International Journal of Aquaculture 2015, Vol.5, No.

36, 1-9
http://ija.biopublisher.ca

Research Report
Research Article Open Access

Effect of an Indigenous Probiotic (Shewanella algae) Isolated from Healthy

Shrimp (Penaeus monodon) Intestine on Clarias gariepinus
Ariole C.N. , Eddo T.T.
Department of Microbiology, University of Port Harcourt, P.M.B. 5323, Port Harcourt, Rivers State, Nigeria
Corresponding author email: cnariole@yahoo.com
International Journal of Aquaculture, 2015, Vol.5, No.36 doi: 10.5376/ija.2015.05.0036
Received: 26 Oct., 2015
Accepted: 10 Dec., 2015
Published: 25 Jan., 2016
Copyright © 2015 Ariole and Eddo, This is an open access article published under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Ariole C.N. and Eddo T.T., 2015, Effect of an Indigenous Probiotic (Shewanella algae) Isolated from Healthy Shrimp (Penaeus monodon) intestine on Clarias
gariepinus, International Journal of Aquaculture, 5(36): 1-9 (doi: 10.5376/ija.2015.05.0036)

Abstract The efficacy of Shewanella algae, isolated from healthy shrimp (Penaeus monodon) intestine, as a biocontrol agent
against Aeromonas hydrophila infection in Clarias gariepinus was evaluated. Spread plated technique was employed for bacterial
isolation. The bacterial isolates were screened for in vitro antimicrobial activity against pathogenic Aeromonas hydrophila using agar
well diffusion assay. The most active isolate (B2) was identified using phenotypic and molecular methods. The safety of the
indigenous probiotic (B2) and the pathogenicity of the Aeromonas hydrophila on Clarias gariepinus were investigated. Feeding
experiment was designed to evaluate the in vivo antibacterial efficiency of the most active strain using three groups of fish
(Shewanella algae- supplemented feed group, control infected and control non-infected groups). At the end of the experiment, five
fish from each group were randomly selected for haematological and serum biochemical response investigation. The parameters
analysed were haemoglobin, packed cell volume (PCV), red blood cell (RBC), white blood cell (WBC), lymphocytes, monocytes,
total protein and albumin. A total of four genera belonging to Pseudomonas, Shewanella, Vibrio and Proteus which were isolated
from shrimp intestine had antagonistic ability against Aeromonas hydrophila with zone of inhibition of 8.0 ± 0.0 mm, 10.0 ± 0.0 mm,
8.0 ± 0.0 mm and 8.0 ± 0.0 mm respectively. Molecular analysis conducted on the most active isolate (B2) revealed that it is closely
related to Shewanella algae strain KJ-W32 gi: 385880930. The virulence test carried out on Clarias gariepinus using Aeromonas
hydrophila which was injected intraperitoneally, showed 50% lethal dose value at 6.4 x 104 cfu ml-1. The result from the probiotic
trial showed that fish fed with incorporated Shewanella algae strain KJ-W32 exhibited better resistance to pathogenic Aeromonas
hydrophila than the control infected fish. The mortality rate during the in vivo challenge experiments observed for Shewanella algae -
supplemented feed group, control infected and control non -infected fish groups were 0%, 50% and 5% respectively. Fish fed with
supplemented diet showed better haematological and serum biochemical performance than those fed with normal fish feed. The study
showed that Shewanella algae strain KJ-W32 is beneficial to Clarias gariepinus. Therefore, this indigenous bacterium could be used
as effective biocontrol agent for management of aeromonasis in aquaculture.
Keywords Probiotic; Aeromonas infection; Clarias gariepinus; Physiological parameters

Introduction
Aquaculture is the farming of aquatic organisms in
order to enhance their production. It allows a selective
increase in the production of species used for human
consumption, industry or sport fishing. As a result of
overfishing of wild populations, aquaculture has
become an economic activity of great importance
worldwide. Some wild fish species such as tilapia
(Oreochromis niloticus), African catfish (Clarias
garipienus), cod (Gadus morhua), turbot (Psetta
maxima) and tuna (Thunnus spp) have become more
and more attractive as potential aquaculture species
(Van De Nieuwegiessen, 2009).
