| Rabbit pasteurellosis,known as rabbit hemorrhagic septicaemia,caused by Pasteurella multocida,is an acute,septic and infectious disease.Most countries and regions around the world had reported the occurrence of rabbit pasteurellosis.The disease caused serious economic losses to the rabbit breeding industry.We established an indirect ELISA method for detecting the antibody of rabbit pasteurella multocida,and applied it to test the clinical serum samples.The results showed that the methed would provide an effective tool to detect and monitor the state of animal disease epidemic and vaccination.The main research contents are as follows:1.Establishment of indirect ELISA method for detecting rabbit Pasteurella multocidaIn this experiment,the broked rabbit Pasteurella multocida was used as a coating antigen.The concentration of coating antigenic and the serum dilution were determined by matrix method.The antigen coating time,antigen blocking time,antigen incubation time,serum incubation time,HRP-labeled antibody dilution,HRP-labeled antibody incubation time and substrate reaction time were optimized.the stability and specificity of the method were detected.The shelf life of the enzyme-labeled plate was measured.The results showed that the optimal conditions were:the concentration of coating antigen was 0.5 ?g/mL,the serum dilution was 1:100,the optimal coating condition was 37? 2 h,the best block liquid was 5%skimmed milk,the best serum incubation time was 60 min,the best dilution of HRP-labeled antibody was 1:20000,the best incubation time of HRP-labeled antibody was 1 h,and the optimal time of substrate reaction time was 10 min.The criterion was determined.The critical value of negative serum was 0.227,and that of positive serum was 0.277.The result of specific test showed that no cross reaction with positive serum of rabbit Bordetella bronchiseptica and clostridium perfringens was observed.The stability of the test plate had achieved satisfactory results.Shelf life of the test plate was three months with preservatives.It is simple to operate the method,and it is suitable for detecting large-scale clinical serum sample.Therefore,the ELISA method established in this experiment has certain clinical application value.2.Primary application of indirect ELISA method for detecting rabbit Pasteurella multocidaThe established ELISA method was used to detect Pasteurella multocida antibody level of rabbit serum samples from four different rabbit farms and analyzed epidemiological state of Pasteurella multocida.The established ELISA method was used to select sensitive rabbits for immunizing inactivated vaccines of Pasteurella multocida.And it was used to detect serum samples of rabbits immunized by Pasteurella multocida inactivated vaccines after 21 d and relations between antibody levels and mortality was analyzed.The ELISA method was also used to detect serum samples of rabbits immunized by Pasteurella multocida inactivated vaccines in immune duration and the changes of antibodies was analyzed.The results indicated that the positive rate of Pasteurella multocida antibodies of rabbits in those rabbit farms was different.The highest positive rate was from the rabbit farm of Chuzhou in Anhui Province(38.89%),the lowest positive rate was from the rabbit farm of Huangmei in Jurong(8.82%),the positive rate in Nanjng A rabbit farm and Nanjng B rabbit farm were 18.39%and 20.39%respectively.After challenged 21 days post-vaccination,the mortality of rabbit with vaccination was significantly lower than control group.The ELISA test results showed that the survival rate was about 80%in serum-positive rabbits.During the immunization period,the antibody levels of rabbits increased from 0.15 to 0.45 in first month,and the antibody levels remained at about 0.45 in the next 6 months.However,there was no obvious change of control group,which always retains at about 0.15.The antibody levels of immunized group were significantly higher than control group at all time points.After immunized by different batches Pasteurella multocida inactivated vaccine,the changes of protective antibody was consistent. |
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