Changes in biochemical, histological and specific immune parameters in Catla catla (Ham.) by Cynodon dactylon (L.)

2.1 Fish

The Indian major carp, C. catla, of similar age groups were obtained from the Golden fish farm Karandhai, Thanjavur, Tamil Nadu, India. Average weight of the fishes used for the experiment was 88.05 ± 4.75 g. All fishes were maintained in fiber reinforced plastic (FRP) tanks. The water was replaced once in two days to maintain the water quality. Fishes were kept at the ambient, uncontrolled temperature of 28 ± 2 °C under the natural photoperiod. Fishes were acclimated for 15 days and fed ad libitum with balanced fish diet prepared in the laboratory.

2.2 Bacterial strain

A. hydrophila (ATCC 49140) was cultured in tryptone soya broth (Himedia) for 24 h at 37 °C. The culture broth was centrifuged at 3000g for 10 min. The supernatant was discarded and the pellet was resuspended in phosphate buffered saline (PBS, pH 7.4) for immunization. The optical density (OD) of the bacterial suspension was adjusted to 0.5 and kept in the water bath at 60 °C for 2 h. This bacterial suspension was used for the challenge test. Sterility was confirmed by lack of growth on the nutrient agar. The stock culture in tryptone soya broth was stored at −70 °C with 0.85% (w/v) NaCl and 20% (v/v) to provide stable inocula throughout the study (Yadav et al., 1992).

2.3 Collection of plant and preparation of ethanolic extract of C. dactylon L.

The plant C. dactylon (L.) was collected from Madurai, Tamil Nadu, India, and transferred to PRIST University, Thanjavur, Tamil Nadu, India. It was taxonomically identified and authenticated by Rev. Dr. S. John Britto SJ, Director, The Rapinat Herbarium and Centre for Molecular systematics, St. Joseph College (Autonomous), Thiruchirapalli, Tamil Nadu, India. The voucher specimen was deposited at the Rapinat herbarium and the voucher number is RHCD BP18.

Fresh plant of C. dactylon (L.) was cleaned and shade dried. The dried plants were pulverized by an electrical blender and passed through 20 μm mesh sieve. A powdered plant (550 g) was extracted successfully with 1:2 w/v in 70% ethanol by using soxhlet apparatus. The extraction was carried out in 24 h at room temperature with mild shaking (Chopra et al., 1992). The extract was filtered and concentrated at 45 °C using rotary vacuum evaporator under reduced pressure. The yield of extract was 12.2% w/w in terms of dried starting material.

2.4 Diet preparation and design of experiment

Artificial diet was prepared by using the ingredients fish meal, fish grower, wheat flour, cod liver oil (Universal Medicare Pvt. Ltd.) and vitamin premix (Vetsfarma Ltd.), which contains 20% carbohydrates; 41% proteins; 15% lipids and 9% ash (Table 1). For experimental diet, various concentrations such as 0.05%, 0.5% and 5% level of ethanolic extract of C. dactylon were mixed with the normal diet before pelletization. The control and experimental diet groups were fed with normal diet at the rate of 2% of the body weight per day for 15 days. Experimental feeding was started four weeks prior to immunization.

Fishes were randomly divided into four groups in triplicate each with 12 fishes (n = 12 fishes/group). They were fed with 0%, 0.05%, 0.5% and 5% (later it will be called as control, group A, group B and group C, respectively) extract of supplemented diet at the rate of 2% of body weight per day for 60 days. Blood was collected for every ten days from each group for assaying particular biochemical parameters.

2.5 Collection of blood samples

For bleeding, each fish was individually caught using a dip net and was bled from the common cardinal vein using 1 ml tuberculin syringe fitted with 24 gauge needle (Michael et al., 1994). In order to collect the blood for serum separation, 200 μl of blood was drawn and whole bleeding procedure was completed within 1 min. The blood was collected in serological tubes and clot was stored at 20 °C overnight. The clot was then spun down at 400g for 10 min. The serum collected by aspiration was stored in sterile eppendorf tubes at −20 °C for further use. After blood collection, the spleen was removed from the fish for analysis of RNA/DNA ratio.

