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Research Article
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Dietary Inclusion of Protease Producing Novel Pontibacter spp. and Bacillus megaterium as a Probiotic Enhances Immune Responses in Labeo rohita |
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C. Sumathi,
V. Dillibabu,
Dash-Koney Madhuri,
D. Mohana Priya,
C. Nagalakshmi
and
G. Sekaran
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ABSTRACT
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This study stresses the key role which can be played by Tannery Fleshing (TF) hydrolyzing probiotic Pontibacter spp. in aqua feed formulation and identifies the probiotic strains in the fish gut capable of enhancing the overall growth and immune responses. Probiotics included are Pontibacter species (Pb) and Bacillus megaterium (BM) wherein Lactobacillus (LB) served as control. Experimental diets includes tannery fleshing (TF1), TF+LB strain (TF2), TF+BM strain (TF3), TF+Pb strain (TF4), Fishmeal+BM(TF5 ), Fishmeal+Pb and Control fish meal based diet (TF6). Compared with control, total weight gain (TWG), Specific Growth Rate (SGR), Feed Conversion Ratio (FCR) and Protein Efficiency Ratio (PER) in fish fed with diets supplemented with probiotics were significantly increased (p<0.05). NBT, lysozyme activity, total protein and globulin content were highest in TF4 diet. After challenge with Aeromonas hydrophila, TF4 recorded highest survival and TF1 lowest survival in comparison with the control. Growth and related parameters reveals the effective utilization potential of tannery fleshing probiotic as a feed source. Comparative studies with standard fish meal diets reveals that the fish fed with Pontibacter sps and Bacillus megaterium included feeds enhanced both assimilating capacity and immunological responses in Labeo rohita.
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How
to cite this article:
C. Sumathi, V. Dillibabu, Dash-Koney Madhuri, D. Mohana Priya, C. Nagalakshmi and G. Sekaran, 2014. Dietary Inclusion of Protease Producing Novel Pontibacter spp. and Bacillus megaterium as a Probiotic Enhances Immune Responses in Labeo rohita. Pakistan Journal of Biological Sciences, 17: 451-461. DOI: 10.3923/pjbs.2014.451.461 URL: https://scialert.net/abstract/?doi=pjbs.2014.451.461
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Received: March 26, 2013;
Accepted: May 08, 2013;
Published: November 26, 2013
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INTRODUCTION
Recently a diverse microbial community has been reported from the guts of various
fish (Bairagi et al., 2002; Clements
and Choat, 1995; Luczkovich and Stellwag, 1993).
However, reports on probiotic activity of protease producing Pontibacter
sps and Bacillus megaterium and their influence on fish growth and immunological
parameters remain unexplored. It is often emphasized that probiotic organism
to be used should be isolated from the gastrointestinal tract of host species
intended to study (Patel et al., 2010; Verschuere
et al., 2000).
Probiotics isolated from mature animals are mostly included in feed of immature
animals of same species (Gomez-Gil et al., 2000).
Nutritional and health aspects of functional foods incorporating probiotic bacteria,
especially Lactobacillus, Bifidobacterium, Bacillus, Streptococcus,
Pediococcus, Enterococcus genera and yeast of the Saccharomyces,
Aspergillus and Torulopsis genera have received considerable attention
and eventually led to numerous claims in the literature (Gomes
and Malcata, 1999; Huis int Veld and Havenaar
1997; Berge and Storebakken, 1996). Probiotic for
fermentation combine the positive images of both probiotics and fermentation
organisms that catalyze the proteolytic activities and facilitate the conversion
of substrate into simpler compounds to aid in easy assimilation (Kunji
et al., 1996). The Bacillus species produce proteases which
break down proteins (Farzanfar, 2006) and aids in digestion
(Sanders et al., 2003). Bacillus genus
are preferred in aquaculture industry as they are non pathogenic and produce
most of the antibiotics, amino acids and enzymes (Moriarty,
1990; Gullian et al., 2004). Bacillus
megaterium was included as a probiotic in feed formulation. Pontibacter
sps. PROLR15, a non motile gram-negative coccobacillus which has 92% similarity
with existing Pontibacter strains has been recently identified and only
a few publications i.e. pond and desert (Wang et al.,
2010; Nedashkovskaya et al., 2005) related
to their occurrences are cited. This is first report on the protease producing
Pontibacter strain PROLR15 used as a probiotic.
