International Journal of Advanced Research in Engineering and Technology

(IJARET)
Volume 6, Issue 10, Oct 2015, pp. 62-75, Article ID: IJARET_06_10_011
Available online at
http://www.iaeme.com/IJARET/issues.asp?JType=IJARET&VType=6&IType=10
ISSN Print: 0976-6480 and ISSN Online: 0976-6499
IAEME Publication
___________________________________________________________________________

DEVELOPMENT OF MARINE DERIVED

PROBIOTIC BACTERIAL CONSORTIUM
FOR THE SUSTAINABLE MANAGEMENT
OF LITOPENAEUS VANNAMEI CULTURE
R Karthik, Angelin C Pushpam, and M. C. Vanitha
Department of Marine Biotechnology and Centre for Marine Bio prospecting,
AMET University (Declared as deemed to be University U/S 3 of UGC Act 1956),
Kanathur, Chennai, 603112, Tamil Nadu, India
D. Yuvaraj
Department of Biotechnology, Vel Tech High Tech Dr. Rangarajan Dr. Sakunthala
Engineering College, Avadi, Chennai, 600 062, Tamil Nadu, India
ABSTRACT
The use of probiotics or beneficial bacteria, which control pathogens
through a variety of mechanisms, is increasingly viewed as an alternative to
antibiotic treatment, which cause attendant problems like drug residue in
tissues, export rejection etc.. The present study was aimed to determine the
probiotic effectiveness of Bacillus stratosphericus (AMET1601), Arthrobacter
sp (AMET1852) and Lactobacillus sp (AMET1506) (isolated from marine
samples) on Litopenaeus vannamei culture for the prevalence of white spot
syndrome virus (WSSV) under laboratory scale conditions. All the three
selected isolates were included in the diet of juvenile shrimp at different
combination and concentration. Two bioassays were conducted with
treatments by triplicate (10 shrimp per replicate). Based on the, initial mean
weight, Mean weight gain, FCR, DWG, Yield, Survival rate and Vibrio load,
in test group T-5, where the shrimps fed with all the three different probiotic
strains (Bacillus stratosphericus (AMET1601) + Lactobacillus sp
(AMET1506) + Arthrobacter sp (AMET1852)) incorporated feed (in the range
of 5106 cfu mL) showed significant changes in regard to the mentioned
biometric parameters than other groups. In bioassay II, after 21 days of
culture, the maximum shrimp survival (46%) was observed in treatment II
comparatively than control (28%). The consortium of three potential
beneficial probiotic bacterial consortiums didnt eliminate the WSSV in
cultured shrimps, but increase the survival rate and decrease the vibrio load
in the culture systems and water.

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Development of Marine Derived Probiotic Bacterial Consortium For The Sustainable

Management of Litopenaeus Vannamei Culture

Key words: Shrimp Culture, Probiotics, White Spot Syndrome Virus, Immune
System, Litopenaeus Vannamei.
Cite this Article: R Karthik, Angelin C Pushpam, and M. C. Vanitha and D.
Yuvaraj. Development of Marine Derived Probiotic Bacterial Consortium For
The Sustainable Management of Litopenaeus Vannamei Culture. International
Journal of Advanced Research in Engineering and Technology, 6(10), 2015,
pp. 62-75.
http://www.iaeme.com/IJARET/issues.asp?JType=IJARET&VType=6&IType=10

