Fish and Shellfish Immunology 76 (2018) 324–332

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Fish and Shellfish Immunology

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Leucaena leucocephala pod seed protein as an alternate to animal protein in T

fish feed and evaluation of its role to fight against infection caused by Vibrio
harveyi and Pseudomonas aeruginosa
Vipin Kumar Vermaa, Kumari Vandana Ranib, Shiva Raj Kumara, Om Prakasha,∗
a
Department of Zoology, Sri Venkateswara College, University of Delhi, Dhaula Kuan, New Delhi, India 110021
b
Department of Zoology, Kalindi College, University of Delhi, Delhi, 110008, India

A B S T R A C T

The laboratory acclimatized Clarias gariepinus (80 ± 10 g) were divided into six groups and five subgroups each
containing 10 fish. A fish feed was reconstituted by adding 33% powder of Leucaena leucocephala seed in place of
fish trash. Group B, C and E were fed on reconstituted feed and group A, D and F were fed on artificial feed
containing animal protein for 7 days prior to start of experiments. Then Group B was challenged with BSA while
other groups were challenged with Vibrio harveyi (Group C, D) and Pseudomonas aeruginosa (Group E, F). Group A
was used as negative control (not challenged with antigen). The fish were challenged on weekly intervals till
28th day. Blood was collected from one subgroup of each group on day 7, 14, 21 & 28 and finally sacrificed on
day 35. Change in body weight, liver function tests (SGOT, SGPT) and serum ALP levels were monitored. The
phagocytic index, percentage phagocytosis and nitric oxide levels were measured in macrophages isolated from
spleen and head kidney. The levels of total fish immunoglobulin were also measured following indirect ELISA.
The results showed improved immune response in fish fed on 33% L. leucocephala pod seed reconstituted feed;
however their specific growth rate was low.

1. Introduction protection and promoting disease resistance against pathogenic bac-

teria, thus preventing heavy losses in aquaculture [10,11].
Plants are the primary producers which serve as major source of Aquaculture of the finfish, crustaceans and mollusks is one of the
nutrition for animals including humans. Seeds/cereals, fruits, flowers, fastest growing foods producing industry where different kinds of
roots and shoots from different plants are used for nutritional purpose marine and fresh water fish have been cultured. This has resulted in
since ancient times. Because of the widespread use of rich medicinal increased worldwide production of cultured fish every year (FAO 2014)
plants in primary health care, many pharmaceutical companies and [12]. In water, fish are continuously exposed to ubiquitous population
medicinal research committees focus on them for drug discoveries [1]. of viruses, bacteria and protozoans, among which few are potentially
Secondary metabolites such as alkaloids, polyphenols, flavonoids, gly- infectious to fish. Pathogens can enter fish body through various routes
cosides, terpenes, tannins and few other pigments provide protection to and cause infection. Infection depends directly on the virulence
plants from diseases and stressful environment [2]. The amount of ac- strength and quantity of potential pathogen, host animal health and
tive constituents varies in different plants, their different parts and in environment [13]. The most common concern of fish farmers is the
different climatic conditions [3–6]. These traditional drugs are effective rapid multiplication of pathogens in host and water bodies which is
with low cost and rare side effects compared to synthetic chemicals. responsible for fast transfer of infection to other individuals, resulting
Majority of present drugs for animals and human are derived from in uncontrollable mortalities in hatcheries, culture farms and natural
ancient herbal medicines [7–9]. habitats [14,15].
Many infections spread through water and food. The infectious Warm-water fish farming is very common and economically im-
agents or pathogens require medium to spread and grow, inside and portant source in many parts of the world especially for Asian countries.
outside the host. Therefore, several attempts are being made to control Because of desirable conditions, pathogens can also grow easily and are
the pathogen by promoting health management strategies from fish to more capable of causing infection. Aeromonas sp., Edwardsiella sp.,
humans. Now days, wide range of herbal compounds are being used for Pasteurella sp., Pseudomonas sp., Flavobacterium sp., Vibrio sp. and

∗
Corresponding author.
E-mail address: oprakash@svc.ac.in (O. Prakash).

https://doi.org/10.1016/j.fsi.2018.03.011
Received 23 March 2017; Received in revised form 18 February 2018; Accepted 4 March 2018
Available online 05 March 2018
1050-4648/ © 2018 Elsevier Ltd. All rights reserved.
V.K. Verma et al. Fish and Shellfish Immunology 76 (2018) 324–332

