Application of Probiotic, Prebiotic and Synbiotic for the Control of Streptococcosis in Tilapia Oreochromis niloticus
One of the fish diseases that is becoming the main problem in tilapia culture is streptococcosis caused by Streptococcus agalactiae. Application of probiotic, prebiotic and synbiotic are expected to be an alternative for controlling the disease. The purpose of this study was to examine the effectiveness of the administration of probiotic, prebiotic and synbiotic through artificial feed to control streptococcosis in tilapia. This study consisted of five treatments with three replications, namely positive control, negative control; 1% probiotic treatment; 2% prebiotic treatment and synbiotic treatment (1% probiotic and 2% prebiotic). Results showed that fish survival rate before the challenge test for all treatments was between 95 and 100%. Growth and feed conversion ratios in probiotic, prebiotic and synbiotic treatments were better than that of the controls. After the challenge test, the fish survival rate in probiotic, prebiotic and synbiotic treatments were 74.08, 74.08 and 85.19%, respectively; whereas, in the positive control it was only 18.52%. Results showed that S. agalactiae bacteria could be found in the brain, kidney, liver and eyes. The number of S. agalactiae bacteria and the damage level of various target organs in probiotic, prebiotic and synbiotic treatments were lower than that of positive control.
Received: October 08, 2014;
Accepted: January 14, 2015;
Published: March 13, 2015
Streptococcus agalactiae is a species of pathogenic bacteria which causes
one of the major problems in tilapia cultivation, causes a high mortality rate
and a huge economic loss. Streptococcus agalactiae can cause disease
with chronic or acute effects, depending on the degree of the infection. Chronic
clinical signs are lesions on the body surface, red spots on the fins, lethargy
and appetite loss. The acute signs which are lethal are assumed to be due to
the loss of fluids from the distal digestive tract. Before the fish dies, it
exhibits lethargy and the tendency to stay at the bottom of the aquarium, shows
low interest in feeding, exhibits whirling, curling its body into a "C",
changes in body color and the operculum opens more quickly (Evans
et al., 2006). Streptococcus agalactiae usually attacks the
brain, eyes and other organs which have high water content (Evans
et al., 2002).
The conventional control of the disease involves chemicals such as drugs, antimicrobials
and disinfectants (Gomez-Gil et al., 2000). Uncontrolled
use of antibiotics for treating diseases cause imbalances in the natural dynamics
of the microorganisms involved in fish cultivation. Therefore, the use of the
chemicals above is not recommended. One of the alternatives to control the disease
is the application of probiotics, prebiotics and synbiotics (the combination
between probiotics and prebiotics). Verschuere et al.
(2000) defined probiotics as live microbial agents which have beneficial
effects on the host by balancing the hosts intestinal micro flora. Probiotics
also give benefit to the host by strengthening the hosts immune system, improving
the quality of the hosts living environment and increasing the nutritional
value of the feed. The success of the application of probiotics has become the
foundation of other concepts such as prebiotics and synbiotics (Nayak,
2010). Prebiotics are feed ingredients which cannot be digested by the host
but give benefit to the host by selectively improving the metabolic activity
and growth of one or more of the bacteria in the intestines (Roberfroid,
2000; Schrezenmeir and de Vrese, 2001), whereas synbiotics
are a combination between probiotics and prebiotics in the effort of supporting
the survival and growth of beneficial bacteria in live organisms digestive
tract (Schrezenmeir and de Vrese, 2001). Various studies
have demonstrated the benefits of applying probiotics and prebiotics in aquatic
animal cultivation (Merrifield et al., 2010;
Nayak, 2010; Ringo et al.,
2010). In several studies, the application of synbiotics has produced better
results compared to the applications of probiotics and prebiotics separately
(Li et al., 2009; Rodriguez-Estrada
et al., 2009; Zhang et al., 2010).
This study was aimed to evaluate the effectiveness of the administration of
probiotic, prebiotic and synbiotic through feed in controlling S. agalactiae
infection in tilapia.
MATERIALS AND METHODS
This study was conducted using the Completely Randomized Design (CRD) which
consisted of 5 treatments, i.e., the administration of feed without the addition
of any probiotic, prebiotic or synbiotic and then challenged by S. agalactiae
(positive control); the administration of feed without the addition of any
probiotic, prebiotic or synbiotic and not challenged by S. agalactiae (negative
control); the administration of feed with the addition of 1% probiotic and then
challenged by S. agalactiae (Pro); the administration of feed with the
addition of 2% prebiotic and then challenged by S. agalactiae (Pre);
the administration of feed with the addition of 1% probiotic+2% prebiotic and
then challenged by S. agalactiae (Syn).
The probiotic used in this study was Bacillus sp. NP5 which had been
isolated from the digestive tract of tilapia and had been tested for its antagonistic
activity against S. agalactiae in vitro (Putra, 2010).
