Fish disease controlling efficacy study of selected Indian medicinal plant

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Journal of Pharmaceutical, Chemical and Biological Sciences ISSN: 2348-7658 Impact Factor (GIF): 0.615 Impact Factor (SJIF): 2.092 June-August 2016; 4(2):176-187

Fish disease controlling efficacy study of selected Indian medicinal plant

Ashokkumar R*, M. Ramaswamy

Department of Zoology, Karpagam University, Coimbatore 21, India

*Corresponding Author: Ashokkumar R, Department of Zoology, Karpagam University, Coimbatore 21, India

Received: 22 October 2015 Revised: 15 July 2016 Accepted: 17 July 2016

Original Research Article

ABSTRACT

The present study is aimed to investigate the disease controlling efficacy of the methanolic leaf extract of Indian medicinal plant, Phyllanthus amarus, amended with manually prepared feed fed to fingerlings of edible freshwater fish, Channa striatus(Bloch) which was exposed to two different concentrations (10^-5& 10^-3) of two species of microorganisms, Pseudomonas fluorescens (bacteria) and Penicillium chrysogenum (fungus)in different rearing tanks. Haematological parameters of control (prefed with normal feed) and experimental (prefed with plant extract/chemical antibiotics) fingerlings revealed that the methanolic extract of Phyllanthus amarushas has got higher controlling efficacy of bacterial infection than fungal infection by way of increasing the lymphocyte count in the blood to stimulate humoral immune response by way of producing immunoglobulins to fight against harmful microorganisms.

Keyword: Phyllanthus amarus; Pseudomonas fluorescens; Penicillium chrysogenum; channa striatus(Bloch); lymphocyte; humoral immune response; immunoglobulins.

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industry, causing heavy loss to farmers. In order to address this problem, several studies have been conducted on the modulation of fish immune system in order to prevent the disease outbreak as reviewed recently by Sakai (1999) [1]. Disease outbreaks are increasingly being recognized as a potential constraint on aquaculture production and trade, and cause massive financial loss through mortality or reduced meat quality, resulting in reduced profit margins, Smith et al., 2003 [2]. The economic loss due to the diseases outbreak in the aquaculture sector can be considerable.

For example, economic loss attributed due to the diseases in the Asian region countries was estimated to be at least US$ 1400 million in 1990 (ADB/NACA 1991). Antimicrobial effects of different extracts of various medicinal plants were studied by a number of workers, Subhadradevi V et al., 2011; Anushia C et al.,2009; Ramakrishnan G et al.,2011; Kaveri Singh et al.,2010; Sathya A V et al.,2012;

Mahesh B and Satish S, 2008; Foysal M J et al.,2011; Chakraborthy G S, 2008; Sathya A and Ambikapathy V, 2012; Ogunjobi A A and Ogunjobi T E, 2011. [3-12].

MATERIAL AND METHODS Collection of plant

Leaf sample of medicinal plant species, Phyllanthus amarus was collected from Kalingarayan canal bank at Bhavani (Erode District, Tamilnadu) Identification of the plant species was done with the help of Dr.R.Gopalan, Professor of Botany Karpagam University (former Scientist, BSI, Coimbatore),Coimbatore.

Preparation of leaf extract

The collected leaves were shade dried at room temperature for 20 days. The dried leaves were powdered in mechanical grinder. 20 grams of leaf powder was weighed, 150ml of methanol solvent was added and kept for 3 days. The

paper and the supernatant was collected. The residue was again extracted two times (with 3 days of interval) and supernatants were collected. The supernatants were pooled and evaporated (at room temperature, 28± 1˚C) till the volume was reduced to fully dried level.

Extract of the leaf powder of the Phyllanthus amarus stored in air tight bottles for further analysis.

Preparation of normal and extract/chemical antibiotics-amended fish feeds.

The normal fish feed was prepared by using known quantities of ingredients such as Anchovy, Jawala (dry fish), soya flour, tapioca flour, wheat flour and rice flour to maintain the required protein level. The selected ingredients were powdered and sieved to get fine particles of uniform size. Then, the ingredients were weighed according to the formulation [13] hand kneaded by adding sufficient quantity of distilled water and finally made into a dough.

