Fish & Shellsh Immunology 34 (2013) 254e264

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Fish & Shellsh Immunology

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Azadirachta indica (neem) leaf dietary effects on the immunity response and
disease resistance of Asian seabass, Lates calcarifer challenged with Vibrio harveyi
Allah Dad Talpur a, b, *, Mhd Ikhwanuddin b
a
b

Department of Fisheries, Government of Sindh, Pakistan

Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, Kuala Terengganu, Terengganu, Malaysia

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 28 June 2012
Received in revised form
26 September 2012
Accepted 2 November 2012
Available online 20 November 2012

The present study was aimed to address the possible evaluation of Azadirachta indica (neem) leafsupplemented diets on innate immune response in Asian seabass, Lates calcarifer ngerlings against
Vibrio harveyi infection. Fish were fed for two weeks diets containing six graded levels of neem leaf at 0 g,
1 g, 2 g, 3 g, 4 g and 5 g per kg feed. Fish fed neem leaf-supplemented diet displayed signicant
differences (p < 0.05) in weight gain, specic growth rate (SGR) and feed conversion ratio (FCR)
compared to the control group fed without neem leaf-supplemented diet. Various innate immune
parameters were examined pre-challenge and post-challenge. Fish was injected intraperitoneally with
a lethal dose of V. harveyi containing 108 cells mL
1. Supplementation of neem leaf diet signicantly
increased phagocytic activity, superoxide anion production, serum lysozyme, serum bactericidal activity,
serum anti-protease activity throughout the experimental period when compared with the control
group. Dietary doses of neem leaf diet signicantly inuenced the immune parameters, haematological
parameters and blood biochemical indices of treated sh. The results suggested that sh fed neem leafsupplemented diet improved the immune system and increased survival rate in L. calcarifer ngerlings
against V. harveyi infection.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Immune response
Intraperitoneally
Phagocytic activity
Dietary
Survival

1. Introduction
The Asian seabass, Lates calcarifer is an eminent commercial sh
species for aquaculture. The culture of L. calcarifer in marine netcages is a popular aquaculture activity worldwide including in
Malaysia. The culture of L. calcarifer in marine net-cages has
suffered due to bacterial infections particularly by the occurrence of
vibriosis caused by Vibrio harveyi, which results in heavy losses and
causes economic loss to sh farmers [1]. Vibriosis due to Vibrio sp. is
one of the main bacterial diseases in mariculture systems [2].
Vibriosis caused by V. harveyi, a halophilic Gram-negative bacterium that is known to cause disease to sh, shrimp and shellsh
either in the culture systems or in the wild aquatic environments
[3]. Vibriosis owing to infection of V. harveyi is responsible for
anorexia, darkening of the whole body of the sh, haemorrhagic
ulcers on the mouth or skin surface, tail and n rot, muscles focal
necrotic lesions and swollen intestine, and eye opacity [4,5]. In

* Corresponding author. Institute of Tropical Aquaculture, Universiti Malaysia

Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia. Tel.: 60 16 950 2627;
fax: 60 9 668 3390.
E-mail address: mrtalpur@yahoo.com (A.D. Talpur).
1050-4648/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fsi.2012.11.003

general, young sh are more susceptible to infection followed by

severe mortality [5]. Intraperitoneal injection of V. harveyi at
5.0  104 cfu mL
1 to L. calcarifer ngerlings demonstrated high
virulence resulting in100% mortality within two days of postchallenge [1]. The control of infections due to V. harveyi could
facilitate effective surveillance and prevention of the disease in
aquaculture. Conventionally, the prevention of disease outbreaks in
aquaculture systems are usually attempted by using antibiotics or
disinfectant chemicals [6]. Antibiotics used in aquaculture have
problems including toxicity, cost and governmental restrictions [7]
and frequent use of antimicrobial drugs, pesticides, and disinfectants in aquaculture has led to drug-resistant microbes [8,9]. In fact,
antibiotic resistance displayed by microbial pathogens has
encouraged more environment-friendly approaches to screening of
plants for their potential antimicrobial activity to control disease
[10,11]. In particular, infections caused by V. harveyi in sh could be
controlled through immunostimulant plants as feed additive [12].
Bricknell and Dalmo [13] dened the immunostimulant as a
naturally occurring compound that modulates the immune system
by increasing the hosts resistance against diseases that in most
circumstances are caused by pathogens. Azadirachta indica known
as Margosa or neem, is an evergreen tree of potential medicinal
value found in most tropical countries [14]. Many authors have

