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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 2; Year 2015; Page: 96 - 111

Research Article
EFFICACY OF PLANT EXTRACTS ADDITIVES ON IMMUNITY AND
RESISTANCE AGAINST Aeromonas hydrophila IN COMMON CARP,
Cyprinus carpio
M. Mariappan*, J. Prakash Sahaya Leon and K. Balakrishnan
Department of Zoology DDE, Annamalai University, Chidambaram 608 002, Tamil Nadu, India
Abstract
Fish farmers used vaccines to control diseases can result in the development of drug resistant
pathogens, environmental pollution and accumulation of residues in fish. In recent years, application of
medicinal valuable plants in aquaculture is considered as a promising alternative to vaccines. The present
investigation was designed to study the eight plant extracts and screened their antimicrobial activities against
Aeromonashydrophila, a bacterial pathogen in common carp, Cyprinuscarpio. Using disc diffusion assays,
five extracts among these ten (Azadirachtaindica, Andrographispaniculata, Allium sativum,
SolanumtricobatumandOcimum sanctum) were selected and equal proportions of them were mixed
thoroughly with the artificial feeds at concentrations of 0.0 (A), 100 (B), 200 (C), 400 (D) and 800 (D) mg
kg-1 of dry diet. The experiment was conducted with the adult fishes for a period of 60 days. The prepared
diets were fed the common carp during experiment and then challenged with A. hydrophila which was given
by intraperitoneal injection. To evaluate the immune response and resistance against A. hydrophila infection
of fish, hematological, biochemical and immunological parameters were investigated at 15, 30, 45 and 60
days of feeding and also 15th day of post-challenge. Results shows that RBC, WBC, Hb, Ht, MCV, MCH,
MCHC, serum total protein, glucose, cholesterol, serum bactericidal activity and lysozyme activity were
increased in fish fed with plant extract mixed diets compared to the control group. At the end of postchallenge test, the survival rates were 42.43% in control group (A) and 62.75, 79.35, 84.65 and 89.57% in
group B, C and D respectively. These results indicate that addition of herbal extract can promote immunity
and can prevent disease in common carp.
Article History
Received: 20.03.2015
Revised : 30.03.2015
Accepted: 04.04.2015

Key words: Aeromonas hydrophila, Cyprinus

carpio and Plant extracts.

1. Introduction

Vaccination is one of the most promising

methods for to enhance the immune system in fish
to prevent diseases. Vaccines, enhance the
acquired immune response of the fish and a single
vaccine is effective only one type of pathogens
* Corresponding author: M. Mariappan
Tel.: +91-8428223898
E-mail: mmkarprsch@hotmail.com

(Laszlo Ardo et al., 2008), but Aeromonas

hydrophila is a heterogenous species, having
variable antigens, that vaccines development is
extremely complex (Ramasamy et al., 2010).
However, the use of vaccine in aquaculture has
received considerable attention because their use
can lead to the development of drug resistant
bacteria and the accumulation in fish cause risk to

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M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015

