The main stages of the phagocytic process after the intraperitoneal injection of colloidal carbon to tilapia (Oreochromis niloticus), was ultra-structurally documented. Carbon particles were initially indiffuse form in the cytoplasm but they gradually formed discrete electron dense bodies that later fused with cytoplasmic granules to form phagolysosomes. Ultra structural results demonstrate that colloidalcarbon is actively phagocytized by cytoplasmic granules (lysosomes) in the spleen of O. niloticus. Direct phagocytic reaction of cells in the presence of an antigen is therefore very important in immunity

Adedeji, OB

Adeyemo, OK

Agbede, SA

Junaid, AU

English

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 African Journal of Biotechnology Vol. 5 (22), pp. 2350-2353, 16 November 2005     Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2006 Academic Journals       Full Length Research Paper  Ultrastuctural study of the phagocytic activities of splenic macrophages in tilapia (Oreochromis niloticus)    Samuel Adewumi AGBEDE1, Olanike Kudirat ADEYEMO1*, Olufemi Bolarinwa ADEDEJI1, Abdulkadir Usman JUNAID2   1Department of Veterinary Public Health and Preventive Medicine, University of Ibadan, Ibadan, Nigeria. 2Department of Veterinary Public Health and Animal Production, Usmanu Dan Fodiyo University, Sokoto, Nigeria.  Accepted 27 May, 2006  The main stages of the phagocytic process after the intraperitoneal injection of colloidal carbon to tilapia (Oreochromis niloticus), was ultra-structurally documented. Carbon particles were initially in diffuse form in the cytoplasm but they gradually formed discrete electron dense bodies that later fused with cytoplasmic granules to form phagolysosomes. Ultra structural results demonstrate that colloidal carbon is actively phagocytized by cytoplasmic granules (lysosomes) in the spleen of O. niloticus. Direct phagocytic reaction of cells in the presence of an antigen is therefore very important in immunity.  Key words: Tilapia, Oreochromis niloticus, ultrastructure, colloidal carbon, phagocytic activities.   INTRODUCTION  Fish are one of the most primitive vertebrates and are an important link between invertebrates and higher vertebrates. They possess the non-specific defence mechanisms of the invertebrates such as the phagocytic mechanisms developed by macrophages and granular leucocytes, and were also the first animals to develop both cellular and humoral immune responses mediated by lymphocytes. The main lymphoid organs of fish are the thymus, the anterior kidney and the spleen. In fish, non-specific immunity is considered as the first line of defence and represents a considerable part of the imm-une response (Dalmo et al., 1997).  The innate (non-specific) immunity is thought to have a major role in disease resistance of fish (Roed et al., 1993; Balfry et al., 1997a,b). The most important cells involved in this defense are the phagocytes (Esteban and Meseguer, 1997; Esteban et al., 1998). Phagocyto-sis is a defense mechanism against infection common to all  metazoans,  including  invertebrates. The phagocytic   *Corresponding author. E-mail: olanikeadeyemo@hotmail.com. Tel: +234-805-545-4544. system in fish consists of resident macrophages present in all organs and tissues of the body. They are the first phagocytes to encounter the invading microorganisms and as such, play a very important role in the initial stages of infections. Phagocytic activity is an important immunological parameter in the study of bacterial infections in fish, since it is related to the nutritional status, age and race of the animals and to preventive or therapeutic treatment. Several parameters of the innate immune response, such as respiratory burst activity, spontaneous haemoly-tic activity, lysozyme activity, complement concentration, and total IgM, are found to be associated with disease resistance in fish (Wiegertjes et al., 1996).  The aim of the present work was to study the phagocy-tic response of splenic macrophages to intra-peritoneal injection of colloidal carbon in tilapia (Oreochromis niloti-cus).   MATERIALS AND METHODS  Fish  Twenty (20)  juvenile  tilapia  (0. niloticus)  weighing  between  45.5         Figure 1. Electron micrograph of an ellipsoid wall (EW) with a carbon laden macrophage closely applied. Note the carbon particles within the cytoplasm. X 8000 (Let X 2000= 0.1cm, Scale bar = 0.4cm)     Figure 2. Electron micrograph of a macrophage with carbon particles diffusely scattered within the cytoplasm. X 10000 (Let X 2000= 0.1cm, Scale bar = 0.5cm)         Figure 3. Aggregation of phagocytosed free carbon particles formed discrete electron dense bodies (arrow) X 15000. (Let X 2000= 0.1cm, Scale bar = 0.75cm).   