Isolation And Identification Of Pasteurella Multocida In Swine And Study Immunogenicity Of PMT Toxin

Pasteurella multocida is an important pathogenic bacterium of livestock and birds, which belongs to Pasteurellaceae. Pm can cause Swine infectious atrophic rhinitis and Pneumonic pasteurellosis. Both diseases have brought with heavy loss of husbandry industry.Swine infectious atrophic rhinitis (AR) caused by Bordetella bronchiseptica (Bb) and toxigenic Pasteurella multocida (T+Pm) is a common disease of pigs. Vaccination is an important way for its control and prevention. The bivalent vaccine contained inactivated Bb and toxin purified from D-type T+Pm showed better immunity effect than the conventional inactivated vaccine or the vaccine contained only toxin. But the procedure of PMT toxin purification and inactivation is not mature. So it is difficult to produce toxin by scale. But with the development of molecular biology, it is possible to express truncated PMT toxin and use the expression product make vaccine by ways of molecular biology.In this study,1140bp fragment of toxA Nterminal gene of T+Pm was cloned into prokaryotic expression vector called pET28a, formed a recombinant plasmid named pET28a-toxAN. pET28a-toxAN was expressed efficiently after inducing in Escherichia coli. The molecular weight of the fusion protein was about 45KDa. Western blot confirmed that this protein could specifically react with antiserum against Pasteurella multocida. The fusion protein and inactivated HH-0809 emulsified with white oil in equal volumes to get a vaccine. We test its safety in mice piglets and pregnant sows. We also test its protection efficiency in mice and piglets. At the same time we isolated and identified Pasteurella multocida from lung, kidney, heart, liver, brain, spleen and other organs in sick pigs.The main contents are as follows:1. Isolate and identity Pasteurella multocidaIn 2010, we isolated doubt Pasteurella multocida from lung, kidney, heart, liver, brain, spleen and other organs in sick pigs. Then we purified the doubt Pasteurella multocida, progressed biochemical reaction and PCR identification with purified bacterium. We have isolated 148 strains Pasteurella multocida from 530 clinical samples from sick pigs. Among 148 isolates, sixty-eight were non-toxigenic Pasteurella multocida of serogroup A, seventy-four were non-toxigenic Pasteurella multocida of serogroup D, three were toxigenic Pasteurella multocida of serogroup D, three were non-toxigenic Pasteurella multocida which could not identified serogroup.2. Study the immunogenicity of the PMT toxin(1)The primers of immunogenic fragment of HN-13 were designed according to the N end fragment of toxA gene (GenBank:AF240778), constructed the recombination plasmid named PET28a-toxAN and induced to express it by IPTG. Western blot confirmed that this protein could specifically react with antiserum against T+Pm. We purified the product.(2)We has largely cultivated Bb(HH-0809) on TSB medium and harvested it then counted it. After inactivation and enrichment, bacterial cells were mixed with equal volumes of purified rtoxAN protein, then the mixture was emulsified them with a certain proportion of white oil by organization stamp mill. In the vaccine, there were 2.0×1010 CFU Bb and 100μg rtoxAN protein per ml.(3)The BALB/c mice showed normal foraging and growth with no abnormal reaction after vaccination. Thus, this vaccine is proved to be safe for mice. Immunization and challenge experiment was applied to test the protection efficiency. The whole 32mice were divided into two groups on average randomly, all mice in group 1 were immunized with the vaccine, the group 2 injected with PBS as a contrast. There will be a boost on 14th day after the first one. Mice were blooded by tail vein on day 14 and 28 after the first immunization, to detect antibody. Two weeks after the second immunization, half mice of each group were challenged intramuscular with 6μg of PMT toxin, the other was challenged intranasally with 5.0×106CFU (10×LD50) of HH-0809 in 50μl PBS. Observing the dead condition, the results showed the vaccine could elicit quite antibody of IgG against Bordetella bronchiseptica and rtoxAN; The vaccine have a protective efficiency of 100% against Bordetella bronchiseptica and 87.5% against PMT toxin, but the mice in contrast group were all died.(4)After inoculated with 4ml vaccine, the piglets and pregnant sows showed normal foraging and growth with no abnormal reaction after inoculated with the vaccine. The pregnant sows showed no records of stillbirth and abortion. So this vaccine is safe for piglets and pregnant sows.The whole 15 piglets of 7 days were divided into three groups on average randomly, all pigs in group 1 were injected with Bb-toxAN vaccine, the group 2 were injected with commercialization vaccine which import from other country, the group 3 injected with PBS as a contrast. There will be a boost on 14th day after the first one. Pigs were blooded on day 14 and 28 after the first immunization to detect antibody. Two weeks after the booster immunization, all pigs were challenged intranasally with 3.0×109 CFU of HH-0809 in 2mL PBS, three days later, all pigs were intramuscular with 1.2mg of PMT toxin. The results showed these two types of vaccine could elicit high antibody of IgG against Bordetella bronchiseptica, but only the Bb-toxAN vaccine could elicit quite high antibody of IgG against rtoxAN. The neutralization test showed these two vaccines could elicit quite high neutralization antibody. After challenged, these two vaccines have a protective efficiency of 80% against Bordetella bronchiseptica and PMT toxin.