Isolation And Identification Of Haemophilus Parasuis In Anyang Area And Preparation Of Propolis-adjuvant Inactivated Vaccine

Haemophilus parasuis is a small Gram-negative bacterium and has been assigned as a species of the family Pasteurella. It occurs sporadically among swine herds and is usually observed in three week to four month-old pigs. Clinical signs may include fever, swollen leg joints, breathing difficulties and polyserositis. Currently, has spread widely in China and worldwide, bringing serious economic losses to the pig industry. There are many serotypes of H. parasuis, but no cross-immunity between each serotype, so it is very easy for them to produce drug resistance, and there is no perfect drug could be used for the effective control of the disease.Isolation and identification of Haemophilus parasuis and resistance research in Anyang:A total of75samples including different tissues and organs were collected from three pig farms in Anyang, He’nan Province and detected by PCR assay in this study. The result showed the prevalence was53.3%in the lungs, and20%in spleen, blood, pleural and pericardial effusion, while no positive sample was found in pleural effusion. The result indicated that the lungs were the most susceptible tissues and had the highest prevalence of. The overall prevalence was32%. The serotype and drug susceptibility test were carried out, and H. parasuis was attempted to isolate from PCR-positive samples. The result showed that the incidence of successful isolation of H. parasuis was33.3%, all of them were type5of H. parasuis. The isolated5H. parasuis strains were inoculated on chocolate agar, blood agar plate, TSA, TSB agar plates respectively. The results showed their characteristics of isolates were in consistent with the biological and biochemical characteristics of H. parasuis. Balb/c mice and weaned piglets were challenged with109CFU of isolated H. parasuis respectively. And the tested of the isolates to genetic stability. The results indicated that the isolations had high virulent, we selected The strains of5th,10th,15th and20th, The results indicated typical biological characteristics and genetic stability is good and could be used for the production of inactivated vaccine strains.After incubation of the species,0.4%formaldehyde was used to inactivated and then added to Propolis Adjuvant, and the inactivated vaccine of propolis was prepared, a final concentration of3×109CFU/mL of bacteria were obtained after adding0.4%solution of formaldehyde to the isolated H. parasuis. And we found the perfect concentration of the Propolis inactivated vaccine was2mL.Research on the physical properties, safety, efficacy, validity period of the propolis inactivated vaccine,and comparison of the effect of propolis inactivated vaccine and the commercially available inactivated vaccines.the physical properties of the laboratory prepared propolis inactivated vaccine was proved to be in common with the characteristics of common inactivated vaccine. Inoculation of14-day-old piglets were carried out on the neck using intramuscular, and the immunization dose is: Single dose, Repeat Single dose and overdose.These results indicated that the propolis inactivated vaccine have good safety.Comparison of the effect of propolis inactivated vaccine and the commercially available inactivated vaccines:propolis inactivated vaccine and commercially available inactivated vaccines were immunized for40pigs. The results showed that after inoculation, the25-day-old piglets were6.49kg, and is higher than those inoculated with the commercially available inactivated vaccine (6.21kg) and the control group (6.10kg). The survival rate of25-day-old to70-day-old of pigs was97.5%, which is higher than the commercially available inactivated vaccine (95%) and the control group (90%). The average weight of pigs inoculated with propolis inactivated vaccine was18.90kg, which is higher than the commercially available inactivated vaccine (18.50kg) and the control groups (18.10kg).The results of the individual weight, from the data analysis is P<0.05.The experimental results showed that propolis inactivated vaccine are better than commercially available inactivated vaccine.Through the research, Determine the advantages of Anyang area of Haemophilus parasuis disease epidemic strains serotype5type, and through separated strains using propolis as adjuvant preparation of inactivated vaccine can the piglets to protect, Anyang area prevention of Haemophilus parasuis disease, provides a new way.