Streptococcus sp. etc. generally cause infections in fish. Many antibiotics, Ampicillin and gentamicin (10 μg per disc) were used as positive con-
vaccines, macromolecules, vitamin/mineral sources, bio-actives and trols. The bacteria cultured on LB agar plates were allowed to grow at
other compounds are used to prevent fish disease or to stimulate fish 37 °C for 14h. The zone of inhibition was measured carefully and an-
immune system so as to minimize infections [16,17]. timicrobial activity was evaluated.
Many different marketed products specifically vaccines and anti- The minimal inhibitory concentration was also determined by serial
biotics offer protection from single or multiple bacterial strains. dilution method of the extracts. The methanol and ethanol extracts
Vaccines are formalin killed bacteria or oil adjuvant products that are were used in serial dilutions at concentration 500, 250, 125, 62.5, 31.2,
administered into fish orally, by injection or through mixing with feeds. 15.6, 7.8, 3.9, 1.9, 1 mg/ml in test tubes. 10 μl of overnight cultured
Vaccination is recommended to healthy fish only which are free from bacteria were placed in each tube. The lowest concentration which did
stress [18,19]. The Antibiotics or chemotherapeutic agents have faster not show any growth after 24 h of incubation at 37 °C was determined
rate of action, but their misappropriation may not only exert selective as MIC.
pressure on micro flora, but may also create resistant bacteria [20]. The
environmental stressors also weaken the immune system of fish which 2.3. Experimental animals and feed preparation
can be reduced by improving water quality and preventing over-
crowding. The most effective method, to prevent fish from disease, is to The fish weighing 80 ± 10g were used in the experiments and
improve resistance in fish against disease or simply to improve their maintained at 25 °C ± 1 °C with light dark regime of 12L: 12D for
immune system (innate and adaptive) using immunostimulants. This acclimatization to laboratory conditions. During these days fish were
has encouraged fish immunologist to investigate some herbal immuno- fed with normal feed. Following this fish were divided into six groups
stimulants which enhance immune response of fish to minimize the with five subgroups (each containing 10 fish). Group A, D and F were
stress due to various pathogens [21]. fed on artificial fish feed while B, C and E were fed with experimental
Approximately 500,000 species of plants are estimated to be present feed containing 33% powder of pod seeds of L. leucocephala along with
on earth and only a small fraction of it is used for consumption by other ingredients (see Table 1) @ 3% of their body weight for 7 days.
humans and animals [22]. Plants can produce many metabolites like After this Group C and D were challenged with V. harveyi, Group E and
phenolics, tannins, quinones, terpenoids, phenolics and essential oils as F were challenged with P. aeruginosa and Group B was immunized with
a response against microorganisms or insects [23]. Voluminous re- BSA at weekly interval (day 0, 7, 14, 21 and 28). Blood was collected
search has been done throughout the world to analyze plant properties for one of the subgroup of all groups at weekly interval i.e. day 7, 14,
for health care of animals [24,25]. The use of herbal immunostimulants 21, 28 and 35. Fish were weighed weekly to monitor growth pattern.
in fish diet has accelerated in last few decades to overcome the im- Finally fish were sacrificed to collect tissue.
munosuppressive effects that occur in normal aquaculture procedures
or unavoidable consequences of high density culture [26–28]. 2.4. Challenge of fish with pathogen
Catfish are widely used in aquaculture because of low input cost in
maintenance and high output production. Clarias gariepinus is an air Bacteria used in the challenge experiments were cultured overnight
breathing, exotic fish from Africa, which was introduced in the Asian in nutrient Luria-Bertani broth under shaking conditions (200 rpm) at
countries and distributed around the world due to its high tolerance to 25 °C. Following day, culture medium was centrifuged (6000 rpm) to
varied conditions (FAO 2012) [29]. obtain cell pellet. Cells were washed three times with phosphate buffer
This study is an effort to control infection in C. gariepinus using saline (pH 7.4), re-centrifuged and pellet was re-suspended in PBS. Cells
novel components present in seed powder of Leucaena leucocephala, were counted and suspension was diluted to a cell density of
which would help to control the spreading of pathogens in fish. The 1 × 108 cfu ml−1 [34]. Groups were challenged intra-peritoneally with
components present in L. leucocephala not only control the pathogens 100 μl of live bacterial cells (1 × 107) at intervals according to design of
but also enhance the defense mechanism of the fish [30,31]. Studies the experiments. 100 μl of PBS was injected in negative control group.
also suggest that the pod seed of L. leucocephala is rich source of pro- Blood was drawn from caudal artery and tissues (spleen and head
teins and can also be used as a nutritional supplement for animals kidney) were collected. Macrophages were also isolated from spleen
[32,33]. This plant is not used as an important source of food by hu- and head kidney of fish [35]. The isolated macrophages were used for
mans and therefore its usage remains unexploited. This elicited our estimation of nitric oxide and to evaluate macrophage activity (per-
curiosity to check the outcome of complete replacement of the animal centage phagocytosis and phagocytic index).
protein in artificial feed of C. gariepinus with plant protein (pod seed
powder of L. leucocephala), which would also help in reduce feed costs.
2.5. Determination of specific growth rate
2. Materials and methodology
Specific growth rate of fish was determined by using standard for-
mula [36].
2.1. Preparation of Leucaena leucocephala seed extract
Specific growth rate
L. leucocephala pod seeds were collected from the Sri Venkateswara (logn Final wt. of fish  − logn Initial wt. of fish) ⎤
college campus (Dhaula Kuan, New Delhi, India) washed and air dried. = ⎡ × 100
⎢
⎣ Time interval ⎥
⎦
The pod seeds were grounded and sieved through 1 mm sieve. 10 mg of
the powder was dissolved in 50 ml of methanol and ethanol solutions
separately and stirred overnight on the magnetic stirrer at 25 °C. The Table 1
slurries were filtered and filtrates were dried using rotary evaporator at Ingredients of fish artificial and plant supplemented feed.
35 °C and stored at RT for further analysis.
Ingredients/100 g of feed Artificial Feed Plant supplemented Feed