It is well documented that a change in diet selects for a different bacterial
community (Olsen et al., 2000; Ringo
and Olsen, 1999) which may have an effect on the Specific Growth Rate (SGR)
and receives the attention of the environmental scientists and biotechnologist
(Al-Hafedh, 1999). The replacement of expensive fish
meal in aqua feed with agro industrial wastes or by products have been studied
earlier (Kaur and Saxena, 2004). Tannery fleshing,
generated from pre-tanning operations constitute about 50-60% among tannery
solid wastes that are highest in protein (50.9%). Furthermore, TF protein contains
all the essential aminoacids required for fish growth (Sumathi
et al., 2012). Hence, in the present study Tannery fleshing was utilized
as protein source and Tannery fleshing degrading bacteria as the probiotics.
Previous researchers have reported on the importance of gut flora on immune
responses (Galdeano et al., 2007). Nutrition
and immunology are inter-related and immunomodulation study based on feed formulation
which implies health status of the fish is the impending areas in fisheries
research. Effect of immune stimulators in animal and plant products as feed
supplements has already been investigated (Iranloye, 2002;
Ji et al., 2007). Hence, gut flora and its metabolic
activities are the determining factors of nutrition and animal welfare. It is
becoming increasingly apparent that the intestinal microflora and its metabolic
activities can be an important contributing factor in nutrition, physiology
and animal welfare. However, significance of the host intestinal microbial community
as a probiotic is limited. Hence, promising prospects are sketched out in accordance
with the isolation of tannery fleshing hydrolyzing fish gut bacteria, inclusion
of probiotic in the tannery fleshing feed and impact assessment through growth
and immune modulating properties.
MATERIALS AND METHODS
Substrate characterization: The Tannery Fleshing (TF) were collected
from a commercial tannery near Chennai (Tamil Nadu, India) and were suspended
in water. The pH was adjusted to 7.0±0.2 and manually excised into small
pieces. The total protein content, fat content and types of amino acids (HPLC)
were determined to assess nutritional quality (Sumathi
et al., 2012).
Screening and isolation of gut bacteria: The fish gut homogenate was
serially diluted in saline and plated on Schaedler HiVegTM Agar (Himedia, India)
and incubated for 48 h at 37°C. The major colonies formed on these plates
were sub cultured and maintained in slants. The isolates were screened for protease
production by inoculating them on skim milk agar plates and gelatin agar plates.
The isolates which produced maximum zone were selected and subjected to hydrolysis
of TF. These isolates were then inoculated in a liquid media containing MgSO4.7H2O
(0.2 g L-1); K2HPO4 (2.0 g L-1);
KH2PO4 (2.0 g L-1) and were assayed for higher
proteolytic activity. The protease activity was determined after 24 h of incubation.
The strains showing higher proteolytic activity were selected, identified and
maintained. Among them ANFLR1 strain of Bacillus megaterium and PROLR15
strain of a new Pontibacter spp. exhibited peak protease activity.
Fermentation of TF by ANFLR1 and PROLR15: Fermentation was carried out
in a 2 L fermentation chamber at room temperature. The production medium for
ANFLR1 contains (g L-1) Sodium chloride, 0.4; Ammonium chloride,
0.015; Dipotassium hydrogen phosphate, 1.25; Potassium dihydrogen phosphate,
0.3 with 5% TF and for PROLR15 was (g L-1) Ferric citrate, 0.1; Sodium
chloride, 19.45; Magnesium chloride, 8.8; sodium sulfate, 3.24; Calcium chloride,
1.8; Potassium chloride, 0.55; Sodium bicarbonate, 0.16; Potassium bromide,
0.08; Strontium chloride, 0.034; Boric acid, 0.022; Sodium silicate, 0.004;
Sodium fluoride, 0.002; Ammonium nitrate, 0.001; Disodium phosphate, 0.008 with
5% TF. The inoculums were added aseptically into the fermentation chamber inside
a laminar air flow. The fermented medium was harvested after 36 h of incubation
time along with the sludge and centrifuged at 8000 rpm for 20 min. The supernatant
was concentrated by vacuum evaporation, lyophilized and then mixed with the
dried sludge.
