Bacillus subtilis spore as an adjuvant in chicken treated with inactivated avian influenza virus, H9N2

Abstract

Bacillus subtilis, a rod-shaped gram-positive and endospore-forming bacterium, is a nonpathogenic species that has been used in a probiotic formulation for both animals and humans. B. subtilis spore is also known as a particulate adjuvant and has remarkable heat resistance properties which can remain dormant to survive under harsh environmental conditions. The objective of the present study was to elucidate the effect of B. subtilis spore as an adjuvant in chickens treated with inactivated avian influenza virus, H9N2 via intramuscular administration. Herein, the adjuvanticity of B. subtilis spores in chicken was demonstrated by enhancing H9N2 virus-specific IgG responses. B. subtilis spores enhanced proportion of B cells and innate cell population in splenocytes from chickens treated with both inactivated H9N2 and B. subtilis spores. Furthermore, the treatment induced significantly high expression of BAFF, CD40, and their receptors coincident with increase of pro-inflammatory cytokines, IL-1β and IL-6 when compared with chickens treated with inactivated H9N2 only. Then, total splenocytes from chickens immunized with inactivated H9N2 in the presence or absence of B. subtilis spores were re-stimulated with inactivated H9N2 in vitro. The results showed that the extent of antigen specific CD4+ and CD8+ T cell proliferation was higher in group co-administered with inactivated H9N2 and B. subtilis spores. Taken together, these data demonstrate that B. subtilis spores, as adjuvant, enhance both H9N2 virus-specific IgG response and CD4+ and CD8+ T cell proliferation, with increased mRNA level of BAFF, BAFF receptor, CD40, CD40L, and pro-inflammatory cytokines. This unique approach to vaccination in chicken together with B. subtilis spores as adjuvant, indicates a significant role in protection against avian influenza virus.