2.6 Haemoglobin content

The haemoglobin content of blood was analysed following the cyanmethaemoglobin method using Drabkins fluid (Qualigens Diagnostics, division of Glaxo Smithkline Pharmaceutical Ltd.). The absorbance was measured using a spectrophotometer (Systronics, UV–Visible spectrophotometer – 118, India) at 540 nm and the final concentration was calculated by comparing with the standard cyanmethaemoglobin (Qualigens Diagnostics). The haemoglobin concentration was calculated by using the following formula: $$\mathrm{Haemoglobin}(\text{g}/\text{dl})=60[\mathrm{OD}(\text{T})/\mathrm{OD}(\text{S})]\times 251/1000$$ where OD (T) = absorbance of test; OD (S) = absorbance of standard.

2.7 Total erythrocyte and leucocyte count

RBC and WBC diluting fluids were used for taking total erythrocyte count and total leucocyte counts. It was done by mixing 20 μl of blood with 3980 μl of appropriate diluting fluid in a clean test tube and was shaken well to suspend the cells uniformly in the solution. A small drop of this mixture was charged to Neubauer’s counting chamber of haematocytometer and counting was carried out (Larsen and Snieszko, 1964). $$\mathrm{Number}\mathrm{of}\mathrm{cells}(\text{cu}.{\text{mm}}^{-1})=\frac{\mathrm{Number}\mathrm{of}\mathrm{cells}\mathrm{counted}\times \mathrm{dilution}}{\mathrm{Area}\mathrm{counted}\times \mathrm{depth}\mathrm{of}\mathrm{fluid}}$$

2.8 Serum glucose, cholesterol, protein, albumin (A), globulin (G) and A/G ratio

Serum glucose level was determined by Nelson as described by Ashwell (1957); serum cholesterol level was estimated by Lieberman-Bourchard method (Naito, 1985).

Serum protein was estimated by biuret and BCG dye binding method (Reinhold, 1953) using the kit (total protein and albumin kit, Qualigens Diagnostics, Division of Glaxo Smithkline Pharmaceutical Ltd.). Albumin was estimated by bromocresol green binding method (Doumas et al., 1971) with slight modification in micro plate adaptation. The absorbance of standard and test was measured against blank in a spectrophotometer (Systronics, UV–Visible spectrophotometer – 118, India) at 630 nm. Globulin was calculated by subtracting albumin values from total serum protein. A/G ratio was calculated by dividing albumin values and globulin values.

2.9 RNA/DNA ratio

The nucleic acid content of the spleen was examined by the method of Schneider (1957). DNA and RNA were determined using diphenylamine and orcinol, respectively.

2.10 Assay of serum anti-trypsin activity

Serum anti-protease activity was performed and calculated by Vasudeva and Chakrabarti (2004): $$\mathrm{Trypsin}\mathrm{inhibition}(\%)=(A1-A2/A1)\times 100$$ where A1 = control trypsin activity (without serum); A2 = activity of trypsin remained after addition of serum.

2.11 Preparation of heat killed A. hydrophila bacterin

A. hydrophila was inoculated in tryptone soya broth and incubated overnight in a shaker for 12 h at 28 °C. The culture was centrifuged at 800g for 15 min at 4 °C. The packed cells were washed three times and then the required dose was prepared in phosphate buffered saline (pH 7.2). The bacterial suspension was prepared to 1 × 10⁶ CFU as determined using a Neubauer haemocytometer. The stock cultures in tryptone soya broth (Himedia) were stored at −70 °C with 0.85% (w/v) NaCl and 20% (v/v) to provide stable inocula throughout the study (Yadav et al., 1992).

Heat killed A. hydrophila bacterin was prepared according to the method of Karunasagar et al. (1997).