A.D. Talpur, M. Ikhwanuddin / Fish & Shellsh Immunology 34 (2013) 254e264

reported that the neem possesses signicant antibacterial [15e17]

antifungal [18] and antiviral properties [19]. Continuing with the
benecial effects of neem, it has been considered to have broadspectrum prophylactic and therapeutic functions, as well as
signicant modulating effect on the humoral and cell-mediated
immune system [20]. In addition, the neem has been reported to
have anti-inammatory, anti-oxidative activity, hepato-protective,
cancer chemo-preventive potential [21e24] and to be effective as
an anti-diabetic agent in animals [25,26].
Food plays a vital role in sh growth and health, therefore the
pertinent feed additive, which has a potent effect against
microbe pathogens, has become very important for feed formulations. The neem has been recognised for its potential broadspectrum prophylactic and therapeutic role; however, there
have been no reports on the neem-supplemented diet to control
disease caused by Vibriosis or other opportunistic pathogens in
L. calcarifer. Based on assumption that the neem may act as an
immunostimulant against pathogenic infection in L. calcarifer.
The present investigation was carried out to evaluate the effects
of dietary supplementation of neem leaf on the innate immune
responses, haematological and biochemical indices of blood/
serum of L. calcarifer and disease resistance against V. harveyi
infection.
2. Materials and methods
2.1. Seabass ngerlings
L. calcarifer ngerlings of average weight 18  2 g were procured
from a local hatchery in Malaysia. The health statuses of sh were
examined instantaneously upon arrival. Fish were quarantine
bathed in 10% formalin for 20 min and were acclimatised for 20
days in 1000 (L) disinfected chlorine-free sea water (salinity
20 ppt). Fish were fed with basal diet at 5% of body weight twice
a day in two equal parts at 9.00 a.m and 4.00 p.m. Water exchange
was done daily at a rate of 50% and water quality was monitored
daily throughout the experiment. Temperature was maintained at
28  1  C, dissolved oxygen concentration >6.0 mg L
1, pH 8.0  0.2
and salinity 20  1 ppt.
2.2. Neem leaves
Fresh neem leaves were collected from the neem in the
surrounding area of the universiti Malaysia Terengganu, Gong
Badak, Kuala Terengganu, Malaysia. Leaves were washed with fresh
tap water and were dried under shade. Leaves were milled into
powdery form and then kept in a dry, clean, airtight jar before being
added to diet.
2.3. Preparation of experimental diets
Ingredients of basal diet are shown in Table 1. Proximate
composition of the basal diet (local feed) comprised crude protein
41.6%, lipids 17.12%, ash 14.6%, moisture 9.7% and bre 3% [12].
Six feed groups were made for experimental trials, neem leafsupplemented (NLS) diet was obtained by incorporating the
neem leaf powder at levels of 0 g, 1 g, 2 g, 3 g, 4 g and 5 g/kg feed for
T0, T1, T2, T3, T4 and T5 respectively. Water was added and the
feedstuffs of basal diet mixed mechanically in (Hobart D300T) for
20 min at a low speed to assure the homogeneity of the ingredients.
Pellets were then prepared using a pellet machine (GZL pellet mill,
China). The pellets were air dried at ambient temperature under
ow hood for 24e48 h, and were stored in labelled screw-cap
airtight containers at room temperature.

255

Table 1
Percentage addition of ingredients for formulated basal diet.
Ingredients

Percent incorporated

Fish meal
Wheat meal
Soybean meal
Fish oil
Vitamins and minerals (pre mixture)
Cornstarch

50
25
15
5
2
3

2.4. Experimental design

Healthy ngerlings of L. calcarifer (n 180) were selected for
the experimental use and equally distributed into six experimental groups based on feed application following a complete
randomised selection using 300 L round tanks lled with disinfected sea water (20 ppt) up to 250 L and equipped with aeration. Control fed with basal diet without NLS diet, treated groups
were fed NLS diet for two weeks (15 days) at 1 g, 2 g, 3 g, 4 g and
5 g/kg of feed during pre-challenge. After challenge sh were fed
NLS diet for two weeks (15 days) at same ratio as above and
control fed with basal diet without NLS diet. Experimental trials
were carried out in duplicate. One group was used for challenge
assay and other group for weight gain, specic growth rate (SGR)
and feed conversion ratio (FCR). The growth performance
including percentage weight gain, specic growth rate (SGR) and
feed conversion ratio (FCR) for each group was determined as
described by Choudhury et al. [29].
Wt. gain% nal wt - initial wt/initial wt  100
SGR log of nal wt-log of initial wt/no. of days
FCR feed given (dry wt)/body wt  gain (wet wt)
2.5. Pathogen bacteria
V. harveyi was isolated from Portunus pelagicus (Linnaeus, 1758)
larviculture, tested for pathogenicity [27,28] and used in this study
as according to Talpur and Ikhwanuddin [12]. Briey, pathogen was
grown in marine broth (Merck, Germany) prepared with sea water
for 24 h at 37  C. Culture broth was centrifuged at 13,000  g for
10 min. The supernatants were discarded and the bacterial cells
were washed twice in sterilised sea water, and the pellets were
resuspended in sterilised sea water for experimental use. The
concentration was adjusted by means of optical density to 0.9 at
OD630 nm, which corresponded to 108 cells mL
1.
2.6. Challenge assay
After feeding sh with NLS diets for two weeks (15 days),
randomly 15 sh from subgroups including control were
Table 2
Growth parameters of L. calcarifer ngerlings fed on different levels of neem leafsupplemented (NLS) diets for 15 days.
Gram of neem leaf
diet/kg feed