1
According to Qi (Qi et al., 2009), fish disease is more
prevalent under intensive management. The crowded
condition of large population of fish would result in
heavy parasitic infection, disease and loss of fish.
Aeromonas hydrophila is a pathogen that can infect
human, animal, bird and fish. As a fish pathogen, it
can infect a wide variety of freshwater and marine fish
causing hemorrhagic septicaemia (Eissa and About
El-Ghiet, 2011). For decades, antibiotics routinely
used for treatment of human infections were also used
for aquatic animals, for therapy, prophylactic reasons
or growth promotion. The potential negative
 International Journal of Aquaculture 2015, Vol.5, No. 36, 1-9
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consequences of using antibiotics in aquaculture, such
as the development of drug resistant bacteria and the
reduced efficiency of antibiotic resistant for human
and animal diseases, have led to suggestions of the use
of non-pathogenic bacteria as disease control agents
(Vaseeharan and Ramasamy, 2003).
The gastrointestinal tracts (GIT) of aquatic animals do
not only habour potentially pathogenic bacteria such
as Salmonella and Escherichia coli but also probiotic
bacteria and other microorganisms. These include
gram-positive bacteria such as Bacillus, The healthy shrimps were cleaned externally with
Carnobacterium and several species of Lactobacillus,
gram negative and facultative anaerobes such as
Vibrio and Pseudomonas as well as certain fungi,
yeasts, and algae of genera Debaryomyces,
Saccharomyces and Tetraselmis respectively (Irianto
and Austin, 2002; Ariole et al., 2014). Some of these
intestinal microbes are known to possess inhibitory
potentials against fish pathogenic bacteria (Ariole and
Kanee, 2013; Ariole and Aso, 2015).
Although the use of probiotics in aquaculture is
relatively recent, interest in them has increased due to
their potential in disease control (Wang et al., 2008).
The application of probiotics is becoming a major
field in the development of aquaculture, based on the
massive advantages of its application. However,
characterization of novel selection criteria for new
strains is needed to allow further probiotic
development. Environmentally friendly methods for
controlling microbial pathogenesis in aquaculture with
probiotic bacteria have gained considerable research
interest and are becoming increasingly preferred as
viable and alternative management practices for
disease prevention as probiotic supplementation
increase the absorbance efficiency of feeds (Haroun et
al., 2006). Therefore, the present study is aimed at
evaluating the efficacy of an indigenous bacterium,
Shewanella algae, isolated from healthy shrimp
(Penaeus monodon) intestine as biocontrol agent
against Aeromonas hydrophila infection in Clarias
gariepinus.
1 Materials and Methods
1.1 Sample collection
Healthy shrimps (Penaeus monodon) were collected
from Sombriero River, Rivers state, Nigeria with the
assistance of a local fisherman and immediately
2
transported in a sterile plastic bag to the laboratory for
analysis.
1.2 Source of pathogen
Aeromonas hydrophila used in this study was
previously isolated from moribund shrimp (Ariole and
Aungwa, 2013) and maintained as part of culture
collection in Microbiology Laboratory, University of
Port-Harcourt.
1.3 Bacterial isolation
ethanol and their gastro-intestinal tracts dissected
under sterile conditions. The gut contents were
weighed and placed in a sterile physiological saline
and then diluted from 10-1 to 10-5 using 10- fold serial
dilution. Sub samples of 0.1ml of the dilutions (10-3 to
10-5) were streaked on sterile nutrient agar plates using
spread plate method. The plates were incubated at
37oC for 24 – 48 hours. Isolates with distinct colony
morphology were picked and streaked repeatedly on
nutrient agar plates until pure. The purified isolates
were identified to generic level based on their
morphological and physiological characteristics (Holt
et al., 1994).