consumers and the environment (Alderman and

Hastings, 1998; Adithepchaikarn et al., 2008).
To overcome the problems of vaccines,
immunostimulants were used to enhance the
innate immune response (Galeotti, 1998; Sakai,
1999; Laszlo et al., 2008). An immunostimulants
is naturally occurring compound that modulates
the immune system by increasing the hosts
resistance against diseases that in most
circumstances are caused by pathogens (Peddie et
al., 2002). The use of immunostimulants for the
prevention of disease in fishes is considered as an
attractive and promising area in the field of
aquaculture (Chinnasamy et al., 2013). The
modulation of immune response by various
substances has been reported, including synthetic,
bacterial, animal and plant products (Vasudeva
Rao et al., 2006). Among this plant extracts have
beneficial effects without any side effects. Some
plants are rich sources of compounds like volatile
oils, saponins, phenolic compounds, tannins,
alkaloids, polypeptides and polysaccharides.
These natural products have various activities like
antistress,
appetizer,
antimicrobials
and
immunostimulant (Citarasu et al., 2002; Citarasu
et al., 2003).
Various studies revealed that plant extracts
have enormous therapeutic potential. Moreover,
they are cheaper, safer, non-toxic, biodegradable
and biocompatible (Subeena and Navaraj, 2013).
Several plant materials or extracts such as
Astragalus membranaceus (Shao et al., 2004),
Azadirachta indica, Oscimum sanctum and
Curcuma longa (Ramasamyet al., 2010), Viscum
album, Urticadioica and Zingiber officinale
(Dugenci et al., 2003; Chinnasamy et al., 2013),
Ocimum tenuiflorum, Zingiber officinale and
Allium cepa (Iruthayam et al., 2014), Eucalyptus
spp. and Plelargoni amroseum. Mehrak et al.
(2013) have been reported to enhance the
immunity and elevated resistance against the
disease causing agents. However, there was no
report in different concentration of plant extracts
in dietary supplementation feed on hematological
and innate immune response of common carp
against A. hydrophila. Pastly, studies have used
hematological indices and immunological assay to
determine the health status of fishes

97

(Harikrishnan, 2003; Harikrishnan, 2009).

Therefore, the aim of the present study was to
investigate the effects of dietary mixed plant
extract supplementation feed on hematological
changes and innate immunity of Cyprinus carpio
against A. hydrophila.
2. Methodology
2.1. Fish
Common carp, C. carpio obtained from a
local fish farm in Cholatharam, Tamilnadu, India
were transported to the laboratory in plastic bags
filled with oxygenated water. The fishes were
acclimatized in cement tank containing 500 l of
water for 2 week under laboratory conditions
(12/12 hrs light/dark cycle), feeding with basal
experimental diet without supplementation of the
plant extracts (Table - 1) at a rate of 2% of their
body weight/day. The observed water quality
parameters were dissolved oxygen concentration
7.0 7.5 mg l-1, pH 7.2 7.6 and temperature at
26 2C during the whole trial. The 50% of water
was renewed daily to remove the unfed and fecal
materials.
2.2. Growth of A. hydrophila
A. hydrophila was obtained from the
Department
of
Microbiology,
Annamalai
University, Chidambaram, Tamilnadu and
maintained in the laboratory under standard
conditions. Sub-cultures were maintained on
Tryptone soya agar (Hi-media, Mumbai) in slopes
at 5C. Stock culture in tryptic soy broth (Himedia, Mumbai) was stored at -70C in 0.85%
NaCl with 20% glycerol to provide stable
inoculate throughout the experiment (Chabot and
Thune, 1991). The broth was incubated overnight
in a shaker for 24 hrs at 20C, and then centrifuged
at 10,000 rpm for 20 min at 4C. The supernatant
was discarded and the bacterial pellet was washed
three times with phosphate-buffered saline (PBS)
at pH 7.2.
2.3. Preparation of Plant extracts
The parts of eight plant species used in this
study are listed in Table - 2. Fresh parts of these
plant species were collected around Annamalai
University campus during October 2012. The
plants were washed in sterile distilled water and

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separately shade dried for 10 days till weight

constancy was achieved. Each plant parts were
finely powdered in an electric blender. Extracts of
the powdered plant parts were prepared using
water, 95% ethanol, as solvents at ratios of 1:10
(w/v). The extractions were carried out at room
temperature. The extracts obtained were
centrifuged at 5,000 rpm for 15 min. The filtrate
obtained from ethanol was evaporated to dryness
at 40C in a rotary evaporator and the water
extract was then freeze-dried by using a freeze
drier system. Finally, the extracts were stored at
4C until use (Arabshah Delouee and Urooj,
2007).
2.4. Screening of the Plant extract against A.
hydrophila
A disc diffusion method (Bauer et al.,
1996) with some modifications was used to detect
antimicrobial activity of plant extracts against A.
hydrophila.
Each 500 g of extract was
impregnated in 5mm diameter sterile paper discs
and allowed to dry. Dried antimicrobial discs with
impregnated herbal extracts were carefully
dispensed with uniform distances over soyabean
casein digest agar surface and correct implantation
was assured by applying gentle pressure over the
disc. The plates were incubated at 30C for 24
hrs, after which the inhibitory zone formation of
antimicrobial extracts around the paper discs were
measured and recorded (Table - 2).
2.5. Fish diet preparation

