In ovo CpG DNA delivery increases innate and adaptive immune cells in respiratory, gastrointestinal and immune systems post-hatch correlating with lower infectious laryngotracheitis virus infection

Abstract

Cytosine-guanosine deoxynucleotides (CpG) DNA can be delivered in ovo at embryo day (ED)18 for the stimulation of toll-like receptor (TLR)21 signaling pathway that ultimately protects chickens against a number of bacterial and viral infections. There is a dearth of information understanding the mechanisms of protection induced by in ovo delivered CpG DNA. The objective of this study was to determine the immune cell changes post-hatch following in ovo delivery of the TLR21 ligand, CpG DNA. In order to quantify changes of percentage of KUL01+, IgM+ B, cluster of differentiation (CD)4+ and CD8α+ cells, trachea, lung, duodenum, large intestine, spleen and bursa of Fabricius were collected on day 1 post-hatch. We found increased recruitments of KUL01+ cells, in organs of these body systems post-hatch following in ovo delivery of CpG DNA. Although IgM+ B cells, CD4+ and CD8α+ cells were increased in lungs and immune system organs, these cells were not quantifiable from the trachea, duodenum and large intestine immediately following the hatch. Furthermore, when CpG DNA is delivered in ovo and subsequently infected with infectious laryngotracheitis virus (ILTV) post-hatch on day 1, CpG DNA reduces morbidity and mortality resulting from ILTV infection. This study provides insights into the mechanisms of host responses elicited following in ovo delivery of CpG DNA in avian species.

Fig 1. In ovo delivery of CpG DNA increases KUL01+ and CD4+ cell numbers in lungs pre-hatch.

SPF ED18 eggs were injected with CpG DNA (n = 9), non-CpG DNA (n = 4) or PBS (n = 6). At ED19, lungs were collected and immunofluorescent assay was performed for KUL01+ and CD4+ cells. The quantitative data for KUL01+ (a) and CD4+ cells (b) are shown. The one-way ANOVA test with Bonferroni's post test for selected comparison was performed to identify group differences and the differences were considered significant at P< 0.05. The bars represent mean ± SEM.

Fig 2. In ovo delivered CpG DNA increases KUL01+ cell numbers in lungs, trachea, duodenum, large intestine and bursa of Fabricius.

SPF ED18 eggs were delivered with CpG DNA (n = 5), non-CpG DNA (n = 4) or PBS (n = 4) in ovo and the eggs were incubated to hatch. At day 1 post-hatch, samples of lungs, trachea, duodenum, large intestine, spleen and bursa of Fabricius were collected in OCT, sectioned and immunofluorescent assay was performed to quantify KUL01+ cells. The quantitative data from the immunofluorescent assay of each organ is shown a) lungs, b) tracheas, c) duodenum, d) large intestine, e) spleen, f) bursa of Fabricius. The one-way ANOVA test with Bonferroni's post test for selected comparison was performed to identify group differences and the differences were considered significant at P< 0.05. The bars represent mean ± SEM.

Fig 3. In ovo delivery of CpG DNA increases IgM+ B cells in spleen, CD4+ cells in lungs, bursa of Fabricius and spleen and CD8α+ cells in lungs post-hatch.

SPF ED18 eggs were delivered with CpG DNA (n = 5), non-CpG DNA (n = 4) or PBS (n = 4) in ovo and the eggs were incubated to hatch. At day 1 post-hatch, lungs, trachea, duodenum, large intestine, spleen and bursa of Fabricius were collected in OCT, sectioned and immunofluorescent assay was performed for the quantification of IgM + B cells, CD4+ cells and CD8α+ cells. The quantitative data from the immunofluorescent assay for IgM+ B cells in spleen (a) for CD4+ cells in lungs (b), bursa of Fabricius (c) and spleen (d) and for CD8α+ cells in lungs (e) are shown. The one-way ANOVA test with Bonferroni's post test for selected comparison was performed to identify group differences and the differences were considered significant at P< 0.05. The bars represent mean ± SEM.

Fig 4. In ovo delivered CpG DNA induces protective host response against ILTV infection encountered post-hatch.

SPF ED18 eggs were delivered with CpG DNA (n = 8) or non-CpG DNA (n = 7) in ovo, the eggs were incubated to hatch and infected with ILTV intra-tracheally at day 1 post-hatch. The chickens were observed for 12 days. At 4 days post-infection, oropharyngeal and cloacal swabs were collected and ILTV genome loads quantified. a) Survival percentage, b) clinical scores, c) ILTV genome loads in cloacal swabs and d) ILTV genome loads in oropharyngeal swabs. Log-rank test was used to identify differences in survival percentages, Mann-Whitney U test was used to identify differences in clinical score data and student's t-test was used to identify differences in ILTV genome loads. The differences were considered significant at P< 0.05. The bars represent median ± inter quartile range in Fig. 4b and mean ± SEM in Fig. 4a, c and d.

Fig 5. ILTV genome loads and KUL01+, IgM+ B, CD4+ and CD8α+ cell numbers in lungs of in ovo CpG DNA and non-CpG DNA treated chickens following ILTV infection post-hatch.

The chickens were delivered with either in ovo CpG DNA or non-CpG DNA and infected with ILTV intra-tracheally at day 1 post-hatch. The chickens were observed for end points. The lungs were collected for determination of ILTV genome loads and cellular responses from chickens that reached humane endpoint at 6 days post-infection and experimental end point at 12 days post-infection. The ILTV genome loads are given separately for 6 and 12 days post-infection (a). The quantitative data of the immunofluorescent assays done for IgM+ B cells (b), KUL01+ (c), CD4+ cells (d) and CD8α+ cells (e) are shown separately for 6 and 12 days post-infection. Statistical analysis was precluded due to low numbers of animals per group per time point. The bars represent mean ± SEM.