2.2. Determination of antimicrobial activity Fish meal (g) 32.88 –

Wheat flour (g) 55.12 55
10 μl overnight bacterial cultures (A. hydrophila, E. coli, E. faecalis, Cod liver oil (g) 10 10
Supradyn Tablets 2 2
P. aeruginosa, S. aureus, V. anguillarum and V. harveyi) were spread
L. leucocephala (pod seed powder) – 33
individually on LB agar plate. Sterile paper discs (6 mm diameter) were (g)
imbibed with 100 and 200 mg of extracts dissolved in 0.2% DMSO.

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V.K. Verma et al. Fish and Shellfish Immunology 76 (2018) 324–332

The fish weight was measured on scientific balance upto two deci- Giemsa. Phagocytosis was determined by counting number of positive
mals on successive time intervals. phagocytes (showing yeast endocytosis) per 100 cells counted. The
phagocytic index was calculated using formula; average number of
2.6. Estimation of enzymes yeast cells engulfed by positive phagocytes multiplied by percentage
phagocytosis (for details see Verma et al., 2012) [35].
2.6.1. Measurement of SGOT and SGPT
Serum glutamic oxaloacetic transaminase (SGOT) and Serum glu- 2.8. Measurement of fish immunoglobulin by sandwich ELISA
tamic pyruvate transaminase (SGPT) were estimated in the serum
samples of fish using diagnostic kits (Autopak, Siemens Healthcare Polyclonal antibodies were raised in male albino rats by intra-
Diagnostics Ltd. Baroda, India; for details see Verma et al., 2012) [35]. peritoneal immunization with BSA and whole cell of V. harveyi and P.
Reagents of the kits were reconstituted as per manufacturer specifica- aeruginosa (100 μl, 106 cell/ml), for four consecutive weeks. Rat-IgG
tions, just before use. For both the assays 10 μl serum sample and 100 μl was purified using Immunoglobulin purification kit (Genei, India). For
of respective reconstituted reagents were added to 96 well assay plate ELISA, 100 μl of diluted fish serum (1:40 in 50 mM carbonate buffer, pH
(Thermo scientific Nunc, Asia and Pacific). Absorbance was recorded at 9.6) was coated in 96 well immunoassay plate (Thermo scientific Nunc,
340 nm in a decreasing kinetic reaction time with 60 s delay time, for 4 Asia and Pacific) and incubated overnight at 4 °C. Unbound serum
times. proteins were removed by three successive washings of 5 min each
(gentle shaking) with PBS-T wash buffer (phosphate buffered saline, pH
2.6.2. Measurement of alkaline phosphatase 7.4 containing 0.05% Tween-20) at RT. The open sites in the plate were
Alkaline phosphatase (ALP) levels were estimated in the serum blocked by adding 300 μl of 1% casein prepared in PBS and incubating
samples by using diagnostic kit (Autospin, Span Diagnostics Ltd. Surat, the plates for 1 h at 37 °C. The wells were washed thrice with PBS-T.
India) in a 96 well assay plate. Two microliter of serum sample and 125 μl of specific antigen [BSA (1 mg/ml) or sonicated V. harveyi and P.
100 μl of reconstituted reagent was dispensed and mixed gently. aeruginosa (1 mg/ml crude protein)] was added to each well and in-
Immediately the absorbance was recorded in increasing kinetic mode at cubated for 2 h. After washing, 125 μl of purified Rat IgG (anti-BSA,
405 nm using plate reader (Micro Scan 5608, ECIL), with 30 s delay anti-V. harveyi and anti-P. aeruginosa) (diluted 1:1000 times in PBS) was
time for 5 times. added to well accordingly and incubated at 37 °C for 2 h. Plates were
again washed and 125 μl of Rabbit anti-Rat IgG, conjugated with HRP
2.7. Macrophage activity (diluted 1:1000 with PBS) was added to each well and incubated for
