Research Article

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
Author    Correspondence author
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: 18 Jan., 2016
© 2015 BioPublisher Publishing Platform
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 

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).
 
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 Abou 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 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, 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 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
The healthy shrimps were cleaned externally with 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 0.1 ml 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 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 108 cfu ml-1 and by subsequent serial dilutions in order to obtain the concentrations used. The control in Batch 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. 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 108 cfu ml-1. Ten-fold serial dilution was done to obtain Aeromonas hydrophila concentrations of 3 x 107 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 temperature for cooling and drying prior to use.
 
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 commercial feed (control infected) and group 3 was injected with sterile normal saline also fed with normal feed diet (control non- infected). All experimental groups were kept under observation for 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 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 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 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 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 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 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 1,200 µl 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 2 µl of deionized water was placed on the lower sample pedestal and blanked to 0.00 mg/ml. The sample pedestal was cleaned with a sample wipe and 4 µl of extracted DNA sample was 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 27F5’AGAGTTTGATCTGGCTCAG 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 the experimental analysis on the fish (Clarias 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, 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 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).
 

 

Table 1 Antibacterial activity of isolates against Aeromonas hydrophila

 

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).
 

 

Figure 1 Phylogenetic tree showing species relatedness of isolate (B2)

 

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).
 

 

Table 2 Safety test of Shewanella algae strain on experimental fish (Clarias gariepinus) after 5 days post inoculation 

 

 2.4 Virulence of Aeromonas hydrophila to Fish
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.
 

 

Table 3 Daily observations of the virulence of Aeromonas hydrophila intraperitoneally injected into Clarias gariepinus for 5 days post inoculation

 

 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).
 

 

Table 4 Daily observation of Clarias gariepinus fed with diet containing 106 cells/g Shewanella algae for 7 days and then challenged intraperitoneally with 0.1 ml (3x105 cells) of Aeromonas hydrophila

 

 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 5 Haematological and serum biochemical response of fish to various treatments

 

3 Discussion
Four bacterial genera Pseudomonas, Shewanella, Vibrio and Proteus isolated from shrimp (Penaeus monodon) intestine were found to possess antibacterial activity in vitro against fish pathogenic Aeromonas hydrophila (Table 1). It has been reported that probiotic microorganisms have the ability to inhibit or even eliminate some potential pathogenic bacteria; this can be accomplished through production of inhibitory biological substances such as antibiotics, 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 (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 that was injected with the pathogen and fed with 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 (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 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 probiotic on the liver cells which activated the 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 bacterium, Shewanella algae, could be used effectively in aquaculture as a biocontrol agent against Aeromonas infection.
 
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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