Weight gain %

0
1
2
3
4
5

62.2
69.9
78.7
89.2
92.3
94.3








4.47
3.01a
3.84a
3.41a
4.25a
6.35a

SGR
1.4
1.6
1.8
1.9
2.0
2.1








FCR
0.2
0.1a
0.3a
0.1a
0.6a
1.0a

1.76
1.75
1.74
1.65
1.65
1.67








0.34
0.13a
0.22a
0.32a
0.32a
0.42a

Data expressed as mean  SE, p < 0.05, n 15. SGR, specic growth rate; FCR, feed
conversion ratio.
a
Values in columns were signicantly different (p < 0.05) from control.

256

A.D. Talpur, M. Ikhwanuddin / Fish & Shellsh Immunology 34 (2013) 254e264

Fig. 1. Effect of neem leaf-supplemented (NLS) diet at different levels on survival of L. calcarifer after challenge with V. harveyi. Data expressed as mean  SE (n 15). Mean values at
bars with different superscript letters were signicantly different (p < 0.05) from the control.

challenged intraperitoneally with a lethal dose of 0.1 mL
1

suspensions of V. harveyi in 0.9% (w/v) saline containing 108 cells
mL
1 according to Talpur and Ikhwanuddin [12]. Mortalities
were recorded over a period of two weeks (15 days) and any dead
or moribund sh examined bacteriologically to conrm the
presence of V. harveyi as per standard procedure. The relative
percentage survival (RPS) was calculated as describe by
Amend [41].
2.7. Collection of blood
Feeding to sh was stopped 24 h before blood samples were
collected. Blood samples were collected from each sh group from
ve randomly selected sh (n 5 pre-challenge and postchallenge), thorough caudal-vein puncture using sterile syringes.
To prevent clotting, samples were transferred into sterile vacuette
tubes containing heparin as anticoagulant. Blood samples were also
collected without heparin, allowed to clot, centrifuged at 3000 rpm
for 15 min at 4  C, and the serum collected was stored at
80  C
until use. Collected samples of blood/serum were used for estimation of immunological, haematological and biochemical
parameters.

2.8. Determination of haematological, biochemical and

immunological parameters
Haematological parameters were determined following the
method previously described [30,31]. Glucose concentration was
estimated according to method described by Trinder [32]. Plasma
protein content was determined using the method described by
Wootton [33]. Total lipids were determined calorimetrically
according to Knight et al. [34]. Triglycerides and cholesterol-level
determination was carried out according to the method
described by Stein [35]. Immunological parameters were
measured as per method described by Brunt and Austin [36] and
Newaj-Fyzul et al. [37]. The kidneys were removed from the
same sh and the control, crushed in a tissue grinder (Sigmae
Aldrich) for study as per methods of Nya and Austin [38].
Macrophages and cell viability were obtained as according to Nya
and Austin [38] and the percentage of viable (unstained) and
dead (stained) cells was scored after Sakai et al. [39]. The
percentage inhibition of trypsin activity was calculated by
comparing the average of the absorbance reading with the value
for 100% enzyme activity according to method described by Zuo
and Woo [40].

Fig. 2. Phagocytic ratio and phagocytic index of the head kidney macrophages of L. calcarifer fed with neem leaf-supplemented (NLS) diet at different levels. Data are expressed as
mean  SE (n 5). Mean values at bars with different superscript letters at same stage were signicantly different (p < 0.05) from the control.

A.D. Talpur, M. Ikhwanuddin / Fish & Shellsh Immunology 34 (2013) 254e264

257

Fig. 3. Superoxide anion production (Respiratory burst) by blood leucocytes of L. calcarifer fed with neem leaf-supplemented (NLS) diet at different levels. Values are expressed as
mean  SE (n 5). Mean values at bars with different superscript letters at same stage were signicantly different (p < 0.05) from the control.

2.9. Statistical analysis

Results for each parameter were expressed as mean  standard
error (SE). Data were analysed using one-way ANOVA and the
comparisons of the mean values were done by using Duncan
multiple range tests using software program SPSS version 16 for
Windows. Differences were considered statistically signicant
when p < 0.05.
3. Results
3.1. Weight gain, growth parameters and feed conversion ratio
Fish fed NLS diet showed signicantly (p < 0.05) higher weight
gain over the control. Specic growth rate (SGR) and feed conversion ratio (FCR) values in sh fed with NLS diet were signicantly
increased as compared with the control. Elevated SGR was
observed in NLS diets fed groups compared with control group.
Highest weight gain, SGR and FCR was observed in those sh
groups fed with NLS diet at 5 g/kg feed (Table 2).
3.2. Fish survival
After challenging sh with V. harveyi, the mortality was recorded for 15 days. There was no mortality of sh up to 24 h. Fish fed
NLS diets showed signicantly (p < 0.05) higher survival for all
treated groups compared with the control. The highest survival was
observed in those sh group that were fed NLS diet at 4 g/kg feed
(Fig. 1). At the end of the experiments sh survivors that received
NLS diets did not show any disease signs.

