2 H3C SO3Na Figure 1: Chemical structure of sodium lauryl sulfoacetate. dimethyl sulfoxide (DMSO) were purchased from Sigma. LaSota NDV vaccine was obtained from QianYuanHao Bio- logical Company Limited. Florfenicol pellets (0.25 g/piece) were purchased from Shanghai Animal Drug Factory. Flu- orescein isothiocyanate- (FITC-) labeled monoclonal anti- body against chicken CD4, phycoerythrin- (PE-) labeled monoclonal antibody against chicken CD8a, and biotin- labeled monoclonal antibody against chicken CD3 were purchased from Southern Biotech (USA). Streptavidin- allophycocyanin was purchased from Becton Dickinson (USA). 2.2. Lymphocyte Proliferation Assay. 10 mL of pooled blood sample was collected from 10 chickens and onefold was diluted with PBS. PBMCs were separated by density gradient centrifugation on 14 mL Ficoll-paque lymphocyte separation medium at 400 g for 15 minutes and resuspended (1×106 cells/mL) in RPMI 1640 medium supplemented with 10% FCS, 100 IU/mL penicillin, and 100 μg/mL streptomycin [19]. Lymphocyte proliferation was measured using MTT method as described [20]. Brieﬂy, 100 μL of the cell sus- pension was dispersed into each well of 96-well plates and then 62.5, 125, 250 or 500 μg/mL SLS was added (n = 12). After incubation at 39.5◦C, 5% CO2 for 44 hours, 10 μL MTT (5 mg/mL) was added into each well and the incubation was continued for 4 hours. Then 100 μL of DMSO was added and incubation was continued for additional 24 hours before measurement for OD570 values using an ELISA reader (Bio-Tek Instruments, VT). The cell suspension without SLS treatment and with 5 μg/mL Con A was used as the negative and positive control, respectively. 2.3. Immunization and Antibody Detection. One hundred forty-four chickens were randomly divided into 4 groups. The three experimental groups were intramuscularly injected with 1, 2, or 4 mg/kg of SLS for 3 consecutive days and the control group was treated in the same way with normal saline. On the third day after injection, all birds were intranasally vaccinated with LaSota NDV vaccine and the immunization was boosted on day 17. On day 10, 17, 24, 31, 38, or 45 after SLS primary treatment, serum samples were collected from all birds and the NDV-speciﬁc antibody was titrated using standard hemagglutination inhibition (HI) assay [19]. 2.4. Determination of CD4+/CD8+ T-cell Ratio and Immune Organ Indices. On day 10, 17, 24, 31, 38, or 45 after SLS treatment, 6 chickens of each group were sacriﬁced and the blood samples were collected for lymphocyte separation. CD4+/CD8+ T-cell ratio in PBMCs was measured using Veterinary Medicine International O HO Table 1: Promotion of chicken PBMC proliferation by SLS. OD570 0.154 ± 0.015∗ 0.171 ± 0.024∗ 0.158 ± 0.017∗ 0.140 ± 0.031∗ 0.149 ± 0.017∗ 0.073 ± 0.012 Group (dose) SLS (500 μg/mL) SLS (250 μg/mL) SLS (125 μg/mL) SLS (62.5 μg/mL) Con A (5 μg/mL) Normal saline The data are expressed as mean ± SD (n = 12) in relation to the blank well. ∗P < .05 was accepted as signiﬁcant diﬀerences compared to the normal saline control. ﬂow cytometry [21]. Brieﬂy, 2×106 cells from each chicken were incubated at 4◦C for 45 minutes with an FITC-labeled monoclonal antibody against chicken CD4, a PE-labeled monoclonal antibody against chicken CD8, and a biotin- labeled monoclonal antibody against chicken CD3. After two washings with PBS, the cells were incubated at 4◦C for 30 minutes with streptavidin-allophycocyanin conjugate. After two additional washings, CD4+/CD8+ T-cell ratio was determined by ﬂow cytometry. At the end of the experiment, the spleen, thymus, and bursa of Fabricius of each chicken were collected for organ index calculation [22]. 2.5. Determination of Anti-Immunosuppressive Eﬀect of SLS in Chickens. One hundred and eight chickens were separated into three groups. Each bird in experimental group I was intramuscularly injected with 2.0 mg/kg SLS and then orally administered with 1.2 g/kg thiamphenicol for 3 consecutive days, while the birds in experimental group II or control group were treated in the same way with 1.2 g/kg thi- amphenicol or normal saline only. On the third day after treatment, all birds were intranasally immunized with LaSota NDV vaccine and boosted on day 17. On day 10, 17, 24, 31, 38, or 45 after SLS treatment, the serum samples and PBMCs were prepared for determining the NDV-speciﬁc antibody response and CD4+/CD8+ T-cell ratio as previously described. 2.6. Statistical Analysis. The signiﬁcant diﬀerence of the averaged data was analyzed using t-test (P < .05). 3. Results 3.1. Enhancement of Lymphocyte Proliferation by SLS. After treatment for 44 hours with 62.5, 125, 250, or 500 μg/mL SLS, the OD570 values of all experimental groups were signiﬁcantly higher than those of the control group, which was comparable to that of Con A-stimulated group (Table 1). Among the four concentrations of SLS tested, the 250 μg group had the highest stimulatory eﬀect on chicken PBMCs, but the diﬀerence was not signiﬁcant. 3.2. Enhancement of Immune Response by SLS. After treat- ment with diﬀerent doses of SLS or normal saline, all chickens were vaccinated twice with live NDV vaccine, and