Multilocus Sequence Typing (MLST) Of Haemophilus Parasuis And Identification Of Its Quorum-Sensing Signal Molecule

Haemophilus parasuis(HPS) is the causative agent of Gl?sser’s disease, with symptoms of polyarthritis, fibrinous polyserositis and meningitis, which caused high morbidity and mortality in swine herds. Although H. parasuis strains can be classified into 15 serovars by the agar gel diffusion method,a large number of isolates cannot be classified and they were identified as nontypable(NT) strains,limiting the analysis of epidemiology and population structure.Due to its ability to form biofilms, which is associated with quorum sensing system, H. parasuis colonies are resilient and exhibit high survival rates. The quorum sensing system of H. parasuis is unknown to date, so the research of quorum sensing system of H. parasuis will contribute to the understanding of the pathogenic mechanism of the bacteria.In this study, multilocus sequence typing(MLST) was used to analyze 50 H. parasuis field strains isolated from China and 15 reference strains. 26 new alleles and 29 new STs were detected to enrich the MLST databases of H. parasuis. Burst analysis indicated that H. parasuis lacked stable population structure and had high heterogeneity; there was no links between STs and geographic area. UPGMA dendrogram showed that these H. parasuis could be divided into two major groups, the isolates in group A mostly belonged to serovars that are generally thought to be avirulent or less virulent, the strains of group B tended to be virulent.CV026 and KYC55 was used as the reporter to analyze the existence of acyl-homoserine lactone(AHL) of H. parasuis, which is the autoinducer of type-ⅠQS system. The HPLC-MS-MS system was used to identify the AHLs that were produced by H. parasuis. It showed that there were two kinds of AHLs synthesized by H. parasuis. They have the same relative molecular with C10-HSL and C12-HSL respectively. Due to the rare production of AHLs, it was difficult to identify the structure of AHLs of H.parasuis. The catechin has the ability to inhibit the effect of quorum sensing system. This study shows that catechin can reduce the biofilm mass of H. parasuis by 25.4 percent.The RbsB gene deficient mutant of H. parasuis H19 and their reverant were constructed by pK18 mobsacB with homologous recombination method. Compared with wild-type H19, the obvious decrease of the ability of biofilm formation in RbsB deficient mutants were observed, the ability of biofilm formation of reverant was improved. That showed the gene RbsB might be involved in the biofilm formation of H. parasuis.