1.4 Determination of antibacterial activity
The isolates were examined for antagonistic activities
against Aeromonas hydrophila. This was carried out
using agar well diffusion assay. The isolates and
pathogen were grown to the log phase in
Muller-Hinton broth for 24 hr at 37°C. Wells were
punched using a cork borer in plates of Muller-Hinton
agar, which were seeded with 0.1ml of 24hr old broth
culture of pathogen. Then 0.1ml of 24 hr old broth
culture of each isolate was introduced into each well.
The plates were allowed to stand for some time to
allow for diffusion before inverting the plates and
incubating them at 37°C for 24 hr. The diameter of
clear zone was measured and recorded expressing the
antibacterial activity.
1.5 Determination of the safety of Shewanella algae
strain
The safety of Shewanella algae that showed
antibacterial activity against the Aeromonas
hydrophila in vitro was evaluated using healthy
Clarias gariepinus weighing approximately 4.0 ± 0.65
g/fish. Fish samples were obtained from a private fish
farm in Port Harcourt, Rivers State, Nigeria. They
 International Journal of Aquaculture 2015, Vol.5, No. 36, 1-9
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were acclimatized for one week before the
experimental trial. Fish were fed daily and left over
feed and faeces were syphoned. About 50% of the
water was changed every day and dead fish removed.
The fish were divided into 6 batches with 20 fish per
batch. Batches 1, 2, 3, 4 and 5 were intraperitoneally
injected with 0.1ml of 3 x103, 3 x104, 3 x105, 3 x106
and 3 x107 cfu/ml Shewanella algae respectively. The
bacterial suspension was prepared with reference to
McFarland standard No1 which is equivalent to 3 x
8 -1
10 cfu ml and by subsequent serial dilutions in order
to obtain the concentrations used. The control in Batch temperature for cooling and drying prior to use.
6 was injected with sterile normal saline. The
experiment was carried out in duplicate and kept
under observation. Mortalities were recorded for five
days post-inoculation.
1.6 Determination of virulence of Aeromonas
hydrophila on fish
About 250 healthy Clarias gariepinus were
acclimatized for one week prior to experimental
analysis. Fish were fed daily and left over feed and
faeces were siphoned. About 50% of the water was
changed every day and dead fish removed. The
virulence of the pathogen was evaluated and the LD50
of the pathogen determined. Fish weighing
approximately 4.0 ± 0.65 g/fish were used for this test. commercial feed (control infected) and group 3 was
The fish were divided into 6 batches with 20 fish per
batch. Dilutions of the bacterial suspension of the
pathogen was prepared and adjusted to McFarland
turbidity standard No1 which is equivalent to 3 x 10
cfu ml-1. Ten-fold serial dilution was done to obtain
7
Aeromonas hydrophila concentrations of 3 x 10 to 3 x
103 cfu ml-1. Batches 1-5 were intraperitoneally
injected with 0.1ml bacterial suspension of 3 x 103, 3
x 104, 3 x 105, 3 x 106 and 3 x 107 cfu/ml respectively,
and sterile normal saline solution was injected into the
6th group as the control. The experiment was carried
out in duplicate. Mortalities were recorded for five
days post-inoculation for the varying concentrations
of the pathogenic bacterium. The LD50 value was
calculated using SPSS version 20, Probit Analysis
Package.
1.7 Preparation of Shewanella algae-supplemented
feed
The safest dilution of the cell suspension of
Shewanella algae strain KJ-W32 was prepared and
added to 1.5 mm Coppens® commercial dry feed
containing 49% crude protein, 1.2% fibre, 13% fat, as
well as vitamins and minerals in the form of pellets.
Shewanella algae strain KJ-W32 was grown on
nutrient broth for 24 hours and harvested by
centrifugation at 1000 rpm for 10 min. The resultant
cell biomass was washed with sterile physiological
saline and re-suspended in saline to 108 cfu ml-1. The
volume was then mixed thoroughly in 100 g of the
commercial dry feed to achieve a dose equivalent to
106 bacterial cells/g of feed (Irianto and Austin, 2002).