1. INTRODUCTION
Shrimp culture represents an important and economically profitable venture and their
production has grown enormously in recent years by intensive and semi-intensive
methods of culture. Penaeid shrimps are one of the most important preferred species
for culture in artificial impoundments (Megarajan Sekar et al., 2014). As of now more
than 5 million metric tons of shrimps are annually produced through aquaculture but
the current global demand for both the wild (naive) and farmed shrimps is more than
6.5 million metric tons per annum (Karthik et al., 2014). To overcome this, many
shrimp farms are being created throughout the world to solve this increasing food
demands (FAO 2012). However, intensive development of these shrimp industries
and extensive culture of these aqua farms has created various ecological, economical
and social problems. During the last few years white spot syndrome virus (WSSV)
disease has spread worldwide and caused large scale mortalities and economic loss in
shrimp culture particularly in Asia (Danya and Jagadish, 2014). Due to the continuous
outbreak of this WSSV disease in Penaeus monodon culture leading to loss of shrimp
culture in India the farmers are seriously looking for alternative shrimp species for
culture. In 2008, the Coastal Aquaculture Authority of India (CAA) introduced a new
shrimp species Litopenaeus vannamei as an alternative to Penaeid species in India to
culture and export. Since the Litopenaeus vannamei exhibits fast growth rate and its
culture period is significantly shorter compared to Penaeus monodon. Several
maritime countries have switched over to Litopenaeus vannamei culture instead of
Penaeus monodon as a prospective species in terms of economical gain and standing
top production in short periods (Karuppasamy, et al., 2013).
In general, shrimp ponds are enclosed cultivation systems, subject to periodic
water renewal to compensate for volume changes (due to evaporation) and salinity
changes (evaporation, precipitation) and to maintain water quality. The excess feed
and faecal matter may result in bacterial decomposition of organic matter in the
sediment and produce excess of toxic compounds like ammonia. In addition abnormal
algal growth (eutrophication) may cause stress to the animal and ultimately end with
microbial diseases and high mortality. Moreover, the effluent from shrimp ponds is
often a water quality hazard, due to higher organic loading. Very limited research has
been carried out on the culture, growth performance and disease management of L.
vannamei. The benefit of probiotics will be long lasting, and the application of
probiotics will become a major field in the development of aquaculture. Probiotics in
aquaculture have been shown to have several modes of action: competitive exclusion
of pathogenic bacteria through the production of inhibitory compounds, improvement
of water quality by detoxicating NH3 and NO2 and enhancement of immune response
of host species; and enhancement of nutrition of host species through the production
of supplemental digestive enzymes (Verchuere et al., 2000; Velmurugan and
Rajagopal., 2009). The present study was aimed to determine the probiotic

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R Karthik, Angelin C Pushpam, and M. C. Vanitha and D. Yuvaraj

effectiveness of Bacillus stratosphericus (AMET1601), Arthrobacter sp.
(AMET1852) and Lactobacillus sp (AMET1506) on Litopenaeus vannamei culture
for the prevalence of white spot syndrome virus (WSSV) under laboratory scale
conditions.

2. MATERIAL AND METHODS

2.1. Isolation and identification of bacterial strains
The bacterial strains namely Bacillus stratosphericus (AMET1601) Arthrobacter
defluvii sp (AMET1852) and Lactobacillus sp (AMET1506) were isolated from the
marine sediment samples and identified by biochemical examination using Bergeys
Manual of Determinative Bacteriology (1989) and also by 16s rRNA sequencing.

2.2. Antibacterial activity of bacterial strains

The antimicrobial activity of all the three bacterial strains was determined by testing
against different seafood borne bacterial pathogens (E.coli, V. cholerae, V.
parahaemolyticus, V. harveyi, V. alginolyticus, V. fischeri, Salmonella sp. and
Shigella sp. (previously isolated from infected seafood samples) and the antagonistic
activity between the bacterial strains also determined by agar well diffusion assay
(Schillinger and Lucke 1989, Karthik et al., 2013).