Before being used in the challenge test, the Koch postulate was applied to increase
the S. agalactiaes virulence. The prebiotic used was oligosaccharides
extracted from sweet potatoes var. sukuh using 70% ethanol (Muchtadi,
1989). The prebiotics Total Dissolved Solids (TDS) were measured using
the method developed by Apriyantono et al. (1989)
to measure the prebiotics dissolved solids concentration.
Tilapia BEST strain weighing 15-20 g were reared in 15 aquariums (60×30×40
cm3) at a density of 10 individuals per aquarium. The in vivo
assay was conducted by mixing the probiotic, prebiotic and synbiotic with egg
yolk amounting to 2% of the feed weight and spraying the mixture thoroughly
onto the fish feed. One percent of Bacillus sp. NP5 (1 g per 100 g feed)
(Putra, 2010) at a concentration of 106 CFU
mL-1 was administered in the probiotic treatment. Two percent of
the prebiotic (2 g per 100 g feed) (Mahious et al.,
2006) with a TDS of 5% (Marlis, 2008) was added.
Tilapia were fed commercial feed three times a day by ad satiation. Probiotic,
prebiotic and synbiotic were administered to the feed and fed to the fish once
a day for 14 days. On the 15th day, tilapia were challenged by injecting them
with S. agalactiae at a dose of 0.1 mL per individual at a concentration
of 105 CFU mL-1 which is the LD50 dose (Taukhid,
2009). After being injected with S. agalactiae, the fish were reared
for 14 days and fed the control feed. In order to maintain water quality in
the aquarium, 10% of the volume of water in the aquarium was siphoned every
The parameters observed during the study were survival rate (Effendie,
1979), Daily Growth Rate (DGR) (Huisman, 1987), Feed
Conversion Ratio (FCR) (Zonneveld et al., 1991),
clinical signs, total S. agalactiae count on target organs and the histopathology.
The tilapias survival rate was calculated at the end of the probiotic,
prebiotic and synbiotic treatment and after the challenge test using S. agalactiae.
The FCR and DGR were calculated after 14 days of probiotic, prebiotic and synbiotic
treatment. Clinical signs, histopathology and the total S. agalactiae
count on target organs were observed after the challenge test using S. agalactiae.
Statistical analysis: The data obtained was analyzed using ANOVA with
the SPSS 14 program and then followed by the Duncan test.
The survival rate of tilapia after 14 days of probiotic, prebiotic and synbiotic
administration was 100%, not significantly different from the controls (95.0-97.5%).
However, after the challenge test with S. agalactiae, there was a quite
a lot of deaths in the positive control, resulting in the lowest survival rate,
i.e., 18.52% (Table 1). The treatment using probiotic, prebiotic
and synbiotic resulted in the higher survival rates, i.e., 74.08, 74.08 and
The daily growth rate in the synbiotic treatment was higher than the other
treatments (Table 1). Moreover, FCR in the positive control,
negative control, probiotic, prebiotic and synbiotic treatments were 2.28, 2.18,
1.82, 1.78 and 1.77, respectively (Table 1).
||Tilapias Survival Rate (SR), Daily Growth Rate (DGR) and
Feed Conversion Ratio (FCR) after treatment with probiotic, prebiotic and
|Different superscript letters in the same row signify significantly
different results (p<0.05)
||Bacterial count of Streptococcus agalactiae in target
organs in (a) Week 3 and (b) Week 4
Observations of clinical signs were done to note the development of the S.
agalactiae infection in tilapia. After the challenge test with S. agalactiae,
there were macroscopic changes in the external organ anatomy such as the external
part of the operculum, eyes and body of tilapia. On the first day of the
S. agalactiae infection, tilapia exhibited changes in color, becoming paler
and vertical black stripes started to show on the fishs body and the pupils
shrank. The next day, tilapia exhibited clear operculum; the operculum first
became yellowish then translucent. The next level of damage was the eyes became
cloudy or purulent then swollen and finally they detached from the eye socket.
Prior to death, tilapia started whirling and their bodies curled into a "C"
The Bacillus sp. NP5 probiotic bacterias ability to suppress the growth
of S. agalactiae in tilapia could be seen from the number of S. agalactiae
in target organs, i.e., the brain, kidney, liver and eyes. The results of this
study demonstrate that in the third week (7 days post-challenge test) S.
agalactiae was found in the brain, kidney, liver and eyes in all treatments
(Fig. 1). The results of histopathological examinations of
the fish infected by S. agalactiae showed that 71.2% of the damage was
found in the brain and the rest were found in the kidney, liver and eyes. In
the fourth week (14 days post-challenge test), S. agalactiae was no longer
found in all the target organs in the fish treated with synbiotic while in the
fish treated with probiotic or prebiotic separately the bacteria was only found
in the brain. However, in the positive control, S. agalactiae was still
found in all target organs.