The supplemented feed was kept in ambient
1.8 In- vivo challenge test
Healthy Clarias gariepinus weighing approximately
4.0±0.3 g/fish were acclimatized for one week prior to
experimental analysis. Fish were fed daily and left
over feed and faeces were syphoned. About 50% of
the water was changed every day and dead fish were
removed. Then they were randomly stacked at a rate
of 20 fish per tank. They were divided into 3 groups.
Group 1 was fed with Shewanella algae supplemented
diet for 7 days and then infected with Aeromonas
hydrophila (Shewanella algae supplemented diet).
Group 2 and 3 served as control. Group 2 was injected
with Aeromonas hydrophila and fed with normal
injected with sterile normal saline also fed with
normal feed diet (control non- infected). All
experimental groups were kept under observation for
8 7 days post-inoculation. Mortalities were recorded
daily for 7 days.
1.9 Haematological and serum biochemical
analyses
At the end of the experiment, 5 fish from each tank
were sacrificed and blood samples collected with
sterile syringe using ethylene diamine tetra acetic acid
(EDTA) as anticoagulant. Blood was allowed to flow
freely from the vein of caudal peduncle to capillary
tube and was allowed to fill the tube by means of
capillary action. Parameters that were analysed
include total leukocytes (WBC), erythrocytes counts
(RBC), hemoglobin (Hb) content, hematocrits (PCV)
and leukocytes differential count. Haematocrits (PCV)
was determined immediately after sampling, using a
microhematocrit centrifugation at 10,500 ×g for 5 min.
Capillary tube was centrifuged for 5 min at 10,500
rpm in a micro-haematocrit centrifuge and PCV was
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measured using microhematocrit reader. The blood
plasma was obtained by centrifuging the heparinized
blood at 4100 × g for 10 min at 4°C and the blood
cells (erythrocytes and leukocytes) were separated
into eppendorf tubes. All analyses of blood parameters
were performed within 12 h. Blood smears were
air-dried and stained by the Giemsa Romanowski
method. Erythrocyte counts were determined using a
Bürker counting chamber and Hayem solution. The
counts were made in 2 × 20 rectangles per sample.
Hemoglobin concentrations (g/dl) were determined by
the cyanhaemoglobin method using a wavelength of
540 nm. The leucocytes were differentiated according
to Svobodova (Svobodova et al., 1991) and the
relative abundance of all cell types was determined by
counting a total of 200 blood cells.
Blood samples for biochemical indices were collected
into another sample bottles without the anticoagulant.
Clotted blood in capillary tubes was centrifuged for 10 sample wipe and 4 µl of extracted DNA sample was
minutes using a high-speed centrifuge (Xiangyi®
Centrifuge Instrument Co., Ltd, Model: TG16-W)
4100 × g for 10 min at 4°C. The separated serum was
then analysed for total protein (TP) and albumin. The
serum biochemical indices were done using the
clinical routine procedures outlined by Olorede
(Olorede et al., 1996).
1.10 Molecular analysis
1.10.1 Chromosomal DNA extraction protocol (Zymo
Research Bacterial DNA MiniPrep Kit)
Sterile straight wire with pointed tip was used to pick 27F5’AGAGTTTGATCTGGCTCAG
a single colony from Petri dish of the test bacterium,
and transferred into a 5 ml tube containing
Luria-Bertani broth and incubated for 24 hrs at 37ºC,
and then 1 ml of the test bacterium was pipetted into a the experimental analysis on the fish (Clarias
ZR bashing bead lysis tube. Then 750 µl lysis solution
was added to the tube. The tube was fitted into a ZR
disrupter ginie holder and processed for 5 minutes.
The sample in the ZR Bashing bead lysis tube was
centrifuged at 10,000 × g for 1 min. Then 400 µl of
supernatant was then transferred into a Zymo-spin IV
spin filter (orang top) collection tube and centrifuged
for 1min at 10,000 × g. Then 1,200µl of Bacterial
DNA Binding Buffer was added to the filtrate in the
collection tube. Then 800 µl of the mixture was
transferred into a Zymo-spin IIC column in a new
collection tube and centrifuged at 10,000 × g for 1 min.
