Isolation And Mlst Study Of Haemophilus Parasuis And Construction Of Aroa Gene Suicide Plasmid

Haemophilus parasuis is a member of the family Pasteurellaceae and the causative agent of Glasser’s disease in pigs, which is pathologically characterized by fibrinous to fibrinopurulent polyserositis and polyarthritis.Hps is a kind of NAD-dependent, non-motion and Grma-negative bacterium. It belongs to the Pasteurella and the genus Haemophilus. The hig prevalence of Hps in intensive pig farms has become the main cause for the high mobidity and mortality of young pigs.Three Parts have been ineluded in this study:1. The operation of pure culture, dye and observation, and biochemical experiment of17isolates of Hps from14pig farms of Guangdong were investigated. The results showed that the cultural characteristics, patterns of structure and basic biological characteristics of the20strains are basically consistent with the published information of Hps.16s rRNA-based PCR confirmed the isolates and demonstrated that the bacteria are assigned to HPs. The results indicated that Hps is prevalent in Guangdong Province. We also studied serotypes of these17H. parasuis,then found that the dominant serotype in Guangdong region is still popular of type5, having8isolaters,47.0%of the total number of strains;followed by serotype8, a total of5strains,29.4%of the total number of strains, but it is puzzling that the previous literature reported that8-type strain, but all of the isolates in this experiment are from Glasser clinical symptoms of pig body, also needs further validation.2.21isolates of Haemophilus parasuis from Guangdong region was genotyped by MLST. And results showed that Haemophilus parasuis of Guangdong region of seven housekeeping genes have different degrees of variation, the highest rate of6pgd variation100%, the average variation of seven genes was52.4%, each bacterium produced a new ST type, the variation rate of77.3%. Gene an average distance of0.732, the IA value of0.824, consistent with previous findings. BURST analysis of the21allele type and ST type of bacteria as well as published by the Hps the MLST database116ST strains and found that these21strains were divided into10groups, there are several strains of bacteria and far in Europe isolates and the Americas have a genetic relationship, and even isolated from a pig farm the same pig isolates, genotype differences prove Haemophilus parasuis high prevalence of the mutation rate is also high. The last of these MLST genotypes and serotypes of21strains, the results validate the previous conclusion, namely, the strain MLST genotypes and serotypes is no direct correspondet ce between.3. The H10strain of Haemophilus parasuis was isolated from piglet’s brain tissue with severe meningitisin. It is serotype5with very strong virulence. So H10is a very representative strains. By PCR amplification of the aroA gene of the bacterium, and through a series of cloning with the suicide nature pEMOC2connection constructed from the molded plasmid pEM△aroA the foundation for the follow-up of the aroA gene knockout.