The number of S. agalactiae in target organs caused some changes that
could be observed by histopathology slides. In the brains of infected tilapia
there were signs of encephalitis, i.e., congestion, hypertrophy and vacuolization,
necrosis and degeneration in the positive controls, whereas, the fish in other
treatments only experienced hyperplasia and hypertrophy (Fig.
2). Moreover, the positive control kidney of tilapia which had been infected
with S. agalactiae underwent pathological changes in the form of hypertrophy,
hemorrhage, degeneration, congestion, exhibiting the presence of inflammation
cells and necrosis. In treatment with probiotic, prebiotic and synbiotic, there
was only hemorrhage and congestion (Fig. 3). In the liver
of the positive control (Fig. 4), there were signs of atrophy,
fatty degeneration, congestion and hemorrhage. In the fish treated with probiotic
and prebiotic there was congestion, hemorrhage and hypertrophy, whereas, in
those treated with synbiotic there was only hemorrhage and congestion. The pathological
changes were also obtained in the eyes of the positive control in the form of
hypertrophy, hyperplasia, vacuolization and necrosis (Fig. 5).
Histopathology of the brain of tilapia
injected with Streptococcus agalactiae, A: Hyperplasia, B: Hypertrophy,
C: Necrosis, degeneration, D: Congestion, E: Vacuolization (1 bar = 50
Histopathology of the kidney of tilapia
injected with Streptococcus agalactiae, A: Normal kidney, B: Vacuolization,
C: Inflammation cells, D: Deposition of hyaline, E: Congestion, F: Degeneration,
necrosis, G: Haemorrhage (1 bar = 50) μm
The treatment using probiotic, prebiotic and synbiotic resulted the higher
survival rates. This was because the probiotic, prebiotic and synbiotic administered
were able to improve the fishs immune response, enabling them to suppress the
growth of S. agalactiae. The highest death rate occurred on the 4th and
5th day post-challenge test in all treatments. This is because the peak of S.
agalactiaes virulence factors are assumed to be on those days. According
to Evans et al. (2004), most of the tilapia deaths
post S. agalactiae infection happened between day 4-7. The study by Taukhid
(2009) also showed that the highest tilapia mortality in the S. agalactiae
LD50 test happened between day 4-5.
Synbiotic treatment showed the better growth performance in this study. The
assumption is that it is because the activity and growth of the probiotic bacteria
increased with the addition of the prebiotic which was then able to improve
feed utilization in tilapia. The study by Putra (2010)
showed that the addition of synbiotic to feed resulted in the highest growth
rate because of the higher bacterial population and digestive tract enzyme activity
compared to the control. In this study, the growth in the individual probiotic,
prebiotic and synbiotic treatments were not significantly different among them
but were significantly different from the controls.
Histopathology of the liver of tilapia
injected with Streptococcus agalactiae, A: Normal liver, B: Atrophy,
C: Fatty degeneration, D: Hypertrophy, E: Congestion, F: Hemorrhage (1
bar = 50 μm)
Histopathology of the eye of tilapia
injected with Streptococcus agalactiae, A: Normal eye, B: Hypertrophy,
C: Vacuolization, D: Hyperplasia, E: Necrosis (1 bar = 50 μm)
This indicates that the addition of probiotic, prebiotic and synbiotic could
improve the tilapias growth performance due to better nutrient utilization
compared to the controls. The synergistic effect in the synbiotic treatment
is not significant; probably because the 1% probiotic and 2% prebiotic combination
is not yet an optimum combination. The low FCR values in the probiotic, prebiotic
and synbiotic treatments showed that the feed utilization was better than the
controls. The FCR value has a positive correlation to the daily growth rate.
The addition of probiotic, prebiotic and synbiotic in feed were able to improve
the fishs ability to utilize feed due to the rise in the number of beneficial
bacteria in the fishs digestive tract. The study by Putra
(2010) demonstrated that Bacillus sp. NP5 is an amylolytic bacteria
strain which could secrete amylase which has an important role in the fishs
digestion process, i.e., hydrolyzing the carbohydrate in feed in the fishs
digestive tract. This bacteria ability to produce amylase can optimize feed
utilization in the tilapias digestive tract. The administration of a prebiotic
could facilitate the growth of beneficial micro flora in the intestines and
in turn improve feed utilization. In the study by Putra (2010),
the administration of probiotic, prebiotic and synbiotic individually could
increase the activity of amylase and protease in tilapias digestive tract.
This increases the digestibility of protein and carbohydrate in feed, increasing
the amounts of protein and energy from the feed that could be absorbed by the
intestines and be utilized by the fish which in the end optimizes feed utilization.