98

2.6. Fish feeding experiment

Fishes were randomly divided into five
groups in triplicate each with 50 fishes (n = 50
fishes/group). They were fed with 0, 100, 200,
400 and 800 mg kg-1 extract mixture (later it will
be called as group A (control), group B, group C,
group D and group E, respectively). The fish were
fed twice a day at 8:00 and 14:00 hrs at 2% of the
body weight until the end of the experiment.
2.7. Blood sampling
Blood samples of five fish/group were
selected randomly from each group and collect
blood from caudal vein using 1 ml tuberculin
syringe fitted with 24 gauge needle (Michael et
al., 1994) on 15, 30, 45 and 60 days after the
feeding. Fishes were anesthetized with 100 mg of
tricaine methane sulphonate (MS-222). Individual
fish were sampled only once to avoid the influence
on the assays due to multiple bleeding and
handling stress on the fish. One half of each blood
sample was immediately used for hematological
examination, while the other half was mixed with
heparin anticoagulant then frozen at 4C using
EDTA serum tubes. The serum tubes were placed
at room temperature and allowed to clot for 2 hrs.
The supernatant serum was separated and
collected from the remaining blood after
centrifugation (1500 rpm for 20 min) and stored at
-70 C for biochemical and immunological
analyses.
2.8. Challenges with virulent pathogen

The formulated fish feed was prepared in

the laboratory using soyaflour and fish meals as
the protein sources (Table - 1). Based on the zone
of inhibition, Azadirachta indica, Andrographis
paniculata, Allium sativum, Solanum tricobatum
and Ocimum sanctum were selected for the present
study. To enrich the normal diet with in 100, 200,
400 and 800 mg kg-1 (w/w) of the five plants
extract was added into the feed slowly, mixing
part in a drum mixer, after which it was air dried
under sterile conditions for 12 hrs. Control diet
was prepared using the same composition of
ingredients except the extract mixture. The pellets
were dried in an oven at 30C for 18 hrs, packed
and stored at -2C in air tight containers until fed.

The susceptibility of the fish fed plant

extracts to a bacterial challenge was examine in
vivo A. hydrophila was used as the challenge
strain. Groups of 25 fishes fed with 0, 100, 200,
400 and 800 mg kg-1 extracts were challenged 60
days after the start of treatments. Carp were
injected intraperitoneally with 100 L of live A.
hydrophila (1 108 cfu mL-1). The fish were
observed regularly at 12 hrs intervals for mortality
upto 15 days. The surviving fish were than
sampled for serum and blood parameters as per the
method described earlier.
2.9. Hematology
Red blood cell (RBC: 106mm-3) and white
blood cell (WBC: 103mm-3) were counted

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M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015

manually by haemocytometry (Samuel, 1986)

