Morphology and cytochemistry of leucocytes and thrombocytes of six species of fish

Andreasen

Menzel

et al. 2005

A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,30,5,50-tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of b-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content. Abstract A direct, rapid, quantitative colorimetric assay to determine neutrophil primary granule degranulation was adapted for use with fathead minnow kidney neutrophils. The assay measures the exocytosis of myeloperoxidase (MPO) using 3,3 0 ,5,5 0 -tetramethylbenzidine as a substrate. The assay was validated by comparing the total myeloperoxidase content of neutrophil populations obtained from adult cattle, as a known positive, and fish; evaluating the effects of calcium ionophore (CaI), phorbol myristate acetate (PMA), aqueous solution of b-glucan (MGAQ) and zymosan (Z) with and without cytochalasin B (cyto B) as stimulants of degranulation; determining the kinetics of primary granule exocytosis and detecting changes in degranulation when fish were exposed to stress and anaesthesia with MS-222. The MPO assay detected MPO activity in fathead minnow neutrophils that correlated to neutrophil numbers, confirmed that degranulation was increased when CaI was used compared to other stimulants, determined degranulation peak at 60 min and confirmed decreased degranulation after exposure to handling and crowding stress, with and without MS-222. Therefore, the MPO assay is capable of detecting important differences that may occur in degranulation of fathead minnow kidney neutrophil primary granules and in total neutrophil myeloperoxidase content.

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Section: Discussionmentioning

confidence: 99%

Section: Neutrophil Separationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A rapid, direct assay to measure degranulation of primary granules in neutrophils from kidney of fathead minnow (Pimephales promelas Rafinesque, 1820)

Andreasen

Menzel

et al. 2005

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“…If independent counts differed for more than 10%, a third count of the leukocytes on the slide in question was performed using double-headed scope. Identification and morphological characterization of leukocytes were done using suggested criteria [4,5,7].…”

Section: Kidney Leukocyte Separationmentioning

confidence: 99%

Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing

Beck

et al. 2011

Studies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diffquick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of lymphocytes, monocytes, platelets/thrombocytes and granulocytes was determined in all fish species. Arctic grayling, June sucker, and cutthroat trout had MPO positive granulocytes, while shovelnose sturgeon heterophils had positive reaction for leukocyte AP, but not MPO. Presence of MPO indicated potential to measure oxidative burst and degranulation of neutrophil primary granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO in shovelnose sturgeon suggested use of different enzyme marker (AP) in degranulation assay for this species. Standardization of cytochemical techniques allowed forrapid screening of leukocyte types, reducing the number of fish, time and effort to select adequate neutrophil function assays to be used in studies of health status in species of special concern. KeywordsFish species of special concern, leukocyte morphology, cytochemical characterization, granulocyte function RightsWorks produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted. b s t r a c tStudies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diff-quick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of ...

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“…Granulocyte characterisation in cyprinid species (common carp, Cyprinis carpio L. and goldfish, Carassius auratus L.) describes the presence of myeloperoxidase in neutrophils [3,4]. Minimal data about leucocytes in fathead minnows suggest that neutrophils are the most abundant granulocytes in this species, since no basophils and very few eosinophils have been found in blood smears [25].…”

mentioning

confidence: 99%

Gradient separation and cytochemical characterisation of neutrophils from kidney of fathead minnow (Pimephales promelas Rafinesque, 1820)

Andreasen

Frank

et al. 2005