Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents and vaccine adjuvants in a variety of bacterial and protozoan diseases in different animal species. The objective of this study was to investigate the immunoprotective effect of formulated CpG-ODN with polyphosphazene, liposome or oil-in-water emulsion against E. coli infections in neonatal chickens. Eighteen-day-old embryonating eggs were inoculated with 50 microg CpG-ODN or formulated CpG-ODN with polyphophazene, liposome or oil-in-water emulsion. Four days after exposure to formulated CpG-ODN or day-1 post-hatch, 1 x 10(4) or 1 x l0(5) cfu of a virulent isolate of E. coli was inoculated by the subcutaneous route in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for eight days following challenge with E. coli. The survival rate of birds following E. coli infection was 0% in groups receiving either non-CpG-ODN or saline. In contrast, birds receiving either CpG-ODN or CpG-ODN formulated with polyphosphazene had significantly higher survival of 55% (P<0.0001). The relative risk of mortality was significantly reduced for birds treated with CpG-ODN formulated in PCPP (0.25), in PCEP (0.33), or unformulated CpG-ODN (0.39) in comparison to the group treated with saline (p<0.01). Although formulation of CpG-ODN with liposomes or oil-in-water emulsion did not increase the immunoprotective effect against E. coli infection, no adverse reactions or poor hatchability were observed in embryos. This is the first time that CpG-ODN formulated with polyphosphazene has been demonstrated to have an immunoprotective effect against an extra cellular bacterial infection in neonatal broiler chickens following in ovo delivery.