Pathological changes associated with fish diseases

A- clinical pathological changes (Fluids = blood and serum)

Hematological studies:• The blood samples for haematological

parameters were collected into the bottle pre-treated with EDTA, an anticoagulant.

• Blood samples for biochemical indices were collected into another sample bottle containing no anti-coagulant. These samples were spurned in the centrifuge at 3,000 rpm and the clearer portion decanted (after centrifugation) into small sample tubes stored in a freezer.

• The haematological indices examined include Red Blood count (RBC), white blood cell (WBC), packed cell volume (PCV), haemoglobin concentration (Hb) and Leucocytes differential count (monocyte, lymphocyte, eosinophil etc.).

• (The packed cell volume (PCV) was determined by spinning about 75 μl of each blood samples in heparinised capillary tube in a haematocrit centrifuge for about 5 min and read on haematocrit reader (Benson et al., 1989).

• Erythrocyte and Leucocytes counts were determined using Neubaur chamber method as described by Lamb (1981).

• The blood sample collected in each treatment was diluted at a ratio of 1: 200 for RBC counts using red cell diluting fluid while a dilution ratio of 1: 20 (blood: white cell diluting fluid) was used for WBC counts. Samples of RBC and WBC counts were obtained using the relationship:

• RBC/μl = Numbers of red blood cells counted × 5 × 10 × 200

• WBC/μ = Numbers of white blood cells counted × 0.25 × 10 × 20.

• Haemoglobin was estimated using cyanomethaemoglobin method. 0.02 ml of blood was expelled into 4 ml drakkins solution. The mixture was allowed to stand for 5 min for full colour development (Jain, 1986).

• Haemoglobin (g per 100 ml) MCH = × 10 RBC count million per cu.mm

Determination of phagocytic activity and phagocytic index:Phagocytic assay was determined according to Kawahara et al. (1991). Fifty µg Candida albicans culture was added to 1 ml of citrated blood collected from fish and shaken in water bath at 25 oC for 5 hours. Smears of the blood were then stained with Giemsa stain. Phagocytosis was estimated by determining the proportion of macrophages which contained intracellular yeast cells in a random count of 300 macrophages and expressed as percentage of phagocytic activity (PA). The number of phagocytized organisms was counted in the phagocytic cells and called phagocytic index (PI). Results were expressed as means ± S.E. and differences were evaluated by Student’s t–test.- Phagocytic activity (PA) =Percentage of phagocytic cells containing yeast cells. Number of yeast cells phagocytized- Phagocytic index (PI) = ----------------------------------------------- Number of phagocytic cells

The biochemical examination of blood serum was performed with the automatic- The following parameters were monitored: urea nitrogen (urea), creatinine (crea), Triglyceride, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), cholesterol (cholest) and glucose (gluc). - The serum total protein was determined by the Biuret method (Reinhold, 1953) using a commercial kit (Randox Laboratories Ltd, U.K), while albumin value was obtained by bromocresol green method (Doumas and Biggs, 1971). The globulin and albumin/globulin ratio were determined according to the method of Coles (1986). The serum creatinine and urea nitrogen were estimated by deproteinisation and Urease Berhelot colorimetric methods, using a commercial kit (Randox Laboratories Ltd, U.K). Also the free cholesterol was determined by nonane extraction and enzymatic colorimetric methods, respectively using commercial kit (Quimica Clinica Applicada, S.A), while the serum enzymes; Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) were obtained using the Randox Laboratories Ltd, UK test kits.- However, measurement of calcium, ph. and Mg. were carried out as the technique described in the references (Brown et al., 1986 and Brown and Taylor, 1995).

- Kits of serum parameters- Kits of Plasma glucose was determined using glucose kits supplied by Boehring Mannheium kit, according to Trinder (1969). - Kits of Total protein content was determined colorimetrically according to Henry (1964). - Kits of Activities of aspartate amninotransferase (AST) and alanine aminotransferase (ALT) were determined colorimetrically according to Reitman and Frankel (1957).- Kits of lactate dehydrogenases (LDH)) were measured by using Diamond diagnostics kits according to the method of Cobaud and Warblewski (1958).

