Oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG-ODN) motifs have been shown to stimulate the innate immune system against a variety of bacterial and protozoan infections in a variety of vertebrate species. The objective of this study was to investigate the immunostimulatory effect of CpG-ODN in neonatal broilers against Salmonella Typhimurium septicemia. Day-old broiler chicks, or embryonated eggs that had been incubated for 18 days, received 50 microg of CpG-ODN, 50 microg of non-CpG-ODN, or saline. Four days after exposure to CpG-ODN or day 2 posthatch, 1 x 10(6) or 1 x 10(7) colony-forming units (cfu) of a virulent isolate of Salmonella Typhimurium was inoculated by the subcutaneous route in the neck. Clinical signs, pathology, bacterial isolations from the air sacs, and mortality were observed for 10 days following challenge with Salmonella Typhimurium. The survival rate of birds in groups receiving either non-CpG-ODN or saline following Salmonella Typhimurium infection was 40%-45%. In contrast, birds receiving CpG-ODN had significantly higher survival rate of 80%-85% (P < 0.0001). Bacterial loads and pathology were low in groups treated with CpG-ODN compared to the groups receiving saline or non-CpG-ODN. Colony-forming units of Salmonella Typhimurium in the peripheral blood were significantly lower in birds treated with CpG-ODN compared to the group that received saline. This is the first time that CpG-ODN has been demonstrated to have an immunoprotective effect against an intracellular bacterial infection in neonatal broiler chickens following in ovo delivery.