Experimental feeds and aqua feed formulation: Six experimental feeds
were formulated and utilized for the study (TF1, TF2, TF3, T F4, TF5 and TF6)
differing in the probiotic inclusion wherein fishmeal based diet served as a
control (Table 1). Aqua feed preparation involved separately
extruding a mixture of the protein hydrolysates or the raw TF and feed additives
into usable form. In experimental feeds raw and fermented TF slurry serves as
a sole protein source (38%). Appropriate quantities of dry ingredients were
weighed, ground and mixed in a food processor.
Inclusion of probiotic: The sterilized powder of B. megaterium and
Pontibacter culture of concentration 108 cells were moistened
with other ingredients. The dough was extruded to about 3 mm diameter and then
stored in a container -20°C until further use. The proximate compositions
of diets were determined according to AOAC protocols (AOAC,
2006).
Table 1: |
Feed composition of probiotic formulated diets |
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Fish and experimental condition: Labeo rohita fingerlings 10±2
g were obtained from nearest farm in Chennai (India) and acclimatized in 1000
L cylindrical tanks for 15 days with control feed. Three hundred and fifty fingerlings
were selected for the study and divided into seven groups. Each group of 50
fingerlings was again divided into two equal duplicate subgroups. Fish were
fed twice daily (9:00 a.m. and 17:30 p.m.) to apparent satiation. The fish were
fed twice a day and their growth parameters were observed. Blood and serum samples
were collected from each group and examined for the following parameters: total
protein, albumin, globulin, albumin/globulin ratio, Serum Glutamate Oxaloacetate
Transaminase (SGOT), Serum Glutame Pyruvate Transaminase (SGPT), alkaline phosphatase
(ALP), serum bactericidal activity, serum lysozyme and Nitroblue tetrazolium
(NBT) activities and WBC.
Growth parameters: Growth and nutrient utilization were monitored and
analyzed in terms of SGR or percent body weight increase per day and FCR for
all the experimental groups were calculated according to Ricker
(1979). Recorded mortality data was used for calculating the Relative Percentage
Survival (RPS) (Amend, 1981).
Determination of serum biochemical and hematological parameters: Blood
was sampled from the caudal vein of the individual fish after anaesthetization.
The whole blood was collected in a syringe, allowed to clot for 1 h in microtubes
at room temperature followed by 5 h at 4°C and then serum was harvested
by centrifuging at 1500xg for 5 min at 4°C. All serum samples were preserved
at -20°C prior to analysis. The different serum samples collected were analyzed
for total protein following the method of Lowry method; albumin content by Doumas
et al. (1971) globulin content (subtracting albumin from the total
protein) and albumin:globulin ratio. Total leucocyte count was determined following
the method of Shaw (1930). Alkaline phosphatase was
estimated according to the method of Bergmayer (1963)
using p-nitrophenyl phosphate as a substrate. SGOT and SGPT were determined
by method of Reitman and Frankel (1957).
Challenge test against Aeromonas hydrophila: After 60 days of
feeding, fish from each subgroup were challenged intraperitoneally with a lethal
dose of 1x108 CFU L-1 of A. hydrophila ATCC 49040
(OD adjusted to 0.5 at 456 nm) and observed for a period of the next 10 days
for mortality. Estimation of serum and blood parameters were carried out in
the surviving fish after day-10 post infection as per the methods described
earlier. From each tank, a minimum of eight and six fish were sampled for serum
and blood, respectively. The control and experimental fish were subjected to
challenge test by inoculating 10 mL of active pathogenic A. hydrophila
strains. The reactions of the fish for next 24 to 48 h were observed and mortality
rate was determined. Experiments were conducted in triplicates.
Phagocytosis assay: The assay was performed following Siwicki
et al. (1990) and Park and Jeong (1996)
with slight modification. The phagocytic cells and phagocytized bacteria were
enumerated. Phagocytic Ratio (PR) and Phagocytic Index (PI) were determined
by enumerating 100 phagocytes per slide under a microscope. An average of three
slides was calculated:
Lysozyme activity: Lysozyme activity was analyzed by the turbidometric
assay method, originally described by Parry et al.