The flow through from the collection tube was
discarded and the bacteria DNA was added and
centrifuged at 10,000 x g for 1 min. Then, 200µl DNA
Pre-Wash Buffer was added to the Zymo-spin IIC
column in a new collection tube and centrifuged at
10,000 × g for 1 min. Then, 500 µl Bacterial DNA
Wash Buffer was added to the Zymo-spin IIC column
and centrifuged at 10,000 × g for 1 min. The
Zymo-spin IIC column was transferred to a clean 1.5
ml microcentrifuge tube and 100 µl DNA Elute Buffer
was added directly to the column matrix and
centrifuged at 10,000 × g for 30 sec. Ultra-pure
bacterial chromosomal DNA was obtained for PCR
amplification.
1.10.2 DNA concentration
The Nanodrop machine was cleaned with a piece of
fabric wipe. Then 2µl of deionized water was placed
on the lower sample pedestal and blanked to 0.00
mg/ml. The sample pedestal was cleaned with a
dropped onto the sample pedestal and covered with
the Nanodrop analyser. The measured nucleic acids
values were recorded on the computer screen in
mg/ml.
1.10.3 Preparation of PCR cocktail mixture for 16S
sequencing
The following components were used in preparation
of PCR cocktail mixture: Extracted DNA template of
bacterial isolates, Universal primers
(1492R5’TACGGYTACCTTGTTACGACTT 3’ and
3’), Taq
Polymerase, Master Mix (nucleotides) and PCR water/
buffer. The following concentrations were used for the
polymerase chain reaction done on the isolate used for
gariepinus) samples:
Master Mix= 12.5 µl, Forward primer=0.5 µl, Reverse
primer = 0.5 µl, Template 3 ml and PCR water 8.5 ml
1.10.4 16S rRNA Amplification and sequencing
The 16S rRNA region of the rRNA genes of the
isolate was amplified using the 27F:
5’AGAGTTTGATCMTGGCTCAG 3’ and 1492R:
5’TACGGYTACCTTGTTACGACTT 3’ primers on
an ABI 9700 Applied Biosystems thermal cycler at a
final volume of 50 microliters for 35 cycles. The PCR
mix included: the X2 Dream taq Master mix supplied
by Inqaba, South Africa (taq polymerase, DNTPs,
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MgCl), the primers at a concentration of 0.4 M and
the extracted DNA as template. The PCR conditions
were as follows: Initial denaturation, 95ºC for 5
minutes; denaturation, 95ºC for 30 seconds; annealing,
52ºC for 30 seconds; extension, 72ºC for 30 seconds
for 35 cycles and final extension, 72ºC for 5 minutes.
The product was resolved on a 1% Agarose gel at 120
V for 15 minutes and visualized on a UV
Transilluminator. The amplified 16S products were
sequenced on a 3500 genetic analyser using the
Bigdye-Termination technique by Inqaba South
Africa.
1.10.5 Phylogenetic analysis
The sequences were edited using the bioinformatics
algorithm Bioedit, similar sequences were 2.4 Virulence of Aeromonas hydrophila to Fish
downloaded from the National Biotechnology
Information Center (NCBI) data base using Blast N,
and these sequences were aligned using Clustal X.
The evolutionary history was inferred using the
Neighbor-Joining method in MEGA 6.0. The
bootstrap consensus tree inferred from 500 replicates
is taken to represent the evolutionary history of the
taxa analysed. Branches corresponding to partitions
reproduced in less than 50% bootstrap replicates were
collapsed. The evolutionary distances were computed
using the Jukes-Cantor method and are in the units of
the number of base substitutions per site.
1.11 Statistical analysis
All data were statistically treated using one-way
ANOVA. Significant differences among means (p <
0.05) were tested by Duncan’s multiple range test.
LD50 of the pathogen was calculated using SPSS
version 20 Probit Analysis.
