IMMUNOPROTECTIVE MECHANISMS ASSOCIATED WITH IN OVO DELIVERY OF OLIGODEOXYNUCLEOTIDES CONTAINING CpG MOTIFS (CpG-ODN) AND FORMULATION OF CpG WITH NANOPARTICLES TO ENHANCE ITS EFFICACY IN NEONATAL BROILER CHICKENS

Abstract

Oligodeoxynucleotides containing CpG motifs (CpG-ODNs) are known for their ability to stimulate vertebral immune system and provide protection against pathogens. Although CpG-ODN provides protection against bacterial infections in chickens mechanisms of immunoprotection remains elusive. The objective of this study was to identify mechanisms of immunoprotection of CpG-ODN following in ovo delivery in chickens . In the second chapter, we provide the mechanistic insights into CpG-ODN induced protection against Escherichia coli (E. coli). Multiplex cytokine gene analysis using QuantiGene Plex 2.0® technique revealed that CpG-ODN upregulates both Th1 and Th2 cytokines, as well as pro-inflammatory cytokines in both spleen and lung. In our study, Lipopolysaccharide-induced TNF factor-alpha factor (Litaf) stands out in the cytokine profiles of spleen and lungs, underscoring its role in CpG-ODN induced protection mechanisms. Flow cytometry analysis showed a marked increase of T lymphocyte and antigen presenting cells (APC) as well as enhanced expression of CD40 by APCs in spleen and lung after CpG-ODN treatment. This study demonstrated for the first time that CpG-ODN provides protection in neonatal chicks against E. coli infections by eliciting cytokine responses and enriching the immunological niches in spleen and lungs. In the third chapter, we show that CpG-ODN induces a dose-dependent influx of macrophages, CD4+ and CD8+ T-cell subsets in spleen and lungs of chicks that correlates with the immunoprotection against E. coli. We observed a dose-dependent enrichment of the immunological niches, wherein 25 µg and 50 µg of CpG-ODN induced significant changes in the immune profile of both spleen and lungs that correlated with their ability to resist E. coli infection. In the fourth chapter, we report that in ovo delivery of CpG-ODN formulated with CNT or lipid-surfactant potentiate the protective effect against E. coli infection. In conclusion, this study provides a greater understanding of cellular and molecular mechanisms for CpG-ODN induced antimicrobial immunity. And this significant advancement in the mechanistic insight will help in utilizing the full therapeutic potential of CpG-ODNs. Our work on CpG-ODN dose-dependent changes and enhanced protection against E. coli infection by nanoparticle formulations demonstrate the possible utilitization of CpG-ODN in the poultry industry as an alternative to antibiotics to prevent bacterial infections of neonatal chickens.