Lymphocyte proliferation response during Eimeria tenella infection assessed by a new, reliable, nonradioactive colorimetric assay.
The application of a tetrazolium salt, WST-8,2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt to the lymphocyte proliferation assay in the chicken system was evaluated. Proliferation of concanavalin (Con A)-induced splenic lymphocytes and peripheral blood lymphocytes (PBL) was evaluated with WST-8 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Coefficients of correlation (r) between these two reagents were 0.98 and 0.97 in splenic lymphocytes and PBL, respectively. In general, the sensitivity of the WST-8 assay was significantly higher than that of the MTT assay, and the standard deviations of the WST-8 assay were significantly lower than those of the MTT assay. The WST-8 assay was fast and highly reproducible and provided a good indication of mitogen-induced proliferation of spleen cells induced by Con A. With the use of the WST-8 assay, splenic mitogenic response of chickens infected with Eimeria decreased transiently at 7 days but increased significantly at 10 days after primary infection compared with that of uninfected chickens. Additionally, the measurement of interleukin (IL)-2 production with WST-8 was highly reproducible and showed a significant increase in IL-2 production upon stimulation of Eimeria tenella-immune spleen cells with Con A. After E. tenella infection, splenic IL-2 production increased significantly at 7 days post-primary and at 2 days post-secondary infection. The WST-8 assay is fast, simple, and more reproducible and sensitive than the MTT assay. This study demonstrates the effectiveness of the WST-8 assay to assess cell-mediated immune response of chickens in normal and disease states.
Miyamoto
Tadashi
T
Parasite Biology, Epidemiology, and Systematics Laboratory, Animal and Natural Resources Institute, USDA, ARS, Beltsville, MD 20705, USA.
Min
Wongi
W
Lillehoj
Hyun S
HS
D000818:Animals; D002452:Cell Count / Q000662:veterinary; D002645:Chickens / Q000469:parasitology*; D003048:Coccidiosis / Q000276:immunology / Q000662:veterinary*; D003124:Colorimetry / Q000379:methods / Q000662:veterinary*; D003208:Concanavalin A / Q000494:pharmacology; D016786:Eimeria tenella / Q000276:immunology*; D007202:Indicators and Reagents / Q000494:pharmacology; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000276:immunology; D011201:Poultry Diseases / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013047:Specific Pathogen-Free Organisms; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013778:Tetrazolium Salts; D013844:Thiazoles
D007202:Indicators and Reagents; D007376:Interleukin-2; D013778:Tetrazolium Salts; D013844:Thiazoles; D003208:Concanavalin A; C022616:thiazolyl blue