method for nucleotide sequencing of S.suis Secreted Nuclease A,and establish a method for detected by PCR ,the result showed this method had advantage of higher sensitivity ,specificity and stability. The S. suis nuclease activity was detected by toluidine blue agar (TBA),this method was convenient and the consequence was accuratissime, it would be usefol in activity detected.To establish a method for nucleotide sequencing of S.suis ADS , and establish a method for detected by PCR ,the result showed this method had advantage of higher sensitivity ,specificity and stability.The SsnA from the 8 Streptococcus suis serotype 2 CHONGQING isolates was amplified by PCR using 3 pairs of primer. Nucleotide sequencing and analysis results revealed that the SsnA gene is 3,126bp. The similarity of SsnA gene and amino acid from 8 CHONGQING isolates was more than 98% and 94%, respectively. The SsnA gene was detected by PCR with the expected size was 301bp and the S. suis nuclease activity was detected. The results showed that 35 pathogenic Streptococcus suis isolates from diseased pigs' viscera, 32 isolates detected by PCR were positive and 24 isolates were the nuclease phenotypes:: 10 isolates detected by PCR were positive and 8 isolates were the nuclease phenotypes in 14 detected Streptococcus suis isolates from pigs' tonsil. Thirty-eight isolates detected by PCR were positive and 32 isolates were the nuclease phenotypes in 44 detected Streptococcus suis serotype 2 isolates. SS1, SS7, SS9, SS14 and SS1/2 detected by PCR were all positive but only SS13 was nuclease phenotypes. The requirement of Ca²⁺ and Mg²⁺ for SsnA activity was determined. S.suis growth study showed that SsnA was expressed throughout all stages.The ADS from 9 CHONGQING Streptococcus suis serotype 2 isolates was amplified by PCR. Nucleotide sequencing and analysis results revealed that the ADS gene is 1,231bp. The similarity of ADS gene and amino acid from 9 CHONGQING isolates was more than 99% and 96%, respectively. The ADS gene was detected by PCR with a 237bp expected fragment. The results showed that in 35 pathogenic Streptococcus suis isolates from diseased pigs' viscera, 30 isolates were positive; 11 isolates were positive in 14 detected Streptococcus suis isolates from pigs' tonsil. SS1, SS7, SS9, SS14 and SS1/2 detected by PCR were all positive.