Antimicrobial mechanisms of fish phagocytes and their role in host defense

Beck

Fish & Shellfish Immunology

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 ...

Section: Discussionsupporting

confidence: 89%

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

Beck

Fish & Shellfish Immunology

et al. 2011

“…The phagocytosis is one of the most effective mechanisms for destroying invasive pathogens and for protection against disease outbreaks due to its immediate action that is a characteristic of innate defense (Neumann et al, 2000b). The leukocyte respiratory burst assay, optimised in this study, is a reliable tool to measure the ROSs produced during phagocytosis and can be applied in any study that evaluates the immune system.…”

Section: Resultsmentioning

confidence: 99%

“…One of the most important mechanisms of fish defense is the phagocytosis and some cells are able to destroy invading particles besides processing and introducing them to the specific cells that will promote immunoglobulin production (Neumann et al, 2000b). Monocytes, macrophages and neutrophils are professional phagocytes; however other cells can perform phagocytosis so that the cell requires the detection of the foreign particle through membrane receptors.…”

Section: Introductionmentioning

confidence: 99%

Leukocytes respiratory burst activity as indicator of innate immunity of pacu Piaractus mesopotamicus

et al. 2013

The present study evaluated the assay to quantify the respiratory burst activity of blood leukocytes of pacu as an indicator of the innate immune system, using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of the production of reactive oxygen species (ROS). In order to assess the accuracy of the assay, fish were challenged by Aeromonas hydrophila and sampled one week after challenge. The A. hydrophila infection increased the leukocyte respiratory burst activity. The protocol showed a reliable and easy assay, appropriate to determine the respiratory burst activity of blood leukocytes of pacu, a neotropical fish, in the present experimental conditions. Keywords: immune methodology, fish immunology, cellular immunity. Atividade respiratória de leucócitos como indicador de imunidade inata de pacu (Piaractus mesopotamicus)Resumo O presente estudo avaliou o ensaio para quantificar a atividade respiratória dos leucócitos do sangue de pacu como um indicador do sistema imune inato, usando a redução do nitroazul tetrazólio (NBT) a formazan como medida da produção de espécies reativas de oxigênio (EROs). Para avaliar a precisão do ensaio, peixes foram desafiados por Aeromonas hydrophila e amostrados uma semana após o desafio. A infecção com A. hydrophila aumentou a atividade respiratória dos leucócitos. O protocolo se mostrou confiável e de fácil aplicação, apropriado para determinar a atividade respiratória de leucócitos do sangue do pacu, peixe neotropical, nas condições experimentais apresentadas.Palavras-chave: metodologia, imunidade de peixe, imunidade mediada por células.

“…Several specifi c and innate immune components have been identifi ed and characterized in different fi sh species. The major innate immune components include the mucus layer on the skin, gills and gastrointestinal tract, phagocytes, non-specifi c cytotoxic cells (Evans et al 2001, Neumann et al 2001, as well as epithelial and dendritic cells (Dalmo et al 1996, Ganassin andBols 1996). Among these, fi sh mucus is considered a primary and a key component of fi sh immune system (Ingram 1980).…”

Section: Introductionmentioning

confidence: 99%

Elucidation of innate immune components in the epidermal mucus of different freshwater fish species

Timalata

Krishnaveni

Rao

et al. 2015

Acta Ichthyol. Piscat.

., Arockiaraj J. 2015. Elucidation of innate immune components in the epidermal mucus of different freshwater fi sh species. Acta Ichthyol. Piscat. 45 (3): 221-230.Background. Fish epidermal mucus is a key component of innate immune system and plays a major role in protecting fi sh against invading pathogenic microbes. The information on the role of epidermal mucus components is insuffi cient for many commercially important freshwater fi sh species. Hence, this study was directed to understand the innate immune components of epidermal mucus in these fi sh species. Materials and methods. Mucus samples were obtained from African catfi sh, Clarias gariepinus (Burchell, 1822); Indonesian snakehead, Channa micropeltes (Cuvier, 1831); striped snakehead, Channa striatus (Bloch, 1793); marble goby, Oxyeleotris marmorata (Bleeker, 1852); Nile tilapia, Oreochromis niloticus (Linnaeus, 1758); and Asian redtail catfi sh, Hemibagrus nemurus (Valenciennes, 1840). The variation in the protein profi le of the mucus were observed using SDS-PAGE analysis. The specifi c activities of various hydrolytic enzymes including lysozyme, alkaline phosphatase, esterase, and protease were analysed in the mucus and the levels of enzyme activities were compared among the experimental fi shes. We also elucidated the effect of various protease inhibitors on the protease activity and identifi ed the type of proteases in the fi sh mucus by azocasein hydrolysis assay and zymography. Results. Signifi cantly (P < 0.05) highest levels of lysozyme-, alkaline phosphatase-, and esterase activities were observed to be 15.77, 18.96, and 10.65 U · mg -1 protein respectively in C. striatus followed by 13.67, 15.06, and 16.67 U · mg -1 protein, respectively in C. gariepinus. The highest level of protease activity was recorded in H. nemurus (1630.97 U · mg -1 protein) and C. gariepinus (1596.4 U · mg -1 protein). A wide range of variation was noticed in the enzyme activities of fi shes, even from the same genus, for example C. micropeltes and C. striatus. Specifi c inhibitors were added to azocasein hydrolysis assay and zymography experiments to characterize the individual proteases present in the mucus of each fi sh species. This result revealed the highest level of serine proteases in mucus of redtail catfi sh and African catfi sh compared to cysteine and metalloproteases. In contrast, Indonesian snakehead and marble goby had similar levels of serine, cysteine and metalloproteases. Conclusion. This study provides an insight into the presence of various epidermal mucus enzymes in fi shes and therefore believed to be the components of innate immune system that may possibly provide protection for the fi sh against invading pathogenic microbes.