• Determination of serum globulin:Serum globulin was determined by subtract the total serum albumin from total serum protein according to (Coles, 1974).• Determination of serum albumin / globulin ratio:Determined by division of serum albumin value on serum globulin value according to the method implied by (Coles, 1986).

• Determination of Serum bactericidal activity: Serum bactericidal activity to Aeromonas hydrophila strain was determined according to Rainger and Rowley (1993).Briefly, a 300 µl of Aeromonas hydrophila suspension (1.5 x 103 cells / ml) and 300 µl of fresh serum were mixed in sterile eppendorf tubes. A blank consisted of 300 µl of bacterial suspension and 300 µl of sterile PBS. The tubes were incubated at 28 oC. A 50 µl sample was removed at 0 , 1 , 2 , 3 , 4h , and different dilutions were plated on nutrient agar for 24 h at 28 oC , and colony forming units (CFU) were counted . The results were recorded as survival index (SI) (Word Low and Unlles, 1978). Values were Calculated as follows: SI = CFU at end / CFU at start x 100.

• Determination of serum lysozyme activity:Serum lysozyme activity was measured with the turbidimetric method described by Engstad et al. (1992); using 0.2 mg / ml lyophilized Micrococcus lysodekticus as the substrate in phosphate buffer adjusted to pH 5.75. Fifty microlitres of fish serum was added to 3 ml of bacterial suspension. The 540 nm absorbance was measured by spectrophotometer after-mixture (Ao) and after incubation for 30 min at 37 oC (A). The result was expressed as one unit of lysozyme activity was defined as a reduction in absorbency of 0.001/min.Lysozyme activity = (A0 - A) / A.

• Nitric Oxide Assay: It was measured using the method described by (Ramadan and Attia, 2003), briefly a total volume of 100 uL of each serum sample was incubated with an equal volume of Griess reagent, the mixture put a 92 micro titer plate well with flat bottom and incubated at 21 oC for 10 minutes, after incubation the optical density was recorded spectophotometerically at 570 nm using an ELISA reader (Model 680, BioRad). Absorbency of test sample was converted to micro molar of nitrite by comparison with absorbance values of sodium nitrite (Sigma Co) standard curve a linear curve fit.

• Mitogen assay:This procedure was done according to a method modified from Burrells and Wells (1977) and Rouse and Babiuk (1974). Briefly, blood was centrifuged at 600 x g to obtain buffy coat cells. These cells were layered onto Ficoll- (Pharmacia Canada Ltd., Dorval, Quebec) hypaque (Winthrop Laboratories, Aurora, Ontario) (density 1.079 g/cm3) to separate mononuclear cells from erythrocytes and granulocytes. The mononuclear cells were resuspended at a concentration of 1 X 106 viable cells/mL in RPMI-1640 (Grand Island Laboratories, Grand Island, New York) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Grand Island Laboratories, Grand Island, New York). Cells (1 mL) were cultured for 48 h at 37°C in the presence of 6.5 ,ug/mL of concanavalin A (CON A) (Difco Laboratories, Detroit, Michigan) or Escherichia coli lipopolysaccharide (LPS) (Difco Laboratories, Detroit, Michigan) before pulsing for 18 h with 1 ,Ci of methyl-[3H]-thymidine (Amersham, Oakville, Ontario).The amount of trichloroacetic acid insoluble radioactivity incorporated was measured as described previously (29) and expressed as mean counts per minute (cpm.).

Enzymatic biomarkers for heavy metals or any pollution:• Superoxide dismutase (SOD) activity was assayed

according to Paya et al. (1992) with minor modifi cations (Peixoto et al., 2006). Nitrotetrazolium blue chloride (NBT) was used as detection molecule instead of cytochrome c. Assays were conducted in the pres ence of potassium phosphate buffer (100 mM, pH 7.0), hypoxanthine (10 mM), and NBT (10 mM). The reaction was initiated by the addition of xan thine oxidase (0.023 U/mol) to enzymatic extract at 25 ºC. Activity was reported by its ability to inhibit 50 % reduction of NBT and the result is expressed as U/min/mg/protein.

