| Haemophilus parasuis(HPS)is a Gram-negative,rod-shaped,NAD-dependent bacterium that belongs to the Pasteurellaceae family.As a facultative pathogen,this bacterium can cause Gl?sser’s disease characterized by polyserositis in pigs under stress or immunocompromised conditions.This disease is associated with high morbidity and mortality,causing significant economic losses to the global swine industry.Therefore,this study aimed to screen potential virulence gene-deleted strains among the 45 strains of HPS with gene deletion and construct corresponding scarless gene-deletion strains for evaluation as gene-deleted attenuated vaccines.The main research findings are as follows.1.Screening of virulence gene-deleted bacterial strains through cell-based assays.We infected PAEC cells with 45 gene-deletion strains of HPS,using the highly virulent strain SH0165 as a positive control and PBS as a negative control.The cell toxicity of the45 gene-deletion strains and the expression of IL-2 and IFN-γ in cells infected by bacteria were detected.To obtain weak virulent strains with good immunogenicity and capable of stimulating high levels of IL-2 and IFN-γ expression,Selected gene-deleted strains with decreased but not extremely significant cell toxicity,and increased expression levels of IL-2 and IFN-γ(p<0.05 indicates a significant difference,and p<0.01 indicates extremely significant difference).We selected SH0165Δ2032 and SH0165Δfic,two gene-deletion strains that met the requirements,from the 45 gene-deletion strains of HPS.2.Generating scarless gene-deletion strainsA recombinant plasmid containing Methanocaldococcus jannaschii Argonaute(Mj Ago)was constructed,which significantly improved the efficiency of homologous recombination in the natural transformation of HPS.As a result,scarless gene-deletion strains of the 2032 and fic genes were successfully screened.3.Experimental study of immune protection of gene-deletion strains in piglets.SH0165Δ2032 and SH0165Δfic were immunized via the intraperitoneal route,and a second immunization was performed two weeks after the first immunization.Commercial vaccine and PBS blank control groups were used as positive and negative controls,respectively.Two weeks after the second immunization,piglets were challenged with a virulent strain of HPS SH0165.The results showed that SH0165Δ2032 and SH0165Δfic could produce positive levels of antibodies and provide solid protection for piglets.We comprehensively evaluated the immunization efficacy of the gene-deleted vaccines from the aspects of induced antibody levels,body temperature changes before and after immunization challenge,the survival rate of piglets after immunization challenge,pathological examination,clinical symptom scoring,tissue bacterial load,tissue inflammatory factors,and tissue pathology sections.Compared with the commercial vaccine,SH0165Δ2032 could induce comparable immune protection,while the protective effect of SH0165Δfic was superior to the commercial vaccine. |