control including post-challenge period. Highest superoxide anion

production was determined in sh groups fed NLS diets at 2 g/kg
feed followed by 3 g/kg feed (Fig. 3).
3.3.3. Lysozyme activity
Signicantly (p < 0.05) higher lysozyme activity was observed in
the serum of those sh groups fed NLS diets including postchallenge, when compared with the control group. Highest lysozyme activity was observed in sh group fed with NLS diet at 3 g/kg
feed. Lysozyme activity after challenge was signicantly higher for
all treated groups over the control (Fig. 4).
3.3.4. Bactericidal activity
Serum bactericidal activity in different NLS diet fed groups was
signicantly (p < 0.05) higher for all samples when compared with
control, including post-challenge. Highest bactericidal activity was
observed in those sh group fed with NLS diet at 5 g/kg feed. Serum
bactericidal activity was dose response in all treatment groups
(Fig. 5).
3.3.5. Anti-protease activity
Results showed that sh fed NLS diet at different levels had
a signicantly (p < 0.05) higher serum anti-protease activity
compared with the control including post-challenge. Elevated
doses raised the anti-protease activity in treated groups (Fig. 6).

3.3. Immunological parameters

3.3.1. Phagocytic activity (ratio) and index
Phagocytic activity (ratio) and index was signicantly
(p < 0.05) higher for all samples of NLS diets fed groups including
post-challenge when compared with the control group. Signicantly higher phagocytic ratio and phagocytic index was observed
in sh fed NLS diet at 5 g/kg feed compared with the control
(Fig. 2).
3.3.2. Production of superoxide anion
The results indicated that superoxide anion production in the
experimental groups was signicantly (p < 0.05) higher than the

Fig. 4. Lysozyme activity in the plasma of L. calcarifer fed with neem leafsupplemented (NLS) diet at different levels. Values are expressed as mean  SE
(n 5). Mean values at bars with different superscript letters at same stage were
signicantly different (p < 0.05) from the control.

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A.D. Talpur, M. Ikhwanuddin / Fish & Shellsh Immunology 34 (2013) 254e264

Fig. 5. Serum bactericidal activity of L. calcarifer fed with neem leaf-supplemented (NLS) diet at different levels. Values are expressed as mean  SE (n 5). Mean values at bars with
different superscript letters at same stage were signicantly different (p < 0.05) from the control.

3.4. Haematological parameters

The results showed that RBC count and WBC count was
signicantly (p < 0.05) higher for all sh fed the different doses of
NLS diet compared with the control group including postchallenge. In general, pathogen V. harveyi affected the RBC and
WBC count in post-challenge. Higher RBC and WBC count was
found in those sh fed with NLS diets at 4 g and 3 g/kg feed
respectively (Fig. 7, Fig. 8). The haematocrit % was signicantly
(p < 0.05) higher over the control including post-challenge except
sh fed NLS diet at 1 g/kg feed had lower haematocrit % than
control (Fig. 9). Haemoglobin content was not signicantly
higher over the control but it slightly increased in post-challenge
(Fig. 10).
Mean corpuscular volume (MCV) was observed signicantly
higher in those sh that received NLS diets at 3 and 4 g/kg feed.
However, mean corpuscular haemoglobin (MCH) and mean
corpuscular haemoglobin concentration (MCHC) values observed in
treated groups were not signicant differences between groups.
There was a decrease in these parameters in post-challenge
(Table 3).

The result showed that there were considerable changes in the

relative proportions of lymphocytes, monocytes, neutrophils,
eosinophils and basophils but not thrombocytes, following feeding
with NLS diets including post-challenge (Table 3). The higher
proportion (%) of lymphocytes was found in sh groups that fed
NLS diet at 3 g/kg feed compared with the control followed by sh
fed NLS diet at 2 g/kg feed. The proportion (%) of monocytes was
higher in NLS diet fed groups except those sh fed NLS diets at 4 g
and 5 g/kg feed. The proportion of neutrophils (%) was signicantly
higher in all groups that were fed with NLS diets including postchallenge. The proportion of eosinophils and basophils was
signicantly higher in all groups fed with NLS diets (Table 3).
3.5. Blood/serum biochemical parameters
Blood glucose level in all treated groups was signicantly lower
compared with the control. In post-challenge sh fed with NLS diet
at 2 g/kg feed had a higher glucose level over the control (Table 4).
Serum total protein content was signicantly different in treated
groups from the control except those sh fed NLS diets at 1 g and
2 g/kg feed (Table 4). The highest protein content was observed in

Fig. 6. Anti-protease activity of L. calcarifer fed with neem leaf-supplemented (NLS) diet at different levels. Values are expressed as mean  SE (n 5). Mean values at bars with
different superscript letters at same stage were signicantly different (p < 0.05) from the control.