The dough was then autoclaved in a closed aluminum container at 105^oC for about 15 minutes and then cooled. To the cooled dough, aqua savor, mono sodium phosphate, vitamin pre mix, mineral pre mix, vitamin C and vegetable oil were added and this served as the normal feed. The normal feed (Plate 13) thus prepared, was divided into five parts. One part was treated as control feed and the remaining 4 parts were used for preparing amended feeds by mixing the methanolic leaf extract of P.

amarus, tetracycline and fluconazole (2.5gm/50kg body weight concentrations).

Preparation of stock culture of microorganisms Stock cultures of Pseudomonas fluorescens was whereas, Penicillium chrysogenum was grown in potato dextrose agar (PDA) nutrient broth at 30˚C. Sub cultures of the microorganisms were also prepared and maintained at 4˚C, *14+.

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Collection of fingerlings

Fingerlings of Channa striatus, the freshwater, Indian murrel, vernacularly called as “Verral” or

“Snake headed fish”. A bulk sample of fingerlings (of 3 ± 0.5 cm length and 10 ± 2 gm Wt) of Channa striatus were obtained from M/S. Murugan fish farm, Palayamkottai. The bulk samples were acclimated for 10 days in large fiber glass tanks with regular feeding with normal fish feed [13].

Collection of blood samples for haematological analysis

For haematological observations, blood sample was collected by direct cardiac puncture of fingerlings using a sterile insulin needle (1ml) pre-rinsed with anticoagulant (heparin) solution. 0.5ml of blood sample was obtained by pooling the blood collected from 10 fingerlings for each experimental set up. Blood samples were collected from control fingerlings prefed with normal feed and microbe exposed, and fingerlings prefed with amended feed and microbe-exposed at the end of 1^st, 7^th, 14^th and 21^st days. The haematological parameters such as total RBC count, haemoglobin content, total leucocyte count (TLC) and differential leucocyte count (DLC) were estimated in the blood of fingerlings of Channa striatus prefed with control/amended feeds and exposed to microorganisms [13].

RESULTS

Haematological parameters in microbe- exposed fingerlings of Channa striatus prefed with control and amended feed

The data on the different haematological parameters of control (prefed with normal feed) and experimental (prefed with plant extract/chemical antibiotics) fingerlings exposed to two different concentrations of microorganisms are presented in Tables 1 to 8.

Comparative analysis of percent changes (from control level) in different haematological parameters in the blood of experimental fingerlings prefed with amended feed and exposed to microorganisms are shown in Figs. 1 to 8.

(a) Total erythrocyte count

The total erythrocyte count in the blood of control (prefed with normal feed and Pseudomonas fluorescens exposed for 6 days) fingerlings at the end of 21 days ranged from 3.30 ± 0.01 to 3.40 ± 0.01 10⁶cells/mm³. The total erythrocyte count of the fingerlings registered elevations (ranging from 0% to 3%) from the control levels (excepting that of 10^-3 concentration of bacteria – exposed fingerlings prefed with PA extract) (Table 1).

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Table 1. Total erythrocyte count (10⁶cells/mm³) in the blood of control (prefed with normal feed) and experimental (prefed with plant extracts/chemical antibiotic) fingerlings exposed to two different concentrations of Pseudomas fluorescences and Penicillium chrvsogenum.

Values are means of 6 observations ± S.E. percent from control level are given in the parenthesis (b) Haemoglobin content

The haemoglobin content of control fingerlings (prefed with normal feed) from 1^st to 21^st days after microbial exposure insignificantly ranged from 12.24 ± 0.06 to 13.90 ± 0.05 g/100ml.