(1965) and modified for microtitre assay by Demers and
Bayne (1997). Serum (25 mL per well) was placed in triplicate in a ninety-six-well
plate and 175 mL of a suspension of Micrococcus lysodeikticus (75 mg
mL-1 in 0•1 M-phosphate buffer with 0•09% (v/v) NaCl,
pH 5•8) was added. After the plate had been shaken, the decrease in absorbance
at 450 nm was recorded for 5 min. Lysozyme activities were converted to lysozyme
concentrations using hen egg white lysozyme as a standard.
Leukocyte metabolic activity assay or respiratory burst activity: The
metabolic activity of the phagocytes measured as the ability to produce superoxide
anion was assessed by NBT reduction. Superoxide anion production by phagocytic
cells was determined by slight modification of the method described by Chung
and Scombes (1988) and Misra et al. (2006a).
Serum bactericidal activity: Serum bactericidal activity was estimated
following the procedure of Kajita et al. (1990).
An equal volume (100 μL) of serum and bacterial suspension (A. hydrophila)
was mixed and incubated for 1h at 25°C. A blank control was prepared by
replacing serum with sterile PBS. The mixture was then diluted with sterile
PBS at a ratio 1:10. The serum-bacterial mixture (100 μL) was pour-plated
in nutrient agar and plates were incubated for 24 h at 37 °C. The number
of viable bacteria was determined by counting the colonies grown in nutrient
agar plates.
Statistical analysis: All data were analyzed by one-way analysis of
variance (ANOVA) using the software of the SPSS 11.0 for Windows. When ANOVA
identified differences among groups, multiple comparisons among means were made
using Duncan's new multiple-range test. The results are presented as Mean±SD
and probabilities of p<0.05 were considered significant.
RESULTS
Analysis of growth parameters: Growth parameters data are presented
in (Fig. 1). In the present investigation, Bacillus
and Pontibacter as probiotic inclusion in Tannery Fleshing feed formulation
has positive effect on growth of Labeo rohita depicting significantly
higher SGR values and lower FCR values when compared to the control group. Similar
results were observed with other immunostimulants (Misra
et al., 2006b). This implies that protease producing probiotic facilitated
digestion of the protein source (TF) in the fish. The net change in weight of
the fish over the control feed was positive (p>0.05) indicating that probiotic
incorporated tannery fleshing diet could substantially replace the control feed.
Total replacement of fish meal with probiotic containing Tannery fleshing diets
had no deteriorating effect and probiotic included in the diet was harmless
to fish.
Survival rate: After injection with Aeromonas hydrophila, the
first mortality was recorded after 14 h. Mortality was recorded up to 7 days
after injection (Table 2). The relative % survival is presented
in Table 2. TF4 and TF6 fed groups showed significantly higher
survival percentage. Significantly lower survival was recorded for TF1fed groups.
After incorporating probiotic with Tannery Fleshing diets, fish species showed
enhanced non-specific immune response activities and resistance to experimental
challenge with pathogenic bacteria. Mortality percentage was only 8 and 9% in
Pontibacter included diets. Relative percentage survival was also high
(78 and 76%) in Pontibacter included diets (TF4 and TF6) and coincides
with the results of immunostimulant levan included diet (Rairakhwada
et al., 2007).
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Fig. 1: |
FCR and SGR values of the fish fed with experimental diets
after the immunomodulation trial days, Mean values bearing same superscript
are not statistically significant, p>0.05, SGR: Ln of final weight-Ln
of initial weight)/t (time interval in days)x100, FCR: Feed given (dry weight)/Weight
gain (wet weight) |
Table 2: |
Aeromonas challenge test |
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Table 3: |
Serum hematological responses observed before and after the
immunostimulation trial period in Labeo rohita fed probiotic included
diets |
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Serum protein, albumin, globulin and A/G ratio: The concentration of
total protein in blood serum is used as a basic index for health status of fish.
Among the serum protein, albumin and globulin are the major proteins, which
play a significant role in the immune responses. There were significant differences
in the serum total protein and globulin content among the different experimental
groups before and after the challenge study (Table 3). There
was marked increase in the WBC count in TF6 fed fish (137-168 103 cells
mm-3) when compared to Control (128-149 103 cells mm-3)
during pre and post challenge period. A significant decrease (p<0.05) in
the serum total protein and globulin content was recorded after challenge with
Aeromonas hydrophila either fed with TF1(1.35 g dL-1) diet
or control diet (1.89 g dL-1).
