| Porcine Contagious Pleuropneumonia(PCP)is a common respiratory infectious disease,which is caused by Actinobacillus pleuropneumoniae.Actinobacillus pleuropneumoniae spread fast and widely,that is seriously hindered the economic development of the industry of pigs.The traditional inactivated vaccine is used frequently.However,inactivated vaccine has low immunity and cross protection.Therefore,it is very important to develop a new vaccine to prevent APP infection.Bacterial ghost(BG)belongs to a new type of vaccine,which has a complete structure of the outer membrane and the empty cell body of the cell.A novel preparation method for APP ghost by using the minimum inhibitory concentratin(MIC)of sodium hydroxide(NaOH)was established.Bacteria was cultured for 12h,24h,48h and 72h respectively and each incubation period of bacteria concentration was adjusted to 1×10~8 CFU/mL and 1×10~9 CFU/mL to determine the MIC of NaOH against APP serotype 1 strain.Four kinds of culture time and two kinds of the concentration of APP serotype 1 strain were mixed corresponding MIC of NaOH,then were incubated at 37?for 75min.The morphology and lysis of bacteria were observed to determine the conditions for the preparation of bacteria.The results showed that MIC of NaOH had same effects on the different concentrations of bacteria in different cultures.It was found that BG had no live bacteria and lysis rate of APP serotype 1 strain reached 100%.After the scanning electron microscope,the cell membrane of the bacteria was observed,and the cell morphology was normal.The hole of outer membrane of BG was significantly and the cell morphology of the BG was normal.To evaluate the immune effect of the BG by animal experiments(BALB/c mice and pigs).Each mouse was immunized 2 times with 14 days interval and immunized vaccine with 1×10~8 CFU by intraperitoneal immunization.228 mice were divided into 19 groups.Group A1(BG of APP serotype 1),A2(Inactivated vaccine of APP serotype 1)and A3(PBS control)were challenged by APP serotype 1 strain shope.Group B1(BG of APP serotype 1),B2(Inactivated vaccine of APP serotype 1),B3(Inactivated vaccine of APP serotype 2)and B4(PBS control)were challenged by APP serotype 2 strain S1536.Group C1(BG of APP serotype 1),C2(Inactivated vaccine of APP serotype 1),C3(Inactivated vaccine of APP serotype 3)and C4(PBS control)were challenged by APP serotype 3strain S1421.Group D1(BG of APP serotype 1),D2(Inactivated vaccine of APP serotype1),D3(Inactivated vaccine of APP serotype 5)and D4(PBS control)were challenged by APP serotype 5 strain K17.Group E1(BG of APP serotype 1),E2(Inactivated vaccine of APP serotype 1),E3(Inactivated vaccine of APP serotype 7)and E4(PBS control)were challenged by APP serotype 7 strain WF83).The weight of mice in each group were measured from 0 day to 28 day of the first immunization,(a total of 5 times,each time interval 7 day).Indirect ELISA detected the specific antibody level and the levels of cytokines(IL-2,IFN-?)in all groups of mice(serum samples were collected from 14 day to 28 day,3 times,each time interval 7 day).After 14 days of challenged,we observed and analyzed the pathological changes of lung tissue.The results of antibody detection showed that the level of specific antibody of APP serotype 1 in group of BG and inactivated vaccine of APP serotype 1 was significantly higher than control group(P<0.01).The level of specific antibodies of APP serotype 3,5,7 were also detected in group of immunized BG,which was slightly lower than group of inactivated vaccine,but significantly higher than group of PBS control.IFN-?and IL-2expression levels of pigs were detected that in the serum of immunized with BG were significantly higher than that of control group at 28 days of the first immunization(28 days of the second immunization).After challenged,the survival rate in group of BG and inactivated vaccine of APP serotype 1 was 100%.The group of inactivated vaccine of APP serotype 2,3,5,7 showed good protection against the infection of homologous strain,the protective rate was 75%,33.33%,25%,50%,respectively.The group of BG showed obvious cross protection to APP serotype 3,5,7 strains,and the protective rates were41.7%,66.67%,respectively.The results of pathological examination of lung was normal in the group of BG.Screening 9 pigs of ApxIV antibody negative were randomly divided into 3 groups:Group 1(BG of APP serotype 1);Group 2(Inactivated vaccine of APP serotype 1);Group3(blank control).Each pig was immunized vaccine with 4×10~9 CFU by muscle immunity and were immunized 2 times with 14 days interval.The blood samples were collected from the jugular vein(from 7 day to 35 day,4 times,each time interval 7 day).Detection of porcine serum ApxII antibody was detected by indirect ELISA and porcine whole blood cytokines(IL-2,IFN-?)was detected by fluorescence quantitative PCR.All the groups were challenged by APP serotype 1 at 14 days of the second immunization.After challenged,we collected the nasal swab,measured temperature of body,recorded number of the death of pigs.Dissected and analysed lung lesions and carrier status.The results of antibody detection showed that the levels of Groups 1 was significantly higher than the blank group.the levels of cytokines(IL-2,IFN-?)of Groups 1 was significantly higher than the other two groups.After challenged,the Groups 1 had no symptoms and the temperature of body was normal.The results of PCR detection of nasal swabs and lung tissue were negative.The results of Lung biopsy showed that all pigs in Group were normal,the Group of 2 and 3 had inflammatory cell exudation and hemorrhagic lesions.The incidence rate of the Group 1 was lower than Group 2 and Group 3.It is a simple and easy method for prepare the BG by NaOH.It shows a good protective effect on mice and pigs.This new preparation method for APP ghost is safe and effective.It provides the foundations for studying ghost vaccine of APP. |
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