The changes in lymphocyte proliferation (A₅₇₀ value) are shown in Table 4. After immunization, the A₅₇₀ value in the SPP adjuvant (10, 30 and 50 mg/mL) groups and in the OA group were significantly higher than those in the BC group (p < 0.05) at all the time points. The A₅₇₀ value in the 30 mg/mL and 50 mg/mL SPP group kept higher level than those in the OA and BC groups at all the time points (p < 0.05). The A₅₇₀ value in the 10 mg/mL SPP group was significantly higher than that in the OA group on the 14th, 21st and 28th days (p < 0.05).

Sargassum pallidum was purchased from Qingdao Hai-jie Aquatic Science and Technology Co., Ltd., Shandong, China. The Sargassum pallidum polysaccharides (SPP) are polysaccharides compound easily dissolve in water, high viscosity, which are extracted from Sargassum pallidum.Ye analyse monosaccharide composition of SPP as rhamnose, xylose, fucose, mannose, glucose and galactose by GC. Polysaccharides in the Sargassum pallidum include outermost skeleton polysaccharide, the intercellular mucopolysaccharide and storage polysaccharides in protoplasts. The recent studies about SPP in the domestic and foreign are the intercellular mucopolysaccharide primarily [18,19]. The SPP we used were prepared as previously described [20]. The net polysaccharide content was 81.2%, as measured using the sulfuric acid-anthrone method. The SPP were diluted with deionized water, sterilized and assayed for endotoxin using the pyrogen test. When the endotoxin content was at the level recommended by the standards of the Chinese Veterinary Pharmacopoeia (less than 0.5 EU/mL) [21], the SPP were stored at 4 °C.

The ND virus vaccine (La Sota strain, EID50, 8.5) and the IB virus vaccine (M41 strain, EID50, 6.5) were provided by the China Institute of Veterinary Drugs Control; the AI virus vaccine (H9, K strain, EID50, 7.5) was provided by Heilongjiang Baizhou Bio-engineering Co., Ltd. (Baizhou, China), and was identified by the Virus Influenza Centre of the Chinese Academy of Preventive Medicine. The SPP adjuvant vaccines were produce by mixing the vaccines with three different concentrations of SPP; the non-adjuvant vaccine was produced by diluting the vaccines with PBS, and the oil adjuvant vaccine was produced by emulsifying the vaccines with white oil, in accordance with a previous report [22]. All of the vaccines contained the same amount of antigen.

Modified RPMI-1640 medium (Thermo Fisher Biochemical Products Co., Ltd., Beijing, China) supplemented with benzylpenicillin (100 IU/mL), streptomycin (100 IU/mL) and 10% (volume fraction) fetal bovine serum (Hangzhou Evergreen Biological Engineering Materials Co., Ltd., Hangzhou, China, No. 090610) was used for washing, re-suspending and culturing the cells and was stored at 4 °C. The concanavalin globulin (Con A; Sigma company, Beijing, China) was prepared at a concentration of 0.025 mg/mL in serum RPMI 1640 culture medium, was filter sterilized, dispensed and stored at −20 °C. The 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit, from the Institute of Biotechnology, was stored at 4 °C in the dark. The lymphocyte separation medium (No. 20110923) was purchased from Beijing Soledad Bao Technology Co., Ltd. (Beijing, China). The FITC-labeled rat anti-chicken CD3, PE-labeled rat anti-chicken CD4, PE-labeled rat anti-chicken CD8a, FITC-labeled rat anti-chicken IgGl, and PE-labeled rat anti-chicken IgGl monoclonal antibodies were obtained from the Jingmei Biotech Co., Ltd. (Shenzhen, China).

The AA broiler chickens, one day old, were purchased from the Zhucheng Foreign Trade Breeder Farm, and were housed in an isolator (220 cm × 85 cm × 180 cm) in an air-conditioned room at 37 °C with light for 24 h per day at the beginning of the pretest period. The temperature was gradually decreased to room temperature, and the light time was gradually decreased to 12 h per day; these conditions were maintained until sacrifice. The chickens were fed with a commercial starter diet from the Qingdao Zhengda Agricultural Development Co., Ltd. (Qingdao, China). All the procedures were performed in strict accordance with internationally accepted principles and the PR China legislation on the use and care of laboratory animals.

The chickens were divided randomly into 5 groups, with 50 chickens per group (250 in total). The chickens were 14 days old, the average maternal ND antibody titer was 4.6 log 2, the IB antibody titer was 3.8 log 2, the AI antibody titer was 4.3 log 2, and the average body weight was 232 g. The chickens were subcutaneously injected with the vaccines containing 10, 30 or 50 mg/mL of the SPP adjuvant, the oil adjuvant (OA), or physiological concentrations of saline (blank control group, BC).

On days 7, 14, 21, and 28, after vaccination, 6 chickens were used to determine the proliferation of peripheral lymphocytes using the MTT assay [27]. Whole blood samples were collected from the immune chickens and were immediately transferred into aseptic capped tubes containing sodium heparin, which was then diluted with an equal volume of Hanks solution and was carefully layered onto the surface of lymphocyte separation medium. After centrifugation at 800× g for 20 min, the cloud-like lymphocyte band was collected and washed twice with RPMI-1640 media without fetal bovine serum. The resulting pellet was re-suspended at 5.0 × 106 cells/mL in improved RPMI-1640 medium containing 10% fetal bovine serum. The cells were added to 96-well flat-bottomed cell culture plates (50 µL per well), and ConA (50 µL, final concentration of 10 μg/mL) was added. Each sample was prepared in triplicate, and the cells grown without ConA were used as the negative control. The plates were incubated in a humidified 5% CO₂ incubator (HF160W, Heal Force Bio-Meditech Holdings Limited., Shanghai, China) at 39 °C for 48 h. The MTT solution (10 µL) was added to each well and the incubation was continued until the wells were covered with deep-purple-colored crystalline formanzan. Formanzan-dissolving liquid (100 µL) was added to each well, and the incubation was continued in the CO₂ incubator until the formanzan was dissolved (visualized using a CKX41 OLYMPUS microscope, OLYMPUS (China) Co., Ltd., Shanghai, China). The plate was read at 570 nm (A₅₇₀ value) using a multi-well absorbance reader (Model 680, BIO-RAD, Shanghai, China).

On days 7, 14, 21, and 28 after vaccination, 6 chickens were used to determine the changes in the lymphocytes subsets. The lymphocytes were separated as described in Section 3.6. The cell number was adjusted to 5.0 × 106 cells/mL with PBS solution. FITC-labeled rat anti-chicken CD3 (10 µL) and PE-labeled rat anti-chicken CD4 (10 µL) were added to one sample, FITC-labeled rat anti-chicken CD3 (10 µL) and PE-labeled rat anti-chicken CD8a (10 µL) were added to a second sample, and FITC-labeled rat anti-chicken IgGl (10 µL) and PE-labeled rat anti-chicken IgGl (10 µL) were added to the control sample. The samples were added to the lymphocyte suspension (50 µL) and were mixed in the dark at 4 °C for 20 min. Next, PBS (500 µL) was added and the samples were mixed in the dark at 25 °C for 10 min. The cells were re-suspended and analyzed using a FACSCaliburTM flow cytometer (BD Bioscience, Beijing, China) [28].

Data analysis was performed with SPSS 16.0 software. One-Way ANOVA with Duncan’s post hoc test was used for multiple comparisons between groups. Values were expressed as the mean ± standard deviation (SD). The results of the comparisons between groups were considered significantly different if p < 0.05.