2.12 Experimental design for specific immunity

Fishes were fed with 0%, 0.05%, 0.5% and 5% (later it will be called as control, group A, group B and group C, respectively) extract of supplemented diet at the rate of 2% of body weight per day for 60 days.

Fishes were randomly divided into four groups (control, group A, group B and group C) in triplicate each with 12 fishes (n = 12 fishes/group) and were fed with experimental diet for 15 days. They were challenged with heat killed A. hydrophila (1 × 10⁶ cells/fish) through intraperitoneal (i.p.) injection. Then, they were fed with the same doses of diet for 7, 14, 21 and 28 days. Total number of fish used in this assay is 144. After the end of every seven days, blood was collected from the fish for assaying specific immunological parameter. Simultaneously, the post-immunized fish spleen of all experimental groups was isolated for histological studies.

2.13 Enzyme linked immunosorbant assay (ELISA)

The fishes were bled at regular intervals of 7 days post immunization to 28 days. Antibody was produced against heat killed A. hydrophila, which were detected using an indirect ELISA according to Binuramesh et al. (2005).

2.14 Plaque forming cell assay (PFC)

Plaque forming cell assay was carried out by the method of Binuramesh et al. (2005, 2006). Six fishes per group were injected i.p. with 0.1 ml of 5% SRBC after 28 days of confinement for evaluating the effect of confinement in all experimental diet groups and control.

2.15 Histological studies

After immunization with A. hydrophila, the experimental fish spleen samples were taken at 7th day and 28th day of feeding trial. Spleen samples were removed and fixed in 10% neutral buffered formalin, embedded in paraffin and stained with hematoxylin and eosin (H&E) for optical examination (Deivasigamani, 2008). Micrograph of spleen was taken from the paraffin sections at a final magnification of 400 using a Nikon Microphot-FXA microscope and an Olympus DP50 camera. For each group, micrographs originating from six fishes (n = 12) were analysed.

2.16 Statistics

The statistical analysis system (SPSS version 17.0) software was used to analyse all the data. The data were expressed as mean ± standard error of mean (SEM) and the data were analysed using the Student’s t-test and one-way analysis of variance (ANOVA) followed by Tukeys post hoc multiple comparison test. Differences were considered statistically significant at P < 0.05 for all the experiments used in this study. All treatments (six fishes in each treatment) were assayed in duplicate for each fish.

3.1 Haemoglobin contents, total erythrocyte count and total leucocyte count

Haemoglobin contents and total erythrocyte counts significantly increased in group A, group B and group C at 30–60 days of feeding (Table 2a and b, respectively). Total leucocyte count of C. catla significantly produced higher number of cells from 10 days of feeding. Particularly, leucocyte count was higher in group B and group C than in group A from 40 to 60 days of feeding with the experimental diet (Table 2c).

3.2 Serum glucose and cholesterol

Serum glucose level gradually increased in all experimental diet groups from 10 to 40 days of feeding (Fig. 1a). Glucose levels of group B (423 ± 12.52 and 441.33 ± 10.9) and group C (427.7 ± 21.5 and 445.7 ± 12.33) reached optimum at 30 and 40 days of feeding, respectively. Then, the glucose level slightly reduced at 50 and 60 days, although it was higher than that in the control and group A. In general, control and group A probably showed the same level of glucose.

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Figure 1

Biochemical values (mean ± SEM) of blood serum in Catla catla fed with different concentrations of C. dactylon mixed diet between normal diet (control). (a) Glucose (Hb: mg dL⁻¹), (b) cholesterol (mmol L⁻¹), (c) total protein (g L⁻¹), (d) albumin (g L⁻¹), (e) globulin (g L⁻¹), (f) albumin/globulin ratio (g L⁻¹), (g) RNA/DNA ratio were obtained at 10, 20, 30, 40, 50 and 60 days of feeding.

