The Study Of Rabbit Bordetella Bronchiseptica Immunogenicity And Pathogenicity

The rabbit Bordetella bronchiseptica disease is a rabbit multiple respiratory diseases caused by Bordetella Bronchiseptica(Bordetella Bronchiseptica, Bb).It has become one of the most important factors that influence the development of the rabbit industry induced high infection rate, long duration carrying bacteria and difficulty treatment. The immunogenicity of Bb was studied in present study including the screening of virulent strains, establishing the challenge model,detecting the immune response and protection efficiency of whole cell inactivated antigen by using indirect ELISA method.Colonization regularities of Bb in chanllenged rabbits were also studied through TaqMan quantitative PCR methods after Bb intranasally challenge. This study provided a theoretical basis for future prevention against Bb infection in rabbits.1.The establishment of rabbit model artificially infected with the Bordetella BronchisepticaIn this study, six strains of Bb isolated and identified from different rabbit farms were used to intraperitoneal challenge experiments in ICR mice to establish the rabbits artificially infected animal model.Karber method was used to calculate the median lethal dose of Bb chanllenge in mice. Strains of FX-1had been screened as the virulent strain. Five challenge ways such as vein injection, thoracic injection, abdominal cavity injection, nose dropping and subcutaneous injection were tested for selecting the best challenge way in future experiment of rabbits artificially infected with Bb.The results showed that intravenous inoculating was a best way to challenge rabbits by observing the clinical symptoms and pathological changes as a result of chanllenge experiment. The median lethal dose of strain FX-1to40-day-old rabbits was6.61×109CFU. The establishment of rabbits artificially infected animal model laid the foundation for the subsequent Bb immunogenicity and pathogenicity study. 2. The study of Bordetella Bronchiseptica immunogenicityIn present experiments, growth curve of strain FX-1were determined. Rabbits were immunized with different doses of inactivated Bb antigen. Antibody regularities of different immunization doses were detected using indirect ELISA method as well as the pretection percentage. Logarithmic growth area of FX-1strain was4-16h, the bacterial turbidity of the stage and the linear relationship of the number of viable cells for1gCFU=1.3716OD600+6.6516, R2=0.9954. After1mL FX-1immunization, immunization antibody continued to rise from21days to105days with the titers remained at the level of213.Then the antibodies decreased from105days until to133days with the titer of211.14. After2mL FX-1immunization, antibody titers continued to rise within the first21days. From21days to133days the antibody titer maintained about21without having any downward trend. The groups (1mL) were intravenous challenged on21st day after the immunization. The protection rate of immunized group on21st day was87.50%(21/24), the death rate of the control group was95.83%(23/24). This study laid the foundation for the Bb vaccine research and development.3. The distribution regularities of Bordetella in infected rabbitsThe regularities distribution of Bb in infected rabbits after intranasal challenge were studied in this experiment. Specific primers and probes was designed according to the gene CyaA-the virulence factor of Bb, extracted Bb genomic DNA were used as template, a specificity, sensitivity and reproducibility TaqMan fluorescent quantification method was established to explore the distribution of Bb in rabbits infected body organs. Rabbits were intranasally challenged with Bb, Different organ samples (heart, liver, spleen, lung, kidney, trachea) were collected and detected6day and15day post-challenged by using the established Bb TaqMan fluorescent quantitative detection method. The results of determination bacterial content in various organs in infected rabbits with intranasally challenged at each time point showed that bacterial infection mainly located in the trachea and lungs with significantly higher than other four organs (P<0.01). The difference of bacterial content in other4organs (heart,liver,spleen,kindy) was not significant (P>0.05). The anti-Bb IgG drop degrees of infected rabbits rose gradually. Pathogenic mechanism was studied in the present experiment providing a theoretical basis for the prevention and treatment against Bb.