using Neubauer haemocytometer after diluting
blood samples by adding of Hayem solution for
RBC and Turk solution for WBC, respectively.
Haemoglobin (Hb: g dL-1) concentration of the
blood was estimated by the method of using
Shalis haemoglobinometer (Samuel, 1986).
Haematocrit (Ht: %) was measured with
microcentrifuge
method,
using
standard
heparinized micro haematocrit capillary tubes (75
mm at 7000 rpm for 10 min). The derived
erythrocytes of mean corpuscular volume (MCV:
m3), mean corpuscular haemoglobin (MCH: pg)
and mean corpuscular haemoglobin concentration
(MCHC: g dL-1) were calculated according to
Dacie and Lewis (1991). The total protein (TP:
mg dL-1), glucose (GLU: mg dL-1) and cholesterol
(CHO: mmol-1) were determined by Hawk et al.
(1954).
2.10. Respiratory burst activity
Respiratory burst activity was measured by
the Nitro blue tetrazolium assay (NBT),
Secombesis (1990) method with the modification
described by Satasiak and Baumann (1996). The
heparinized blood was collected in silica-coated
Eppendorf tubes and the buffy coat was separated
by centrifuging at 10,000 rpm for 10 min. Fifty
microlitres of the blood coat was placed in each of
the 96 wells of U bottom microtitre plates and
incubated at 37 C for 1 hour, to facilitate
adhesion of cells. Then, the supernatant was
removed and 50 ml of 0.3% NBT was added.
After incubation, NBT was removed. The cells
were then fixed with 100% methanol and washed
thrice with 70% methanol. The plates were airdried. Sixty microlitres of 2N KOH and 70
microlitres dimethyl sulphoxide were added to
each well to dissolve the formazan blue
precipitate. The turquoise-blue coloured solution
was then read in an ELISA reader at 655 nm.
2.11. Bactericidal activity
Serum bactericidal activity was estimated
by following the procedure of Kajita (1990). An
equal volume (100 L) of serum and bacterial
suspension was mixed and incubated for 1 hour at
25 C. A blank was prepared by replacing serum
with sterile PBS buffer. The mixture was then

99

diluted with sterile PBS at a ratio of 1:10. The

serum-bacterial mixture (100 L) was pour-plated
in nutrient agar and the plates were incubated for
24 hrs at 37 C. The number of viable bacteria was
determined by counting the colonies grown on
nutrient agar plates. Data on bactericidal activity
were calculated as follows
Positive control CFU Sample CFU /
(Positive control CFU/100)
2.12. Lysozyme assay
Plasma lysozyme activity was measured
spectrophotometrically by the method of Sankaran
and Gurnani (1972). The lysozyme substrate was a
0.02% (w/v) suspension of Micrococcus
lysodeikticus made up in phosphate buffer (0.05
M, pH 6.2). Lyophilized hen egg white lysozyme
was used as a standard. A new standard curve was
prepared for each assay. Standard solutions as
well as samples were added to the substrate at
25C. The results were expressed as mg mL-1
equivalent of hen egg white enzyme activity.
2.13. Statistics
Results are presented as the average (
standard error) for five fish, and were compared at
each time point using one-way ANOVA and
Dunns multiple range tests (SAS package).
Significant differences between experimental
groups were expressed at a significant level of P <
0.05.
3. Results
3.1. Detection of antimicrobial activity of plant
extracts against A. hydrophila
Extracts of eight plant species in 95%
ethanol were tested for antimicrobial activity
against A. hydrophila (Table - 2). The maximum
inhibitory activity was recorded for A. indica (20.5
1.5) followed by A. paniculata (17.5 1.3), A.
sativum (16.1 1.9), S. tricobatum (13.5 0.5)
and O. sanctum (11.2 1.7).
3.2. Hematological profile
The RBC levels significantly increased (P
< 0.05) in 100, 200, 400 and 800 mg kg-1 of mixed
plant extract enriched diets feeding groups from
15 - 60 days when compared to the control. The