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Figure 1

Biochemical values (mean ± SEM) of blood serum in Catla catla fed with different concentrations of C. dactylon mixed diet between normal diet (control). (a) Glucose (Hb: mg dL⁻¹), (b) cholesterol (mmol L⁻¹), (c) total protein (g L⁻¹), (d) albumin (g L⁻¹), (e) globulin (g L⁻¹), (f) albumin/globulin ratio (g L⁻¹), (g) RNA/DNA ratio were obtained at 10, 20, 30, 40, 50 and 60 days of feeding.

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Figure 1

Biochemical values (mean ± SEM) of blood serum in Catla catla fed with different concentrations of C. dactylon mixed diet between normal diet (control). (a) Glucose (Hb: mg dL⁻¹), (b) cholesterol (mmol L⁻¹), (c) total protein (g L⁻¹), (d) albumin (g L⁻¹), (e) globulin (g L⁻¹), (f) albumin/globulin ratio (g L⁻¹), (g) RNA/DNA ratio were obtained at 10, 20, 30, 40, 50 and 60 days of feeding.

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Figure 1

Biochemical values (mean ± SEM) of blood serum in Catla catla fed with different concentrations of C. dactylon mixed diet between normal diet (control). (a) Glucose (Hb: mg dL⁻¹), (b) cholesterol (mmol L⁻¹), (c) total protein (g L⁻¹), (d) albumin (g L⁻¹), (e) globulin (g L⁻¹), (f) albumin/globulin ratio (g L⁻¹), (g) RNA/DNA ratio were obtained at 10, 20, 30, 40, 50 and 60 days of feeding.

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Serum cholesterol level of fish was significantly higher than that in the control in all experimental diet groups (Fig. 1b). Cholesterol level of group A was about similar to that of control at 30 and 40 days of feeding (2.89 ± 0.01 and 2.892 ± 0.01), respectively. Cholesterol level was very lesser in control at 20 and 50 days of feeding (2.702 ± 0.04 and 2.72 ± 0.01).

3.3 Serum protein, albumin globulin and A/G ratio

Total protein level increased in all experimental diet groups compared with the control, especially the level of total protein significantly increased from 30 to 60 days of feeding (Fig. 1c).

The albumin level also increased in all experimental groups compared with the control (Fig. 1d). However, the level of albumin in all experimental diet groups was relatively equal to that in the control at 10 and 20 days of feeding. Albumin level immediately raised in group B (1.58 ± 0.004) and group C (1.613 ± 0.001) compared to group A (1.0503 ± 0.00233) and control (1.012 ± 0.002) at 30 days of feeding. Then, gradually increased levels of albumin in group A, group B and group C were seen from 40 to 60 days of feeding compared to the control.

The globulin level progressively increased in all experimental diet groups (Fig. 1e). Level of globulin slightly reduced in group A (1.8543 ± 0.002) and group B (1.8553 ± 0.01) at 60 days compared with 50 days (2.083 ± 0.0311, 2.1623 ± 0.02154, respectively) of feeding. However, group C sustained their level of globulin at 50 (2.1953 ± 0.02) and 60 (2.191 ± 0.04) days of feeding.

The A/G ratio showed a relationship with the increasing percentage of C. dactylon extract (Fig. 1f). Among the control, high difference (1.21 ± 0.013) was found at 50 days of feeding. Significantly, low level of difference was found in group B (0.54 ± 0.002) and group C (0.5431 ± 0.0003) at 20 days feeding; group A showed the values 0.7132 ± 0.002 and 0.7925 ± 0.02 at 30 and 40 days of feeding, respectively.

3.4 RNA/DNA ratio

The RNA/DNA ratio in the spleen of experimental diet group fishes was highest at 60 days of feeding. There was a trend of increase in RNA/DNA ratio correspondingly with the number of days and experimental diet concentration (Fig. 1g). RNA/DNA ratio gradually decreased at 20 and 30 days and then significantly increased at 50–60 days of feeding. On the other hand, RNA/DNA ratio significantly increased at 40–60 days in group C (0.399 ± 0.031, 0.424 ± 0.022, 495 ± 0.031, respectively), when compared with the control (0.335 ± 0.0134, 0.384 ± 0.013, 0.389 ± 0.013, respectively).