and 60.2 g were purchased from a fish farm (Zartech farms, Ibadan).  Clinical,  parasitological  and bacteriological examinations Agbede et al.      2351    showed the fish to be free of infectious diseases. The fish were acclimatized at 24.5-25.5oC for fourteen days and fed with commercial fish pellet at 4% body weight. The natural photoperiod was maintained during the acclimation and experimental periods.   Preparation and injection of carbon  A 50% suspension of Rowney Kandahar 28 black Indian ink was prepared in distilled water. Fish, were lightly anaesthesized with 0.2 mg benzocaine dissolved in 5 ml of acetone and added to 8 litres of water (Roberts, 1978). In the test group, each fish was injected intraperitoneally with 0.2 ml of the carbon suspension. Control fish were injected with 0.2 ml of distilled water. Two (2) test fish and one (1) control fish were sacrificed (after being anaesthesized as previously described) at each of the following intervals after injection: 1, 4, 8, 12 and 24 h. The abdominal cavities were then opened; spleens were removed.   Transmission electron microscopy  1 mm3 sized first fixed in 2.5% glutaraldehyde in sodium cacodylate buffer at pH 7.2–7.4 for 1 h at room temperature, then for 8 h at 4°C. Splenic tissues were post fixed in 1% osmium tetroxide for 1 h and dehydrated in graded series of ethanol. They were transferred via propylene oxide into epoxy resin embedding medium to produce medium hardness blocks prior to sectioning in a LKB Ultrotome III ultra-microtome.  Ultra thin sections were stained with uranyl acetate and lead citrate (Chiu et al., 1993). Stained sections were observed with a Jeol-Jem 100S Transmission electron microscope.   RESULTS AND DISCUSSION  Immunology is the study of the complex cellular and chemical reactions that occur in a host animal in response to contact with a foreign entity, such as a pathogen. An immune response involves recognition of the pathogen by the host as foreign and the subsequent immune response to eliminate it (Roitt, 1998). In recent years, there has been growing concern about the effect of environmental pollutants on the immune system. It is known that phagocytosis is the principal function of macrophages in teleosts. The non-specific immunity in fish, similarly to those in higher vertebrates, mainly depends on the activity of monocytes/macrophages, melano-macrophagic centres, neutral granulocytes and thrombocytes (Adeyemo et al., 2002).  In this study, histological sections post injection showed that carbon particles progressively accumulated in the spleen. In the experimental fish sacrificed after 4 h post injection, carbon particles were diffusely distributed in the cytoplasm (Figures 1 and 2), but they gradually form discrete electron dense bodies after 12 h (Figures 3 and 4) that could fuse with some cytoplasmic granules (lysosomes) to form phagolysosomes 24 h post injection of the carbon suspension (Figures 5 and 6). This is in accordance with  the  study  conducted  by  Esteban  and    2352         Afr. J. Biotechnol.        Figure 4. High power electron micrograph of fully formed electron dense bodies of aggregated carbon particles. Note that carbon particles are not uniformly distributed in each electron dense body. X 30000 (Let X 2000= 0.1cm, Scale bar = 1.5cm).         Figure 5. Electron micrograph of a phagolysosome (P), which resulted from the fusion of a carbon-laden macrophage with a lysosome. X 30000 (Let X 2000= 0.1cm, Scale bar = 1.5cm).   Meseguer (1997) and Esteban et al. (1998), which indicate that fish monocyte-macrophages may be considered the most active phagocytic cell type. The phagocytic process of O. niloticus splenic leucocytes showed similar ultrastructural features to those described for sea bass ((Dicentrarchus labrax L.) head-kidney leucocytes (Esteban and Meseguer, 1997) and gilthead seabream (Sparus aurata L.) head-kidney leucocytes (Esteban et al., 1998). Macrophages and neutrophilic granulocytes were the two cell types with significant phagocytic capacity in sea bass, which is similar to what has been previously described for rainbow trout (Afonso et al., 1997, 1998). Granulocytes and thrombocytes have also been described as phagocytic cells in some fish species (Rowley et al., 1988). The results reported in this study demonstrate that macrophages and granulocytes are the leucocytes responsible for phagocytic activities in the spleen of this tilapia, in agreement with results from other fish species (Pedrera et al., 1992; Bandin et al.,       Figure 6. High power electron micrograph of carbon particles concentrated in a phagolysosome. X 60000 (Let X 2000= 0.1cm, Scale bar = 3cm).   