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WBC values in all the experimental periods shows

a gradual significant increase which reached a
maximum in group C and the values restored in
group D and E. The Hb content was enhanced in
all the experimental groups. The highest
significant Hb level (P < 0.05) was observed in
group E on day 45, 60 and post-challenge period
(day 75). The Ht significantly increased in all
treated groups of common carp on days 15, 30, 45,
60 and 75. The MCV significantly differ when
fish treated with 100, 200, 400 and 800 mg kg-1 of
mixed plant extract enriched group B-E when
compared to control. The MCH was significantly
decreased from 15 to 60 days of mixed plant
extract enriched diets and in post-challenge test
groups. On the other hand, the MCHC values did
not show significant change in 800 mg kg-1 of
mixed plant extract diets treated group of all the
assay periods compared to control (Fig. 1).
3.3. Biochemical parameters
Total serum protein level was shown in Fig
- 2. The result indicates that the total serum
protein level was significantly increased (P < 0.05)
in experimental groups compared to control group.
The maximum level of total protein was recorded
in group E for all the assay duration. Similarly,
cholesterol content was significantly higher in
group E as compared to other groups on different
treated days. The level of glucose was shown in
Fig. 2. Compared to the control group, a gradual
significant decrease (P < 0.05) was noted in the
glucose value in entire assay.

100

In addition the highest level of bactericidal

activity was observed in group E.
3.6. Lysozyme activity
Lysozyme activity was showed in Fig - 5.
The result shows that the lysozyme activity was
significantly increased in all treated groups
compared to control group. The lysozyme activity
was gradually increased throughout the
experimental period compared to initial value.
The groups B, C, D and E shows the highest
increase of the lysozyme activity than the control
group due to the addition of the different
concentration of plant extract in the feed.
3.7. Challenges with virulent pathogens
After 60 days of feeding fish were
challenged with A. hydrophila and survival
percentage was registered for 15 days (Fig - 6).
All four treated groups showed reduced mortality
compared to the control. After 10 days there was
no mortality upto day 15. The survival percentage
was found highest (89.57%) in the group E
followed by group D (84.65%) and lowest
(42.43%) in control group (A).

3.4. Respiratory burst activity

The respiratory burst activity showed an
increasing trend from group B to E in all the assay
periods and it was decrease in post-challenge test
(Fig - 3). The highest range of respiratory burst
was recorded at 60th day in group E (1.21 0.03)
and the lowest range was recorded at initial stage
in group A (0.37 0.00).
3.5. Bactericidal activity
The results of the serum bactericidal
activity were shown in Fig - 4. Bactericidal
activity in the four experimental groups was
significantly (P < 0.05) higher than control group
at all the treated periods including post-challenge.
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101

Table - 1:Composition of basal diet with plant extract for common carp
DIET

Components
(g kg-1)
Fish meal
Ground nut oil cake
Sesame oil cake
Soya flour
Rice bran
Tapioca flour
Vitamin and mineral premix
Plant extract

A
(Control)
195
250
160
120
150
105
20
0.00

195
250
160
120
150
105
20
0.1

195
250
160
120
150
105
20
0.2

195
250
160
120
150
105
20
0.4

195
250
160
120
150
105
20
0.8

Table - 2: List of plants and their inhibitory activity against A. hydrophila as

determined by disc diffusion assay
Common
Names
(in Tamilnadu)
Neem
Sirunangai
Garlic
Thoodhuvalai
Tulasi
Omavalli
Keezhanelli
Malli

Scientific Names

Family

Azadirachtaindica
Andrographispaniculata
Allium sativum
Solanumtricobatum
Ocimum sanctum

Meliaceae
Acanthaceae
Alliaceae
Solanaceae
Lamiaceae

Coleus aromaticus

Lamiaceae

Phyllanthusniruri

Euphorbiaceae

Coriandrumsativum

Apiaceae

Sources of
active
compound
Leaf
Leaf
Bulb
Leaf
Leaf
Stem and
Leaf
Leaf
Whole
plant

Inhibition
Zone (mm)
20.5 1.5
17.5 1.3
16.1 1.9
13.5 0.5
11.2 1.7
9.2 0.6
8.4 0.3
8.2 1.6

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M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015