Though the patterns of changes in haemoglobin

content showed variation up to 14^th day, after 21^st day, the haemoglobin content registered elevations (ranging from 0% to 14%) in the blood of fingerlings of all the experimental conditions (Table 2)

Fingerling of Channa striatus

Concentration of microorganism

Days after 6 days microbial exposure

‘F’ value

1^st 7^th 14^th 21^st

Pseudomonas fluorescens – exposed

Control (prefed with normal feed)

10^-3 3.30 ± 0.01 3.31 ± 0.01 3.36 ± 0.01 3.34 ± 0.01 0.42 NS 10^-5 3.04 ± 0.02 3.05 ± 0.02 3.05 ± 0.02 3.08 ± 0.02 0.86 NS

Prefed with PA extract

10^-3 3.04 ± 0.01 (-8) NS

3.10 ± 0.01 (-6) NS

3.25 ± 0.01 (-3)NS

3.26 ± 0.01 (-2)NS

1.25 NS 10^-5 3.08 ± 0.01

(-1) S

3.23 ± 0.01 (+6) HS

3.32±0.01 (+10)HS

3.40±0.01 (+10) HS

15.65 S

Prefed with Tetracycline

10^-3 3.31 ± 0.01 (0) S

3.14 ± 0.01 (-5) NS

3.19 ± 0.01 (-5)NS

3.29 ± 0.01 (-1) NS

8.52 S

10^-5 3.10 ± 0.01 (+2) S

3.18 ± 0.01 (+5) S

3.25±0.01 (+7)HS

3.40±0.01 (+12)HS

25.42 HS

Penicillium chrysogenum- exposed

10^-3 3.33 ± 0.01 3.30 ± 0.01 3.35 ± 0.01 3.38 ± 0.01 0.28 NS 10^-5 3.05 ± 0.02 3.07 ± 0.02 3.01 ± 0.02 3.09 ± 0.02 0.46 NS

10^-3 3.11 ± 0.01 (-7) NS

3.09 ± 0.01 (-6) NS

3.20 ± 0.01 (-4)NS

3.35 ± 0.01 (-1) NS

6.48 S 10^-5 3.19 ± 0.01

(+5) HS

3.00 ± 0.01 (-2) NS

3.20±0.01 (+6)HS

3.38 ± 0.01 (+9) HS

12.56 S

Prefed with Fluconazole

10^-3 2.91 ± 0.0(- 13) HS

2.80 ±0.01 (-15) HS

2.98 ±0.01 (-11)HS

3.30 ± 0.01 (-2) HS

3.52 S

10^-5 3.20 ± 0.01 (+5) HS

3.33 ± 0.01 (+8) HS

3.32±0.01 (+10)HS

3.43±0.01 (+11)HS

14.86 S

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Table 2. Haemoglobin (g/100ml) count in the blood of control (prefed with normal feed) and experimental (prefed with plant extracts/chemical antibiotic) fingerlings exposed to two different concentrations of Pseudomonas fluorescens and Penicillium chrysogenum.

Values are means of 6 observations ± S.E. Percent changes from control levels are given in the parenthesis.

(c) Total leucocytes count (TLC)

The total leucocytes count of control fingerlings of Channa striatus up to 21 days ranged between 12.00 ± 0.04 to 12.40 ± 0.0510³cells/mm³ which were found to be statistically insignificant. However, following 21 days, fingerlings (prefed with extracts and

chemical antibiotics and microbe exposed) registered significant elevations (over control level) in the total leucocytes count ranging from 0% to 3% excepting that of fingerlings prefed with fluconazole and fungus–exposed, which showed -1% reduction from control level (Table 3).

.

Days after 6 days microbial exposure ‘F’ value

1^st 7^th 14^th 21^st

10^-3 12.24 ± 0.06 12.23 ± 0.06 12.25 ± 0.06 12.24 ± 0.06 0.36 NS 10^-5 12.30 ± 0.05 12.42 ± 0.05 12.38 ± 0.05 12.32 ± 0.05 0.14 NS

