Lymphocyte proliferation response during Eimeria tenella infection assessed by a new, reliable, nonradioactive colorimetric assay

Avian Dis. Jan-Mar 2002;46(1):10-6. doi: 10.1637/0005-2086(2002)046[0010:LPRDET]2.0.CO;2.

Abstract

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.

MeSH terms

  • Animals
  • Cell Count / veterinary
  • Chickens / parasitology*
  • Coccidiosis / immunology
  • Coccidiosis / veterinary*
  • Colorimetry / methods
  • Colorimetry / veterinary*
  • Concanavalin A / pharmacology
  • Eimeria tenella / immunology*
  • Indicators and Reagents / pharmacology
  • Interleukin-2 / biosynthesis
  • Lymphocyte Activation*
  • Lymphocytes / immunology
  • Poultry Diseases / immunology*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Specific Pathogen-Free Organisms
  • Spleen / cytology
  • Spleen / immunology
  • Tetrazolium Salts
  • Thiazoles

Substances

  • Indicators and Reagents
  • Interleukin-2
  • Tetrazolium Salts
  • Thiazoles
  • Concanavalin A
  • thiazolyl blue