2 h at 37 °C. Unbound antibodies were washed and color was developed
2.7.1. Nitrite oxide estimation by adding 125 μl of substrate solution (0.05% o-phenylenediaminedi-
Nitric oxide production in different group of fish phagocytes was hydrochloride in 0.1M citrate buffer, pH 5 containing 0.09% H2O2 in
estimated following the method of Ding et al. (1998) [37]. 150 μl of 10% methanol). The reaction was stopped after 15 min by adding 35 μl
spleen and head kidney macrophages (104 cells) suspended in complete of 1M oxalic acid. Absorbance was recorded at 495 nm using ELISA
medium (RPMI-1640, pH 7.8) were added to 96 well assay plate plate reader (ECIL, India).
(Thermo scientific Nunc, Asia and Pacific) and incubated for 48 h (time
selected based on pilot experiments). 100 μl of supernatant was trans- 2.9. Statistical analysis
ferred to fresh assay plate. Equal volume of Griess reagent (1% sul-
phanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride in The experiments were conducted three times to confirm the re-
2.5% H3PO4) was added and incubated for 10 min. Absorbance was producibility of the results. Experimental data was analyzed by ANOVA
measured at 540 nm. followed by Newman Keuls' multiple range tests. The values were re-
presented as mean ± standard error, and p value 0.05 or less were
2.7.2. Determination of phagocytic activity taken as statistically significant.
A cell suspension of yeast was prepared by incubating
Saccharomyces cerevisiae (Baker's yeast) for 20 min at 80 °C. Cells were 3. Results
passed through 26 gauge needles, centrifuged at 1000 g for 5 min and
suspended in RPMI-1640 (3 mg/ml). Monolayer of phagocytes was Antibacterial activity of ethanol and methanol extracts of pod seed
prepared by incubating the 200 μl of spleen suspension (106 cells/ml) powder of L. leucocephala against common fish pathogenic bacteria (A.
for 1 h at 25 °C. Non adherent cells were removed by washing with PBS. hydrophila, E. coli, E. faecalis, P. aeruginosa, S. aureus, V. anguillarum and
200 μl of heat killed yeast was added to adhered phagocytes and in- V. harveyi) at two different concentrations (100μg/disc and 200μg/disc)
cubated for 60 min. Non-phagocytosed yeast cells were removed by is represented in Table 2. The zone of inhibition in methanol extract
washing with PBS. Phagocytes were fixed in methanol and stained with was more than the ethanol extract of L. leucocephala pod seed. The

Table 2
Antibacterial activity of methanol and ethanol extracts of L. leucocephala against gram positive (E. faecalis and S. aureus) and gram negative (A. hydrophila, E. coli, P. aeruginosa, V.
anguillarum and V. harveyi) bacteria by disc diffusion assay.

Bacterial Strain Ethanol Extract Methanol Extract

200 μg/disc 100 μg/disc 200 μg/disc 100 μg/disc

a a a
A. hydrophila 14.25 ± 0.20 10.10 ± 0.20 13.30 ± 0.08 12.60 ± 0.25a
E. coli 10.33 ± 0.05a 8.25 ± 0.67b 11.20 ± 0.17b 10.60 ± 0.05b
E. faecalis 15.33 ± 0.67b 12.75 ± 0.33c 23.50 ± 0.33c 16.50 ± 0.17c
P. aeruginosa 16.50 ± 0.50c 13.33 ± 0.15c 19.33 ± 0.10d 12.33 ± 0.40a
S. aureus 19.10 ± 0.44d 17.33 ± 0.67d 16.67 ± 0.15e 15.80 ± 0.18c
V. anguillarum 20.10 ± 0.14d 18.33 ± 0.26d 22.35 ± 0.67b 21.25 ± 0.17d
V. harveyi 21.50 ± 0.10e 19.25 ± 0.56d 17.75 ± 0.89e 15.25 ± 0.67c

Values are represented as Mean ± SEM. ANOVA employing Newman-Keuls’ multiple range test was performed to calculate p value. p values less than 0.05 were only considered
significant. Dissimilar superscripts (a-e) on the mean values represent that they are statistically different.

