JIA-2019-11

2601 ZHANG Jun-qin et al. Journal of Integrative Agriculture 2019, 18(11): 2598–2604 carried out. Red fluorescence was detected in cells infected with the recombinant baculoviruses (Fig. 1-C) but not in non-infected cells (negative control). Thus, the recombinant proteins could be efficiently expressed in insect cells. 3.3. Detection of antibodies To monitor post-immune antibody response, serum samples were analyzed through a commercial ELISA Kit, of which the cutoff value was the S/P ratio of 0.5. Sera from groups A and B were positive for FAdV antibody on day 14 after the first immunization (Fig. 2). All birds immunized with the recombinant protein were seropositive one week after booster immunization. Notably, the antibody titers of groups Aand B peaked at 21 days post immunization(dpi), and that of group C peaked at 28 dpi. However, no antibodies were detected in the challenge control and negative control group. 3.4. Protection of recombinant proteins against FAdV-4 challenge After the challenge, data on morbidity and mortality were collected (Table 2). Birds immunized with penton and penton+fiber-2 exhibited complete protection. However, one of the ten birds vaccinated with fiber-2 died on day 4 post challenge (p.c). All the birds in the challenge control group (group D) started to show depression on day 2 p.c. and began to die on day 3 p.c, resulting in a mortality of 90% within seven days p.c. 3.5. Clinical and histopathological lesions There were no obvious clinical symptoms in birds of the inoculation group and in the negative control group. All dead and euthanatized birds were subjected to necropsy. Compared with groups A, B, C, and E, group D displayed severe gross lesion with accumulated straw-colored fluid in the pericardial sac and friable liver with multifocal necrotic patches (data not shown). Histopathological analysis showed no apparent clinical lesion in the heart and liver of chickens immunized with penton, fiber-2, and penton+fiber-2 (Fig. 3). The negative control group showed no clinical symptoms and no lesion (Fig. 3). The challenge control group manifested lesions. Lymphocyte infiltration was Table 2 Protective efficiency of recombinant proteins against virulent fowl adenovirus serotype 4 (FAdV-4) challenge Group Vaccination 1) Dosage (200 μL/per bird) Age at challenge (days) Morbidity Mortality Protection (%) A Penton 50 μg 42 0/10 0/10 100 B Fiber-2 50 μg 42 2/10 1/10 90 C Penton base+Fiber-2 25 μg+25 μg 42 0/10 0/10 100 D Challenge control Cell lysates 42 10/10 9/10 10 E Negative control Sterile PBS Sterile PBS 0/10 0/10 100 1) Challenge control, chickens in this group were immunized with Sf9 cell lysate supernatant at 14 days of age and were challenged at 42 days of age; negative control, chickens in this group were immunized and challenged with PBS at 14 and 42 days of age. Fig. 2 Antibody responses after immunization and histological lesions in chickens after fowl adenovirus serotype 4 (FAdV-4) challenge.  A, commercial Fowl Adenovirus Group 1 Antibody Test Kit (Biochek, Netherlands) was used to detect serum antibody levels for all birds from 0, 1, 2, 3, 4 weeks post first immunization. Sample with an S/P≥0.5 is considered positive. Data represent the mean±SD ( n =3). Different letters mean the differences are significant between groups ( P <0.05). 0 0.25 0.50 0.75 1.00 1.25 1.50 Penton (group A) Fiber-2 (group B) Penton base+Fiber-2 (group C) Challenge control (group D) Negative control (group E) Positive cutoff a a a a a a b b c d d b b c c Weeks post first immunization Mean S/P ratio 0 1 2 3 4