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Oligodeoxynucleotides containing CpG motifs (CpG-ODN) induce innate immunity against bacterial infections. Despite recent advances, how CpG-ODN alone protects against bacterial infections remained elusive. Here, we report for the first time, to our knowledge, that CpG-ODN orchestrates anti-microbial protective immunity by inducing a rapid enrichment of various immune compartments in chickens. In this study, eighteen-day-old embryonated eggs were injected with either 50 µg of CpG-ODN or saline (~n = 90 per group). In the first experiment, four days after CpG-ODN treatment, chicks were challenged subcutaneously with a virulent strain of Escherichia coli (E. coli) and mortality was monitored for 8 days. We found significant protection, and reduced clinical scores in CpG-ODN treated chicks. To gain insights into mechanisms of protection induced by CpG-ODN, first we investigated cytokine expression kinetics elicited by CpG-ODN. The spleen and lung were collected from embryos or chicks (n = 3–4 per group) at 10 time points post-CpG-ODN inoculation. Multiplex gene analysis (interleukin (IL)-1, IL-4, IL-6, IL-10, IL-18, interferon (IFN)-γ, IFN-α, and lipopolysaccharide induced tumor necrosis factor (LITAF), revealed a significantly higher expression of pro-inflammatory cytokines following CpG-ODN treatment compared to the saline controls. In our study, LITAF stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN-induced protection. The third experiment was designed to examine the effects of CpG-ODN on immune cell populations in spleen, lungs, and thymus. Flow cytometry analysis was conducted at 24, 48 and 72 hrs (thymus only collected at 72 hr) after CpG-ODN administration to examine the changes in CD4+ and CD8+ T-cell subsets, monocyte/macrophage cell populations and their expression of maturation markers (CD40 and CD86). Flow cytometry data indicated a significant enrichment of macrophages, CD4+ and CD8+ T-cell subsets in both spleen and lungs of CpG-ODN treated embryos and chicks. Macrophages in spleen and lungs showed an upregulation of CD40 but not CD86, whereas thymocytes revealed significantly high CD4 and CD8 expression. Overall, the present study has demonstrated that CpG-ODN provides protection in neonatal chicks against E. coli infection not only by eliciting cytokine responses and stimulating immune cells but also through enriching immunological niches in spleen and lungs.

Synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine phosphodiester guanine (CpG) motifs (CpG-ODN) are effective immunostimulatory agents against a variety of viral, bacterial, and protozoan diseases in different animals including poultry. We have recently demonstrated that in ovo injection of CpG-ODN confers protection in neonatal chickens against bacterial septicemias. The objective of this study was to investigate the effectiveness of needle-free intrapulmonary (IPL) delivery of CpG-ODN microdroplets against Escherichia coli infection in neonatal chicks. In the present study, we used 880 chicks in total keeping 40 chicks per group. Chicks were delivered CpG-ODN or saline by IPL at the day 1 of hatch. Three days later, chicks were challenged with two doses (1 × 10 CFU, n = 20 or 1 × 10 CFU, n = 20) of E. coli. Chicks treated with CpG-ODN by the IPL route had significantly lower clinical signs and bacterial load compared to the group treated with saline ( P < 0.05). CpG-ODN-treated groups were significantly protected against E. coli septicemia. We observed dose- and exposure time-dependent immunoprotective effects of IPL CpG-ODN in chicks. We found that IPL delivery of CpG-ODN can induce protective immunity as early as 6 hr that remains effective at least until day 5 post-treatment. Moreover, there were no adverse effects of IPL delivery of CpG-ODN on growth or mortality up to 42 days of age. Based on these findings, it can be suggested that CpG-ODN delivery by IPL route can be a promising alternative to antibiotics for inducing protective immunity in chicks during the critical first week of neonatal life.

the transition to antibiotic-free poultry production in the face of pathogenic threats is a very challenging task. We recently demonstrated that mucosal delivery of cpG-oDn alone by the intrapulmonary route (IPL) has potential as an effective alternative to antibiotics in neonatal chicks against Escherichia coli septicemia. How exactly mucosal delivery of cpG-oDn elicits, protective antibacterial immunity remained poorly understood. in this study, cpG-oDn or saline was delivered via the intrapulmonary route to day-old chicks (n = 80/group) using a compressor nebulizer in an acrylic chamber (1 mg/mL CpG-ODN for 15 minutes). In the first part of the study, two days after mucosal CpG-ODN delivery, 40 chicks from each group were challenged subcutaneously with 1 × 10 5 cfu (n = 20) or 1 × 10 6 cfu (n = 20) of E. coli and the mortality pattern was monitored for seven days. We found significantly higher survival, better clinical conditions and lower bacterial loads in chicks that received mucosal CpG-ODN. To explore the mechanisms behind this protective immunity, we first looked at the kinetics of the cytokine gene expression (three birds/ group/ time for 10 time-points) in the lungs and spleens. Multiplex gene analysis demonstrated a significant elevation of pro-inflammatory cytokine genes mRNA in the CpG-ODN group. Interleukin (IL)-1β robustly upregulated many folds in the lung after CpG-ODN delivery. Lipopolysaccharide-induced tumor necrosis factor (LITAF) and IL-18 showed expression for an extended period in the lungs. Anti-inflammatory cytokine IL-10 was upregulated in both lungs and spleen, whereas IL-4 showed upregulation in the lungs. To investigate the kinetics of immune enrichment in the lungs and spleens, we performed flow cytometry, histology, and immunohistochemistry at 24, 48 and 72 hrs after CpG-ODN delivery. CpG-ODN treated lungs showed a significant enrichment with monocytes/macrophages and CD4 + and CD8 + t-cell subsets. Macrophages in CpG-ODN treated group demonstrated mature phenotypes (higher CD40 and MHCII expression). Importantly, mucosal delivery of CpG-ODN via the intrapulmonary route significantly enriched immune compartment in the spleen as well, suggesting a systemic effect in neonatal chicks. Altogether, intrapulmonary delivery of aerosolized CpG-ODN orchestrates protective immunity against E. coli septicemia by not only enhancing mucosal immunity but also the systemic immune responses.

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