Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Characterization of Blood Leukocytes in Fish Species of Special Concern by Rapid Cytochemical Staining Dušan Palić1, Jelena Palić2 , Linda S. Beck3 Department of 1 Biomedical Sciences, 2Veterinary Pathology, The College of Veterinary Medicine ,Iowa State University; 3US Fish & Wildlife Service –Malherur Wildlife Refuge Abstract Materials and methods Studies of innate immunity in fish species of special concern are essential for better understanding of their health status. The cytochemical and morphological characterization of blood leukocytes has been used to provide information about phylogenetic differences and determine potential for use of neutrophil functional assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian leukocytes have been used to characterize leukocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Three fish from each species were sampled and six blood smears were prepared from each fish. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), Sudan Black B (SBB), Periodic acid-Schiff (PAS), alkaline phosphatase (AP), alpha-naphthyl acetate esterase, and Diff-quick stain; examined using bright field and differential interference contrast microscopy, and leukocytes on blood smears evaluated based on the cell morphology 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 did not. Sturgeon granulocytes showed positive reaction for leukocyte AP. 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, as well as use of phagocytosis or bacterial killing assays as method of choice for this species. Standardized cytochemical techniques allowed for rapid screening of leukocytes, 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. Leukocytes from four fish species of special concern were evaluated: Arctic grayling (Thymallus arcticus), cutthroat trout (Oncorhynchus clarki lewisi), June sucker (Chasmistes liorus), and shovelnose sturgeon (Scaphirhynchus platorynchus). Shovelnose sturgeon were used as surrogates for the endangered pallid sturgeon (S. albus). Three fish from each species were euthanized in MS-222, blood was collected in heparinized microhematocrit tubes from severed caudal peduncle, and six blood smears per fish were prepared. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), Sudan Black B (SBB), Periodic acid-Schiff (PAS), alkaline phosphatase (AP), alpha-naphthyl acetate esterase (α-NAE) (all from Sigma), and Diff-quick (Fisher) according to manufacturer’s instructions, with minor modifications. Leukocytes were examined using bright field and differential interference contrast microscopy microscopy. Cell morphology and presence or absence of the specific chromogen were evaluated. For granulocyte function assays kidney tissue from four individual cutthroat trout and shovelnose sturgeon was aseptically collected, and granulocytes were separated using a previously described technique [1]. Degranulation (measured by detection of MPO) and oxidative burst (measured by reduction of cytochrome C) were determined as previously described [1, 2]. The release of AP from shovelnose sturgeon granulocytes was determined using AP detection kit (Biosciences) in a setup similar to [1]. The percent release of MPO and AP was calculated using following formula: % release = [(ODstimulated - ODbackground) / (ODlysed – ODbackground)] x 100 * Figure 1. Characteristic morphology of blood neutrophils. Cells with multi-lobed or indented nucleus with moderate nuclear to cytoplasmic ratio were determined by Diff quick staining in Arctic grayling (A), cutthroat trout (B), and June sucker (C). Presence of myeloperoxidase in neutrophils from Arctic grayling (D), cutthroat trout (E), and June sucker (F) was determined using Peroxidase 391 staining kit. Bar = 20 µM. * Figure 3. Degranulation detected as release of myeloperoxidase in cutthroat trout (blue) and shovelnose sturgeon (red) head kidney leukocyte cell suspension after stimulation for 40 min with 1 µg mL-1 Lipopolysaccharide from E. coli (LPS); 1 µg mL-1 Phorbol myristate acetate (PMA); 5 µg mL-1 Calcimycin A23187; 200 µg mL-1 purified β-glucan from baker’s yeast; with (+) and without 2.5 µg mL-1 of Cytochalasin B; Hank’s Balanced Salt Solution with Calcium and Magnesium (HBSS) was used as non-stimulated control, and cells (100% enzyme activity) were lysed with 0.02% CTAB. *Significant differences (P<0.01) between stimulated and non-stimulated cells. Results * * Introduction Neutrophils are an important component of host defense against many bacterial, viral and fungal infections, and the evaluation of neutrophil function is valuable tool for assessment of the health status of individuals and animal populations. Severe or chronic stress is often associated with poor performance and has long been associated with immunosuppression in cultured fish. The decline in population and ecological relevance have placed fish species of special concern under federal or state conservation programs, including restocking of the hatchery spawned fish to natural habitats. Artificial reproduction d ti off the th species i off special i l concern and d culturing lt i ffry tto th the stocking size in hatchery environment has been performed with limited success. Optimal water quality, nutrition, and spawning conditions for these species are poorly known, and inadequate environment may lead to stress that could undermine efforts for successful re-introduction of adults and fingerlings to their native habitats, and weaken immune system of the brood stock. Cytochemical characterization of leukocytes, and especially granulocytes, with rapid staining kits detecting different enzymes has been used in mammalian species to provide insight in leukocyte function. Presence or absence of granular enzymes can reveal differences in function and type of the leukocyte, and help in making the decision about which assays show more promise in evaluation