Construction Of Haemophilus Parasuis Mutant And Research On Mutant's Biological Characterization

Haemophilus parasuis (Hps) is the etiological agent of polyserositis and polysynovitis (Glasser's disease), one of the new infectious diseases in swine. Hps can infect pigs at age of from two-week to four-month old, resulting in large economic losses in pig industry. Fifteen serotypes and no cross-protection between Hps serotypes have been documented, hampering development of vaccine with cross-protection. Although some molecular studies are in progress, the function of some important genes was still unclear. Construction of mutants with different phenotypes will pave the way to understand the function of important Hps genes.The transposon technology has three unique features: (1) easy to identify the mutants based on antibiotic resistance; (2) there was a known DNA fragement in the muton so the location of the inserted gene could be identified by molecular hybridization; (3) the mutant has polarity, so that the mutated gene which was in the downsteam of the same operon had lost the function. Therefore, the transposon technique has been used in gene functional analysis.In this experiment, chromosomal DNA of the Hps serotype 5 was mutated in vitro by using Tn7 transposon, followed by electrotransformation with wild type Hps strain to yield mutants by homologous recombination. From 543 transformatants, twenty mutants were obtained through screenning with the Kan^r phenotype and amplification of the inserted sequences. Mice were used to evaluate the mutant's virulence, some attenuated strains were found while some strains with increased virulence were observed. One mutant (No.173 strain) which completely losted virulence to mice was isolated and its LD₅₀ was 2.93 × 10⁹CFU. Compared with the parent strain, the virulence of the mutant greatly decreased. Mice were immunized with mutant at concentration of 9.5 ×10⁸CFU, and challenged with wild type serotype 5 and serotype 4 strain two weeks post immunization.The fact that the immunized mice had gained some protection against wild type Hps challenge indicated the potential of the attenuated strain in vaccine research.