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In Vitro Phagocytic Study of Blood Leucocytes and Peritoneal Macrophages of Walking Catfish Clarias Batrachus ( Ham.) Against Aeromonas Hydrophila and Escherichia Coli.

M. Mamnur Rashid
Department of Aquaculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

M. Sardar
Department of Aquaculture, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh

M.R. Islam
Department of Pathology, Bangladesh Agricultural University, Mymensingh

Abstract/ Executive Summary:

In observation of in vitro phagocytic activity against Aeromonas hydrophila isolate 34k (a virulent form) and Escherichia coli (an avirulent bacteria) of neutrophil- and monocyte-like cells of walking catfish Clarias batrachus showed phagocytosis. Neutrophils and monocytes phagocytized the avirulent form of bacterial isolate more than the virulent one. Other blood leucocytes did not show phagocytosis. Peritoneal macrophage of the fish were separated by glycogen elicitation and the macrophages were being adhered on plastic cover slips for studying their in vitro phagocytic activity. Most of the cells were alive after adherence and showed phagocytosis against the virulent and avirulent bacteria. The percent phagocytosis and phagocytic index were higher against the avirulent E. coli than the virulent A. hydrophila.

Keywords: in vitro phagocytosis, Clarias batrachus; Leucocytes, Bacteria, Catfish

Location: Bangladesh Agricultural University, Mymensingh

Start Date: 00-00-2002

End Date: 00-00-2002

Program Area: Animal Health and Management

Commodity: Cat fish

Objectives:

To report the phagocytosis of virulent and avirulent bacteria by blood leucocytes (monocytes and neutrophils) and peritoneal macrophages.

Methodology:

Experimental fish: Clarias batrachus of about 70-80 g body weight were used to observe the activity of their phagocytic cells. They were acclimatized in aquaria containing tap water for 7 days prior to the experiment with aeration and feeding at alternative day with SABINCO fish feed. Seventy percent water was changed everyday. The fish were not fed during the experiment. Bacteria: Aeromonas hydrophila 34k, a virulent isolate and Escherichia coli, an avirulent bacteria, were used. These bacteria were cultured on TSA plates at 25°C for 48 hrs. A suspension of 50 mg/ml of both the bacteria were prepared in PS for using in whole blood and 10 mg/ml for using in peritoneal macrophage suspension. Staining procedure: Routine method of staining was performed for staining of blood smear on glass slide and adherent macrophages on cover slip by Wright and Giemsa stain. Isolation of peritoneal macrophages Fish were injected intra-peritoneally with 2.5 ml of 2.5% w/v glycogen in PS. After 3 days peritoneal macrophage were collected. As much blood was taken out as possible with a syringe with anticoagulant from the caudal vein to avoid possible contamination of peritoneal macrophage with red blood cells. Abdominal region of the fish was disinfected with 70% ethanol-cotton and 5-8 ml of calcium and magnesium free Hanks balanced salt solution (HESS, Sigma Chemical Co. USA: pH 7.2) was injected intraperitoneally with a 26 gauge needle. After 10 min the peritoneal macrophage were collected after making a small incision on the ventral position of the abdomen with one ml sterilized pipette in 15 ml plastic centrifuge tube. The abdomen was rinsed with another 5-8 ml of HESS and peritoneal macrophage suspension was collected. After centrifuging the tube at 1500 rpm for 15 min the supernatant was discarded and the peritoneal macrophage was resuspended in minimal essential medium with Earle's salt and L-glutamine (MEM, GIBCO laboratories, USA). The peritoneal macrophage-MEM suspension was centrifuged again for avoiding the effect of glycogen and the supernatant was discarded. The cell suspension was then passed through 26 gauge needle for several times to release any possible clot of the cells. Viability of the cells was checked by staining with 0.2% trypan blue and counting in haemocytometer. Almost 100% of the cells were alive at this stage. Cell concentration was adjusted to 4.5 x 10⁵ cells/mm³^.Phagocytosis in whole blood culture One mililiter of blood was aseptically withdrawn from caudal vein of walking catfish with a syringe containing one drop of anticoagulant (3.6% sodium citrate) as before. The blood was divided into two allocates of 0.5 ml into two vials. About 50 micro liter suspension (50 mg/ml) of Aeromonas hydrophila 34k and E coli were added to each vial separately. This mixture of blood and bacteria were incubated under shaking condition at 20°C for 2 h. Smears were prepared from the vial of blood and stained with Wright and Giemsa stain. The phagocytic index was calculated by counting at least 100 bacteria that were phagocytized by certain number of phagocytic cells/macrophages.

Source of Information: Bangladesh J. Fish. Res. 6(1), 2002: 35-41

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The blood leucocyte plays an important role in primary defense mechanism because of their engulfing nature against foreign particles.

Knowledge Management: Journal