102

Fig 1: Cyprinuscarpio: Changes in hematological profile of different experimental groups (n = 5 in each

group). Control-fed with normal diet and fish feed with 100, 200, 400 and 800 mg kg -1 of mixed plant extract
supplementation feeds. RBC: red blood cell, WBC: white blood cell, Hb: hemoglobin, Ht: hematocrit, MCV:
mean corpuscular volume, MCH: mean corpuscular haemoglobin, MCHC: mean corpuscular haemoglobin
concentration, *Indicates statistically (P < 0.05) significant difference compared to control group at the same
sampling days.
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M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015

103

Respiratory Burst Activity

Fig 2:Cyprinuscarpio: Changes in biochemical profile of different experimental groups (n = 5 in each group).
Control-fed with normal diet and fish feed with 100, 200, 400 and 800 mg kg -1 of mixed plant extract
supplementation feeds.*Indicates statistically (P < 0.05) significant difference compared to control group at the
same sampling days.
*
*

*
*

*
*

Control

*
*

100 mg

Days
200 mg

400 mg

800 mg

Fig 3: Respiratory burst (NBT) activity of Cyprinuscarpio, fed on plant extract supplementation
diets.*Indicates statistically (P < 0.05) significant difference compared to control group at the same sampling
days.

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Bactericidal activity
(cfu/control%)

M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015

**

**

*
**

**

**
*

104

**

Days

Control

100 mg

200 mg

400 mg

800 mg

Lysozyme activity

Fig 4: Bactericidal activity of Cyprinuscarpio, fed on plant extract supplementation diet.*Indicates statistically
(P < 0.05) significant difference compared to control group at the same sampling days.

**

Control

**

***

100 mg

Days
200 mg

**

400 mg

*
***

800 mg

Survival (%)

Fig - 5: Plasma Lysozyme activity of Cyprinuscarpio, fed on plant extract supplementation diet.*Indicates
statistically (P < 0.05) significant difference compared to control group at the same sampling days.

Groups

Fig 6: Survival rate of Cyprinuscarpio fed on plant extract supplementation diets (B-E) and control
diet (A) after challenged with A. hydrophila

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M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015

4. Discussion
Generally,
monocytes,
granulocytes,
neutrophils, macrophages and humoral elements
like lysozyme, agglutinin and metalion binding
proteins are the main components of the nonspecific immune system (Secombes and Fletcher,
1992; Ardo et al., 2008; Sudagar and Hajibeglou,
2010). The immunostimulating effect of plant
extracts were investigated in this study. All the
composition of plant extracts was able to enhance
the non-specific immune response to common
carp. Similarly, Jinish (2002), who extracted the
plants Terminalia ballerica, Ocimum sanctum,
Daemia extensa, Andrographis paniculata,
Solanum suratense,
Tinospora
cardifolia,
Withania somnifera and Myristica fragrans
extracts against the gut microbiota isolates from
the Grouper Epinephelu stauvina have strong
immunostimulant activity. Punitha et al. (2008)
showed that benzene and petroleum ether plant
extracts from Cynodond actylon, Piper longum,
Phyllanthus niruri, Tridax procumbens and
Zingiber
officinalis
posses
strong
immunostimulant activity against Vibrio harveyi
infection in E. tauvina.
Blood is a physiological factor of
organisms, hematological tool have been used as a
diagnostic parameter for investigation of disease
and physiological disorders (Nancy et al., 2000).
Changes in blood oxygen transport capacity reflect
the health status of fish like striped mullet
(Cameron, 1970). In our study, red blood cell
significantly increased in the entire treated, but
declined in post-challenge test. Similarly, such
reductions have been confirmed in Limanda
ferruginea infected with various pathogens (Allen
et al., 2003). Further, these observations also
verify the findings of other investigations by
Gopalakannan and Arul (2006) who found that
there is an increase in the RBC count in the
common carp after exposure to chitin and chitosan
extract. The white blood corpuscles level