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V.K. Verma et al. Fish and Shellfish Immunology 76 (2018) 324–332

Table 3 respect to negative control (Group A) was noticed in Group D whereas

MIC of Pod seed of L. leucocephala. nitric oxide levels were significantly elevated in other groups B, C, E &
F. Highest nitric oxide levels in spleen as well as kidney macrophages
Test organism Ethanol Extract Methanol Extract
were observed in group E.
Aeromonas hydrophila 15.6 7.8 The immunoglobulin levels were observed to increases successively
E. coli 31.2 15.6 in all groups except Group A. Group E showed highest immunoglobulin
E. faecalis 7.8 3.9
levels throughout the experiment (Fig. 4).
P. aeruginosa 7.8 7.8
S. aureus 3.9 7.8
V. anguillarum 1.9 1.0
V. harveyi 7.8 3.9 4. Discussion

Minimum inhibitory concentration of methanol and ethanol extracts of L. leucocephala Leucaena leucocephala is a mimosoid leguminous tree commonly
against gram positive (E. faecalis and S. aureus) and gram negative (A. hydrophila, E. coli, called as lead tree which belongs to family Fabaceae (Leguminosae) and
P. aeruginosa, V. anguillarum and V. harveyi) bacteria. is grown for variety of uses, including as green manure, livestock fodder
and for soil conservation. Some studies have revealed that L. leucoce-
highest zone of inhibition of methanol extract was observed against E. phala has anti-diabetic and anti-nematicidal potential [38,39]. All plant
faecalis, while lowest zone was observed with ethanol extract against E. parts have high amount of secondary metabolites which signifies its
coli. The values calculated for minimal inhibitory concentration of the importance for various means. L. leucocephala seed has high nutritional
ethanol and methanol extracts of pod seed of L. leucocephala that ex- value because of high protein content (24.5–46%) containing arginine,
tracts were most effective against V. anguillarum in both methanol and alanine, cysteine, glutamic acid, isoleucine, leucine, lysine and me-
ethanol extracts (Table 3). thionine as their essential amino acids [40,41]. The pods of L. leuco-
The specific growth rate of Group A was highest throughout the cephala also contain non-protein free amino acid named mimosine (β-
experiment. Group B, C and E showed significantly lower specific (3-hydroxy-4-pyridon-1-yl)-L-alanine) which have insecticidal and
growth rate in comparison to Group A, while Group D and F have pesticidal activity [42]. Mimosine is toxic to non-ruminant animals and
significantly higher specific growth rate (Fig. 1). humans which can arrest the dividing cells in the late G1 phase by
There were no marked changes in SGOT, SGPT and ALP levels in the inhibition of DNA replication initiation. Mimosine can be removed by
serum of Group A and Group B at any time (Table 4). While Group C, D, boiling or soaking the leaves overnight in water [43]. Brown seed from
E and F showed significant differences in SGOT, SGPT and ALP levels the plant has not been exploited and can be used as natural anti-oxi-
when challenged with respective bacteria. But, the levels of enzymes in dant. Benjakul et al. (2013) explored the antioxidant potential of water
Group C and E were within physiological limits. However in Group E extract of pod seed of L. leucocephala using various methods [44]. The
and F the levels of these enzymes were very high after challenge with V. seeds are reported to contain galactomannan and lectin galactomannan
harveyi and P. aeruginosa respectively. that constitutes a glycoside, which has an anti-diabetic role [39].
The phagocytic activity of macrophages isolated from the head The leaves and seeds are also used as human food in Central
kidney and spleen was examined by calculating percentage phagocy- America, Indonesia, and Thailand [45]. The seeds may also be used as
tosis and phagocytic index. The percentage phagocytosis was highest in concentrates for dairy animals, as manure [46,47], as a protein source
Group C and Group E. Group D and Group F showed lower phagocytic [48], as an oil seed and as a potential source of commercial gum
activity with respect to Group C and E. Marked changes were also ob- [49,50].
served in percentage phagocytosis of Group B (positive control) com- Leaves of L. leucocephala also contains high protein (29%) [51]. This
pared to Group A (negative control, Fig. 2A). The values of phagocytic property of L. leucocephala plant leaves was evaluated by Wee and
index represented by Group D & F were lower than Group C & E but Wang (1987) on Nile Tilapia [43]. They suggested that replacement of
were significantly higher than the control groups A & B (Fig. 2B). fish protein in feed with 25–50% with mimosine free L. leucocephala
Nitric oxide levels in the macrophages isolated from head kidney leaf did not show any adverse effect on health of fish; however it slowed
and spleen of C. gariepinus is represented in (Fig. 3). No change with the growth rate of fish. Bairagi et al. (2004) also suggests that

