Experimental Fish
Haematological changes in infected Thai pangus Thai pangus, Pangasius hypopthalamus, were collected from Bangladesh Fisheries Research Institute" (BFRI) Mymensingh for artificial infection with Edwardsiella tarda bacteria. Fish were acclimatized in aquaria containing tap water for 7 days before experiment with aeration and feeding at alternate day with a commercial catfish feed (Saudi-Bangla Fish Feed Ltd.). Seventy percent water was changed every day. Mean length and weight of the fish were 7.5 ± 0.21 cm and 28.21 ± 0.49 g respectively.
Bacterial Inoculation and Sampling
The bacteria, Edwardsiella tarda stocked in fish disease Laboratory as a pathogenic form for P. hypopthalamus was used. It was isolated from kidney of P. hypopthalamus by the method of Mamnur Rashid et aL, (1999). Ten-fold serial dilutions were made from a fresh culture of this stock by using sterile physiological saline (90% NaCl = PS). A volume of 0.05 ml from the 10.2 dilution was injected intramuscularly to each of 20 fish. Thus infected fish were divided into 4 post inoculation groups: 1 day, 3 day, 7 day and moribund group each having 5 fish. A control group of 5 fish were injected with equal volume of PS. Fish were sampled at 1 day, 3 day and 7 day intervals. Sampling of the fish from control group was also performed at the above intervals. Moribund fish were collected at 9 day, 11 day, 13 day and 20 day post inoculation of bacteria.
Blood Collection
Fish were caught gently with a small scoop net avoiding stress and transferred into a plastic bowl containing the same water where they were stocked. After anesthetizing the fish with 5 ppm quinaldine (Sigma Chemical Co. USA; Mamnur Rashid et aL, 2002) blood samples were collected from the caudal vein with a heparinized sterile disposable plastic syringe. To avoid contamination with mucus and water the area of insertion of needle of syringe was wiped with cotton soaked in 70% alcohol before and after the blood collection. The collected blood was gently pushed into a sterilized small glass vial containing anticoagulant (potassium salt of ethylenediamine tetra-acetic acid, EDTA), to give final concentration of 5 mg EDTA per ml of blood. Sampled bloods were mixed gently but thoroughly and discarded if any difficulty was encountered in taking them or if clots were seen in the vial during inspection at the laboratory. This blood samples were used for determining the erythrocyte sedimentation rate (ESR), packed cell volume (PCV) or haematocrit value and haemoglobin concentration (Hb g%).
Blood Assessment
Blood was assessed according to Mamnur Rashid et al (2002) as follows: Total erythrocyte and leucocyte counts: To count total erythrocyte and leucocyte, Dacie's fluid was used. A 1 in 50 dilution of the blood in Dacie's fluid was prepared by mixing 20 mm3 of blood with 0.98 ml of the diluent. The suspension was introduced into an improved Neubaur ruling haemocytometer counting chamber (Precicolor, UBG, Germany) and the cells were counted by eye under microscope with high power objective (x45). Hand counter was used during cell count. For the erythrocyte count 1/5 mm2 was counted, while for the leucocyte count 4 mm2 were counted.
Erythrocyte sedimentation rate and haernatocrit value: Collected blood with anticoagulant was pushed into dry Wintrobe's haematocrit tube by Pasteur pipette exactly up to 0 or 100 mark. Care was taken to prevent air bubble formation in the tube. The tube was placed in a special rack in vertical position for 1 hour. The erythrocyte sedimentation rate (ESR) was calculated by measuring the distance of the erythrocyte that had sedimented in the scale, from the top of the tube and the result was expressed in mm/h. The tube was then spun in haematocrit centrifuge at 3000 rpm for 30 minutes for determining the haematocrit value (packed cell volume, PCV). After centrifuge three layers were observed: top, middle and lower layer. The top layer was plasma layer, the middle layer was leucocyte layer (buffy coat) and the lower layer was PCV value or haematocrit value. The volume at the top of the red column (i.e. erythrocyte layer) was measured from the scale from the bottom of the tube and expressed in ml %.
Haemoglobin concentration: Haemoglobin concentration was measured by the haematin method. Sahli haemoglobinometer (Resistance LW, Germany) was used for determining the haemoglobin concentration. Prepared 0.1 N hydrochloric acid (HCI) was placed up to the 20- marked graduation into the perfectly cleaned and dried haemoglobinometer tube. Blood sample was drawn into the Sahli pipette exactly up to 20 cm mark, side of the pipette was wiped with absorbent cotton and blood of the pipette was transferred immediately into a specialized graduated tube for haemoglobin estimation containing 0.1 N HCI. At that time pipette was rinsed 2 to 3 times by sucking water. The tube was shook until the blood was well mixed with the hydrochloric acid and water and the mixture became uniformly dark brown in colour. After about 5 min water was added into the brown colour solution drop by drop with a dropper and each time the solution was mixed with a stirrer until the colour of the solution matches the standard colour of haemoglobinometer. After matching, the result was taken in day light from the scale of the measuring tube by observing the graduation mark at the lower edge of the meniscus at the top of the liquid column and expressed in g %.