• Catalase (CAT) activity was assayed by the method Claiborne (1985). The reaction mixture consisted in potassium phosphate buffer 50 mM, pH 7.4, hydrogen peroxide19 mM and PMS 10 %. The reaction was carried out at 25 ºC and the change in absorbance was recorded at 240 nm. CAT activity was calculated in terms of μmol H2O2 consumed/ min/mg/protein.

• Glutathione reductase (GR) activity was as sayed by the method of Carlberg and Mannervik (1975) as modified by Mohandas et al. (1984). The reaction system consisted of potassium phosphate buffer (100 M, pH 7.4), EDTA 0.5 mM, oxidized glutathione (GSSG) 1 mM, NADPH 0.1 mM and PMS 10 %. Enzyme activity was quantified at 25 ºC by measuring the disappearance of NADPH at 340 nm and expressed as nmol NADPH oxidized/min/ mg/protein.

• Glutathione S-transferase (GST) activity was measured according to Habig et al. (1974) with minor modification. Reaction mixture contained 2 mL of potassium phosphate buffer 100 mM, triton X-100 10 %, 1-chloro-2, 4-dinitrobenzene (CDNB) 100 mM, and GSH 100 mM. Reaction was started at 25 ºC by adding the sample and the absorbance was monitored at 340 nm. The GST activity was expressed in nmol CDNB/min/mg/protein (Uguz et al., 2003).

• Reduced glutathione (GSH) was determined by us ing the method of Jollow et al. (1974). PMS 10 % was precipitated with sulfosalicylic acid 4 % in 1:1 ratio. The samples were kept at 4 ºC for 1 h and centrifuged at 1500 rpm for 15 min at 4 ºC. The supernatant was used for GSH estimation. The assay mixture contained supernatant, phosphate buffer (100 mM, pH 7.4) and 5 5’ dithiobis 2 nitrobenzoic ‑ ‑ ‑ ‑acid, DTNB (stocks 100 mM in 100 mM sodium phosphate buffer, pH 7.4) in total volume of 3 mL. GSH activity was deter mined spectophotometerically by measuring reaction product at 412 nm and expressed as nmol of GSH consumed/mg/protein.

• Assay of Malondialdehyde (MDA): This was carried out according to Soon and Tan (2002). 1.0 ml of sample was combined with 2.0 ml of TCA-TBA-HCl reagent and thoroughly mixed. The mixture was heated for 15 minutes in a boiling water bath. After cooling, the flocculent was removed by centrifugation at 1000g for 10 minutes. Absorbance of the supernatant was determined at 535 nm against a blank containing all the reagents except the sample. The MDA concentration in the sample was calculated using extinction co-efficient of 1.56 x 10-5 M CM.

In shrimp: Hemolymph sampling

Hemolymph was sampled individually at the beginning of the study from all collecting shrimp. Hemolymph (100 µl ) was withdrawn from the ventral sinus of each shrimp into a 1 ml sterile syringe (25 gauge) containing 0.9 ml anticoagulant (30 mM trisodium citrate, 0.34 M sodium chloride, 10 mM EDTA, 0.12 M glucose, pH 7.55, osmolality 780 mosM kg /1).

• Phenoloxidase activity measurement:Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome produced from l-dihydroxyphenylalanine (l-DOPA) following the procedures of Hernandez- Lopez et al. (1996). The details of the measurements were described previously (Liu and Chen, 2004). The optical density of phenoloxidase activity was measured by recording the formation of dopachrome produced from L-dihydroxyphenylalanine (L-DOPA) as described by Hernandez-López et al. (1996). Briefly, the diluted hemolymph was centrifuged at 300 × g at 4°C for 10 min. The supernatant fluid was discarded and the pellet was rinsed, resuspended gently in 1 ml cacodylate-citrate buffer (sodium cacodylate 0.01 M, sodium chloride 0.45 M, trisodium citrate 0.10 M, pH 7.0) and then centrifuged again. The pellet was then resuspended with 200 μl cacodylate buffer (sodium cacodylate 0.01 M, sodium chloride 0.45 M, calcium chloride 0.01 M, magnesium chloride 0.26 M, pH 7.0).