Fig. 1. Effect of reconstituted artificial feed (33% L. leucocephala pod seed) on specific growth rate of C. gariepinus. Specific growth rate is calculated using for-
(log Final fish wt.. − log Initial fish wt .)
mula.Specific growth rate = ⎡ n n ⎤ × 100 Values are expressed as Mean ± SEM. P value calculated by ANOVA followed by Newman-Keuls’ multiple range
⎣ Time interval ⎦
test (p < 0.05).
p values less than 0.05 were considered as significant. Error bars bearing different superscripts (a-c), between groups at a specific time point and (p-q), within groups at different time
points] differ significantly.

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V.K. Verma et al. Fish and Shellfish Immunology 76 (2018) 324–332

Table 4
SGOT, SGPT and ALP levels in serum of African catfish, Clarias gariepinus after feeding on L. leucocephala (pod seed)supplemented feed with complete replacement of fish trash (Group B, C
and E) with respect to fish feed on the non-supplemented artificial feed. Group A = non-challenged, Group B = immunized with BSA, Group C & D = challenged by Vibrio harveyi and
Group E & F = challenged by Pseudomonas aeruginosa. Group B, C and E fed on 33% L. leucocephala supplemented feed while group A, D and F were fed on normal artificial feed.

Assays Days Group A Group B Group C Group D Group E Group F

SGOT 7 29.76 ± 2.26a,p 34.08 ± 1.36a,p 40.16 ± 1.26b,p 28.71 ± 1.32a,p 43.94 ± 1.64b,p 29.78 ± 1.68a,p
14 31.54 ± 2.79a,p 32.19 ± 1.13a,p 44.33 ± 2.66b,p 49.08 ± 2.40b,q 42.97 ± 1.30b,p 50.05 ± 1.69b,q
21 32.95 ± 3.77a,p 34.92 ± 1.44a,p 38.70 ± 1.57a,p 72.65 ± 2.97b,q 37.64 ± 1.09a,q 52.19 ± 1.81c,q
28 31.35 ± 2.66a,p 35.5 ± 1.21a,p 44.81 ± 1.03b,p 67.9 ± 2.99d,q 37.05 ± 0.85a,q 50.83 ± 2.19c,q
35 32.25 ± 3.52a,p 35.31 ± 1.03a,p 39.87 ± 0.81a,p 71.88 ± 13.8b,q 37.64 ± 0.98a,q 63.44 ± 1.95b,r
SGPT 7 29.13 ± 1.24a,p 34.53 ± 1.16b,p 32.30 ± 0.96a,p 37.64 ± 1.66b,p 33.76 ± 0.75a,p 39.09 ± 2.18c,p
14 31.41 ± 1.55a,p 31.23 ± 2.80a,p 33.95 ± 1.60a,p 43.46 ± 0.82b,p 32.47 ± 0.71a,p 36.76 ± 3.99a,p
21 30.18 ± 1.25a,p 31.33 ± 0.87a,p 38.22 ± 1.23b,p 50.92 ± 2.99c,q 41.61 ± 1.76b,q 46.17 ± 3.83c,p
28 28.88 ± 0.94a,p 30.35 ± 1.98a,p 41.52 ± 2.83b,q 57.81 ± 2.14c,q 39.19 ± 2.36b,q 54.61 ± 1.75c,q
35 29.84 ± 1.34a,p 34.53 ± 0.98a,p 42.29 ± 1.52b,q 52.96 ± 3.18c,q 39.28 ± 2.01b,q 57.04 ± 1.20c,q
ALP 7 23.23 ± 1.84a,p 21.99 ± 0.80a,p 22.6 ± 0.52a,p 24.11 ± 1.09a,p 21.09 ± 0.30a,p 22.6 ± 1.38a,p
14 24.55 ± 1.56a,p 23.5 ± 0.52a,p 24.11 ± 1.09a,p 18.68 ± 0.80b,q 23.20 ± 0.80a,p 26.52 ± 1.31a,p
21 23.58 ± 0.68a,p 22.90 ± 0.80a,p 23.80 ± 1.31a,p 18.08 ± 1.04b,q 24.41 ± 0.52a,p 30.74 ± 0.52a,q
28 24.98 ± 1.78a,p 22.6 ± 0.52a,p 20.79 ± 0.52b,p 12.36 ± 0.80c,r 28.67 ± 0.80d,q 39.17 ± 1.68e,r
35 22.74 ± 1.56a,p 24.41 ± 0.52a,p 21.39 ± 1.09a,p 11.15 ± 0.80b,r 27.72 ± 1.83a,q 52.43 ± 2.39c,s

