Abstract: 【Objective】There are many commercial inactivated vaccines against the H9 subtype avian influenza approved for use in China, and their protection efficacy and selection have been widely concerned by farmers. Evaluation of protection efficacy of main commercial vaccines against H9N2 viruses recently isolated in the field are of great significance for guiding the prevention and control of H9 subtype AI by immunization in China.【Method】According to the vaccine batch release of the National Veterinary Drug Basic Information Database in China, 4 commercial vaccines (labeled as A-D) with large batches were selected from 40 kinds of sold H9 commercial vaccines. The four H9N2 subtype AIV, CK/XJ/S1204/2015, DK/JX/S4512/2017, CK/YN/S1666/2020 and CK/NX/S4590/2020, belong to h9.4.2.5 branch, isolated at different times and places were used in this study to evaluate the protection efficacy of selected commercial H9 subtype AI vaccines. The 50% chicken embryo  infection (EID₅₀), 50% chicken infection (CID₅₀), and 50% cell infection (TCID₅₀) of the four viruses were tested to determine the challenge dose of the animal experiment and the infection dose of the cell test. 40 3-week-old SPF chickens were injected intramuscularly with 4 kinds of inactivated vaccine, and groups of 10 similar SPF chickens were inoculated with PBS as control group. 3 weeks post vaccination(p.v.), the serum of all experimental chickens were collected before challenge, hemagglutination inhibition (HI) and neutralization (NT) antibody titers were detected by HI test and neutralization test; The 40 immunized chickens of each vaccine were randomly divided into four groups, 10 chickens of each vaccine and 10 control chickens were infected intranasally with the 10CID₅₀.of each H9N2 virus. Oropharyngeal and cloacal swabs were collected on days 3 and 5 post challenge(p.c.) for virus shedding detection and calculation of the protective rate of each vaccine. 【Result】The CID₅₀ of the four strains were 10^3.5 EID₅₀/0.1 mL, 10^2.5 EID₅₀/0.1 mL, 10^2.5 EID₅₀/0.1 m and  10^3.5 EID₅₀/0.1 mL, respectively. At three weeks p.v., the mean HI antibodies titers against the commercial H9 subtype HI test antigen (CK/SH/10/2001) in 4 groups of vaccinated chickens ranged from 9.4log2 to 11log2, the mean HI antibodies titers against challenge virus in 4 groups of vaccinated chickens ranged from 4.6log2 to 10.8log2, and significant differences of HI antibodies titers were observed among different vaccine groups, the maximum difference of mean HI antibodies titers were 64-fold among them. The mean NT antibodies titers against challenge virus in 4 groups of vaccinated chickens ranged from 6.7log2 to 12.2log2, the maximum difference of mean HI antibodies titers were 32-fold among them, the HI antibody and NT antibody of the control group were negative. After intranasal infection with different H9 viruses, the immune effects of the four vaccines were quite different. In CK/XJ/S1204/2015 challenged groups, 3 vaccines (B-D) could provide more than 80% protection. In DK/JX/S4512/2017 challenged groups, 1 vaccine (B) can provide more than 80% protection. In CK/YN/S1666/2020 challenged groups, 2 vaccines (A and B) can provide more than 80% protection. In CK/NX/S4590/2020 challenged groups, the protection efficacy of the four vaccines (A-D) were all less than 80%. In the same period, the virus shedding rate of chickens in the control group was more than 8 /10. 【Conclusion】There was a great difference in the immune protection efficacy of the four kinds of commercial vaccines after challenge with recently isolated H9 strains, and the difference of antigenicity between vaccine antigens and isolates was the main reason for the decrease of immune protection efficacy of the commercial vaccines. The titers of HI antibody and NT antibody against H9N2 prevalent strains could be as important data for evaluation of the commercial H9N2 AI vaccines. This study provides scientific references for selection and usage of the commercial vaccines against H9N2 AI.

Key words: H9N2 subtype, AIV, Commercial vaccine, Protective efficacy