Preparation And Application Of The Oil Emulsion Inactivated Vaccine Of Pasteurella Multocida

The research slected a strain isolated from the sick ducks of farms in Sichuan province of China. The strain was diagnosised to be Pasteurella multocida (PM) by isolation and identification of bacteria, then was used to be as a vaccine-making bacterium to prepatation oil emulsion inactivated vaccine of Pasteurella multocida by inactivated with formaldehyde and high-speed mixing with mineral oil emulsion adjuvant. The research detected the seurity of the vaccine through the physical properties testing of the vaccine and the sterility testing of vaccine. The research studied the immunogenicity of the vaccine by the immunity efficiency test of immuned ducks which were challenged by homologous of PM. The research used Indirect Enzyme-linked Immuosorbent Assay (ELISA) to test the changes of PM’s antibody responses in test ducks and the ducks of farm inoculated with vaccine.1. Isolation and identification of the bacteria:The research select a particular strain of the sick duck of large-scale duck farms, picked the liver, blood of heart were inoculated on the blood agar plates to culture. Through the cultural characteristics of bacteria, Biology, API 20NE biochemical test stip and 16s PCR rRNA gene identification results showed that the strain was pasteurella multocida. Biolog antomated microbial identification system identification results showed that the strain was Pasteurella multocida subsp, multicida. The PCR of capsular genotyping indentification results show the strain belongs to A menrbane type of Pasteurella multocida.2. Physical properties and sterility testing of vaccine:the vaccine physical proterties tests through examine formulations, viscosity, stability of the appearance vaccine. The results showed that the vaccine meets the basic criteria of oil adjuvant vaccine prepared. Then take a small amount vaccine on blood agar plate, TSA plate, each per 0.2ml, observed 24～72h, the results showed no bacterial growth on the medium within 72h to prove that the vaccine sterile test was qualified.3. Vaccine safety testing:the physical properties testing of the vaccine and sterility testing were qualified, than passing on the 30-day-old ducks intramuscular injection 1ml each and observed 7 days, the results showed that the ducks did not indicate any adverse reactions after vaccination.4. Challenge protection test:oil vaccine sterility and safety testing were qualified, than 30-day-old ducks were picked which each intramuscular injected 1ml vaccine. After the immune 14d,21 d conducted challenge protection test respetively. The PM challenge contained approximately 4.5×108 CFU in 1 ml. The homologous challenge with PM demonstrated that immunoprotective rates were 80%,100% recpectively when 14,21 days post vaccinated with PM inativated vaccine in oil adjuvant.5. Indirect ELISA antibody levels changes:The ducks Pasteurella multocida bacteria ultrasonic lysis of whole antigen coating antigen, determine the best work concentration of antigen and HRP- goat anti-duck IgG by the square method, the results show 1.1μg/ml, 0.33μg/ml respectively. After the sterile and safety testing of the vaccine were qualified, picked six 30-day-old ducks each intramuscular 1mL vaccine. The ducks post vaccination 0,3,6,9,12,15,21,28,35,42,49,56,63,70,77,84,91,98, and 104 days detected the serum antibody titers. The results showed that 6 days after vaccination PM antibody were significantly higher than control group, then increased gradually, specific antibody peaked 28 days post vaccination in titer, then antibody levels decline gradually, till 91 days days post vaccination, the titer is similar or significantly higher than the same group’s 12th day’s level,104 days post vaccination remains able to detect the presence of antibodies titer.6. Practical application of the vaccine:The vaccine was proved to be safe and effective by laboratory testing. And the vaccine production meets the basic requirements of the system. Then a large number of the prepared vaccine inoculation particular 30-day-old adult duck for the application, observed the clinical effects and the serum samples were taken back to the lab to detect the antibody levels. The results showed that the duck after immunization vaccine were significantly reduced the incidence of the disease, the higher antibody levels produced by the ducks, and can last longer. The ducks farm post vaccination can effectively control the epidemic of the disease.