A.D. Talpur, M. Ikhwanuddin / Fish & Shellsh Immunology 34 (2013) 254e264

259

Fig. 7. RBC count observed in sh fed with neem leaf-supplemented (NLS) diet at different levels. Data are expressed as mean  SE (n 5). Mean values at bars with different
superscript letters at same stage were signicantly different (p < 0.05) from the control.

the group that received NLS diet at 4 g/kg feed. However, a signicant decrease in protein contents was found in all treated groups in
post-challenge (Table 4).
Total lipid level at all samplings was signicantly lower in sh
fed with NLS diets at 4 g and 5 g/kg feed. However, sh groups that
fed with NLS diet at 3 g/kg feed had a higher lipid level compared
with the control. A decrease in lipid level was found in all postchallenge samples. There were no signicant differences in lipid
levels (Table 4). Moreover, triglycerides and cholesterol level was
not signicant different in all treated groups including postchallenge when compared with the control (Table 4). Feeding of
different levels of NLS diet did not increase in albumin content
including post-challenge. There were no signicant differences in
albumin content (Table 4). However, a signicantly (p < 0.05)
higher globulin level was found in all NLS diets fed groups except
that sh group fed NLS diet at 1 g/kg feed had shown low globulin
level when compared with the control. In post-challenge period,
globulin level was signicantly higher in treated groups except that
sh group fed NLS diets at 1 g and 3 g/kg feed (Table 4). Albumin :
globulin ratio was found signicantly higher in sh group fed NLS
diet at 2 g/kg feed; there was no signicant (p > 0.05) increase in
remaining treated groups. In post-challenge, a decrease in albumin
: globulin ratio was found in those sh groups that were fed NLS
diets at 3 g and 4 g/kg feed respectively.

4. Discussion
Vibriosis led to severe losses in aquaculture production [42].
Potential herbal plants at certain dosages can reduce the mortalities
against pathogenic challenges [43,44]. Neem contains active
compounds that have antiviral, antibacterial and antifungal properties [45,46]. Many reports in literature suggest that herbs tested in
aquatic animals signicantly increased the growth [47,48]. To the
best of our knowledge, the present study is the rst report to
investigate the role of neem leaf-supplemented (NLS) diet as
potential of immunostimulant in L. calcarifer. Growth improvement
can provide benets for aquaculture by decreasing production
times, increasing FCR, and productivity. Results of this study showed
that signicantly (p < 0.05) higher weight gain, SGR and FCR
observed in those sh that were fed with NLS diet when compared
with the control. This study demonstrated that feeding of NLS diet
signicantly increased the survival rate of L. calcarifer and increased
resistance against V. harveyi when challenged. It was clear from the
results of this study that the application of neem leaf powder at
certain levels in the diet of sh could improve the non-specic
immunity mechanisms of sh and demonstrated positive health
benets as a result of a reduction in mortalities after challenge.
Proximate composition of neem leaf includes alkaloids, tannins,
saponins, avonoids, phenols, carbohydrates, terpenoids, glycosides,

Fig. 8. WBC count observed in sh fed with neem leaf-supplemented (NLS) diet at different levels. Data are expressed as mean  SE (n 5). Mean values at bars with different
superscript letters at same stage were signicantly different (p < 0.05) from the control.

260

A.D. Talpur, M. Ikhwanuddin / Fish & Shellsh Immunology 34 (2013) 254e264

Fig. 9. Haematocrit (%) observed in sh fed with neem leaf-supplemented (NLS) diet at different levels. Data are expressed as mean  SE (n 5). Mean values at bars with different
superscript letters at same stage were signicantly different (p < 0.05) from the control.

antiquinones [49,50]. Neem leaf also contains protein, fat, bre,

minerals, vitamin C and amino acids [51]. The compounds found in
neem leaf directly inuence sh health by trigging immune system.
The immunomodulatory effects of NLS diet in sh follow an
increased level of phagocytic activity, which is a vital module of the
non-specic immune system of sh [52]. Neem is well-considered
to have broad-spectrum activity including activation immunostimulation owing to its more than 100 bioactive compounds, which
have been reported to have biological activities such as antiinammatory, antiarthritic, antipyretic, hypoglycaemic, antigastric
ulcer, spermicidal, antifungal, antibacterial and diuretic [53]. The
neem leaves possess potential immunostimulant activity as evidenced by both humoral and cell-mediated responses [54,55]. In
the present investigation, phagocytic activity was higher in treated
groups compared with the control which clearly indicate that
addition of neem leaf in the diet signicantly improved the nonspecic immunity of sh. Many studies have indicated that
extracts of neem plant may possess signicant antioxidant properties that can boost the blood antioxidant status [21] and signicantly protect the animals against oxidative stress [56]. It has been
reported that immunostimulant plants forages superoxide anion
[43] which is an effective scavenger to provide a possible protective
mechanism against autotoxicity and fatality [38,57,58]. The

