| Infectious Bronchitis(IB)is an acute and highly contagious respiratory disease caused by the Infectious Bronchitis Virus(IBV)that occurs only in chickens.The prevalence of the disease in China is on the rise,especially Renal pathological type IB has spread to chicken-raising areas throughout the country,causing serious economic losses,and currently the antibody test method that used to evaluate the immune effect of the Infectious Bronchitis vaccine is mainly the HI test.Hemagglution(HA)antigens need to be prepared with trypsin or phospholipase C in the HI test.This process has a certain damage to the fibrin protein of IBV and it doesn’t fully reflect the IBV antibody level.In addition,there are many serotypes and its vary greatly in IBV.It is questioned whether the HI test can reflect the antibody level after immunization with different serotype IBV vaccines.This study aimed to establish an Indirect Hemagglutination test(IHA)for detection of IBV antibodies on the erythrocyte carrier of IBV.Determine the difference between IHA antibodies and HI antibodies through cross-correlation of W93and M41 antigens or field immunity serum antibody levels.Using IBV W93 strain live vaccine,live vaccine strain M41 and M41 Vaccine strain chicken serum antibody levels may reflect changes for different serotypes of vaccine in chickens IHA antibody and HI antibody.Test results are as follows:1.IHA method for detecting IBV antibodies was established.Different strains of IBV were inoculated with 10-day-old non-immune chicken embryos.After 48 hours,allantoic fluid was harvested and inactivated with 0.2%formaldehyde.The IHA antigen preparation was inactivated IBV-containing allantoic fluid with 2.5%citrate decomposed chicken erythrocytes.According to 1:2 sensitization,the best sensitization time is 1 h,IHA price of IBV positive serum up to 2~8.The preparation of HA antigen was prepared by 1%trypsin treatment with IBV allantoic fluid.The optimal ratio of pancreatin and antigen was 1:3.The best time for antigen treatment was 2 h,and the blood coagulation value(HA)was up to 2~8-2~9.2.The establishment of IHA method and HI method are practical.There were used to detect IBV W93 positive serum,M41 positive serum,IBV negative serum and Avian Influenza(AI)H9,Avian Influenza(AI)H5,Newcastle disease(ND),and Infectious Bursal Diseases(IBD),egg drop syndrome(EDS-76)and other viral positive serum.The results showed that the IHA and HI methods established with the antigens prepared from the IBV W93 and M41 strains had good specificity.The titers of 10 immunized chickens were detected by IHA and HI methods respectively.The titer of IHA was 2~6 to 2~8,and the titer of HI was 2~8 to 2~9.The HI titer is 2 to 4 times higher than the IHA method.The IHA antigen and HA antigen made from W93 and M41 were used to detect the IHA and HI antibodies in 6 chickens immunized with the vaccine respectively.The HI results of the IBV W93 and M41 strains were identical,whereas results of the IHA test showed that there were difference of 1 to 2 fold antibody drops.3.There are differences in the regularity of IHA and HI antibody length in IBV-vaccinated chickens.The healthy chicks were immunized with IBV W93 live vaccine,M41 live vaccine,and M41 inactivated vaccine.Blood was collected at different times after immunization,meanwhile the serum was separated.IHA and HI tests were used to detect the growth and distribution of immunized chickens.The results demonstrated that the duration of antibody maintenance was short and the peak value was not high after the immunization with W93 and M41 live vaccines.Although the peak of antibody level was not high,the maintenance time was longer after immunization with M41 inactivated vaccine.Although both IHA and HI can be used to evaluate the effect of immunization against infectious bronchitis in chickens,there is little difference in individual antibodies detected by the HI test,but there is a large difference in individual antibodies detected by the IHA test. |
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