106

increased in all the treated groups and in post

challenge test it show significant moderate level
indicating the growth of the pathogen has been
suppressed due to the plant extract enriched diet.
Monitoring these values and gathering information
on the profile of leucocytes can reflect general
immune system status in fish (Ramasamy et al.,
2010). The findings of the present study is in
coincide with the work of Sahu et al. (2007) and
Subeena Begum and Navaraj (2012) who reported
that WBC counts are higher in Labeo rohita
fingerlings treated with Mangifera indica when
compared to control. The alterations of WBC,
RBC and Hb could possibly be due to activation
of the immune system in the presence of herbal
extracts, which in turn may be an adaptive
response of the organism resulting in a more
effective immune defense (Barreto et al., 2005).
Hematocrit level is an indicator for fish health
which gives clue on fish health and explains
abnormalities caused by immunostimulants
(Dorucu et al., 2009). Scott and Rogers (1981)
suggested that the spleenic contraction increased
Ht level due to introduction of erythrocytes into
the circulating system. In the present study, Ht
values increased in all the feeding experiments
and also in the post-challenge test. The same mode
of increase in Ht levels has been showed after
exposure to black cumin seed in rats (Zaoui et al.,
2002). Increase levels of MCV, MCH and MCHC
have been reported in C. carpio (Harikrishnan et
al., 2005) and rainbow trout (Rehulka, 1998) in
response to A. hydrophila. The general concept
that the use of herbals in aquaculture may produce
various beneficial effect proved by Harikrishnan et
al. (2003) and Harikrishnan et al. (2009).
The immunostimulant plant extracts
supplemented diets helped to increase the humoral
elements in the serum. In the present study, the
total serum protein and cholesterol level were
significantly increased in experimental groups
compared with the control group. Mohsen Abdal-

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M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015

Tawwab et al. (2010) observed an increase in

serum protein level in Tilapia fed with Green tea
incorporated diet and infected with A. hydrophila.
Similar results were also observed by Sudagar and
Hajibeglou (2010) in C. carpio fed with feed
incorporated with mixed plant extracts for 60 days
and infected with A. hydrophila. Increases in the
levels of serum protein content might be in part
due to an increase in the WBC, which is a major
source of serum protein production such as
lysozyme, complement factors and bacterial
peptides (Misra et al., 2006). The glucose level
was increases in the infected or stressed animals to
overcome the stress or infection (Citarasu et al.,
2006). In the present study, an inverse relationship
between glucose level and value of plant extract
mixture in the diet was observed. As the amount
of extract mixture increased in the diet, the level
of glucose decreased. The increment in blood
glucose might result from an increase in plasma
catecholamine and corticosteroid hormones
(Pickering, 1981). The findings of the present
study agree with Citarasu et al. (2006) and
Mehrak et al. (2013) who found that glucose
levels are reduced in the aquatic animals affected
by herbal immunostimulants.
Phagocytosis and killing activity by
phagocytic cells is an important defense
mechanism against pathogenic bacteria (Mac
Arthur and Fletcher, 1985; Guojun et al., 2006).
Phagocytes also produce superoxide anion (O2)
during a process called respiratory burst
(Neumann et al., 2001). Since, O2 is the first
product to be released from the respiratory burst,
the measurement of O2 has been accepted as a
direct and accurate way of measuring respiratory
burst activity (Secombes, 1990; Secombes, 1997).
In the present study, the nitrobluetetrazolium
(NBT) assay, was used which measures the
amount of intracellular superoxide anions
(Vasudeva Rao et al., 2006; Ardo et al., 2008;
Subeena and Navaraj, 2012). Herbal based

