| Avian influenza virus(Avian influenza virus,AIV)and Newcastle disease virus(Newcastle disease virus,NDV)can infect a variety of poultry and birds,have caused serious economic losses to the development of china and the world poultry industry.The H9 subtype are the main AIV subtypes,endanger China's poultry industry,co-infection with NDV generally,not only cause economic loss,but also bring difficulties to the prevention.Therefore,implement combined Immunization strategy of AIV and NDV,can improve the efficiency of immunization,prevent cross-infection,and reduce economic losses.In the current study,we optimized the productive technology,prepared three AIV-NDV vaccines used different novel adjuvants and common adjuvants.We compared the physical properties,safety,immune effect,immunization period,and shelf life of three vaccines.The research lay the foundation for selecting the optimal adjuvants,developing the commercialization of NDV-AIV inactivated vaccine.This study includes the following aspects:1.In this experiment,NDV La Sota strain and AIV H9 subtype X-19 strain were inoculated 9-day-old SPF chicken embryos,respectively.Two virus were collected,and the titer were were 108.5EID50 / 0.1m L and 107.5EID50 / 0.1m L,respectively.The two viruses were concentrated 3-4 times using ultrafiltration membrane after inactivated by formaldehyde solution,respectively.The two viruses HA titers were 1:1024 and 1: 1024.Three AIV-NDV vaccines were successfully prepared use different adjuvants,and the quality were conducted a preliminary evaluation,its physical properties and safety inspection are conform to the relevant provisions.2.1-week-old SPF chickens wer randomly allocated to four groups with 30 per group.Each groups were injected through chest muscles at 0.6m L/chicken with different vaccines,continuously observed for 8weeks,all survived,did not appear local and systemic adverse reactions.Regular eye and microscopic observation were conducted of different adjuvants in the local tissue of absorption and histopathologicchanges.Partial lesions and non-absorption phenomenon were observed in the injection site of chicken in each group after vaccination.The group of ISA71 VG and ISA71 R VG recovered quickly,while Hangzhou white oil group absorb somewhat slowly.3.Blood were collected at certain days,serum separated,and the antibody specific for NDV and AIV were tested to investigate the antibody variation.Half of all the chicken were challenged with NDV strain Beijing-F48E9 through chest muscle.Fourteen days later,100% protected in the Immunized groups,while all chicken died in control group.Another part of the chickens were challenged with AIV strain X-19 through intravenous.The AIV isolation ratio from immunized groups was 80%,while control group was 100%.These results showed that,the HI titer of NDV and AIV H9 subtypes were higher than 1:64 in the groups inoculated with vaccines,while the control group serum HI titer is negative,21 days post immunization.Antibody was still at the level of protection,20 weeks after vaccination.ISA71 VG group produced specific antibodies five days after immunization,specific antibodies in the group induced rapidly,shorter time needed for antibodies to achieve ideal level,and a longer duration of antibodies.4.Monitor the quantity of peripheral blood T lymphocytes and its subgroups of the immune groups and control group by fluorescein labeled monoclonal antibody specific for chicken CD3+,CD4+,CD8+T lymphocytes.Results show that,the CD4+,CD8+ and CD3+ T lymphocytes in the peripheral blood of experimental chickens have least percentage in the age of 7 days.The CD4+,CD8+ T lymphocytes of immune group was increased compared with the control group after immunized at 7 days age,wherein,CD4+ and CD8+ T lymphocytes in the peripheral blood of ISA71 VG group were significantly higher than the control group,indicated that the vaccine can stimulate the humoral immunity and cellular immunity to a certain extent. |
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