Abstract: To investigate the effect of double-expression DNA vaccine of SS and CST (hereinafter referred to as double-expression DNA vaccine) immunized calves through different routes, 27 dairy buffalo calves aged 2-6 months were randomly divided into four groups. The first three groups were orally (group O), nasally (group N), and orally plus nasally (group ON) vaccinated with dual-expression DNA vaccine at a dose of 3×10⁷ CFU·mL^-1, respectively. The control group (C) was orally plus nasally immunized with PBS, and boosted immunization once after 2 weeks. The results showed that all three immunization methods could induce calves to produce antibody of SS and CST. After 2 weeks of primary immunization, the positive rates in N and ON groups were the highest, both at 85.71%. At the 6^th week, the positive rates in each group decreased, the levels of antibody also decreased in O and N groups, but the levels of antibody increased in ON group. At the 10^th week, the positive rates in ON group decreased to 14.29%, and no antibodies were detected in the O and N groups. With respect to weight gain, at the 2^nd week, the average daily gain in the ON group was significantly higher (P < 0.05) than that in the O, N, and C groups. During the entire experimental period (0-10 weeks), compared with the C group, the O, N and ON groups were respectively increased by 5.26%, 10.53%, and 15.79%. After 10 weeks of primary immunization, the chest girth and leg-hip circumference in the ON group were significantly higher (P < 0.05) than those in the C group. Hormone measurement results showed that the concentration of GH, IGF-1, and T₃ in each group were as ON>N>O>C group. The IGF-1 concentration of O, N and ON group was significantly higher (P < 0.05) than that of C group at the 2^nd week. At the 6^th week, the concentrations of T₃ and T₄ in the ON group were significantly higher (P < 0.05) than those in the O and C groups. The cytokine test results showed that the three immune methods could stimulate the body to produce humoral and cellular immunity. At the 6^th week, the concentration of INF-γ in the ON group was significantly higher (P < 0.05) than that in the C group, and there was no significant difference (P>0.05) between the other groups. The detection of blood biochemical indexes showed that there was no significant difference (P>0.05) in the contents of total protein, glucose, non-esterified fatty and urea nitrogen in the serum of calves between the groups. In summary, oral, nasal and oral plus nasal could induce good immune effect in calves, especially oral + nasal spray combined immunization had the best effect.

Key words: immunization route, calf, somatostatin, cortistatin, DNA vaccine