2 Results
2.1 Isolation of antagonistic bacteria
A total of four genera belonging to Pseudomonas,
Shewanella, Vibrio and Proteus, isolated from shrimp
intestine, had antagonistic ability against the pathogen
with zones of inhibition of 8.0 ± 0.0 mm, 10.0 ± 0.0
mm, 8.0 ± 0.0 mm and 8.0 ± 0.0 mm respectively
(Table 1).
2.2 Molecular Identification of the most active
Isolate (Shewenella sp.)
The obtained 16S rRNA sequence from the isolate
(Shewanella sp.) produced an exact match during the
mega blast search for highly similar sequences from
the NCBI non-redundant nucleotide (nr/nt) database.
The 16S rRNA of the isolate showed a percentage
similarity to other species at 99%. The evolutionary
distances computed using the Jukes-Cantor method
were in agreement with the phylogenetic placement of
the 16S rRNA of the isolate within the Shewanella sp.
and revealed a closely relatedness to Shewanella algae
strain KJ-W32 (gi: 385880930) than the others (Figure
1).
2.3 Safety of Shewanella algae strain
The Shewanella algae strain was safe to fish when
compared with the control which had 10% mortality
(Table 2).
Mortality was observed in groups 1 - 5 and other
clinical signs such as lethargy, skin ulcers, swimming
abnormality, lack of appetite and then death. The fish
in control group showed no clinical signs and there
was no mortality recorded (Table 3). The estimated
effective dose that would lead to 50% mortality is
approximately 6.4x104cfu ml-1.
2.5 Challenge test
The group (group 1) infected with the pathogen and
fed with the supplemented diet showed no mortality
(0%) after 7 days post inoculation. The control group
infected with the pathogen and fed with the normal
feed (group 2) had the highest mortality (50%), while
the other control group (group 3) injected with
sterile physiological saline and fed with normal feed
recorded one death (5% mortality) (Table 4).
2.6 Physiological analysis
The haematological and biochemical parameters
analysed were significantly (p < 0.05) higher in the
group fed with Shewanella algae supplemented diet
than in the control groups (Table 5).
Table 1 Antibacterial activity of isolates against Aeromonas
hydrophila
Isolate code Inhibition (mm) ±S.D
Aeromonas hydrophila
B1 Pseudomonas sp. 8.0 ±0.0
B2 Shewanella sp. 10.0 ±0.0
B3 Proteus sp. 8.0 ±0.0
B4 Vibrio sp. 8.0 ±0.0
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Table 2 Safety test of Shewanella algae strain on experimental Table 4 Daily observation of Clarias gariepinus fed with diet
fish (Clarias gariepinus) after 5 days post inoculation containing 106 cells/g Shewanella algae for 7 days and then
challenged intraperitoneally with 0.1 ml (3x105 cells) of
Route of Number of Number
Groups Dosage Aeromonas hydrophila
injection fish dead
Studied Number of days post Total number
0.1ml sterile Mortalit(%)
Control I.P 20 2 groups inoculation of deaths
saline
1 2 3 4 5 6 7
0.1ml (3x103
1 I.P 21 Nil Shewanella
cfu/ml)
algae-
0.1ml (3x104 0 0 0 0 0 0 0 Nil 0
2 I.P 22 Nil supplemented
cfu/ml)
feed
0.1ml (3x105
3 I.P 23 Nil Control
cfu/ml) 1 1 2 2 2 1 1 10 50
infected
0.1ml (3x106
4 I.P 24 Nil Control non
cfu/ml) 0 0 1 0 0 0 0 1 5
infected
0.1ml (3x107
5 I.P 25 Nil Table 5 Haematological and serum biochemical response of
cfu/ml)
fish to various treatments
Table 3 Daily observations of the virulence of Aeromonas Control
hydrophila intraperitoneally injected into Clarias gariepinus Shewanella
Control Non
for 5 days post inoculation Parameters algae-supplemente