10^-3 12.12 ± 0.06(- 1) NS

12.21 ± 0.05 (0) NS

12.25 ± 0.05 (0)NS

12.35 ± 0.06(+1) S

4.85 S

10^-5 13.55

±0.05(+10) HS

13.40 ± 0.06(+8)HS

13.65 ± 0.05(+10)HS

13.89

±0.04(+13) HS

16.25 HS

10^-3 12.16 ± 0.05 (-1) NS

12.12 ± 0.05(-1) NS

12.25 ± 0.04 (0) NS

12.40 ± 0.03 (+1) S

30.46 HS

10^-5 13.45 ± 0.03 (-9) HS

13.65±0.03(- 10) HS

14.02 ± 0.3 (+13) HS

14.10 ± 0.03(+14)HS

25.16 HS

10^-3 12.20 ± 0.06 12.26 ± 0.06 12.28 ± 0.06 12.25 ± 0.06 0.74 NS 10^-5 12.42 ± 0.05 12.38 ± 0.05 12.34 ± 0.05 12.36 ± 0.05 0.28 NS

10^-3 12.20 ± 0.05 (0) NS

12.06 ± 0.05(-2) NS

12.20 ± 0.02 (- 1) NS

12.29 ± 0.06 (0) S

42.15 HS

10^-5 12.80 ± 0.05 (+3) HS

12.92 ± 0.05(+4)HS

13.69 ± 0.05(+11)HS

13.90 ± 0.05(+12)HS

36.14 HS

10^-3 12.18 ± 0.06 (0) NS

12.15 ± 0.08(-1)NS

13.05 ± 0.05 (+6) HS

13.00 ± 0.05 (+6)HS

12.86 HS 10^-5 12.89 ±0.08

(+4) HS

13.30 ± 0.05(+7)HS

13.60 ± 0.08(+10)HS

13.95 ± 0.02(+13)HS

25.42 HS

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Table 3. Total leucocyte count (10³cells/mm³) in the blood of control (prefed with normal feed) and experimental (prefed with plant extracts/chemical antibiotic) fingerlings exposed to two different concentrations of Pseudomonas fluorescens and Penicillium chrysogenum.

Values are means of 6 observations ± S.E. Percent changes from control levels are given in the parenthesis

(d) Differential leucocyte count (DLC) Basophil count

The basophil count in the blood of control fingerlings insignificantly ranged from 3.00 ± 0.04 percent to 2.00 ± 0.02 percent following 1^st to 21^st days. Unlike previously mentioned parameters, the basophil count uniformly

showed significant reduction (from control level) ranging from 0% to -67% up to 21 days under various experimental conditions. This reduction in basophil content, inspite of elevated TLC level (Table 4) is noteworthy for discussion.

‘F’ value

1^st 7^th 14^th 21^st

Control (prefed with normal feed)

10^-3 12.00 ± 0.04 12.01 ± 0.04 12.02 ± 0.03 12.00 ± 0.05 0.14 HS 10^-5 12.30 ± 0.03 12.34 ± 0.04 12.30 ± 0.03 12.35 ± 0.04 0.16 NS

10^-3 11.70 ± 0.06 (-3) NS

11.50 ± 0.07 (-4) NS

11.80 ± 0.03 (-2) NS

12.30 ± 0.05 (0) S

12.85 HS

10^-5 11.20 ± 0.02 (-9) NS

11.75 ± 0.02 (-5) NS

12.20 ± 0.05 (-1) NS

12.40 ± 0.05 (0) S

16.46 S

10^-3 11.00 ± 0.08 (-8) NS

11.10 ± 0.07 (-8) NS

11.50 ± 0.07 (-4) NS

12.00 ± 0.05 (0) S

12.48 S

10^-5 11.10 ±0.05(- 10) NS

11.20± 0.05 (-9) NS

11.80 ± 0.02 (-5) NS

12.40 ± 0.02 (0) S

22.45 HS

10^-3 12.08 ± 0.02 12.07 ± 0.03 12.11 ± 0.03 12.09 ± 0.02 0.19 NS 10^-5 12.28 ± 0.04 12.32 ± 0.06 12.35 ± 0.02 12.40 ± 0.03 0.15 NS