respiratory burst activity is recognised as an indicator of the status

of macrophage and neutrophil activation. The respiratory burst
activity of the neutrophils observed in this study was in agreement
with previous work [12,38,59e61] in which an increase in NBT
(Nitro Blue Tetrazolium) activity over controls was recorded in sh
fed with garlic-added and ginger-added diets [12,38,60] at certain
levels. In the present study following challenge, the production of
superoxide anions by the neem in treated sh provided resistance
to V. harveyi infection by enhancing immune system.
Immunostimulants are considered to increase serum lysozyme
activity [59,62], and rising of lysozyme following immunostimulation has been demonstrated in a number of sh species
[12,38,59e61]. Lysozyme activity is relevant as a rst line barrier of
defence system thus resulting in the diminution of disease by
preventing bacterial pathogens [63]. In the present study, sh fed
diets supplemented with different levels of neem leaf showed
signicantly higher lysozyme activities when compared with the
control group. At present, no information on the NLS diet effects on
serum lysozyme activity in L. calcarifer has been reported. Data of
the present study demonstrates that lysozyme activity was raised
signicantly in the groups of sh fed with NLS diet. As a result,
a defence capability of sh against induced pathogen was ultimately increased.

Fig. 10. Haemoglobin (g%) observed in sh fed with neem leaf-supplemented (NLS) diet at different levels. Data are expressed as mean  SE (n 5). Mean values at bars with
different superscript letters at same stage were signicantly different (p < 0.05) from the control.

A.D. Talpur, M. Ikhwanuddin / Fish & Shellsh Immunology 34 (2013) 254e264

Table 3
Haematological parameters for L. calcarifer fed different levels of neem leafsupplemented (NLS) diet pre-challenge and post-challenge.
Parameter

Neem leaf diet

g/kg feed

Pre-challenge

MCV (fL)

0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5

93.8
92.3
90.1
97.3
94.2
93.2
21.2
21.3
19.2
20.3
16.2
15.2
26.4
21.1
26.1
26.3
26.3
18.2
22.8
20.2
29.2
31.3
22.5
23.2
16.1
22.3
27.1
19.2
13.1
10.2
43.5
51.0
44.2
50.2
48.4
52.4
11.4
6.8
10.2
10.4
9.2
10.2
0.20
0.22
0.25
0.28
0.30
0.28
0.12
0.11
0.19
0.18
0.14
0.17

MCH (pg)

MCHC (%)

Lymphocytes %

Monocytes%

Neutrophils%

Thrombocytes%

Eosinophils (103/mL)

Basophils (103/mL)
























































1.4
1.1
1.2
1.3a
1.2 a
1.2
1.1
1.3
1.2
1.0
1.0
1.4
0.2
1.2
2.1
1.3
1.2
1.3
1.2
1.5
1.1a
0.2a
2.4
1.0a
1.2
1.4a
1.2a
1.6a
1.2
1.6
1.4
2.1a
2.2a
4.2a
5.1a
3.4a
2.2
2.1
3.1
1.4
1.2
1.2
1.1
0.2a
0.1a
0.3a
1.1a
1.1a
0.1
0.2
1.1a
1.0a
0.2a
1.2a

Post-challenge
66.3
67.1
72.0
66.3
83.4
74.6
16.1
14.1
17.3
17.2
16.2
14.1
21.5
20.4
21.6
18.2
21.1
17.6
16.5
18.7
18.9
25.4
20.1
22.6
13.0
11.8
17.1
10.0
9.1
7.4
45.3
46.2
49.1
48.3
49.7
54.6
10.1
11.2
8.2
11.1
10.1
8.2
0.15
0.16
0.20
0.20
0.21
0.18
0.10
0.11
0.10
0.12
0.10
0.11
























































1.2
1.1
1.1
1.2
1.5
1.2
1.3
1.0
1.4
1.1
1.2
1.5
0.4
1.3
1.2
1.2
1.0
1.6
1.1
2.1
1.1
1.1
1.3
1.2
1.0
1.6
1.1
2.1
1.4
1.0
1.6a
2.1a
3.2a
2.5a
2.6a
1.8a
1.3
1.2
0.1
1.2
2.3
1.3
0.2
1.1
1.3
0.4
1.0a
2.1
1.0
0.1
1.1
1.1
1.2
1.1

Data are represented as mean  SE (n 5). MCV, mean corpuscular volume; MCH,
mean corpuscular haemoglobin; MCHC, mean corpuscular haemoglobin
concentration.
a
Values of means were signicantly different (p < 0.05) from the control.

