Carvacrol nanoemulsion as a novel approach to augment H9N2 influenza vaccine efficacy

Avian influenza vaccines are essential for preventing seasonal influenza outbreaks and limiting pandemics, but their effectiveness has declined due to antigenic drift and variable immune responses. We hypothesized that carvacrol, particularly in nano-emulsion form, could enhance the avian influenza H9N2 vaccine and reduce the impact of viral challenge. This study evaluated the effect of carvacrol and carvacrol nano-emulsion on avian influenza vaccine efficacy by assessing immunological responses and immunohistopathology in vivo in broiler chickens. Our results showed that carvacrol, especially in its nanoform, significantly enhanced immune responses, as evidenced by increased phagocytic activity, higher hemagglutination titters, and improved immune organ indices. Biochemical analysis revealed significantly elevated levels of the immunomodulatory cytokines IL-10, IL-12, and IFN-γ, along with other immunity-related parameters, and molecular analysis showed upregulation of genes associated with IFN-γ, IL-12, IL-10, TLR4, TLR3, and TLR1. A notable increase in CD3 expression was also observed in key immune organs; the bursa of Fabricius, thymus and spleen suggesting enhanced T-cell-mediated immunity and improved pathogen recognition. The enhanced immunogenicity of influenza vaccine via carvacrol nano-emulsion resulted in stronger protection against avian influenza H9N2 virus ameliorating clinical picture and preserving the structural integrity of lung and trachea post viral challenge. These results emphasize the potential of nano-emulsion technology to strengthen immune responses in poultry and boost the efficacy of avian influenza vaccines, thereby supporting more effective control against influenza virus. Therefore, avian influenza control programs can incorporate carvacrol nano-emulsion as an immune-boosting adjuvant, since nano-emulsion delivery offers a practical tool to enhance existing vaccines without changing vaccination schedules.