In contrast, a significantly higher serum globulin level in TF4-TF6 (1.72,
1.73 and 1.76 g dL-1) groups was noticed during post challenge period.
No marked changes in the serum albumin content were recorded either pre-or post-challenge
studies in experimental and control diets. The A/G ratio was significantly (p<0.05)
increased in the post-challenge period than the pre-challenged period irrespective
of the type probiotic inclusion. TF1 group recorded a significantly lower A/G
ratio after challenge. During the post-challenge period, a significantly (p<0.05)
higher A/G ratio was recorded in the groups fed with Lactobacillus and
Pontibacter included Tannery fleshing diets.
Serum lysozyme activity: Changes in lysozyme activity are considerably
influenced by the dose and the type of stressors to which fish are exposed.
Serum lysozyme activity can be increased using immunostimulants, which may either
be due to an increase in the number of phagocytes secreting lysozymes or an
increase in the amount of lysozymes synthesized per cell (Edahiro
et al., 1990). There was a significant difference (p<0.001) in
lysozyme activity among the various treatment groups in the pre and post-challenge
period (Table 3). Lysozyme activity was significantly higher
(p<0.05) in TF4 groups than the TF3 fed groups during both the pre-and post-challenge
period. Similarly, post-challenged fish showed significantly higher lysozyme
activity (p<0.05) than the pre-challenged fish fed with probiotic included
diets. B. magetarium or Pontibacter Species fermented Tannery
Fleshing had higher lysozyme activity in the post-challenge period than in the
pre-challenge period. Similar, elevation of lysozyme following dietary incorporation
of immunostimulants has been demonstrated in a number of fish species (Paulsen
et al., 2003).
Total Leukocyte count: Leukocyte count is considered as an indicator
of the health status of fish because of its role in nonspecific or innate immune
and inflammatory responses. In the present study, increased WBC count was observed
in TF diets. This may be due to metabolic stress mediated by the new protein
(Secombes, 1996). The post-challenge increases in leukocyte
and monocyte count irrespective of the Tannery fleshing inclusion signify a
possible increased infection and inflammatory response mediated by leukocyte
against bacteria.
NBT assay or Respiratory burst activity: Respiratory burst activity
of phagocytes was measured by reduction of Nitro Blue Tetrazolium (NBT) by intracellular
superoxide radicals produced by leukocytes. The production of superoxide radicals
as examined by NBT reduction was significantly influenced by the probiotic diets.
Maximum increase in the NBT reduction value was observed in Pontibacter
incorporated diets and was similar in response to Lactobacillus and Bacillus
formulated diets. This further confirms the immunomodulatory properties of Tannery
fleshing hydrolyzing probiotics. Similar observations are reported with inclusion
of turmeric enriched diet and levan supplemented diets in Cyprinus carpio
(Rairakhwada et al., 2007). This result further
reveals the positive influence of Probiotic on the survival of Labeo rohita
following A. hydrophila infection.
Serum bactericidal activity: Many investigators have reported enhanced
bactericidal activity by the phagocytic cells in different fish species (Jorgensen
et al., 1993; Sharp and Secombes, 1993).
Serum bactericidal activity was lowest in the control group and highest in the
experimental groups. The highest activity was observed in probiotics included
diet. This shows a positive correlation with the results of NBT assay in which
the highest activity was observed in the probiotic incorporated diets. In the
remaining test groups (TF1) and control the serum bactericidal activity was
very low. The bacterial counts were only slightly lower than the control group
and there was no significant difference. Serum bactericidal activity in TF3
to TF4 diets fed fish was significantly (p>0.05) higher than control fish
on all the assay days (Table 3). The serum bactericidal activity
was higher in the group of fish fed with Pontibacter incorporated diets.
In our study, survival was marked to be negatively correlated with total plate
count of bacteria, thus indicating a positive correlation of bactericidal power
of serum (lysing more number of bacteria and reducing total plate count) with
high survivability. The increased serum bactericidal activity in probiotics
included diets confirms that various humoral factors involved in innate and/or
adaptive immunities are elevated in the serum to protect the host effectively
from infection.