3.5 Serum anti-trypsin activity

Serum anti-protease production significantly increased in all experimental diet groups from 10 to 60 days of feeding (Fig. 2). There was no significant enhancement of anti-protease activity observed in group A (69.9 ± 1. 37) compared with control (65.6 ± 1.64) at 10 days of feeding. Among the experimental diet groups, anti-protease activity was higher in group C and low in group A during all experimental periods. Among all the experimental groups, group B was more or less similar to group C except 50 and 60 days of feeding. These results proved that the anti-protease activity directly depends on the concentration of experimental diet.

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Figure 2

Effect of C. dactylon mixed diet on serum anti-protease activity in C. catla. Each value represents the arithmetic mean ± SEM (*P < 0.05) of six fishes.

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3.6 ELISA

ELISA result showed that a significant response in the production of antibody against heat killed A. hydrophila bacterin was observed in group B and group C than in group A and control (Fig. 3). Especially, group B (1.002 ± 0.03) and group C (1.22 ± 0.1) showed optimum response against A. hydrophila at 14 days of feeding and the level of antibody was also higher at 14, 21 and 28 days than group A (0.89 ± 0.01, 0.82 ± 0.001 and 0.65 ± 0.002, respectively) and control (0.89 ± 0.03, 0.81 ± 0.01 and 0.59 ± 0.01, respectively). A minor response of antibody was observed in group A (0.65 ± 0.002) when compared with control (0.59 ± 0.01) against A. hydrophila at 28 days of feeding. Furthermore, the results of group B and group C were more or less similar, which confirmed that the higher concentration of C. dactylon mixed diet provides a better response.

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Figure 3

Effect of antibody response to heat killed A. hydrophila in C. catla (n = 6 fishes/group) by ELISA. Each value represents the arithmetic mean ± SEM (*P < 0.05) of six fishes.

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3.7 Plaque forming cell assay

The number of antibody producing cells in response to SRBC was observed from 7 to 28 days of feeding in all experimental diet groups (Fig. 4). Among the experimental diet, a higher number of plaque forming cells showed in group C and it reached the maximum (61 ± 4.4) at 21 days of post immunization. The least number of antibody producing cells was observed in control (11 ± 1.5) than group A (38.7 ± 2.33), group B (45 ± 3.4) and group C (47.3 ± 5.9) at 28 days of feeding.

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Figure 4

Effect of the number of antibody producing cells against SRBC in C. catla (n = 6 fishes/group) by plaque forming cell assay. Each value represents the arithmetic mean ± SEM (*P < 0.05) of six fishes.

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3.8 Histological studies

The spleen of C. catla is a reddish-brown, elongated, thick and flattened structure, lying along the intestine and near the pancreas, trunk-kidney. The spaces within the connective tissue framework are filled with splenic pulp, the distinction of which into red pulp and white pulp is difficult in histological preparations. The pulp in general consists of large venous sinuses and their branches, representing the red pulp (RP), white pulp (WP), nodule-like ellipsoids of lymphoid tissue and dark-staining melanomacrophage centres (MMC) (Fig. 5). Heat killed A. hydrophila challenged fish spleen showed loosely packed red and white pulp (arrow) (Fig. 5a and c); macrophage aggregation can be viewed in Fig. 5b and collapsed MMC and progression of MMC are observed in Fig. 5d–h, which showed that the challenged fishes get immunized and develop their immunological parameters. Through this histological evidence it is proved that C. catla increased their immune response depending on the C. dactylon extract concentration.

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Figure 5

Light micrographs of spleen of Catla catla. Aeromonas hydrophila infected spleen shows loosely packed red and white pulp and collapsed MMC, which shows heavy infection. Arrows: melanomacrophage centres i.e., cluster of macrophage cells. RP: red pulp; WP: white pulp; MMC: melanomacrophage centre; BV: blood vessels.

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