The single chain chicken interleukin-12 (chIL-12)- or mature chIL-18-encoding region was cloned into the nonessential gene F1 1L of fowlpox virus (FPV) to generate the recombinant (r) FPV/chIL-12 (rFPV/chIL-12) or rFPV/chIL-18 for rchIL-12 or rchIL-18 production. Splenocytes of chickens were cultured with various dilutions of binary-ethylenimine (BEI)-inactivated rFPV/chIL-12 or rFPV/chIL-18-infected cell lysate for 48 h for interferon-gamma (IFN-gamma) determination. It was found that 1:10,000 chIL-12 or 1:10 chIL-18 cell lysate stimulated the highest levels of IFN-gamma production. When chickens given BEI-treated 1:10 rchIL-12 or rchIL-18 cell lysate, intraperitoneally, or rFPV (0.2 x 10(5) TCID(50)) by wing-web puncture, the highest level of IFN-gamma was detected in sera on day 3 postinoculation (dpi). In the splenocyte culture supernatants, the highest level of IFN-gamma was detected at 14 dpi or 21 dpi in responses to rchIL-12 or rchIL-18, respectively. The results indicated that rchIL-12 or rchIL-18 could induce IFN-gamma production both in vitro and in vivo assays, suggesting that both are biologically active and may allow them to be used in the future as the biological adjuvant in the poultry vaccine development, particularly co-administering with vaccine antigens. (C) 2010 Elsevier Ltd. All rights reserved.