Interleukin-18 (IL-18) is an important cytokine involved in innate and acquired immunity. In this study, we cloned the fulllength chicken IL-18 (ChIL-18) gene from specific-pathogen-free (SPF) chicken embryo spleen cells and provided evidence that the ChIL-18 gene in a recombinant plasmid was successfully expressed in chicken DT40 cells. ChIL-18 significantly enhanced gamma interferon (IFN-␥) mRNA expression in chicken splenocytes, which increased IFN-␥-induced nitric oxide (NO) synthesis by macrophages. The potential genetic adjuvant activity of the ChIL-18 plasmid was examined in chickens by coinjecting ChIL-18 plasmid and inactivated Newcastle disease virus (NDV) vaccine. ChIL-18 markedly elevated serum hemagglutination inhibition (HI) titers and anti-hemagglutinin-neuraminidase (anti-HN)-specific antibody levels, induced the secretion of both Th1-(IFN-␥) and Th2-(interleukin-4) type cytokines, promoted the proliferation of T and B lymphocytes, and increased the populations of CD3؉ T cells and their subsets, CD3 ؉ CD4 ؉ and CD3 ؉ CD8 ؉ T cells. Furthermore, a virus challenge revealed that ChIL-18 contributed to protection against Newcastle disease virus challenge. Taken together, our data indicate that the coadministration of ChIL-18 plasmid and NDV vaccine induces a strong immune response at both the humoral and cellular levels and that ChIL-18 is a novel immunoadjuvant suitable for NDV vaccination. N ewcastle disease (ND) is a serious avian disease that causes substantial economic loss and remains a major threat to the poultry industry (1, 2). Outbreaks of ND among poultry occur worldwide, and the pathogenic form of the virus is a disease listed in the World Organization for Animal Health (OIE) Terrestrial Animal Health Code and must be reported to the OIE (3), which results in severe trade limitations (4, 5). Currently, vaccination is the major tool for controlling infection by Newcastle disease virus (NDV). The NDV vaccine strains LaSota, B1, Mukteswar, and V4 are used widely in China. However, virulent NDV strains are still frequently isolated in vaccinated birds, indicating that NDV remains an ongoing threat to commercial flocks of birds (6). Therefore, it is necessary to develop more efficacious vaccines to prevent NDV infection.Many techniques have been developed to increase the immunogenicity of vaccines. Among these, cytokines are effective immunomodulators in animal models or in clinical testing (7-10). Among the large number of cytokines, interleukin-18 (IL-18) is a strong stimulator of T helper type 1 (Th1) responses and activates natural killer (NK) cells, stimulates the synthesis of other immunoactive cytokines from Th1 cells, monocytes, and NK cells, and synergizes with IL-12 in the maturation of Th1 cells and the suppression of IgE synthesis by B cells (11-13). Thus, IL-18 functions as an adjuvant (14, 15). However, depending on the cytokine environment, IL-18 may also promote Th2-type responses (16,17) and antibody formation (18).The isolation and characterization of chicken IL-18 (ChIL-18) ...