Fish disease controlling efficacy study of selected Indian ... · Ashokkumar R et al 177 J Pharm...
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176
J Pharm Chem Biol Sci , June-August 2016; 4(2): 176-187
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
INTRODUCTION
Medicinal plants are of great importance for the
health of individual and the society. The
medicinal value of these plants lies in some
chemical substances that produce a definite
physiological action on the human and animal
body. The Indian and Chinese people depend
on medicinal plants as an alternate medicine
remedy against infections. Among various
kinds of cultivated organisms, many marine and
freshwater finfish and shellfish species
constitute an important industry with their
production increasing every year. Recently, due
to intensive farming practices, infectious
diseases pose a major problem in aquaculture
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
extract was filtered using Whatman No.1 filter
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 105oC 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 1st, 7th, 14th and
21st 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 1st to 21st 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 14th day, after
21st 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
1st 7th 14th 21st
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
Control (prefed with normal feed)
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
Prefed with PA extract
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).
.
Fingerling of Channa striatus
Concentration of
microorganism
Days after 6 days microbial exposure ‘F’ value
1st 7th 14th 21st
Pseudomonas fluorescens –exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Tetracycline
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
Penicillium chrysogenum-exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Fluconazole
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 1st
to 21st 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.
Fingerling of Channa striatus
Concentration of
microorganism
Days after 6 days microbial exposure ‘F’ value
1st 7th 14th 21st
Pseudomonas fluorescens –exposed
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
Prefed with PA extract
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
Prefed with Tetracycline
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
Penicillium chrysogenum-exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Fluconazole
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.
Values are means of 6 observations ± S.E. Percent changes from control levels are given in the
parenthesis.
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).
Fingerling of Channa striatus
Concentration of
microorganism
Days after 6 days microbial exposure ‘F’ value
1st 7th 14th 21st
Pseudomonas fluorescens –exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
3.00 ± 0.01 (-1) NS
4.86 S
Prefed with Tetracycline
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
Penicillium chrysogenum-exposed
Control (prefed with normal feed)
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
Prefed with PA extract
10-3 3.00 ± 0.01
(0) NS 2.00 ± 0.01
(-35) NS 2.00 ± 0.01
(-34) 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
Prefed with Fluconazole
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.
Values are means of 6 observations ± S.E. Percent changes from control levels are given in the
parenthesis.
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).
Fingerling of Channa striatus
Concentration of
microorganism
Days after 6 days microbial exposure ‘F’ value 1st 7th 14th 21st
Pseudomonas fluorescens –exposed
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
Prefed with PA extract
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
Prefed with Tetracycline
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
Penicillium chrysogenum-exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Fluconazole
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.
Values are means of 6 observations ± S.E. Percent changes from control levels are given in the
parenthesis.
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
Table 7, it could be observed that the monocyte
count in the blood of experimental fingerlings
showed reductions (of different magnitudes)
following 1st, 7th, 14th and 21st days after
microbial exposure. The reduction percentages
of different experimental fingerlings
significantly ranged from -25% to -51% (Table
7).
Fingerling of Channa striatus
Concentration of
microorganism
Days after 6 days microbial exposure ‘F’ value
1st 7th 14th 21st
Pseudomonas fluorescens –exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Tetracycline
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
Penicillium chrysogenum-exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Fluconazole
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.
Values are means of 6 observations ± S.E. Percent changes from control levels are given in the
parenthesis.
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 1st, 7th, 14th and 21st 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).
Fingerling of Channa striatus
Concentration of
microorganism
Days after 6 days microbial exposure ‘F’ value
1st 7
th 14th 21
st
Pseudomonas fluorescens –exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Tetracycline
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
Penicillium chrysogenum-exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Fluconazole
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.
Values are means of 6 observations ± S.E. Percent changes from control levels are given in the
parenthesis.
DISCUSSION
Among the hematological parameters,
significant increases were observed in the
population of neutrophils and lymphocytes in
the blood of fingerlings exposed to
microorganisms and fed with feed amended
with the methanol extract of the Phyllanthus
amarus medicinal plant compared to absence
of significant change in the above said
parameters in fingerlings treated with
microorganism and fed with feed amended
with chemical antibiotics. This probably
indicates the possible immunostimulant
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
microorganisms and fed with feed amended
with plant extract probably indicate the
absence of cell mediated immune response in
the fish.
CONCLUSION
From the results obtained, it is concluded that
the haematological parameters of control
Fingerling of Channa striatus
Concentration of
microorganism
Days after 6 days microbial exposure ‘F’ value 1st 7th 14th 21st
Pseudomonas fluorescens –exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Tetracycline
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
Penicillium chrysogenum-exposed
Control (prefed with normal feed)
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
Prefed with PA extract
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
Prefed with Fluconazole
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
Ashokkumar R et al 187
J Pharm Chem Biol Sci , June-August 2016; 4(2): 176-187
(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.