10^-3 11.40 ± 0.06 (-6) NS

11.30 ± 0.05 (-6) NS

11.90 ± 0.05 (-2) NS

12.20 ± 0.01 (+1) S

14.25 S 10^-5 11.50 ± 0.05

(-6) NS

11.10 ±0.01 (-10) NS

11.70 ± 0.01 (-5) NS

12.30 ± 0.02 (+1) NS

6.45 S

10^-3 11.20 ± 0.01 (-7) NS

11.50 ± 0.05 (-5) NS

11.91 ± 0.03 (-2) NS

12.50 ± 0.03 (+3) HS

24.52 HS 10^-5 11.50 ± 0.02

(-6) NS

11.90± 0.06 (-3) NS

12.11 ± 0.03 (-2) NS

12.20 ± 0.05 (-2) NS

8.52 S

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Table 4. Basophil count (percentage) in the blood of control (prefed with normal feed) and experimental (prefed with plant extracts/chemical antibiotic) fingerlings exposed to two different concentrations of Pseudomonas fluorescens and Penicillium chrysogenum.

Neutrophil content

The neutrophil count of control fingerlings insignificantly ranged from 40.02 ± 1.12 percent to 33.00 ± 1.12 percent following 21 days of experimental period. On the other hand,

fingerlings prefed with leaf extract/fluconazole and exposed to Penicillium chrysogenum showed significant reduction (from control level ranging from -3% to -18%) in the neutrophil content after 21 days (Table 5).

‘F’ value

1^st 7^th 14^th 21^st

10^-3 3.00 ± 0.04 3.06 ± 0.04 3.04 ± 0.04 3.05 ± 0.04 0.46 NS 10^-5 3.12 ± 0.01 3.08 ± 0.01 3.02 ± 0.01 3.03 ± 0.01 0.28 NS

10^-3 3.00 ± 0.01 (0) NS

2.00 ± 0.01 (-35) NS

3.00 ± 0.01 (-1) NS

3.00± 0.02 (-2) NS

3.62 S

10^-5 3.00 ± 0.01 (-4) NS

2.00 ± 0.01 (-35) NS

3.00 ± 0.01 (-1) NS

4.86 S

10^-3 2.00 ± 0.01 (-33)NS

2.00 ± 0.02 (-35) NS

1.00 ± 0.01 (-67) NS

2.00 ± 0.01 (-34) NS

12.58 HS

10^-5 3.00 ± 0.01 (-4) NS

4.00 ± 0.01 (-30) HS

2.00 ± 0.01 (-34) NS

3.00 ± 0.01 (-1) NS

3.64 S

10^-3 3.00 ± 0.04 3.08 ± 0.04 3.02 ± 0.04 3.04 ± 0.04 0.52 NS 10^-5 3.10 ± 0.01 3.09 ± 0.01 3.04 ± 0.01 3.06 ± 0.01 0.65 NS

10^-3 3.00 ± 0.01 (0) NS

2.00 ± 0.01 (-35) NS

2.00 ± 0.01 (-34) NS

42.64 HS

10^-5 3.00 ± 0.01 (-3) NS

3.00 ± 0.01 (-3) NS

2.00 ± 0.02 (-34) NS

2.00 ± 0.01 (-35) NS

36.48 HS

10^-3 2.00 ± 0.03 (-33)NS

2.00 ± 0.03 (-35) NS

2.00 ± 0.05 (-34) NS

1.00 ± 0.02 (-67) NS

48.42 HS

10^-5 2.00 ± 0.04 (-35)NS

2.00 ± 0.03 (-35) NS

2.00 ± 0.03 (-34) NS

2.00 ± 0.06 (-35) NS

26.36 HS

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Table 5. Neutrophil (percentage) count in the blood of control (prefed with normal feed) and experimental (prefed with plant extracts/chemical antibiotic) fingerlings exposed to two different concentrations of Pseudomonas fluorescens and Penicillium chrysogenum.

Eosinophil count

The eosinophil count of control fingerlings insignificantly ranged from 2.00 ± 0.06 percent to 1.00 ± 0.01 percent following 21 days of experimental period. Unlike the neutrophil count, both under Pseudomonas fluorescens

and Penicillium chrysogenum exposures, the experimental fingerlings uniformly showed - 50% reductions (from control level) following 21 days under all the experimental conditions (Table 6).