group Infected
Total d feed group
Mortality group
Group Dosage Number of dead fish number
%
dead Hgb (g/dl) 10.3a ± 0.44 9.0b ± 0.04 9.2c ± 0.14

Day1 Day2 Day3 Day4 Day5 PCV (%) 32.6a ± 1.67 25.2b ±1.92 27.4c ±0.89

0.1 ml RBC ((106/mm3)) 1.4a ± 0.04 1.1b ± 0.05 1.2c ± 0.04

Control sterile 0 0 0 0 0 Nil 0% WBC (103/mm3) 6.4a ± 0.16 5.1b ± 0.16 5.2c ± 0.20
saline Lymphocytes
35.8a ± 3.49 31.4b ±2.19 33.6c ±2.19
0.1ml (103/mm3)
1 (3x103 2 1 1 0 0 4/20 20% Monocytes
3.8a ± 0.44 1.8b ± 0.44 3.4c ± 0.89
cfu/ml) (103/mm3)
0.1ml Protein (g/100 ml) 68.2a ± 2.94 62.8b ±1.78 67.6c ±2.50
2 (3x104 2 2 2 1 0 7/20 35% Albumen (g/100
44.8a ± 0.44 41.4b ±1.51 42.0c ±1.00
cfu/ml) ml)
0.1ml Note: Means on the same row followed by different
3 (3x10 3 5
3 2 1 1 10/20 50% superscripts are significantly different (p < 0.05)
cfu/ml)
3 Discussion
0.1ml Four bacterial genera Pseudomonas, Shewanella,
4 (3x106 4 3 3 3 1 14/20 70% Vibrio and Proteus isolated from shrimp (Penaeus
cfu/ml) monodon) intestine were found to possess
0.1ml
antibacterial activity in vitro against fish pathogenic
Aeromonas hydrophila (Table 1). It has been reported
5 (3x107 5 5 3 3 2 18/20 90%
that probiotic microorganisms have the ability to
cfu/ml)
inhibit or even eliminate some potential pathogenic
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bacteria; this can be accomplished through production
of inhibitory biological substances such as antibiotics, that was injected with the pathogen and fed with
antibacterial substances, siderophores, bacteriolytic
enzymes, proteases and protease inhibitor, lactic acid
and other organic compounds like bacteriocins,
hydrogen peroxide (Lee et al., 2000) and butyric acid
production (Pan et al., 2008). Previous studies have
also demonstrated that bacteria produce inhibitory
substances that inhibit the bacterial pathogens in
aquaculture systems (Chythanya et al., 2002;
Vaseeharan and Ramasamy, 2003; Ravi et al., 2007;
Shakibazadeh et al., 2008; Janarthanam et al., 2012;
Ariole and Anugwa, 2013; Ariole and Nyeche, 2013;
Ariole and Oha, 2013; Ariole and Aso, 2015). This
microbial antagonism plays a major role in reducing
or eliminating the incidence of opportunistic
pathogens in the gastrointestinal tract of aquatic
animals (Balcázar et al., 2006).
The phylogenetic placement of the 16S rRNA of the
isolate revealed a close relatedness to Shewanella
algae strain KJ-W32 (gi: 385880930) (Figure 1).
Members of the genus Shewanella are widely
distributed in nature, especially in aquatic
environments such as freshwater and the ocean (Bozal
et al., 2002). Advanced taxonomic techniques such as
PCR technology have contributed to an increase in the
number of described Shewanella species (Satomi et al.,
2003).
The safety of Shewanella algae strain KJ-W32 during
in vivo experiment (Table 2), affirms that Shewanella
algae strain KJ-W32 is a beneficial bacterium and has
no pathogenic effect on Clarias gariepinus. On the
other hand, Aeromonas hydrophila was toxic to the
test fish (Table 3). This reveals Aeromonas hydrophila
as a pathogen to Clarias gariepinus. Pathological
conditions attributed to members of the motile
aeromonad complex may include dermal ulceration,
tail or fin rot, ocular ulceration, hemorrhagic
septicaemia and scale protrusion disease (John and
Hatha, 2013). Liles et al. (2011) stated that the
outbreaks of motile aeromonad septicaemia can reach
epidemic proportions in farmed aquatic animals with
high rates of mortality.
