Single oral immunization of an attenuated Salmonella Gallinarium formulation consisting of equal quantities of strains secreting H9N2 hemagglutinin-HA1, HA2, and M2eCD154 induces significant protection against H9N2 and partial protection against Salmonella Gallinarium challenge in chickens

The present investigation describes a formulation of a live attenuated Salmonella Gallinarium (SG) vaccine candidate against H9N2 influenza and SG infections in chickens. The formulation consists of an equal ratio of three strains, JOL2158, JOL2113, and JOL2074, which deliver hemagglutinin; HA1, HA2, and matrix protein 2 (M2e):: CD154 fusion (M2eCD154) antigens designed for broad protection against the field-matched H9N2 serotypes. The vaccine was completely safe at the average inoculation doses of 10⁸ and 10⁹ CFU/bird/0.2 mL in phosphate-buffered saline (PBS) used in the study. Bird immunization as a single oral inoculation could significantly engage humoral IgG, mucosal IgA, and cell-mediated immune responses against each immunized antigen, compared to the PBS control group (P < 0.05). The immunological correlates were comparable with the level of protection derived against the H9N2 and SG challenge, which resulted in significant protection against the H9N2 but only partial protection against the SG challenge as we compared against the PBS control group. The level of protection against H9N2 was investigated by determining the viral copy number and histopathological assessment of lung tissues. The results indicated a significant reduction in viral activity and recovery of lung inflammation towards the 14th-day post-challenge in a dose-dependent manner. Upon SG challenge, birds in the PBS control group experienced 100 % mortality, while 40 % and 70 % protection was observed in the SG-immunized groups for each respective dose of inoculation. The present SG-mediated immunization strategy proposes a rapid and reliable vaccine development process that can be effectively used against influenza strains such as H9N2 and holds the potential to minimize fowl typhoid caused by SG strains, mitigating two economically important diseases in the poultry industry.