‘F’

value

1^st 7^th 14^th 21^st

Control (prefed with normal feed)

10^-3 40.02 ± 1.12 40.06 ± 1.12 40.04 ± 1.12 40.05 ± 1.12 0.16 NS 10^-5 40.04 ± 2.47 40.10 ± 2.47 40.08 ± 2.47 40.06 ± 2.47 0.25 NS

10^-3 44.00 ± 1.32 (+10)S

39.00 ± 1.14 (-3) NS

43.00 ± 1.11 (+7)S

46.00 ± 1.20 (+15) S

4.28 S 10^-5 49.00 ± 1.55

(+22)S

48.00 ± 2.05 (+19) S

49.00 ± 1.12 (+22) S

51.00± 1.11 (+27) HS

22.65 HS

10^-3 44.00 ± 1.13 (+10)S

50.00 ± 1.43 (+25) HS

59.00 ± 1.32 (+47) HS

54.00 ± 1.33 (+35) HS

35.48 HS

10^-5 55.00 ± 1.66 (+37) HS

52.00 ±1.21 (+30) HS

53.00 ± 1.22 (+32) HS

48.00± 2.02 (+20) S

22.87 HS

10^-3 40.05 ± 1.12 40.07 ± 1.12 40.08 ± 1.12 40.11 ± 1.12 12.46 S 10^-5 40.02 ± 2.47 40.04 ± 2.47 40.08 ± 2.47 40.06 ± 2.47 8.52 S

10^-3 36.00 ± 2.00 (-10) NS

44.00 ± 2.01 (+9)S

37.00± 1.13 (-8) NS

33.00 ±1.53 (-18) NS

12.56 S

10^-5 39.00 ± 1.45 (-3) NS

34.00 ± 2.00 (-15) NS

35.00 ± 1.99 (-13) NS

33.00 ± 1.12 (-18) NS

14.62 S

10^-3 39.00 ± 2.04 (-3) NS

36.00 ± 1.33 (-10) NS

39.00 ± 1.32 (-3) NS

41.00 ± 1.41 (+2) S

6.65 S

10^-5 40.00 ± 1.11 (0) NS

39.00 ± 1.20 (-3) NS

38.00 ± 1.08 (-5) NS

37.00 ± 1.12 (-8) NS

7.28 S

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Table 6. Eosinophil (percentage) count in the blood of control (prefed with normal feed) and experimental (prefed with plant extracts/chemical antibiotic) fingerlings exposed to two different concentrations of Pseudomonas fluorescens and Penicillium chrysogenum.

Monocyte count

The monocyte count in the blood of control fingerlings of Channa striatus after 21 days of experimental period following microbial exposure insignificantly ranged from 4.02 ± 0.02 percent to 2.00 ± 0.02 percent (Table 7). From

count in the blood of experimental fingerlings showed reductions (of different magnitudes) following 1^st, 7^th, 14^th and 21^st days after microbial exposure. The reduction percentages of different experimental fingerlings significantly ranged from -25% to -51% (Table

‘F’ value

1^st 7^th 14^th 21^st

10^-3 2.00 ± 0.06 2.04 ± 0.06 2.05 ± 0.06 2.03 ± 0.06 0.46 NS 10^-5 2.08 ± 0.03 2.05 ± 0.03 2.09 ± 0.03 2.08 ± 0.03 0.35 NS

