| Porcine contagious pleuropneumonia(PCP)is a highly contagious disease caused by actinobacillus pleuropneumoniae.At present,the prevalence of actinobacillus pleuropneumoniae in foreign countries is mainly caused by serum type 2,while the main domestic type is mainly serum type 7.Moreover,it has been found that serum type 12 porcine pleuropneumoniae is more prevalent in some areas of China.Nowadays,there are mainly serotype inactivated vaccines of 1,2,7,and 1,3,and 7,actinobacillus pleuropneumonia on the market,however there is no corresponding vaccine for the currently more popular serotype 12.Therefore,serum 2 type QS-1 strain,serum type 7 NJ-2 strain and serum type 12 AH-18 strain with good stability,antigenicity and pathogenicity are filtered out by the preliminary laboratory as strains for preparing vaccines.The main experiments in this study are as follows:(1)The OD600 values are determined by bacterial counting method and 600 nm wavelength,and the growth curve characteristics of the three vaccine strains are determined.(2)Formaldehyde is added at a concentration of 0.1%,0.2%,and 0.3%after mixing with TSB-YE,and inactivated at 3 h,4 h,6 h,10 h,and 14 h after inactivation.Then test to screen out the best inactivation time.(3)Three vaccine strains are set up with 6 different concentration gradient groups,and PBS control group is set up.Each group of 10 6-week-old SPF Kunming female mice,each mouse attacked 0.2 mL of the corresponding concentration of bacterial liquid.The half-lethal dose of mice is calculated based on the death of mice after challenge.(4)The vaccine strain is inactivated in large quantities in vitro,inactivated,centrifuged,washed with PBS,adjusted for bacterial concentration as a vaccine stock solution,1 part of aqueous phase,three parts of oil compared to the case of seedlings,through finished product inspection,sterility test and safety test The prepared vaccine is evaluated for compliance with the standard.(5)In order to compare the immune effects of the prepared trivalent inactivated vaccine(8801 vaccine)and the commercial inactivated vaccine(1,2,7 serotype),150 are randomly divided into 10 groups,15 in each group,of which 1-group 3 is the 8801 immunization group;4-6 group is the commercial immunization group;7-9 group is the PBS control group;and the 10th group is the blank control group.Subcutaneous multi-point injection is used for one immunization and secondary immunization.After 14 days of secondary immunization,the strains of actinobacillus pleuropneumoniae 5 LDso,strain QS-1,strain NJ-2 and strain AH-18 are challenged respectively.In addition,the effects were evaluated by antibody titer detection,protection rate,organ tissue count and pathological changes.The results showed that the QS-1 strain has the highest bacterial viable count at 4 h,and the OD600 value is 0.41;the NJ-2 strain has the highest viable count at 10 h,the OD600 value is 0.78;the AH-18 strain has the viable count at 10 h.The highest,OD600 value is 0.91,and the three vaccine strains has more viable bacteria at this time.It is screened by formaldehyde inactivation conditions that the formaldehyde concentration is 0.2%,it is inactivated at 37 0 C for 1 h at 150 r/min,is suitable for inactivation of the three strains.Moreover,the QS-1 LD50 is 3.35×108 CFU/0.2 mL,the NJ-2 LD50 is 3.49×108 CFU/0.2 mL,and the AH-18 LD50 is 1.06×109 CFU/0.2 mL according to the modified kous method.After the finished product inspection,sterility test and safety test,the prepared APP trivalent inactivated vaccine meets the standard.In terms of antibody titer,the titer of the two immune antibodies is increased once more than once,but the commercial vaccine did not produce antibodies against the coated type 12 antigen,and the antibody titer did not increase.In terms of protection,the QS-1 strain is challenged,and the immunoprotective power of the 8801 vaccine group,the commercial immunization group,and the PBS control group are 70%,80%,and no protective force respectively;the NJ-2 strain is challenged,8801 The immunoprotective power of the vaccine group,the commercial vaccine group and the PBS control group are 80%,70%and non-protective respectively;the AH-18 strain is challenged,and the 8801 vaccine group is 80%.However,the commercial vaccine group and the PBS control group are used with no protection.In terms of the number of bacteria,QS-1 and NJ-2 strains are challenged.The number of bacteria in the lung,liver,spleen and kidney of the 8801 immunized group and the commercial immunized group is not significantly different(P>0.05),however,there is a big difference in the control group(P<0.01)with PBS.The AH-18 strain is also challenged,and the number of bacteria in the lung,liver,spleen and kidney of the 8801 immunized group and the commercial immunized group and the PBS control group are significantly different(P<0.01).In terms of pathological changes,it can be found that QS-1 and NJ-2 strains are attacked.Moreover,there are no serious pathological changes in the organs of the 8801 immunization group and the commercial immunization group.In addition the AH-18 strain is challenged,and the pathological changes of the organs in the 8801 immunization group are not obvious whlie the commercial immunization group has serious pathological changes.The trivalent inactivated vaccine prepared by our laboratory has immune effect on serum type 2 QS-1 and serum type 7 NJ-2,and also has immune effect on serum type 12 AH-18,which can resist APP infection to a certain extent.It has good protection against the 2,7,12 porcine infectious pleuropneumonia which is currently more popular.This provides a scientific reference for the development of vaccines against epidemic serotypes and the prevention of APP diseases. |
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