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United States Department of Agriculture

Agricultural Research Service

Title: Fluorescein Isothiocyanate Staining and Characterization of Avian Heterophils

Authors
item RATH, NARAYAN
item HUFF, GERALDINE
item Balog, Janice
item HUFF, WILLIAM

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 19, 1998
Publication Date: N/A

Interpretive Summary: Heterophils are a type of white blood cell in birds that protect against bacterial infections. They engulf bacteria and eventually kill them. In order to do that they go through a variety of biochemical maneuvers which are termed as "activation processes." There are different biochemicals which can stimulate these processes in the absence of bacteria. In this study we find that the heterophils of chickens and turkeys can be identified by staining them with a fluorescent dye called fluorescein isothiocyanate or FITC which binds to certain parts of the cells. In order to confirm that these fluorescent cells are heterophils we conducted several tests to show that indeed these cells are heterophils. We also showed that the blood levels of these fluorescent cells are significantly increased in turkeys that were infected with bacteria. The staining technique will be useful to localize these cells in different tissues in order to study their involvement in various disease processes in poultry.

Technical Abstract: Fluorescein isothiocyanate (FITC) was found to stain cytoplasmic granules of avian heterophil-granulocytes. In tissue sections, the fluorescent granulocytes were predominantly distributed adjacent to trabecular bones. The fluorescein stained granulocytes were abundant in synovial fluids of chickens with synovitis. A significant correlation was observed in the percent of fluorescein labeled granulocytes in blood smears and the percen of heterophils determined using an automated counting method, in unstained blood from normal and Escherichia coli-infected turkeys. The fluorescein-binding heterophils purified from chickens showed time dependent increases in the oxidation of 2', 7'-dichlorofluorescin diacetate (DCF-DA) and the reduction of nitroblue tetrazolium (NBT) which were indicative of changes in oxidative burst in response to phorbol 12-myristate 13-acetate (PMA), Salmonella typhimurium lipopolysaccharide (LPS), and zymosan A (ZA). These heterophil-activating agents also caused significant degranulation at 16 h post-treatment, as indicated by the loss of fluorescence. There were microscopically visible alterations in cell shapes and a decrease in the density of granules due to treatment with LPS, PMA or ZA. In addition, these cells also showed phagocytic response which was evident at 30 minutes of incubation with fluorescent latex particles. Both chicken and turkey heterophils produced interleukin-6 in vitro at 24 h in response to LPS but not to PMA, FMLP or ZA. The chicken heterophils showed spontaneous production of matrix metalloproteinases (MMP) which was significantly enhanced by treatment with LPS, PMA and ZA; however, LPS appeared to be most effective in inducing MMP production.

Last Modified: 8/19/2014
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