Screening Candidate Attenuated Vaccine Strains Of Haemophilus Parasuis

The Gram-negative bacterium Haemophilus parasuis（HPS）is a strictly swine pathogen.As a commensal colonizing the upper respiratory tract of pigs,HPS is also an opportunistic etiologic agent that can cause serious systemic disease,namely Gl?sser’s disease,which is characterized by arthritis,meningitisand fibrinous polyserositis.HPS is one of the most important bacterial pathogens in swine industry and it often co-infects with porcinere productive,respiratory syndrome viruse（PRRSV）and porcine circovirus（PCV）which makes relevant respiratory tract infection becomes more complicated.So far at least fifteen serotypes of HPS have been characterized,but individual serotypes show apparent differences in virulence and there is little-to-no cross-protection between different serotypes.Control of HPS infections can be achieved by use of commercial vaccine.However,the protection efficiency of inactivated vaccine is not enough,because it can only provide protection for homologous serotypes but little cross protection for other serotypes infection.The aim of this study is to screen the attenuated strain of SH0165 which can reduce toxicity and induce strong immunological protection in pigs through serial passage in vitro.And then providing material for the development of HPS attenuated vaccine.1.Continuous passage in vitro and growth characteristics.Based on serum type 5HPS virulent SH0165,the strain were cultured with BHI+NAD medium at 37℃and 40℃respectively.The growth curves of the strains SH0165,subcultured strains H-100,H-150,H-250 and H-200 at 37℃and 40℃were measured separately and their linear regression equation between the OD₆₀₀value of the above strains and the CFU/m L was obtained by the bacterial counting method.Our results indicated that the growth rates of subcultured strains were significantly faster than that of primary strains.2.Detection of passage strains.Our previous animal experiments with guinea pigs suggested that the virulence of the subcultured strains H-150,H-200 and H-250 were significantly attenuated compared with SH0165.In this study,piglets were used as the animal experiment model to further study the virulence of H₃₇-150,H₃₇-200,H₃₇-250 and H₄₀-150,H₄₀-200,H₄₀-250.The results demonstrated that the virulence of H₃₇-250,H₄₀-200 and H₄₀-250 were significantly attenuated and they could be used as candidate strains for immunogenicity test experiments.3.Immunogenicity evaluation of passage strains.The dose for both primary immunization and secondary immunization were 1×10⁹ CFU per pig,the dose for challenge is 2×10⁹ CFU;the time interval for immunization and challenge was 2 weeks.The mortality,clinical symptoms,histopathological degree and tissue load of piglets were evaluated synthetically,our results showed that the all three subcultured strains reserved potent immunogenicity and could induce piglets to induce immune protection.