107

immunostimulants can enhance the respiratory

burst activity of fish. A. hydrophila bacterins
significantly enhanced the intracellular respiratory
burst activity in leukocytes of Indian major carps
(Catla catla, Labeo rohita and Cirrhinus mrigala)
(John et al., 2002). Rao et al. (2006) reported that
superoxide anion production by the blood
leucocytes was enhanced in Labeo rohita after
feeding the fish with Achyranthes aspera seed.
Similarly, the plant extracts we used in this study
could enhance respiratory burst activity in all
treatment groups compared to control.
The increased serum bactericidal activity
in plant extract treated groups indicates that
various humoral factors involved in innate or
adaptive immunities are elevated in the serum to
protect the host effectively from infection.
According to Sivaram et al. (2004) reported that
serum bactericidal activity was enhanced in
juvenile greasy groupers (E. tauvina) fed
antibacterial active princioles of O. sanctum and
W. somnifera. Similarly, Catla catla fed with
diets containing different doses of Z. officinale
showed a significant increase in their serum
bactericidal activity (Chinnasamy et al., 2013).
As a first line of defense, various peptides,
such as lysozymes are present in serum where they
prevent adherence and colonization by microorganisms. Lysozyme is a cationic enzyme that
breaks -1, 4 glycosidic acids and N-acetyl
glucosamine in the peptidoglucan of bacterial cell
walls. This action is known to attack mainly Gram
positive bacteria as well as some Gram negative
bacteria in conjunction with complement
(Alexander and Ingram, 1992). In the present
study, the plant extract supplemented feeds
enhanced the lysozyme activity in all the
treatments. The increased levels of lysozyme
could be the result of an increment in the number
of phagocytes which secrete more amount of
lysozyme. When L. rohita was fed with feeds
having extract of Achyranthes apera (Rao et al.,

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M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015

2006) and n-PUFA (Misra et al., 2006), the serum

lysozyme activity was increased considerably.
Other immunostimulants, such as chitosan,
levamisole and chitin also elevated lysozyme
levels in plasma of carp (Gopalakannan and Arul,
2006). Similarly, grouper (E. tauvina) juveniles
fed with diets containing different doses of extract
mixture of some herbs showed a significant
increase in their serum bactericidal activity
(Punitha et al., 2008).
In this study fish which fed with plant
extract supplemented diet challenged with A.
hydrophila, the mortality were significantly
reduced compared to control, with the lowest
mortality in 400 and 800 mg kg-1 diet groups. It
was considered that the plant extract mixed at 400
and 800 mg kg-1 of supplemented feeds gives
more protection than 100 and 200 mg kg-1 of
supplemented feeds in common carp against A.
hydrophila. There was an inverse relationship
between the mortality rate and the amount of plant
extract in the diet. As the amount of plant extract
increased in the diet, the rate of mortality
decreased i.e. the survival rate increased. This
might be due to the enhancement of the nonspecific immune system of the fish by plant
extracts. Similar findings were reported after
feeding C. carpio with Chinese herbs Astragalus
radix and Gamoder malucidum and challenging
with A. hydrophila (Guojun et al., 2009). Earlier
studies revealed that dietary supplementation of O.
sanctum leaf extract enhanced disease resistance
against
A.
hydrophila
in
Oreochromis
mossambicus (Logambal et al., 2000). Sahu et al.
(2007) observed that mango kernel stimulated the
immunity and made L. rohita more resistant to A.
hydrophila infection like as observation in
common carp made in our present study.
In conclusion our results suggest the
protective ability of A. indica, A. paniculata, A.
sativum, S. tricobatum and O. sanctum extract
mediated through cellular and may be non-cellular

108

immune mechanism, as evident from the enhanced

hematological parameters, biochemical and
immunological parameters such as respiratory
burst activity, bactericidal activity and survival
rate against A. hydrophila. Furthermore, the use
of such plant extracts as immunostimulants in
aquaculture systems may also have environmental
value because of their biodegradability.
Acknowledgement
The first author is indebted to University
Grant Commission, New Delhi, India for the
financial assistance for conducting this research
work (Minor research grant
F. No. 40503/2011) and also thankful to the authorities of
Annamalai University, Annamalainagar for
providing all facilities to carry out the work.
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