10^-3 1.00 ± 0.01 (-50) NS

2.00 ± 0.01 (-2) NS

1.00 ± 0.01 (-51) NS

1.00 ± 0.01 (-51)NS

28.42 HS 10^-5 1.00 ± 0.01

(-52) NS

2.00 ± 0.02 (-2) NS

1.00 ± 0.01 (-52) NS

1.00 ± 0.01 (-52)NS

16.52 S

10^-3 2.00 ± 0.01 (0) NS

1.00 ± 0.01 (-51)NS

1.00 ± 0.02 (-52) NS

1.00 ± 0.06 (-52)NS

27.48 HS

10^-5 2.00 ± 0.01 (0) NS

2.00 ± 0.01 (0) NS

1.00 ± 0.01 (-50) NS

1.00 ± 0.01 (-50)NS

18.52 HS

10^-3 2.02 ± 0.06 2.12 ± 0.06 2.08 ± 0.06 2.06 ± 0.06 0.92 NS 10^-5 2.04 ± 0.03 2.05 ± 0.03 2.03 ± 0.03 2.05 ± 0.03 0.85 NS

10^-3 2.00 ± 0.01 (-1) NS

2.00 ± 0.01 (-6) NS

1.00 ± 0.01 (-52) NS

1.00 ± 0.01 (-51)NS

22.15 HS

10^-5 1.00 ± 0.01 (-51) NS

2.00 ± 0.01 (-2) NS

1.00 ± 0.01 (-51) NS

1.00 ± 0.01 (-51)NS

39.46 HS

10^-3 1.00 ± 0.01 (-50) NS

2.00 ± 0.05 (-6) NS

1.00 ± 0.01 (-52) NS

1.00 ± 0.03 (-51)NS

46.45 HS 10^-5 1.00 ± 0.05

(-51) NS

1.00 ± 0.03 (-51)NS

1.00 ± 0.05 (-51) NS

1.00 ± 0.04 (-51)NS

29.74 HS

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Table 7. Monocyte (percentage) count in the blood of control (prefed with normal feed) and experimental (prefed with plant extracts/chemical antibiotic) fingerlings exposed to two different concentrations of Pseudomonas fluorescens and Penicillium chrysogenum.

Lymphocyte count:

The lymphocyte count in the blood of control fingerlings up to 21 days after microbial exposure insignificantly ranging from 51.00 ± 2.03 percent to 62.00 ± 1.42 percent.

Experimental fingerlings exposed to Pseudomonas fluorescensuniformly showed reductions (from control level) in the

lymphocyte count ranging from 0% to - 47%

following 1^st, 7^th, 14^th and 21^st days. On the other hand, experimental fingerlings exposed to Penicillium chrysogenum registered elevations in the lymphocyte count ranging from 0% to +22% following all the experimental periods (Table 8).

‘F’ value

1^st 7^th 14^th 21^st

10^-3 4.02 ± 0.02 4.06 ± 0.02 4.09 ± 0.02 4.05 ± 0.02 0.46 NS 10^-5 4.03 ± 0.04 4.05 ± 0.04 4.00 ± 0.04 4.02 ± 0.04 0.92 NS

10^-3 2.00 ± 0.01 (-50) NS

3.00 ± 0.01 (-26)NS

2.00 ± 0.01 (-51)NS

2.00 ± 0.01 (-51) NS

40.25 HS

10^-5 2.00 ± 0.01 (-50) NS

3.00 ± 0.01 (-26)NS

2.00 ± 0.02 (-50)NS

2.00 ± 0.02 (-50) NS

32.46 HS

10^-3 1.00 ± 0.01 (-75) NS

2.00 ± 0.01 (-51)NS

2.00 ± 0.02 (-50)NS

3.00 ± 0.02 (-26) NS

12.46 S 10^-5 1.00 ± 0.01

(-75) NS

2.00 ± 0.01 (-51)NS

2.00 ± 0.01 (-50)NS

3.00 ± 0.02 (-25) NS

10.52 S

10^-3 4.03 ± 0.02 4.16 ± 0.02 4.09 ± 0.02 4.05 ± 0.02 0.28 NS 10^-5 4.02 ± 0.04 4.04 ± 0.04 4.08 ± 0.04 4.06 ± 0.04 0.42 NS

