Gamma-irradiated fowl cholera vaccines formulated with different adjuvants induced antibody response and cytokine expression in chickens

Abstract

Fowl cholera is one of the most serious and economically important infectious diseases of poultry caused by Pasteurella multocida. Formalin-inactivated vaccine, administered intramuscularly, is widely used in Ethiopia with a low success rate. Gamma irradiation is an effective approach to inactivate pathogens for vaccine development. In a previous study, we reported the feasibility of developing gamma-irradiated vaccines that induced both systemic and mucosal antibody responses with complete protection against homologous lethal challenge. In the present study, we aimed to broaden our understanding of the immunogenicity of the gamma-irradiated vaccines by including peripheral blood mononuclear cells (PBMC) response analysis. A total of 156 eight-week-old fowl cholera-specific antibody negative Bovans Brown chickens were utilized in this experiment. The performances of gamma-irradiated P. multocida vaccines formulated with different adjuvants, Montanide Gel 01 PR (G-1), Carbigen^® (G-2), Emulsigen-D^®+aluminum hydroxide gel (G-3), and Emulsigen-p^® (G-4) were evaluated in comparison with the formalin-inactivated vaccine (G-5) and unvaccinated control (G-6). Chickens received two doses of the vaccines at days 0 and 21. Sera, tracheal, and crop lavage were collected at days 0, 21, 35, and 56 to assess IgG and IgA levels using indirect and sandwich ELISA, respectively. PBMC proliferation was compared between vaccinated and unvaccinated controls. In addition, vaccination-induced expression of cytokine genes was analyzed in PBMC using qPCR. Chickens were challenged with 2.5x10⁷ CFU/ml of P. multocida biotype A intramuscularly one day after day-56 sampling. Significant serum IgG titers were detected three weeks after primary vaccination in G1, G3, and G5. IgG titer substantially increased in all vaccinated groups two weeks post-booster dose. IgA response was induced by gamma-irradiated vaccines but not formalin-inactivated vaccines. Only PBMC from vaccinated chickens proliferated in response to re-stimulation with P. multocida antigen, indicating vaccine-specific priming. Interestingly, gamma-irradiated vaccines resulted in a higher fold change in mRNA transcripts of IFN-γ (>1000-fold change) IL-6 (>500-fold change), and IL-12p40 (>200-fold change), which are hallmarks of a Th1 dominant response, which is essential to combat intracellular infection. Lastly, the candidate vaccines demonstrated various levels of protection, with Emulsigen-D^® containing vaccine rendering complete protection against homologous lethal challenge. In conclusion, gamma-irradiated vaccines can induce broad immune responses, humoral and cellular, and protect against severe outcome of fowl cholera. Therefore, this study has contributed to growing knowledge on the immunogenicity and efficacy of gamma-irradiated vaccines and has shown the potential of such a vaccine platform for field application in extensive as well as intensive farm settings.

Keywords: P. multocida; adjuvants; chicken; cytokines; fowl cholera; gamma-irradiated vaccines; humoral immunity; mucosal immunity.

Figure 1

Experimental design of the study. A total of 156 chickens were classified into six groups depending on the type of vaccine administered. Individual chickens received 2 doses of their respective vaccines 3 weeks apart. Blood was sampled at days 0, 21, 35, and 56 for PBMC and serum analysis. In addition, seven days after the challenge, blood was obtained for cytokine gene expression analysis. Tracheal and crop lavage were also collected by repeatedly euthanizing four chickens. Ten chickens from each group were challenged with a lethal dose of avian P. multocida intramuscularly to assess the efficacy of the vaccines.

Figure 2

Serum IgG response to the candidate vaccines at days 0, 21, 35, and 56. Serum IgG titer was analyzed using indirect ELISA at 450nm. The mean ± SEM is shown for each group on sampling day. A cut-off value of 0.2 was used according to the manufacturer's instructions. Single (*) and double asterisk (**) represent p<0.05, Cohen's effect size value (d = .62) and p<0.01 (d = .72), respectively.

Figure 3

Mucosal IgA response to the candidate vaccines at days 0, 21, 35, and 56. Mucosal IgA titer was analyzed from tracheal and crop lavage samples using sandwich ELISA at 450 nm. Four chickens from each group were euthanized on each sampling day. The mean ± SEM is shown for each group on each sampling day. A cut-off value of 0.1 was used according to the manufacturer's guide.

Figure 4

Isolated PBMCs from vaccinated (A) and non-vaccinated chickens (B) were pooled and stimulated with gamma-irradiated P. multocida antigen. Lymphocyte activation cocktail (LAC) was used to induce proliferation and served as a positive control. PBMCs treated with LAC (C) and PBMCs from vaccinated chickens treated with gamma irradiated P. multocida antigen (D) exhibited a significant proliferation as compared to unvaccinated (E) controls indicating vaccination-induced antigen specific priming.

Figure 5

Cytokine gene expression analyses using RT-qPCR. Chickens were classified into 6 groups as Montanide Gel (A), Carbigen (B), Emulsigen-D+Alum (C), Emulsigen-P (D), Formalin inactivated vaccine (E), Unvaccinated control (F). PBMCs were analysed at days 0, 21, 35, 56 and seven days after challenge (AC). Relative fold change was initially normalized to the expression of the reference gene glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and subsequently expressed as a fold change relative to expression levels of control group. Fold change was calculated using the 2^-AACt method.

Figure 6

Cytokine gene expression analyses using RT-qPCR. IFN-γ (A), IL-12p40 (B), IL-1β (C), IL-6 (D), IL-22 (E), IL-4 (F) in chicken PBMC at days 0, 21, 35, 56 and seven days after challenge (AC). Relative fold change was initially normalized to the expression of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and subsequently expressed as a fold change relative to expression levels of control group. Fold change was calculated using the 2^-ΔΔCt method.

Figure 7

Survival curve analysis of the occurrence of death of chickens in each experimental group: each group comprises 10 chickens that had been followed for the period of 14 days. The chickens in the vaccinated and control groups were given 0.5 ml of avian P. multocida biotype A. The data were used to determine the kaplan-meier estimates (the product limit estimate) of both the control and the vaccinated groups.

Figure 8

PCR using specific primer targeting capsular biosynthesis gene (capA). Lane 1, vaccine with Emulsigen®-D with aluminum hydroxide gel (Alum); lane 2, Montanide/01 PR gel; lane 3, Emulsigen®- P; lane 4, Carbigen®; lane 5, formalin killed vaccine; lane 6, unvaccinated control; lanes 7 and 8, P. multocida master seed strain; M, Molecular marker; N, Negative control; and P, positive control.