The In Ovo Delivery of CpG Oligonucleotides Protects against Infectious Bronchitis with the Recruitment of Immune Cells into the Respiratory Tract of Chickens

Abstract

The in ovo delivery of cytosine-guanosine (CpG) oligodeoxynucleotides (ODNs) protects chickens against many bacterial and viral infections, by activating the toll-like receptor (TLR)21 signaling pathway. Although the delivery of CpG ODNs in ovo at embryo day (ED) 18 has been shown to reduce infectious bronchitis virus (IBV) loads in embryonic chicken lungs pre-hatch, whether in ovo delivered CpG ODNs are capable of protecting chickens against a post-hatch challenge is unknown. Thus, our objectives were to determine the protective effect of the in ovo delivery of CpG ODNs at ED 18 against IBV infection encountered post-hatch and, then, to investigate the mechanisms of protection. We found significantly higher survival rates and reduced IBV infection in the chickens following the pre-treatment of the ED 18 eggs with CpG ODNs. At 3 days post infection (dpi), we found an increased recruitment of macrophages, cluster of differentiation (CD)8α+ and CD4+ T lymphocytes, and an up-regulation of interferon (IFN)-γ mRNA in the respiratory tract of the chickens. Overall, it may be inferred that CpG ODNs, when delivered in ovo, provide protection against IBV infection induced morbidity and mortality with an enhanced immune response.

Keywords: CD4+ cell; CD8α+ cell; CpG oligonucleotide; In ovo; avian macrophage; infectious bronchitis virus.

Figure 1

In ovo delivery of cytosine-guanosine (CpG) oligodeoxynucleotides (ODNs) is protective against infectious bronchitis virus (IBV) infection encountered post-hatch. Specific pathogen free (SPF) embryo day (ED) 18 eggs were delivered in ovo with class B CpG ODNs (n = 7) or control ODNs (n = 6), and on day 1 post-hatch, the chickens were infected with IBV M41 strain at a dose rate of 2.75 × 10⁴ embryo infectious dose (EID₅₀) per bird, and were monitored until 11 days post infection (dpi). (a) Survival percentage and (b) clinical scores. (c–h): SPF ED 18 eggs were delivered in ovo with class B CpG ODNs (n = 21), control ODNs (n = 29), and phosphate buffered saline (PBS) (n = 11). The eggs were incubated until hatching, and on day 1 post-hatch, a subset of in ovo CpG ODN-treated birds was challenged with IBV M41 strain at a dose rate of 2.75 × 10⁴ EID₅₀ per bird (n = 12), and the rest were kept as in ovo CpG pre-treated uninfected controls (n = 9). Similarly, a subset of birds in the in ovo control ODN-treated birds was infected with IBV (n = 18), and the remaining birds were kept as in ovo control ODN-treated uninfected controls (n = 11). The in ovo PBS treated birds were kept as uninfected controls (n = 11). A subset of birds from each group was sacrificed at 3 dpi (n = 5-9 per group), and the remaining birds were sacrificed at 7 dpi (n = 3–9) in order to obtain lung tissue. (c) IBV genome loads in oro-pharyngeal swabs at 3 and 7 dpi, (d) IBV genome loads in cloacal swabs at 3 and 7 dpi, and (e) IBV genome loads in 3 and 7 dpi lung. (f–g) The quantitative data and representative figures from the immunofluorescent assay of the trachea for IBV N antigen is presented. Scale Bar = 200 μm (h) Representative images of histological observations of trachea are given. Control ODNs – IBV: Severe epithelial metaplasia with severe cellular infiltration, germinal center formation is seen (A), superficial epithelial layer has become squamous with complete loss of cilia (B) and mucus glands not detected. CpG ODNs-IBV: pseudostratified simple columnar epithelium and intact ciliated epithelia (arrow) is evident where some have become rounded, and a few mucus secreting glands have been distorted and elongated (arrow head). CpG ODNs-control, Control ODNs-control, and PBS-control: No lesions, normal pseudostratified ciliated columnar epithelium (C) with mucus secreting glands. Log-rank test was used to identify the differences in the survival rate, and the Kruskal–Wallis test followed by the Mann–Whitney U test were used to identify the differences in the clinical scores at selected time points. The student’s t test was performed to identify group differences in the oropharyngeal and cloacal genome loads, and one-way analysis of variance (ANOVA) followed by the Students–Newman–Keuls post hoc test was used to identify the differences in the lung IBV genome loads and IBV N antigen amount in the trachea. The differences were considered significant at * = significant at p ≤0.05, ** = significant at p ≤0.01 *** = significant at p ≤0.001. c–h: the animal numbers and results represent the pooled data of the two independent experiments.

Figure 2

In ovo delivery of CpG ODNs is capable of recruiting key cells of the innate and adaptive arms of the immune system responsible for enhanced immune responses in the respiratory tract. The quantitative data following immunofluorescent assays done for the trachea (a) macrophages, (b) cluster of differentiation (CD)4+ T cells, and (c) CD8α+ T cells are given. The quantitative data following the immunofluorescent assays done for lung (d) macrophages, (e) CD4+ T cells, and (f) CD8α+ T cells are given. One-way ANOVA followed by the Students–Newman–Keuls post hoc test were used to identify the group differences. The differences were considered significant at * = significant at p ≤ 0.05, ** = significant at p ≤ 0.01 *** = significant at p ≤ 0.001. The results represent the pooled data of two independent experiments.

Figure 3

In ovo delivery of CpG ODNs is capable of inducing pro-inflammatory mediator mRNA expression levels in the lungs. (a) Fold change in the interferon (IFN)-γ mRNA expression, (b) fold change in the IL-1β mRNA expression and c) iNOS mRNA expression of 3 dpi lung. One-way ANOVA followed by Students-Newman-Keuls post hoc test was used to identify differences in mRNA expression levels. The differences were considered significant at * = significant at p ≤ 0.05, ** = significant at p ≤ 0.01 *** = significant at p ≤ 0.001. The results represent pooled data of two independent experiments.