Electron-beam Irradiation Inactivation of Salmonella: Effects on Innate Immunity and Induction of Protection Against Salmonella enterica serovar Typhimurium Challenge of Chickens

Abstract

Our laboratories are investigating the use of High Energy (10 MeV) Electron-Beam (E-beam) irradiation for is potential use in vaccine development. Ionizing radiation inactivates microorganisms by “direct and indirect” effects on nucleic acids and other cellular components. Though the cells are inactivated, the surface antigenic properties of the microorganisms remain unaltered. We hypothesized that electron-beam (E-beam) inactivated Salmonella enterica serovars could be used as a potential immune modulator to activate the innate immune response and thus reduce Salmonella intestinal colonization and shedding in neonatal chickens. Three replicate experiments were designed to evaluate the efficacy of a high energy E-beam irradiated Salmonella enterica serovar Typhimurium (ST) administered in ovo to: (a) induce a functional innate immune response and (b) reduce ST colonization in the ceca of chicks three-weeks post-hatch. We have previously shown that unmethylated CpG motifs of bacteria DNA oligodeoxynucleotides (CpG-ODN) given in ovo stimulates innate immune responsiveness of chicken heterophils and increases resistance of young chickens to SE colonization; thus were used as positive controls in these experiments Eighteen-day-old chicken embryos were equally divided into four independent treatment groups: (1) a negative control (sham injected, no challenge) group, (2) an infected control (sham injected, challenged) group, (3) a CpG-ODN injected, challenged positive control, and (4) an E-beam ST-injected, challenged group. All treatment groups contained 100 birds, half of the animals from each treatment group were euthanized on day 4 post-hatch so that peripheral blood granulocytes (heterophils) could be collected to evaluate the functional innate immune response. The remaining birds where reared under normal housing conditions for the remainder of the experiment. On day 18 post-hatch the birds were challenged with the homologous ST strain and five days later (day 23 post-hatch), the experiment was terminated to evaluate the colonization of ST in the ceca of the birds. Differences in the leukocyte function and in the log10 cfu of ST counts among treatment groups were determined by analysis of variance. Significant differences were further separated using Duncan's multiple range tests. Here, heterophil function was measured using in vitro assays for (1) oxidative burst and (2) degranulation. Heterophils from the CpG-ODN and E-beam ST-treated birds exhibited a significant increase (P < 0.05) in both the oxidative response and degranulation when compared to all other treatment groups with no differences in heterophil functions between the CpG-ODN and e-beam-treated groups. ST colonization of the ceca was significantly reduced (P < 0.05) in both the CpG-ODN and the E-beam ST-treated birds when compared to the non-vaccinated control birds. These results demonstrate that in ovo administration of E-beam irradiated Salmonella induced a primed heterophil-mediated innate immune response and provide a protective intestinal colonization-inhibition effect against a homologous Salmonella challenge.