Experimental animals
The animal experiments were conducted with the approval of the Animal Care and Ethics Committee of Yangzhou University. HY-line white chicken eggs were hatched and the chickens were detected for freedom from any clinical signs of enteric disease and negative for Salmonella. Two-day old chickens were used in this study and given antibiotic–free food and water throughout the experimental period.
Bacterial strains
S. Pullorum S06004 (accession No. CP006575.1), a nalidixic acid-resistant (Nalr) clinical isolate obtained from chickens with Pullorum disease in the Jiangsu Province of China in 2006 [15], and the virulent wild type S. Gallinarum strain SG9 (Nalr), supplied by Dr. Barrow [16], were used as challenge strains. S06004ΔSPI2 (Nalr, the whole SPI2 (~40 kb) deleted mutant of S. Pullorum S06004), constructed using the one-step inactivation method described by Datsenko and Wanner [17, 18], was used as the vaccine candidate for this study. Bacterial strains were stored as frozen cultures in Luria-Bertani (LB) broth with 20 % glycerol at −70 °C before use. LB broth, LB solid (15 g/L agar) and XLT4 (Difco) agar were used for culturing bacteria at 37 °C. The media were supplemented with Nal (40 μg/ml) as required.
Bacterial inoculation in chickens
One hundred 2-day old chickens were randomly assigned to 2 groups: vaccinated group (n = 45) and control group (n = 55). The vaccinated group was intramuscularly immunized with 2 × 107 CFU S06004ΔSPI2 in 100 μl phosphate buffered saline (PBS), while control group was unimmunized and only received equal amounts PBS.
Changes of body weight and clinical symptoms after vaccination
Body weights of these chickens were measured at 5, 12 and 19 days post vaccination (dpv), and they were monitored for 19 days for clinical signs of disease, which included anorexia, diarrhea and depression, etc.
Bacterial persistence and clearance from internal organs
Liver and spleen samples of five chickens from each group were aseptically collected at 5, 7, 10, 14 and 21 dpv for bacterial recovery. Then they were weighed and suspended in 1 ml PBS and homogenized individually. Homogenates (100 μl) of different dilutions were inoculated on XLT4 agar (containing 40 μg/ml Nal) for enumeration and incubated for 20 h at 37 °C. The bacterial number in the sample was counted and expressed as log10 CFU/g, negative samples were indicated as 0 CFU/g.
Immune responses induced by the vaccine strain
Humoral immune responses were evaluated through determination of Specific antibody IgG levels by Enzyme-linked immunosorbent assay (ELISA), using heat-killed whole S. Pullorum bacteria as coating antigen as previously described [19]. Serum samples were collected from five chickens of each group at 3, 7, 14 and 21 dpv, and diluted 1:50 to be used as the primary antibody. The secondary antibody was Horseradish peroxidase (HRP)-conjugated rabbit anti-chicken IgG (1:10,000 dilution). The bound HRP activity was determined using o-phenylenediamine dihydrochloride (Sigma), and the OD492 was determined with an ELISA reader after the reactions were stopped by 2 M H2SO4.
Cellular immune responses were evaluated by the peripheral mononuclear cell proliferation assay as previously described [20, 21]. Soluble antigen was prepared from the wild type S. Pullorum strain S06004. Peripheral lymphocytes were separated from blood of five birds per group using the Histopaque®-1077 (Sigma) at 7, 14 and 21 dpv. After trypan blue dye exclusion testing, a viable mononuclear cell suspension (100 μl) at 1 × 106 CFU/ml in RPMI-1640 medium with 10 % fetal calf serum, 2 mM L-glutamine, 50 U/ml of penicillin and 50 μg/ml of streptomycin was incubated in triplicate in 96-well tissue culture plates with 50 μl of medium alone or medium containing 4 μg/ml of soluble antigen at 41 °C (in a humidified 5 % CO2 atmosphere for 48 h). The proliferation of stimulated lymphocytes was measured using adenosine triphosphate (ATP) bioluminescence with the ViaLight® Plus Kit (Lonza Rockland, ME, USA). The blastogenic response against soluble antigen was expressed as the mean stimulation index (SI) as previously described [20].
Evaluation of immune protection
Protective efficacy of S06004ΔSPI2 against challenges with S. Pullorum and S. Gallinarum were assessed, based on survival rates and clinical symptoms (including anorexia, diarrhea, depression, high morbidity and mortality). At 10 dpv, twenty chickens from vaccinated group were randomly divided into two groups of 10 animals (group A and C), thirty chickens from control group were randomly divided into three groups of 10 animals (group B, D and E). Group A and B were challenged intramuscularly with 2 × 109 CFU S06004 in 100 μl of PBS. Groups C and D received equal amounts of SG9. Group E only received 100 μl PBS. The surviving birds were counted at 21 days post challenge, and clinical symptoms were recorded every day from 1–35 dpv.
Statistical analysis
All data were expressed as mean ± standard error of the mean (SEM) values unless otherwise specified and analyzed with GraphPad Prism. P values less than 0.05 were considered significant when using one-way analysis of variance (ANOVA).