Serum bactericidal activity which is involved in eradication of

pathogenic microbes in sh [64], was elevated in all treated groups
over the control in the present study. Neem is recognised to have
bioactive antimicrobial compounds which kill the microbes
including those resistant to drugs [65]. Results of the present study
correspond with the investigations of previous work of Nya and
Austin [38,60] who reported increased serum bactericidal activity
in rainbow trout after feeding with garlic-added diet and gingeradded diet, Sahu et al. [59] in Labeo rohita and Talpur and Ikhwanuddin [12] in L. calcarifer after feeding with garlic-supplemented

261

Table 4
Blood/serum biochemical parameters of L. calcarifer fed different levels of neem leafsupplemented (NLS) diet pre-challenge and post-challenge.
Parameter

Neem leaf diet

g/kg feed

Pre-challenge

Glucose (mg L
1)

0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5

114.2
114.7
112.2
112.8
111.2
111.6
1.5
1.4
1.2
2.2
2.5
2.4
6.8
6.8
6.8
6.9
6.7
6.7
64.53
65.54
63.72
64.56
64.16
64.28
102.80
102.24
102.76
102.82
102.78
102.78
0.3
0.3
0.2
0.3
0.3
0.3
1.3
1.2
1.5
1.4
1.7
1.4
0.2
0.2
0.3
0.2
0.2
0.2

Total protein (mg L
1)

Total lipid (g/L)

Triglycerides mg/dl

Cholesterol mg/dl

Albumin (mg L
1)

Globulin (mg L
1)

Albumin:globulin
ratio (mg L
1)


















































2.4
1.6a
3.4 a
3.3a
1.1a
1.4a
1.1
0.7
1.0
1.6a
1.2a
2.1a
1.2
0.2
0.6
0.2
1.2a
1.2a
1.5
1.0
1.1
1.2
1.1
1.0
1.0
1.3
1.2
1.2
1.0
1.4
0
0.1
0.1
0
0.1
1.4
0.6
0.1
1.1a
0.6a
1.2a
1.6a
1.0
0
0a
0.4
1.2
1.2

Post-challenge
114.3
113.3
116.1
112.1
113.4
110.5
1.5
1.2
1.1
2.0
2.2
2.2
6.8
6.8
6.8
6.8
6.7
6.7
63.26
63.27
63.34
63.82
64.18
63.48
102.38
102.42
102.19
101. 96
102.52
102.36
0.3
0.3
0.3
0.2
0.3
0.3
1.3
1.1
1.4
1.2
1.7
1.4
0.2
0.2
0.2
0.1
0.1
0.2


















































2.2
2.6a
1.3a
1.7a
2.5a
3.1a
0.1
1.2
1.1
0.5a
1.4a
1.0a
0.7
1.2
0.1
1.2
1.1a
1.2a
1.6
1.4
1.2
3.7
3.6
2.6
2.1
1.4
1.2
1.4
2.3
2.1
1.1
1.0
0.2
0.6
1.2
1.2
1.3
1.2
0.4a
0.2
1. 0a
0.2a
1.4
0.2
1.4
0.0.3
0.1
1.6

Data are represented as mean  SE (n 5).

a
Values of means were signicantly different (p < 0.05) from the control.

diet. Serum bactericidal activity in this study was correlated with

dose potency. Highest serum bactericidal activity as a result of
antimicrobial compounds of neem was observed in those sh that
were fed NLS diet at 5 g/kg feed.
Numbers of protease inhibitors are found in the serum and other
body uids of sh which maintain body-uid homeostasis [66]. It
has been reported that the hydrolysis of protein in vivo is regulated
as a result of trypsin inhibition activity which further stimulates the
immune defence mechanism against pathogens [67,68]. Elevated
anti-protease activity observed in this study was dose response,
which support the previous work involving sh fed with garlicadded diet [12,38] and ginger-added feed [60] thus increasing
immune response in sh against pathogens.
The white blood cell (WBC) counts were signicantly increased
in sh (Oreochromis niloticus) challenged with Aeromonas hydrophila and treated with neem leaf extract aqueous [69]. Blood is
a patho-physiological reector of the entire body and the counts of