10^-3 3.00 ± 0.01 (-26) NS

2.00 ± 0.01 (-52)NS

2.00 ± 0.01 (-51)NS

3.00 ± 0.01 (-26) NS

8.52 S

10^-5 2.00 ± 0.01 (-50) NS

3.00 ± 0.01 (-26)NS

2.00 ± 0.01 (-51)NS

2.00 ± 0.01 (-51) NS

42.56 HS

10^-3 2.00 ± 0.02 (-50) NS

2.00 ± 0.01 (-52)NS

2.00 ± 0.03 (-51)NS

2.00 ± 0.08 (-51) NS

47.45 HS

10^-5 2.00 ± 0.03 (-50) NS

2.00 ± 0.03 (-50)NS

2.00 ± 0.06 (-51)NS

2.00 ± 0.04 (-51) NS

23.56 HS

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Table 8. Lymphocyte (percentage) count in the blood of control (prefed with normal feed) and experimental (prefed with plant extracts/chemical antibiotic) fingerlings exposed to two different concentrations of Pseudomonas fluorescens and Penicillium chrysogenum.

DISCUSSION

Among the hematological parameters, significant increases were observed in the population of neutrophils and lymphocytes in the blood of fingerlings exposed to

potential of the plant extract by way of stimulating humoral mediated immune response towards microbial disease control in fish. Absence of significant increase in monocytes in fingerlings exposed to

‘F’

value

1^st 7^th 14^th 21^st

10^-3 51.00 ± 2.03 51.04 ± 2.02 51.06 ± 2.02 51.10 ± 2.06 0.28 NS 10^-5 51.04 ± 2.02 51.08 ± 2.02 51.09 ± 2.02 51.03 ± 2.02 0.42 NS

10^-3 50.00 ± 2.13 (-2) NS

54.00 ± 1.16 (6)S

51.00 ± 1.22 (0) NS

48.00 ± 2.18 (-6) NS

3.56 S

10^-5 45.00 ± 1.15 (-12) NS

45.00 ± 1.16 (-12)NS

45.00 ± 2.00 (-12)NS

43.00 ± 1.89 (-16) NS

6.42 S

10^-3 51.00 ± 1.22 (0) NS

45.00 ± 1.12 (-12)NS

37.00 ± 1.04 (-28)NS

40.00 ± 2.00 (-22) NS

25.42 HS

10^-5 39.00 ± 1.12 (-24) NS

40.00 ± 1.11 (-22)NS

42.00 ± 2.00 (-18)NS

45.00 ± 2.10 (-12) NS

22.58 HS

10^-3 51.02 ± 2.02 51.04 ± 2.02 51.06 ± 2.02 51.09 ± 2.02 0.45 NS 10^-5 51.05 ± 2.02 51.03 ± 2.02 51.04 ± 2.02 51.01 ± 2.02 0.16 NS

10^-3 56.00 ± 2.21 (+10) S

50.00 ± 2.33 (-2) NS

58.00± 2.11 (+14)S

61.00 ±1.32 (+19) S

12.62 HS 10^-5 55.00 ± 1.82

(+8) S

57.00 ± 1.13 (+12) S

60.00 ± 1.32 (+18) S

62.00 ± 1.42 (+22) HS

14.52 HS

10^-3 56.00 ± 2.00 (+10) S

58.00 ± 1.21 (+14) S

56.00 ± 1.12 (+10) S

55.00 ± 1.31 (+8) S

3.58 S 10^-5 55.00 ± 1.10

(+8) S

56.00 ± 1.43 (+10) S

57.00 ± 2.01 (+12) S

58.00 ± 1.02 (+14) S

6.72 S

(12)

(prefed with normal feed) and experimental (prefed with plant extract/chemical antibiotics) fingerlings revealed the methanolic extract of Phyllanthus amarusto to controlling efficacy of bacterial infection than fungal infection and the increases of lymphocyte to stimulate humoral immune response by way of producing immunoglobulins to fight against harmful microorganisms.

CONFLICT OF INTEREST STATEMENT The authors declare that they have no conflict of interests.

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Cite this article as:

Ashokkumar R, M. Ramaswamy. Fish disease controlling efficacy study of selected Indian medicinal plant. J Pharm Chem Biol Sci 2016; 4(2):176-187.