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United States Department of Agriculture

Agricultural Research Service

Title: Lymphocyte Proliferation Response During Eimeria Tenella Infection Assessed by a New, Reliable, Non-Radioactive Colorimetric Assay

Authors
item Miyamoto, Tadashi - USDA:ARS:ANRI:PBESL
item Min, Wongi - USDA:ARS:ANRI:PBESL
item Lillehoj, Hyun

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 18, 2001
Publication Date: N/A

Interpretive Summary: Avian coccidiosis costs the US poultry industry > $ 600 million annual economic losses. Due to an increasing drug resistance of coccidia parasites and the escalating consumer's concerns over the chemical residues in the poultry meat, development of chemical-free control strategy is urgently needed for avian coccidiosis. Understanding complex network of host immune system and the immunobiology of host-parasite interactions would lead to a better control strategy. Because of increasing evidence suggesting an important role of cell-mediated immunity (CMI) in disease resistance against coccidiosis, thorough investigation of avian T-cell function is crucial in the development of vaccination control strategies. For the poultry immune system, a good lymphocyte proliferation assay to evaluate CMI in normal and disease states in vitro is not available. Although the conventional radioactive assay is still considered as the gold standard for the assessment of cell growth because of its sensitivity, reliability, and dynamic range , the use of this assay is restricted in recent years because it poses a biological hazard to personnel and the environment. Therefore, ARS scientists developed a non-radioactive alternatives using a non-radioisotopic, colorimetric assay which is faster, cost efficient, and safe. This assay has been successfully used to measure cellular response during coccidia infection and would enhance the capability of poultry scientists to assess cellular function of poultry in various disease situations.

Technical 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 A (Con A)- induced splenic and peripheral blood lymphocyte (PBL) was evaluated using WST-8 and MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). Coefficient of correlation (r) between these two reagents was 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. . Additionally, the measurement of interleukin (IL)-2 production using WST-8 was highly reproducible and showed a significant increase in IL-2 production upon stimulation of E. tenella-immune spleen cells with Con A.. The WST-8 assay is safe, 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.

Last Modified: 10/22/2014