Preparation And Evaluation Of Immune Efficacy Of Haemophilus Parasuis Ghosts

Haemophilus disease, also known as"Gl?sser's disease"is caused by a opportunistically pathogenic Haemophilus parasuis (Hps) which is common in porcine upper respiratory tract, it invade porcine in the specific conditions, characterized by causing serous, fibrinous, multiple serositis, difficulty breathing, high fever and high mortality rate. Now, with the development of large-scale pig farming industry, the harms of Haemophilus parasuis disease on the swine industry is expanding and it has brought huge economic losses. Now, the prevention and the control of this disease mainly with inactivated vaccine. Through the use of inactivated vaccine, some serotypes of the disease can be well controlled, but because it has more Hps serotypes and significant local features, inactivated vaccine's cross-protection is very limited or invalid for different serotypes Hps, so the development of new vaccines has become a hot field.Bacterial ghost (bacterial ghost, BG) is a empty cell without contents of the cell, retaining only the outer membrane structure which same as live cell and has high immunogenicity and the role of adjuvant. In the control of infectious disease research, the bacterial ghost attracted the attention of domestic and foreign researchers. To develop Bacterial ghost vaccine of Haemophilus parasuis, the primers were designed by lysis gene E coding sequence of phage PhiX174 provided by GenBank and the gene was amplified by PCR. After identification, the gene was cloned toλPL/PR-cI857 repressor system of pBV220 which form a temperature-sensitive lysis boxes E-λPL/PR-cI857.Then this box was subcloned into pMD18-T vector to construct punched plasmid of Haemophilus parasuis and transformed into E.coliβ2155 by Electroporation. The punched plasmid was transferred into Haemophilus parasuis serotype 5 strains by conjunctions of two bacteria. Haemophilus parasuis which contain punched plasmid grow under 28℃to logarithmic phase, and bacterial ghost of Haemophilus parasuis was successfully prepared through E gene expression which induced by 42℃. Electron microscopy results showed that the bacterial contents are released into the extracellular and form a complete Bacterial ghost. This study presents a useful data for the further development of effective vaccine and adjuvant for Haemophilus parasuis.Bacterial ghost vaccine's immune efficacy of the Haemophilus parasuis was tested by animal experiments. Healthy young pigs without Haemophilus parasuis were randomly divided into 6 groups and inoculated with different antigen in different ways: groupⅠ(bacterial ghost vaccine intramuscularly immunization), groupⅡ(bacterial ghost vaccine Nasal immunization), groupⅢ(bacterial ghost vaccine intramuscularly immunization with adjuvant), groupⅣ(bacterial ghost vaccine intranasal immunization with adjuvant), groupⅤ(inactivated vaccine intramuscular immunization), groupⅥ(control group with sterile PBS immunization). Each group inoculated dose was 5×10~9 CFU, second immunization with the same dose and way after three weeks of first immunization. Intraperitoneal injection with Haemophilus parasuis serotype 5 strains with 5×10~8 CFU 10 days later, protecting rate, pathological autopsy, antibody detection, and indirect immunofluorescence were used to evaluate vaccine effects. The results showed that intramuscular injection of the Bacterial ghost vaccine with adjuvant is better than other groups. This study provides a new approach for prevention and control of Haemophilus parasuis disease.