262

A.D. Talpur, M. Ikhwanuddin / Fish & Shellsh Immunology 34 (2013) 254e264

haematological parameters in blood give an indication to the health

status of sh by determining any abnormality occurring owing to
the use of immunostimulants [70]. The increase in RBC, WBC
counts, lymphocytes, monocytes, neutrophils, eosinophils and
basophils counts following NLS diet feeding indicate the immunostimulant effects and anti-infection properties of neem. The
presence of avonoids in neem leaves are known to act as an
antioxidant which neutralises highly unstable and extremely
reactive molecules, called free radicals, which attack the normal
cells of the body and cause a variety of health problems [71].
Furthermore, the presence of terpenoids in neem leaf, considered
as vital antioxidants, exhibit an important role for health by inuencing the immune function [72]. Moreover, it is widely accepted
that immunostimulant plants do possess broad-spectrum properties [59,73]. The results of this study correspond with those by
[38,60] who reported the counts of WBC and RBC were signicantly
higher in rainbow trout fed with the garlic-added and ginger-added
diets and in broiler chicken which were fed neem-added diet [74].
Surprisingly, a reduction in RBC and increase in WBC was found in
post-challenge sh groups. However, these cells were signicantly
higher than the control. The results of the present study suggests
that decrease in RBC count in sh in post-challenge is as a result of
leucocytosis activity and/or stress developed by the induced
pathogen following erythroblastosis, but any anaemic condition
had not developed in sh. This was because of enhanced immune
system due to immunostimulant effect of NLS diet as a result of
bioactive compounds present in leaf, particularly avonoids and
terpenoids. WBC serves as one of the front lines of body defence
and is believed to increase quickly when infections occur and the
presence of tannins in neem leaves helps in infection healing [75].
WBC and neutrophils counts in NLS diet fed sh increased in postchallenge, because of V. harveyi infection that might have damaged
the organs of sh. Increase in number of WBC and neutrophils in
challenged sh may serve as a defence barrier against pathogen.
Indication of low haematocrit (%) reects on the anaemic condition of sh. This happens when sh stop feeding as a result of any
disease or stress. In this study, haematocrit (%) was decreased in postchallenge which indicate sh were not feeding well due to suffering
the infection or stress of induced V. harveyi. When sh is under stress,
the content of haemoglobin and oxygen consumption increases, and
thus, the haemopoietic organs release immature RBCs in bulk quantity; consequently, haemoglobin concentration in blood increases
[63]. In the present study, haemoglobin content was not signicantly
different in treated groups over the control; this demonstrates the
sh was not under stress. However, haemoglobin content was slightly
higher but not signicant in post-challenge groups, which indicates
the sh were under stress owing to V. harveyi infection.
There was a decrease in haematological indices, including MCH,
MCHC and MCV in Carassius auratus after intraperitoneal injection
of extracted microcystins which affected oxygen uptake due to gill
damage [76]. A decrease in MCH, MCHC and MCV was found in
post-challenge, suggesting that the decline in haematological
indices may be due to V. harveyi stress/infection which might have
affected the gill or damaged the tissues.
The hypoglycemic and anti-hyperglycemic properties of neem
plant are well documented. Khosla et al. [77] demonstrated that
neem leaf extract and neem seed oil administration in rabbits
prevented the increase in blood glucose levels when compared to
control diabetic animals. The use of neem in animals has clearly
demonstrated the hypoglycemic potential, either alone [78,79], or
in combinations with other herbs [25]. Presence of terpenoid and
saponins in neem leaf, play an important role in reducing complications associated with diabetes and lowers sugar level in blood
[49,80]. It was clearly observed in this study that feeding of NLS diet
to sh, led to decrease glucose values in sh of treated groups

during the experimental trials. This might have increased the level
of serum insulin because of terpenoid and saponins compounds in
NLS diet. No obvious effect of NLS diet on triglycerides and
cholesterol of sh were observed in the present study.
Results of present study demonstrates that increases in serum
total protein and globulin coincides with previous investigations
using immunostimulants, namely garlic and ginger [38,59,60].
Wiegertjes et al. [81] reported that elevations in serum protein and
globulin levels are believed to be related with a stronger innate
immune response of sh and are vital fractions for sustaining
healthy immune system [82].
Higher doses of immunostimulant suppress the immune
responses [83] and overdoses for long-term administration of
immunostimulants may reduce their efcacy [84]. Although
immunostimulants are known to trigger body immunity, regular
consumption of immunostimulants may cause intolerance that
might lead to overstimulation of the immune system by disrupting
the normal metabolic processes in the body. In the present study,
doses of NLS diet that were fed to sh did not show any overstimulation and/or suppression of immunity during experimental
trials. It could be explained that doses of NLS diet administrated to
sh were not adverse but had shown positive effect by enhancing
immune response and disease resistance to sh.
Supplementation of specic immunostimulant to feed can
improve animal health and thereby reduce management costs.
Among immunostimulants, neem leaf can be used as a feed additive
to replace the antibiotic/or disinfectant in order to develop a new
concept of organic biotechnology for sustainable aquaculture. It is
concluded that therapeutic potential of the neem leaf as dietary
supplement would improve the non-specic immunity of sh, as
evident in this study by the reduced mortality of L. calcarifer after
challenge with V. harveyi. Present investigation has provided new
insight into immunostimulatory capacity of the neem leaf incorporated in sh diet to prevent disease and possibly contribute to an
enhancement in sh welfare and economic growth in the aquaculture industry. However, it remains for further work to consider NLS
diet as sole and/or with other potential plants for disease control
strategies in aquaculture, appropriate eld trials are recommended.
Acknowledgement
This study was nancially supported by the Department of Fisheries, Government of Sindh, Pakistan under capacity building project.
Corresponding author would like to thank Mr. G.M. Mahar Director
General Fisheries and Mr. G. M. Wadahar Director Fisheries Sindh
Inland, Government of Sindh, Pakistan for their extended support for
the present study. Authors would like to thank the Director and the
staff of the Institute of Tropical Aquaculture (Aquatrop) and marine
hatchery, universiti Malaysia Terengganu, Malaysia for their help.
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