• Respiratory burst activity of hemocytes quantification

The respiratory burst activity of hemocytes was quantified using the reduction of nitroblue tetrazolium (NBT) to formazan as a measure of superoxide anion, as described in other studies (Bell & Smith 1993, Song & Hsieh 1994). Briefly, an aliquot of 100μl hemolymph in anticoagulant solution was deposited on microplates previously coated with 100 μl poly-L-lysine solutions (0.2%) to improve cell adhesion. Microplates were centrifuged at 300 × g for 15 min. Plasma was removed and 100μl zymosan (0.1% in Hank’s solution minus phenol red) was added and allowed to react for 30 min at room temperature. The zymosan was discarded and the hemocytes were washed 3 times with 100 μl of Hank’s solution, and then stained with 100 μl of NBT solution (0.3%) for 30 min at room temperature. The NBT solution was removed and the hemocytes were fixed and washed 3 times with 100 μl of 70% methanol and air-dried. Formazan was dissolved by the addition of 120 μl of 2 M KOH and 140 μl of dimethyl sulfoxide. The OD at 630 nm was measured in triplicate using an enzyme-linked immunosorbent assay reader (Dynex Mrx II). The respiratory burst was expressed as NBT reduction by 10 μl of hemolymph.

• Superoxide dismutase (SOD) activitySuperoxide dismutase (SOD) activity was measured by its ability to inhibit superoxide radical dependent reactions using the Ransod Kit (Randox, Crumlin, UK). The details of the measurements were described previously (Liu and Chen, 2004). The optical density was measured at 505 nm, 37 oC, and the rate of reaction was estimated from the absorbance readings 30 s and 3 min after adding xanthine oxidase. A reference standard SOD was supplied with the Ransod Kit. One unit of SOD was defined as the amount required inhibiting the rate of xanthine reduction by 50%. Specific activity was expressed as SOD units / ml /1.

• Phagocytic activity For phagocytic activity tests was carried out after collection of 100 µl of hemolymph from the ventral sinus, and mixed with 100 µl and 900 µl of sterile anticoagulant for the measurement of phagocytic activity and clearance efficiency, respectively. Phagocytic activity was measured following the method described by Weeks-Perkins et al. (1995).Briefly, 200 µl of the diluted hemolymph sample was mixed with 0.2 ml 0.1% paraformaldehyde for 30 min at 4 oC to fix the hemocytes, and then centrifuged at 800/g (Model 5403, Eppendorf, Hamburg, Germany) at 4 oC. The details of the measurements were described previously (Liu and Chen, 2004). Two hundred hemocytes were counted.

• Phagocytic activity, defined as percentage phagocytosis was expressed as:

• Percentage phagocytosis = {(phagocytic hemocytes) / (total hemocytes) {x100.

Histopathological studies of hepatopancreas

Specimens for histopathological techniques were freshly taken from infected hepatopancreas. Samples in Davidson’s fixative and prepared for histological examination by standard procedures for paraffin sections stained with hematoxyline and eosin (H&E) and examined microscopically according to (Roberts, 2001).

Table Hematology (reference interval or mean ±S.D.) in teleost fishesAnalyses Species C. carpio C. auratus O. hybrid (mean ±S.D) (mean ±S.D) (mean ±S.D)

HCT (%) 33.4±1.51 22.3±1.04 27–37 Hemoglobin (g/dl) 8.2±0.36 6.7±0.25 7.0–9.8 MCV (fl) 202±5.5 137±2.6 115–183 MCH (pg.) 49.1 42±1.4 28.3–42.3 MCHC (g/dl) 22–29 22–27 30.32±0.8(%) RBC (mm3 x106) 1.67±0.08 1.61±0.81 1.91–2.83 WBC(mm3 x103) 52.3±4.88b 21.5–154.7 Lymphocytes(×103 _l−1) 32.26–35.15 26.7±2.89 6.8–13.64 Heterophils(×103 _l−1) 1.13–3.78 2.3±0.56 0.5–9.873 Monocytes(×103 _l−1) 0.19–0.7 6 0.2±0.1 0.4–4.3 Eosinophlis(×103 _l−1) 0.19–0.38 0.1±0.1 0.035–1.645 Thrombocytes(×103 _l−1) 12.6±3.5 19.4±3.9 25–85.2 References Groff and Zinkl (1999) Groff and Zinkl(1999) Hrubec et al. (2000) WBC included thrombocytes.