The more protein and energy stored by the fishs body will show the higher protein
retention and the better growth rate.
The experimental fish in this study showed some external anatomy changes after
the challenge test with S. agalactiae, in which those signs were consistent
with the study by Evans et al. (2006). Streptococcus
agalactiae injected into the fish would enter blood vessels and would be
carried by the blood stream to the brain. Hernandez et
al. (2009) stated that the brain is the main target of S. agalactiae
after entering the blood flow. On the other hand, the results demonstrated that
the probiotic, prebiotic and synbiotic administered were able to suppress the
number of S. agalactiae. This is postulated to be due to tilapias increased
immune response in the form of the increased number and activity of macrophages
in tissues, causing an increased phagocytosis and an increased number of bacteria
Besides changes external organs, there were also changes in internal organs
such as the tilapias liver, kidney and brain. The changes were apparent
in the tilapias histopathology. According to Cheville
(1999), focal necrosis could be in the form of liquidization of tissues
as a result of an enzymatic reaction due to the introduction of toxins. Based
on the microscopic observation, S. agalactiae infection caused degeneration
of the brain in the positive control. According to Roberts
(2001), bacterial infections could cause disturbances in cell metabolism
(degeneration) which is signified by intercellular accumulation which can be
seen microscopically as numerous cells packed together, distended cells, paler
color and the discovery of vacuoles and necrosis. The degeneration of the brain
cells is the cause of the fishs loss of balance, whirling and their tendency
to swim to the surface. According to Hardi (2011), fish
which exhibit signs of whirling show signs of degeneration and necrosis of the
cranial cerebellum at histopathological examination. Microscopically, vacuolization
is seen as empty spaces in the brain which occurs as a result of cell damage
which led to cell destruction. Vacuolization is thought to be caused by infections
carried through the blood flowing to the brain, causing the damage to the organs
tissues. The kidney plays a role as an excretory organ which filters waste materials
from the blood. The kidney also actively fights the entry of foreign microorganisms
(pathogens) through the presence of macrophages and lymphocytes in the kidney.
If there is an infection, the kidney will demonstrate resistance mechanisms
such as the formation of white blood cells such as monocytes, lymphocytes and
granulocytes. Rombout et al. (2005) stated that
in teleostei fish, the kidney plays a role in the formation of various white
blood cell groups such as the monocytes and granulocytes (neutrophils, basophils
and eosinophils). High intensity attacks from pathogenic bacteria causes the
kidney to work in overdrive, causing cell damage. In addition, the bacteria
which succeed in attacking the kidney will secrete exotoxins which have the
ability to cause hemorrhages in the epithelial cell of the tubules.
Inflammation of the liver was indicated by infiltration of inflammation cells
which showed that the pathogens had infected liver cells. The migration of inflammation
cells is the indication of a defense reaction toward toxic materials entering
the body in order to destroy the infectious agent. According to Ressang
(1984), inflammation could be triggered by bacteria which could potentially
secrete toxins. Liver cell damage was found in all treatments in week 3 (7 days
post S. agalactiae infection). In week 4, damage was still found in the
positive control, but no longer found in treatments with probiotic, prebiotic
and synbiotic. This showed that the fish in these treatments have already recovered,
approaching the normal condition. This was also shown by the results of the
bacterial count of the S. agalactiae in the kidneys; in fish treated
with probiotic, prebiotic and synbiotic, there were no S. agalactiae
Hypertrophy is the increased volume of a certain organ or tissue due to increased
cell size. Hyperplasia is the increased number of cells but the cells are normal
sized. Even though hypertrophy and hyperplasia are two different processes,
but they often coincide. According to Hardi (2011), hypertrophy
and hyperplasia in the choroidal zone cause the fish to experience exophtalmia
(the eyes bulge both laterally and bilaterally). Hypertrophy and hyperplasia
were observed in positive control in week 3 and 4 but were not found in fish
treated with probiotic, prebiotic and synbiotic. This is probably because the
probiotic, prebiotic and synbiotic were able to increase the tilapias immune
response, making the treated fish more resistant compared to positive control.
S. agalactiae which proliferate in the eye enter through the blood stream
and secrete exotoxins which damage the choroidal zone causing the changes found
there. In this study, hemorrhage was found in the positive control, signifying
that S. agalactiae is septicaemic, capable of spreading its virulence
factor through blood vessels and reach the eye. Macroscopic observations showed
damage to the eyes (exophtalmia) between day 4-7 and this was in line with the
findings of the study by Evans et al. (2006).
The administration of probiotic, prebiotic and synbiotic through feed could
control streptococcosis in tilapia by suppressing the number of S. agalactiae
and the level of damage on the target organs, i.e., the brain, kidney, liver
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