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Fig. 2: |
Effect of probiotic included diets on Serum enzymes, The values
represent the Mean±SE of triplicates |
Serum ALP, SGOT and SGPT levels: Increased activity of ALP was marked
in the group of fish fed with probiotics on different days. Alkaline phosphatase
is associated with the absorption of glucose, lipid, calcium and inorganic phosphate
(Eguchi, 1995). Increased phosphatase activity indicates
higher breakdown of the energy reserve, which is utilized for the growth and
survival of fish (Ghosh et al., 2008). It is
known that the liver is rich in GOT and GPT and damage to the liver may result
in high serum GOT and GPT activities (Fig. 2). Low serum GOT
and GPT activities of probiotic formulated diets suggest healthy liver with
negligible damage, supporting suppressed hepatic amino acid utilization. Generally,
decreased AST and ALT activities in the serum indicates that oxaloacetate and
glutamate were not available to the Krebs cycle through this route of transmission
(Shakoori et al., 1996).
Phagocytic responses: Most of the immunostimulants used in aquaculture
are believed to enhance innate immunity in fish by stimulating leucocyte activity.
Leucocytes derive their bactericidal power from a vast range of cellular functions,
including phagocytosis, pinocytosis and production of superoxide anions and
hydrolytic enzymes (Ellis, 1999). Phagocytic ratios
(Fig. 3) and phagocytic indices (Fig. 4)
in the fish fed with different probiotic included Fleshing diets were significantly
(p>0.05) higher than control fish during the assay period. Diets with Bacillus
megaterium and Pontibacter inclusion (TF3 and TF4) possessed highest
phagocytic ratio and phagocytic index.
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Fig. 3: |
Phagocytic ratio observed on different immunomodulation trial
days, The values represent the Mean±SE of triplicates |
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Fig. 4: |
Phagocytic index observed on different immunomodulation trial
days, The values represent the Mean±SE of triplicates |
DISCUSSION
Recent data on probiotic applications indicate that non-viable microbial components
act in a beneficial manner which aid in development of the immune system, digestive
processes and provide nutritional benefits (Ramirez and
Dixon, 2003; Rowland et al., 1998). In the
present study, the proteolytic strains Bacilllus megaterium and Pontibacter
species that facilitates the breakdown of proteins were isolated from
Labeo rohita gut (Nibedita and Ghosh, 2008).
Previous researchers have reported the diets with a probiotic supplement exhibited
greater growth than those fed with the control diet (Lara-Flores
et al., 2003). Various authors have shown that lactic acid bacteria
are part of the normal intestinal flora of fish (Ringo and
Gatesoupe, 1998; Havenaar et al., 1992) that
are commonly associated with nutritious environments (Walstra
et al., 1999; Ringo and Olsen, 1999). The
administration of L. acidophilus as a probiotic induces a growth promotion
effect in, Arctic charr (Salvelinus alpinus), Atlantic salmon (Salmo
salar) and rainbow trout (Oncorhynchus mykiss) (Gonzalez
et al., 2000). Similar results in the present work suggest that the
strain of B. megaterium and Pontibacter species can also be used
effectively as probiotic in aquafeed.
Although there are few reports on the effects of exogenous enzyme supplementation
in diets for fish, a number of studies on the use of proteases to improve the
digestibility of feed ingredients in poultry, pigs and cattle have been published.
Exogenous enzymes extracted from bacteria like Bacillus circulans or
fungi incorporated into larval feeds were found to aid in digestion and promote
better growth and survival in broiler chickens (Ghazi et
al., 2002) diets resulted in improved digestibility of protein and increased
production of milk and eggs. Correspondingly, protease activity of the probiotic
strains enhanced growth and immune responses in Labeo rohita.
Immunostimulants can increase the host's nonspecific immunity by either increasing
the number of phagocytes or activating phagocytosis and respiratory burst (Shoemaker
et al., 1997) to fight against the microbial pathogen. Innate immunity
due to lysozyme is caused by lysis of bacterial cell wall and these stimulate
the phagocytosis of bacteria. Many researchers have reported the enhancement
of the lysozyme activity, complement activity and bactericidal activity (Selvaraj
et al., 2005) due to administration of immunostimulants. Respiratory
burst activity of phagocytes measured by reduction of NBT by intracellular superoxide
radicals produced by leucocytes showed enhanced activity with diet incorporated
with probiotic and the highest activity was recorded in the diet supplemented
with Pontibacter included diets. In the present study, increased respiratory
burst activity can be correlated with increased bacterial pathogen killing activity
of phagocytes.