1993; Lamas and Ellis, 1994).  Previous studies of fish toxicology have targeted ecological parameters such as population dynamics. More recently; however, attention has focused on toxicological pathways and other parameters at the cellular or tissue level (Wester et al., 1994). Toxic effects on the immune system (immunotoxicity) may serve as an excellent tool and as a biomarker to monitor pollution and water quality since most environmental pollutants are known to alter immune system function even at doses that do not produce organ toxicity. Thus, fish immunotoxicology may offer new tools to assess environmental water quality.   REFERENCES  Adeyemo OK, Agbede SA, Magaji AA (2002). Clearance of colloidal carbon from the blood of tilapia (Oreochromis niloticus L.). Vet. Archiv. 72: 109-118. Agbede SA, Adeyemo OK (2001). Penetration and fate of intraperitoneally injected colloidal carbon in the lympho-haemopoietic organs of tilapia (Oreochromis niloticus L.). Trop. Vet. 19: 37-43.  Afonso A, Ellis AE, Silva MT (1997). The leucocyte population of the unstimulated peritoneal cavity of rainbow trout (Oncorhynchus mykiss). Fish and Shellfish Immunol. 7: 335-348. Afonso A, Lousada S, Silva J, Ellis A, Silva MT (1998). Neutrophil and macrophage responses to inflammation in the peritoneal cavity of rainbow trout Oncorhynchus mykiss. A Light and electron microscopic cytochemical study. Dis. Aquat. Org. 34: 27-37. Balfry SK, Heath DD, Iwama GK (1997a). Genetic Analysis of Lysosyme Activity and Resistance to Vibrosis in Farmed Chinook Salmon, Oncorhyncus tshawytscha (Walbaum).  Aquacult. Res. 28: 893-899. Balfry SK, Shariff M, Iwama GK (1997b). Strain Differences in Non-specific Immunity of Tilapia (Oreochromis niloticus) Following Challenge with Vibro parahaemolyticus.  Dis. Aquat. Org. 30: 77-80. Bandin I, Ellis AE, Barja JL, Secombes CJ (1993). Interaction between rainbow trout macrophages and Renibacterium salmoninarum in vitro. Fish and Shellfish Immunol. 3: 25–33. Chiu W, Schmidt MF, Prasad BVV (1993). Teaching electron diffraction and imaging of macromolecules. Biophys. J. 64: 1610-1625. Dalmo RA, Ingebrigtsen K, Boegwald J (1997). Non-specific defence       mechanisms in fish with particular reference to the reticulo-endothelial system. J. Fish Dis. 20: 241-273. Do Vale A, Afonso A, Silva MT (2002).  The professional phagocytes of sea bass (Dicentrarchus labrax L.): cytochemical characterization of neutrophils and macrophages in the normal and inflamed peritoneal cavity. Dis. Aqua. Org. (In press). Esteban MA, Meseguer J (1997). Factors influencing phagocytic response of macrophages from the sea bass (Dicentrarchus labrax L.): an ultrastructural study. Anat. Rec. 248: 533–541. Esteban MA, Mulero V, Muñoz J, Meseguer J (1998). Methodological aspects of assessing phagocytosis of Vibrio anguillarum by leucocytes of gilthead seabream (Sparus aurata L.) by flow cytometry and electron microscopy. Cell Tissue Res. 293: 133-141.  Lamas J, Ellis A (1994): Atlantic salmon (Salmo salar) neutrophil responses to Aeromonas salmonicida. Fish and Shellfish Immunol. 4: 201–219. Pedrera IM, Collazos ME, Ortega E, Barriga C (1992). In vitro study of the phagocytic process in splenic granulocytes of the tench (Tinca tinca, L.). Develop. Comp. Immunol. 16: 431–439. Agbede et al.      2353    Roberts RJ (1978). The patho-physiology and systemic pathology of Teleost. In: RJ Roberts (ed.) Fish pathology. pp. 55-91. Roed KH, Fjalestad KT, Stromsheim A (1993). Genetic Variation in LysozymeActivity and Spontaneous Haemolytic Activity in Atlantic Salmon (Salmo salar).  Aquaculture 114: 19-31. Roitt I, Brostoff J, Male D (1998). Cell cooperation in the antibody response In: Immunology, 4th edn. Mosby, London, UK.  p. 132. Rowley AF, Hunt TC, Page M, Mainwaring G (1988). Fish. In: Rowley AF, Ratcliffe NA (eds) Vertebrate blood cells. Cambridge University Press (Pub.), Cambridge. pp. 19-127. Wester PW, Vethaak AD, Van Muiswinkel WB (1994). Fish as Biomarkers in Immunotoxicology. Toxicol.  86: 213-232. Wiegertjes GF, Stet RJM, Parmentier HK, Van Muiswinkel WB (1996).  Immunogenetics of Disease Resistance in Fish: A Comparative Approach. Develop. Comp. Immunol. 20: 365-381.    

Ultrastuctural study of the phagocytic activities of splenic macrophages in tilapia (Oreochromis niloticus)

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Ultrastuctural study of the phagocytic activities of splenic macrophages in tilapia (Oreochromis niloticus)

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