The feeding experiment revealed that Clarias
gariepinus which fed on supplemented diet with
Shewanella algae strain KJ-W32 resisted Aeromonas
hydrophila infection when compared to the control
normal commercial feed diet (Table 4). The high
mortality rate in the control infected and low mortality
rate in the probiotic supplemented diet could be
attributed to the inclusion of probiotics in the fish feed
and non-inclusion in the control. During the
experiment the inclusion might have enhanced the
production of inhibitory substances such as
bacteriocins against the pathogenic organism. Smith
and Davey (Smith and Davey, 1993) found reduction
in diseases caused by Aeromonas salmonicida in
Atlantic salmon after challenged with Pseudomonas
fluorescens and Wanga (Wanga et al., 2009) detected
strong protection against Aeromonas hydrophila
infections in Japanese flounder injected with
Pseudomonas fluorescens. A study conducted by Eissa
and About (Eissa and About El-Ghiet, 2011) revealed
that Oreochromis niloticus fed on incorporated diet
with Pseudomonas fluorescens biovar I, II and III
resulted in resistance against Aeromonas hydrophila
infection. Futhermore, the application of probiotic
bacteria (Halomonas aquamarina and Shewanella
algae) was able to inhibit the population growth of
Vibrio harveyi (Suantika et al., 2013). The
administration of probiotics appears to be a very
promising research area for nutrition, biological
control and disease prevention in aquaculture
(Balcázar et al., 2006).
The result of haematological analysis showed that
there were significant increases in the values of RBC,
WBC, monocytes, lymphocytes, PCV, and Hgb in the
group fed with supplemented diet, in comparison with
control groups (Tables 5). The use of haematological
values as indices and stress induced conditions as well
as for feed assessment is a well documented protocol
(George, 2007). The improved blood parameters could
be attributed to hemopiotic stimulation (Sarma et al.,
2001; Rajesh et al., 2006). The result of this finding is
also in agreement with Irianto and Austin (Irianto and
Austin, 2002) who found increase in the RBCs in fish
supplemented diet with probiotic bacteria than the
control group. There was also a significant increase in
total protein and albumin in group treated with
supplemented diet compared with those fed with
normal commercial diet (p < 0.05). This could be
attributed to the immunomodulatory effect of the
7
 International Journal of Aquaculture 2015, Vol.5, No. 36, 1-9
http://ija.biopublisher.ca
probiotic on the liver cells which activated the Ariole C.N., Nwogu H.A. and Chuku P.W., 2014, Enzymatic activities of

anabolic capacity of the hepatocytes to produce blood
proteins (Nayak, 2010). Information on the health
status and management of cultural fish are usually
provided through blood biochemical analysis which
helps to provide details on the health assessment of
fish. These results suggest that the indigenous
intestinal bacteria isolated from farmed Clarias gariepinus,
International Journal of Aquaculture, 4(18): 108-112
Balcazar J.L., De Blas I., Zarzuela-Ruiz I., Cunningham D., Vendrell D. and
Mu´ zquiz J.L., 2006, The role of probiotics in aquaculture, (Review)
Veterinary Microbiology, 114:173–86
http://dx.doi.org/10.1016/j.vetmic.2006.01.009
Bozal N., Montes M. J., Tudela E., Jime´ nez F. and Guinea J., 2002,

bacterium, Shewanella algae, could be used Shewanella frigidimarina and Shewanella livingstonensis sp. nov.
isolated from Antarctic coastal areas, International Journal of
effectively in aquaculture as a biocontrol agent against
Systematic and Evolutionary Microbiology, 52: 195–205
Aeromonas infection. http://dx.doi.org/10.1099/00207713-52-1-195
Figure 1 Phylogenetic tree showing species relatedness of
isolate (B2)
Authors’ Contributions
CNA contributed during conception and design, analysis and
interpretation of results and write-up of the manuscript. TTE
contributed during design, sample collection and analysis as
well as acquisition of data and analysis. All the authors read
and approved the final manuscript.
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