Isolation, Identification Of Bordetella Bronchiseptica From Rabbits And Study On Immune Effect Of Outer Membrane Protein

Bordetellosis in rabbit caused by Bordetella bronchiseptica is a serious respiratory tract diseases. It can cause lactation rabbit and adult rabbit to be dead. Rhinitis, bronchitis and pustular pneumonia can be caused by Bordetella. From 2006, all of rabbits(from 20to30 days old) of a livestock farm appeared low sprit. we can also find lots of purulent secretion in clothing hair and nasal. At last, The clinical symptoms incuding weediness, washy and snoring could be appeared.In order to study on the reason of causing respiratory tract diseases in scale rabbit farm, we isolated the main pathogen and studied the molecularbiology characterisitcs. At the same time we extracted the outer membrane protein of main pathogen and studied on immunogenicity of it. So, the results of this study lay good foundation for controlling serious respiratory tract diseases of rabbits in scale rabbit farm.The research divide two parts:Part One: Isolation, Identification And Molecularbiology Characterisitcs of Bordetella bronchiseptica from RabbitIn 2006, the 20, 30-day-old young rabbits of a large-scale farm in Shandong were diseased suddenly and were caused to death. The rabbits were deressed with most outfolw of purulent nasal in early phase, and with the process of disease, the rabbits showed the diffculties in respiration, the snore, feebleness, and diarrhea. Four bacteria were isolated, and through morphological, cultural, biochemical characteristics and seral reactional traits, the bacteria were identified as Rabbit bordetella Bronchiseptica. The G+C mol% of isolated bacteria was from 61.7% to 62.4%, which accorded to the results of Kereters(61.6-62.6%); the homology of gene sequnence of 16SrRNA from the isolated strains with the corrensponding parts of Avian bordetella Bronchiseptica recored in Genebank was 82.7%; and the homology of gene sequnence of 16SrRNA with the corrensponding parts of Avian strian and Rabbit bordetella Bronchiseptica Reference strian AF177666 was higher, which was 99.7% and 99.9%, respectively. The immunogens were prepared using the isolated bacteria, and the female rabbits were vaccinated before pregnancy. The new-born rabbits showed the high titer abtibody and the good protection against Rabbit Bordetella Bronchiseptica.Part Two: Extraction and Detect on Immune Effect of Outer protein of Bordetella bronchisepticaIn order to evaluate the immunogenicity and their protection of outer membrane protein of Bordetella bronchiseptica and effective antibody(OMP68, we established a mice model.The research improved on the method of Wooldridge in order to extract OMP of Bordetella bronchiseptica(P13, P11),and the differences of OMP were analyzed by SDS-PAGE. The P13-OMP was analyzed with Western blotting and OMP68 was gained by the way of electrophoretic elution .then we maked the effective antibodies of P13-OMP be known.Then P13-OMP, OMP68 and whole-cell bacterin (WCB)immunizing antigen was prepared 70 mice were equally assigned to 7 groups at random. They were immunized by P13-OMP, OMP68 and P13-WCB of different dose respectively. The indirect ELISA was used to detect antibody responses.80 mice were equally assigned to 8 groups at random.Group a and b were immunized by P13-OMP (25μg) respectively. Groups c and d were immunized by OMP68(25μg) ; Groups e and f were immunized by WCB(P13). Ten days after the immunizations, group a, c, e and f were challenged with P13. The other groups were challenged with P11. Then we registered the rate of protective potency and evaluated protection given by P13-OMP, OMP68 and P13-WCB[Result] Results showed that at 42th day, the titer of serum reach the top: P13-OMP(2^15.3)> OMP68(2^14.8). Then, the titer of serum started the slow-moving descent. At 70th day, the titer of serum reach the OMP68 (2^10.2)> P13-OMP (2^9.1). P13-OMP and OMP68 group challenged with P13 and P11 can be efectivly protected; P13-WCB group challenged with P13 and P11 can not be efectivly protected; the control group was died out. The P13-OMP and OMP68 of Bordetella bronchiseptica has good immunogenicity and protection, so the results of this study lay good theoretical foundation for OMP subunit vaccine.