Values are expressed as Mean ± SEM (n = 8). p value is calculated by Student's t-test (p > 0.05) is significant. The Error bars bearing different superscripts (a–c, at specific time point
between different groups) and (p-s within groups at different time points) differ significantly. Statistical significant difference was calculated by ANNOVA followed by student Newman-
Keuls’ multiple range test.

supplementation of fish feed with fish intestinal bacteria (Bacillus sub- SGPT indicated that the fish challenged with bacteria may have infected
tilis and Bacillus circulans) to evaluate the nutritive value of leaves of L. liver or kidney. ALP is correlated with the stress levels of an organism
leucocephala added to the fish meal of fingerlings of Labeo rohita (20%, and is therefore an important hydrolase enzyme. It is mainly secreted
30% and 40%) [52]. They have reported that feed containing 30% L. from liver/bones and helps in dephosphorylation of proteins and nu-
leucocephala leaf inoculated with B. circulans and 40% L. leucocephala cleotides. Its level varies from juveniles to adults, but the lower and
leaf with B. subtilis had shown best performance of rohu on the basis of higher levels of this enzyme imply some harmful diseases. According to
their growth response and diet efficiency. Studies suggests that in- Van Hoof and De Broe (1994), the lower levels of ALP are also an in-
corporation of L. leucocephala leaf meal in the feed of Clarias gariepinus dicator of fish disease, and suggest that fish may be suffering from
upto 30% to replace the expensive and imported fish meal as a crude chronic or acute myelogenous leukemia or paroxysmal nocturnal he-
protein source [53]. Ground seed powder is yellow in color viz. similar moglobinuria [57]. There was no alarming change in ALP levels in
in color and texture to gram seed powder. Total protein content in the various experiments conducted in this study (Table 3).
pod seed of L. leucocephala approximately 43% in sun dried seeds, Macrophages play an important role to mediate innate immunity.
therefore the fish trash in the artificial fish feed was replaced with pod They can phagocytize the bacteria or other foreign particles and kill
seed powder of L. leucocephala. Fish artificial feed contains 30% protein them by various pathways. The non-specific immune parameters such
in the form of high trash meal. So we were interested to see if this as phagocytic activity of macrophage have been widely used to evaluate
protein source (present in L. leucocephala pod seeds) could be used to the heath/immune function in different fish species [34,58–60]. This
completely replace the animal protein in the artificial feed of fish. Study revealed that the incorporation of 33% pod seeds of L. leucoce-
Few studies on the supplementation of L. leucocephala in the feed for phala in the artificial feed replacement for fish trash caused increased
prawns have also been reported. In Macrobrachium rosenbergii, success macrophage activity resulting in higher percentage phagocytosis and
in growth using L. leucocephala leaves were achieved [54]. Penaeus phagocytic index (Fig. 2). Nitric oxide (NO) is another important sig-
monodon showed unaffected growth and survival on incorporation of naling molecule and has been recognized as one of the most versatile
mimosine free leaves of L. leucocephala leaf in feed [55]. Santiago et al. players in the immune system and has been released by the macro-
(1988) also showed decrease in weight of Nile Tilapia (Oreochromis phages. Various studies also demonstrate the increased Nitric oxide
niloticus) when fed on artificial feed supplemented with L. leucocephala levels as a result of administration of plant in their feed [61–64].
but no change in reproductive performance was observed even after 24 Likewise, in all experiments the higher nitric oxides levels were also
weeks when administered upto 40% in feed [56]. Previous studies also observed in treated group fed on plant supplemented diet in compar-
suggest the anti-bacterial effect of pod seeds of L. leucocephala [30]. In ison to positive control and negative control group (Fig. 3).
addition to this in we have also evaluated the minimal inhibitory Acquired immunity is triggered by innate immune response, which
concentration of ethanol and methanol extracts of pod seed of L. leu- involves activation of lymphocytes, and it also exhibits a memory ef-
cocephala for various bacteria. We found the methanol extract has fect. Antibodies form the major component of the humoral immune
better antibacterial potential than the ethanol extract of L. leucocephala system and they have been well documented to play an adaptive role in
(Tables 2a and 3). neutralizing and destroying the invading pathogens in all class of or-
The treatment fish were checked for any adverse effects on liver and ganisms from fish to mammals. Increase in the titer of these specific
kidney that may have occurred; due to 33% supplementary feed. SGOT antibodies helps in neutralization and speedy removal of antigen in-
and SGPT serves as an indicator of liver or kidney disease which usually troduced in the host body. Many findings also revealed similar increase
gets elevated as a result of infection. However, there was no marked in the serum immunoglobulin [65–70]. Group B, C and E showed higher
change in SGOT and SGPT in the serum of control and treated fish antibody titer when compared with their respective A, D and F group,
(Table 3), indicating that the feed does not have any toxic effect on the respectively (Fig. 4).
fish. No mortality was observed in fish however, the visual symptoms
like skin rashes/infection, slight reduced activity and acrid smell in
5. Conclusion
excreta of group A, D & F were observed during the study. But group
which were challenged with respective bacteria had higher levels of
Through above findings we have found that the methanol and
enzymes SGOT and SGPT (Group D and F). Higher levels of SGOT and
ethanol extracts of pod seed of L. leucocephala showed antibacterial