of specific cell type function. Narrowing the choice potential function assays y can reduce the number of fish used in the assay y of p optimization. * Figure 2. Characteristic morphology and cytochemical staining of the shovelnose sturgeon blood granulocytes. Presence of eosinophils and heterophils was detected in blood using Diff quick stain (A). Absence of MPO (B) and presence of AP (C) in granulocytes indicated the potential for use of AP in the degranulation assay. Presence of abundant granules in sturgeon leukocytes was detected by PAS staining (D). Bar = 20 µM. Figure 4. Oxidative burst detected as Cytochrome C reduction in cutthroat trout (blue) and shovelnose sturgeon (red) head kidney leukocyte cell suspension after stimulation for 40 min with 1 µg mL-1 Lipopolysaccharide from E. coli (LPS); 1 µg mL-1 Phorbol myristate acetate (PMA); 5 µg mL-1 Calcimycin A23187; 200 µg mL-1 purified β-glucan from baker’s yeast; with (+) and without 2.5 µg mL-1 of Cytochalasin B; Hank’s Balanced Salt Solution with Calcium and Magnesium (HBSS) was used as non-stimulated control. *Significant differences (P<0.01) between stimulated and non-stimulated cells. Conclusion Statement of problem The effects of captivity and spawning stress on innate immunity in species of special concern has not been studied, and basic characterization of granulocytes in these species has not been performed. The limited population and high value of individual specimens poses a limitation to the sample size to be used in optimization and determination of the appropriate function assays in evaluation of the innate immune status of the fish. Cytochemical staining of granulocytes was performed with commercially available leukocyte staining kits to determine presence or absence of enzymes characteristic for granulocytes in other species. The differences in granular enzymes indicated potential differences in function of the examined cells, assisted in selection of the assays, and reduced number of fish used to determine appropriate function assays. The microscopic analysis of blood smears revealed presence of several leukocyte cell types in all examined fish species: lymphocytes, monocytes, thrombocytes, and granulocytes (Tab. 1). Granulocytes in Arctic grayling, cutthroat trout and June sucker have characteristic neutrophil morphology, kidney shaped, indented, or multi-lobed nucleus with a moderate nuclear to cytoplasmic ratio (Fig. 1A-C). The presence of MPO in the cytoplasmic granules confirmed the presence of neutrophilic granulocytes in Arctic grayling, cutthroat trout and June sucker blood (Fig. 1D-F), while eosinophilic granulocytes, and multi-lobed, neutral stained granulocytes were detected in sholvelnose sturgeon (Fig. 2A). The absence of eosinophil peroxidase and MPO in sturgeon blood and head kidney granulocytes was indicative of heterophils heterophils, rather then neutrophils (Tab (Tab. 1 1, Fig Fig. 2B) 2B). Presence of MPO in neutrophil granules of Arctic grayling, cutthroat trout and June sucker indicated functional capabilities for degranulation and oxidative burst, that was confirmed when cutthroat trout head kidney cells were assayed (Figs. 3, 4). Absence of MPO in shovelnose sturgeon suggested reduced capabilities for oxidative burst and indicated the need for a different enzymatic marker to be used in the degranulation assay (Figs. 3, 4). The presence of AP and granules was detected (Fig. 2C-D), and AP as a marker in the degranulation assay resulted in the detection of 55% degranulation after 30 min stimulation (Fig. 5). Table 1. Overview of different leukocytes found in blood of four fish species. All species had thrombocytes, monocytes/macrophages, and lymphocytes present in the blood. Myeloperoxidase positive granulocytes were considered neutrophils, and MPO negative granulocytes were considered heterophils. Eosinophils were detected by Diff quick in shovelnose sturgeon, and no basophils were detected in any of the fish. The small number of samples and low numbers of blood eosinophils and basophils could have prevented detecting those granulocyte types in other fish species. Figure 5. Degranulation detected as activity of alkaline phosphatase in the supernatant of shovelnose sturgeon head kidney leukocyte cell suspension stimulated with 5 µg mL-1 Calcimycin A23187; HBSS with Calcium and Magnesium g was used as non-stimulated control. Enzyme y activityy in supernatants of cells lysed with 0.02% CTAB was used as 100%. Rapid cytochemical staining can be used as an efficient method for differentiation of various leukocyte types in fish. Information about presence or absence of the myeloperoxidase is indicative of the capability of the granulocytes to develop an oxidative burst response to stimulation, while presence of granules and detection of granular enzymes indicates the most appropriate marker to be used in the degranulation assays. Therefore, cytochemical staining performed on limited number of individual fish can reduce the overall number of fish used to select appropriate granulocyte function assays for measuring effects of stress on species of special concern held in captivity. References 1. 2. Palić D, Andreasen CB, Menzel BW, and Roth JA (2005). A rapid, direct assay to measure degranulation of primary granules in neutrophils from kidney of fathead minnow (Pimephales promelas Rafinesque, 1820). Fish and Shellfish Immunology 19 (3), 217-227. 217 227. Palić D, Andreasen CB, Herolt DM, Menzel BW, and Roth JA (2006). Immunomodulatory effects of β-glucan on neutrophil function in fathead minnows (Pimephales promelas Rafinesque, 1820). Developmental and Comparative Immunology 30(9), 817-830. This work was sponsored in part by Western Area Power Administration grant and Summer Research Scholar Program, The College of Veterinary Medicine, Iowa State University www.sf.adfg.state.ak.us www.tmurphywild.com www.hoglezoo.org John G. Shedd Aquarium