Characterisation Of The Haemolytic Phenotype Of Bovine Pasteurella Multocida Serotype A And Screening Of The Inactivated Vaccine Candidate Strain

The bovine Pasteurella multocida serogroup A is an opportunistic pathogen, causing bovine pneumonia. It is epidemic in china and seriously affected the development of the cattle industry. Nowadays, our country didn’t have the available vaccine, it is brought great difficulties to the prevention and control of P. multocida. P. multocida was found to have haemolytic phenotype under anaerobic conditions. But few studies about the characteristics and reasons of the hemolysis could be available.1Characterisation of the haemolytic phenotype of P. multocida and the investigation of the ahpA geneIn this study, the strains P. multocida CQ1-6(abbreviate "PmCQ1-6") were streaked onto agar plates containing10%rabbit, goat or avian whole blood and were compared the haemolytic phenotype. Cloning and blasting PmCQl-6ahpA genes, which relate with the haemolysis, and building a phylogenetic tree. The upstream regulatory sequences of the PmCQl-6were cloned and blasted. The Real-time quantitative PCR method was established for the detection of the ahpA genes expression level when cultured under aerobic and anaerobic conditions. The recombinant plasmid was constructed by inserting ahpA into plasmid pET-32a(+). The pET-32a-ahpA was transformed into E.coli BL21(DE3), and the haemolytic phenotype of the recombinant bacteria was identified. The reactogenicity and immunogenicity of the ahpA protein was tested by western blotting and immune protection test respectively. The results demonstrated that PmCQ2PmCQ4and PmCQ6lysed all erythrocytes tested. PmCQl was found to cause haemolysis on rabbit and avian erythrocytes. PmCQ5only lysed rabbit erythrocytes. PmCQ3was unable to cause haemolysis. The homology of the PmCQ1-6ahpA genes was100%, and the gene was conservative. The upstream regulatory sequences were consistent. The ahpA gene expressed only under anaerobic conditions, and the expression levels were significant differences among different strains. The recombinant bacteria exhibited a haemolytic phenotype under anaerobic conditions, but not under the aerobic conditions. Western blotting analyses and immune protection test demonstrated that the expressed protein was a good immunogen. The finding showed that the differential of hemolysis of P.multocida,.associated with the expression levels of ahpA gene. The expressed protein had a good immune activity.2Screening of the P. multocida inactivated vaccine candidate strainLD50of the PmCQ1-6was calculated by the method of Reed and Muench in mice. The LD50of PmCQ1-6in Kunming mice were3.18×105CFU,2.2×105CFU,6.54×105CFU,3.45×105CFU,3×105CFU and1.14×108CFU respectively, the virulence of PmCQ6was significantly lower than other strains. The immunogenicity was tested by cross immunization protection in mice. The result showed that the protective rates of PmCQ1-6groups were88.57%,85.71%,77.14%,80%,60%and80%, respectively. The best protective effect was PmCQl, followed by PmCQ2, PmCQ5was the worst. PmCQ2was cultured continuously. The genetic stability, including biochemistry and virulence, was analyzed in the PmCQ2of the primary,90generation and150generation. The result showed that both the biochemistry and virulence of genetic stability were good. So, the PmCQ2was screened as a candidate strain used for inactivated vaccine production.3Preparation and examination of the inactivated vaccine250mL inactivated vaccine with a density of4×109CFU/mL was obtained after a series of processes:inoculating, viable counting, formaldehyde inactivation, oil phase preparation and aqueous phase preparation. Sterility of the experimental vaccine preparations was tested by inoculating200uL of vaccine on Martin Broth Agar and then examining viable P. multocida. Safety trials of experimental vaccines were done by immunizing5mL vaccine to each experimental rabbit and observed10days. The experiment groups were immunized with0.5mL, lmL and1.5mL vaccine in rabbits respectively and given PBS alone as control group. Efficacy of the vaccines was tested after21d of immunization by1MLD dose of P. multocida A challenge of all immunized rabbits and observed daily for survival and morbidity till20d post-challenge. Cross immunization protection was to be done between bovine P. multocida serogroup A and serogroup B. Completeness of inactivation of P. multocida was ascertained by the lack of any bacterial or fungal growth on Martin Broth Agar. All rabbits used in safety trial were healthy at the completion of safety trial on10days. Rabbits in the lmL andl.5mL immunized groups showed100%protection after20d post-challenge. Based on the results obtained, the inactivated vaccine prepared in our study was safe and effective. The vaccines of bovine P. multocida serogroup A and serogroup B have cross immunization protection.