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Fig. 2. Effect of reconstituted artificial feed (33% L. leucocephala pod seed) on macrophages isolated from Spleen and Head Kidney of C. gariepinus after 28 days. “A” and “B” represents
the percentage phagocytosis and Phagocytic index respectively. Group A = non-challenged, Group B = immunized with BSA, Group C & D = challenged by Vibrio harveyi and Group E &
F = challenged by Pseudomonas aeruginosa. Group B, C and E fed on 33% L. leucocephala supplemented feed while Group A, D and F fed on normal artificial feed. Values are expressed as
Mean ± SEM. P value calculated by ANOVA followed by Newman-Keuls’ multiple range test (p < 0.05). P values less than 0.05 were considered as significant. Error bars with different
superscripts (a-d between groups) are significantly different.

activities against A. hydrophila, E. coli, E. faecalis, P. aeruginosa, S. specific growth rate (Fig. 1). While this feed increases humoral and cell
aureus, V. anguillarum and V. harveyi. We also conclude that the in- mediated immune response. However, we suggest that this feed can be
corporation of L. leucocephala enhances the innate and acquired im- used as a preventive measure to control fish disease.
munity without showing any harmful effect on the fish. This feed can be
fed to the farmed fish to reduce the cost of feed. The feed would help
Acknowledgements
the infected fish to recover and it could also be fed to fish as a pro-
phylaxis measure against pathogenic bacteria.
The research Project was funded by University of Delhi, India (SVC-
However, it does not although contribute to increase in their
305) (under Delhi University Innovation Projects). Vipin Kumar Verma

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V.K. Verma et al. Fish and Shellfish Immunology 76 (2018) 324–332

Fig. 3. Effect of reconstituted artificial feed (33% L. leucocephala pod seed) on Nitric oxide production in macrophages isolated from Spleen and Head Kidney of C. gariepinus on day 35th.
Group A = non-challenged, Group B = immunized with BSA, Group C & D = challenged by Vibrio harveyi and Group E & F = challenged by Pseudomonas aeruginosa. Group B, C and E fed
on 33% L. leucocephala supplemented feed while Group A, D and F fed on normal artificial feed. Error bars with different superscripts (a-c, day 35) are statistically different.

Fig. 4. Effect of reconstituted artificial feed (33% L. leucocephala pod seed) on serum Immunoglobulin levels of C. gariepinus challenged with BSA (Group B), V. harveyi (Group C & D) and
P. aeruginosa (Group E & F). Group A = non-challenged, Group B = immunized with BSA, Group C & D = challenged by Vibrio harveyi and Group E & F = challenged by Pseudomonas
aeruginosa. Group B, C and E fed on 33% L. leucocephala supplemented feed while Group A, D and F fed on normal artificial feed. Values are expressed as Mean ± SEM (n = 8). p value
calculated by ANOVA followed by Newman-Keuls’ multiple range test (p < 0.05). p values less than 0.05 were considered as significant. Values represented by different superscripts [(a-
d) between groups at a specific time, and (p-t) within groups at various time points] differ statistically.

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