| Swine infectious atrophic rhinitis (AR) is a kind of respiratory diseases in pigs, mainly caused by D-toxigenic Pasteurella multocida (T+Pm) and Bordetella bronchiseptica (Bb) only or co-infected in pigs. This disease causes significant economic losses in the swine industry worldwide, as it results in serious atrophy of the turbinate bones, inefficient feed conversion and reducing growth rates of the swine. Filamentous hemagglutinin (FHA) and Bordetella pertussis adhesion (PRN) of Bordetella bronchiseptica is considered to be the most important adhesion factor and antigen composition; T+Pm major pathogenic-virulence factor is skin necrosis toxin (DNT) encoded by the toxA gene. Studies have shown that C-terminal catalytic sites and N-side adhesion sites of the toxA gene have great immunogenicity.On the basis of attenuated Salmonella and attenuated Bordetella as the carrier, we have already restructuring toxA gene into QH0814AaroA, recombinant vaccines QH0814AaroA/toxAC, QH0814AaroA/toxAN.And we also already restructured toxA gene/FHA-PRN gene into C501, recombinant vaccines C501(PYA-F1P2) and C501(PYA-toxA).In this study, as candidate vaccines, the four swine atrophic rhinitis vaccine of genetic engineering restructuring strains were inoculated weaned piglets in different immune pathways. The comprehensively effection of this4vaccines were evaluated, in order to filter out a better genetically engineered vaccine for prevention and control of swine atrophic rhinitis and purification to support programs and data. The main contents are as follows:1. Process optimization of freeze drying for genetically engineered vaccine strainsSkim milk was added into’5%sucrose solution to the concentration of10%,12.5%,15%, respectively, to be lyoprotectant. Then with the lyoprotectant vaccines were pre-freezed3-5h respectively at-40℃. The survived strains were accounted in plates to compare the survival rate between the same batch of vaccines with different concentration of lyoprotectant, and between the same batch of vaccines with one concentration of lyoprotectant freezed in different time. The result shows, as lyoprotectant, the best concentration of skim milk was12.5%in5%sucrose solution. In these condition the survival rate can be up to84.3%, the number of vaccine strains survival after freeze drying reached5.22x1010CFU/ml.2. The safety of recombinant vaccine strainsThe four recombinant strains were were grouped into3candidate vaccine: C501(PYA-F1P2)&C501(PYA-toxA), QH0814AaroA/toxic, QH0814AaroA/toxin. Each group was inoculated into28-day-old weaned piglets in the best immunization dose. Then the temperature, mental status, disease infection after immunization of pigs were observed and measured, and the average daily gain of piglets was calculated. The results showed that the vaccinated pigs were in good health, have no significant adverse reactions, the average daily gain was not different significantly, and compared with the control. Therefore, the candidate vaccines were saving to piglets. In addition, piglets were immunities by intranasal and intramuscular with each candidate vaccines, and then nasal swabs, stool and shed water samples were collected everyday. Samples were processed for PCR amplification. The results showed that the recombinant strains can not be detected until seventh days in nasal swabs of pigs immunized by intranasal, and those strains can be detected all the time, in nasal swabs of pigs immunities by intramuscular and in stool and sheds water samples of each group. Therefore recombinant vaccines survived only in the short-term nasal colonization, not to release in the surroundings.3. Immune effectiveness of Recombinant vaccine strains on pigs28-day-old weaned piglets were inoculated with3group recombinant vaccines, respectively, in the best immunization dose; Second immunity was carried out after14d. Then nasal swabs and and blood samples were collected to detect level of the nasal mucus antibody IgA and serum antibody IgG to T+Pm and Bb. The results showed that the levels of T+Pm and Bb antibody increased significantly after vaccination, while the control group were still negative.4. Determination of the optimal immune doseEach group of the four recombinant strains was diluted into different gradient dose to inmune28-day-old piglets by intranasal and intramuscular immunization, respectively. The nasal swabs and blood samples were collected at7d,14d,21d and28d after immunization, to detect the T+Pm and Bb antibody levels by ELISA; to measure temperature and to observed physical condition of the immune piglets within a week. The results show that the best immunizing dose of C501(PYA-F1P2)&C501(PYA-toxA) is5×lO9CFU/head, by neck intramuscular immunization; the best immunizing dose of QH0814AaroA/toxAC and QH0814AaroA/tox-AN is6×lO9CFU/head, by intranasal immunization; and3×109CFU/head, by neck intramuscular immunization.5. The comparative tests between the different recombinant vaccines and imported vaccines, bivalent whole cell inactivated vaccine.As compared with imported vaccines and inactivated whole bacteria seepage, recombinant vaccines were used to inoculate to28-day-old weaned piglets in the best immunization dose, and body temperature and weight of pigs were measured, the situation of intake, using of the other vaccines and drugs and the incidence were observed and record. Average daily gain and feed conversion of piglets immunized were calculated. The results showed that body temperature of the piglets after immunization with the inactivated vaccine had slightly elevated within24hours, the other vaccinated pigs were all in good health. The T+Pm and B antibody levels in nasal and serum of piglets in recombinant vaccine groups were slightly lower than those of piglets in imported vaccine group and whole-cell inactivated vaccine group. And the difference of average daily gain of the piglets between each group is not significant. Compared with the non-immunized control group, feed conversion ratio and the cost of medicines is slightly lower. The results showed that the effects of recombinant vaccines were similar with other vaccine groups, and they have a certain clinical effect. |
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