Variations in lysozyme activity appear to be related to the ability of probiotic
strains to adhere to the intestinal mucus. Lysozyme is a humoral non-specific
defence protein widely distributed in nature, including fish. Although its exact
physiological role is not yet understood, there is a general acceptance that
lysozyme is involved in the defence against micro-organisms. Lysozyme hydrolyses
N-acetylmuramic acid and N-acetylglucosamine, which are constituents of the
peptidoglycan layer of bacterial cell walls. Lysozyme activity in fish serum
has been reported to increase after injecting a bacterial product, in response
to bacterial infection and after probiotic supplementation (Moyner
et al., 1993). Panigrahi et al. (2004)
showed significantly higher serum lysozyme activity in rainbow trout fed with
Lb. rhamnosus JCM 1136 at 109 CFU g-1 feed for
30 days. The results coincide with the above researchers indicating the potential
application of Pontibacter spp. as imuunostimulating probiotic.
These results suggest that probiotics included Tannery Fleshing diet when fed
at a medium dose for 60 days could enhance immunity, growth and survival of
L. rohita fingerlings which can be attributed to the availability of
good quality protein and essential amino acids. Total protein content, globulin
content and lysozyme level increased in the fish after feeding with probiotic
diets. Increase in albumin/globulin percentage after Tannery fleshing administration
in this study corroborates with Mondal et al. (2008).
Such enhancement in the lysozyme activity could also be correlated with enhanced
phagocytic activity. The in-vivo activation of phagocytic cells by Tannery
fleshing probiotics might have also induced other antimicrobial mechanisms,
which include release of lysosomal enzymes, cationic peptides and production
of reactive oxygen species (Kwak et al., 2003)
thereby preventing the adherence and colonization of microorganisms (Alexander
and Ingram, 1992) leading to prevention of infection and disease. In the
present study, an increased growth rate was observed in Labeo rohita
fed feed containing B. megaterium and Pontibacter compared with control
and the high rates of establishment of bacterium in the gastro-intestinal tract
of fish treated with probiotics may have suppressed the A. hydrophila
infection, which ultimately resulted in the higher survival (Kumar
et al., 2006; Ziaei-Nejad et al., 2006).
The alkaline phosphatase levels were drastically decreased during the infection
in control group fish, but probiotic restored the level to normal as in the
uninfected fish. As a result of infection with A. hydrophila, SGOT and
SGPT were increased significantly in the infected control fish. However, these
elevated levels were normalized by probiotic diets and were similar to the uninfected
controls. All these results indicate that probiotic included Tannery fleshing
diet increases the resistance of L. rohita so that it can withstand the
adverse conditions of a challenge.
Mortality following challenge with A. hydrophila was decreased in the
group of fish fed with probiotic incorporated diets. There was an inverse relationship
between the mortality rate and the probiotic inclusion in the diet. Probiotics
have an important role in disease control strategies for aquaculture and may
provide an alternative to the use of antimicrobial compounds. The result suggests
that the probiotics used in this study can be used effectively as a commercial
product for use in aquaculture. Ongoing study is to find metabolic pathways
used by these microorganisms in the alimentary tracts of fish, to explain the
lifelong host-microbe homeostasis and evaluate the potential therapeutic applications
of the bacterial metabolites.
CONCLUSION
In the present study, an increased growth rate was observed in Labeo rohita
fed feed containing B. megaterium and Pontibacter compared with control
diet. Pontibacter sps and Bacillus megaterium as probiotic included
in Tannery Fleshing diet when fed at a medium dose for 60 days could enhance
immunity, growth and survival of L. rohita fingerlings indicates that
Tannery fleshing utilizing Pontibacter spp. and Bacillus megaterium
isolated from Labeo rohita enhanced overall survival and boosted up the
immunity. The observed improvement in fish immune parameters as well as growth
and survival using novel Pontibacter spp. and Bacillus megaterium
as probiotic bacteria may open a new chapter for screening new strains of probiotic
bacteria from fresh water fish Labeo rohita for extensive use in aquaculture.
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