2.3. Mass culture of bacterial strains and preparation of probiotic feed

The bacterial strains namely Bacillus stratosphericus (AMET1601) Arthrobacter
defluvii sp (AMET1852) and Lactobacillus sp (AMET1506) were separately grown in
nutrient broth in a shaking incubator at 30C for 24 hours. After the incubation
period, the cells were harvested by centrifuging at 2000 rpm and the obtained pellet
was washed twice with phosphate-buffered saline (pH 7.2) and re-suspended in the
same buffer. Then, the absorbance at 600 nm was adjusted to 0.25 0.05 in order to
standardize the number of bacteria (105 CFU mL-1) by dilution plating method. The
commercial shrimp feed (Blanca feed pellets, obtained from CP Aquaculture India Pvt
Ltd) was taken for the supplementation of each bacterial strains. In order to reach a
final concentration (105 CFU g-1) the bacterial suspension was slowly sprayed onto
the feed for mixing, the feeds were named as Feed 1, Feed 2 and Feed 3 respectively
according to the bacterial strains. The amount of bacterial load in the feed was
determined by standard plate count method (Ajitha et al., 2004; Karthik et al., 2014).

2.4. Shrimp acclimation for experimental conditions

The healthy shrimps (PL 25) (confirmed negative for the White Spot Syndrome Virus
(WSSV) were collected from a commercial shrimp hatchery located in Marakanam,
Kanchipuram District, Tamil Nadu, India. The shrimps were acclimated to culture
conditions for five days in 120-L indoor plastic tanks. During the first five days of
experimental condition, animals were fed with commercial feed only. Two bioassays
were conducted to evaluate the effect of feed supplemented with different probiotic
bacteria to evaluate in terms of growth performance, survival, immune response and
prevalence of WSSV in experimental shrimp.

2.5. Bioassay I
Total four trials (sub-trials under each trial total 12 trials) were conducted in aerated
120-L indoor plastic tanks containing 80 L of filtered (20 m) sea water (34 to 35 g/l)

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Development of Marine Derived Probiotic Bacterial Consortium For The Sustainable

Management of Litopenaeus Vannamei Culture

and constant aeration in groups of 50 animals per tank. All treatments including
controls had three replicates. The first bioassay was conducted for 60 days with
shrimp weighing 1.4 0.31 g. At days six to 60, animals were fed with a probiotic
bacteria supplemented feed (mixture of two or three probiotic bacteria at ratio 1:1 at a
final concentration of 1x106 cfu mL1, 5.0106 cfu mL1 and 1.0107 cfu mL1) and
one control against each without any probiotic (Table 1). Shrimp were fed twice daily
at 8:00 and 16:00 hrs. Half of the water was exchanged at day three and the uneaten
food and waste matter were removed daily before feeding. Every two weeks, 20
shrimps were scoped out randomly for body weight measurement and shrimp survival
was measured in each treatments. The growth parameters were calculated according
to Robertson et al. (2000), Felix and Sudharsan (2004) and Venkat et al. (2004):

Weight gain (g/shrimp) = Final weight (g) Initial weight (g)

Weight gain (%) = Final weight (g) initial weight (g) X 100
Initial weight (g)

Food conversion ratio (FCR) = Total feed g iven (g)

Wet weight gain (g)

Daily weight gain (DWG; g/days ) = Final weight (g) x initial weigh t (g)
Days

Yield of shrimps (g) = Mean body weigth (g) x Total viable shrimps at harvest

Survival rate (%) =Total number of larvae survived

Initial number of larvae stocked X100
Table 1 Experimental setup for probiotics administration (Bioassay I)

Treatments
T-1
T2

T3

T4

T-5

Feed incorporated with mixture of

Experiments
multi probiotic isolates @ ratio 1:1
Commercial feed
Control
Bacillus stratosphericus (AMET1601) + E - 1
Arthrobacter sp (AMET1852)
E-2
E-3
Bacillus stratosphericus (AMET1601) + E - 4
Lactobacillus sp (AMET1506)
E -5
E-6
Lactobacillus sp (AMET1506) + E - 7
Arthrobacter sp (AMET1852)
E-8
E-9
Bacillus stratosphericus (AMET1601) + E -10
Lactobacillus sp (AMET1506) + E - 11
Arthrobacter sp (AMET1852)
E - 12

Dose cfu
mL-1
1x106
3106
5106
1x106
3106
5106
1x106
3106
5106
1x106
3106
5106

2.6. Bioassay II
The second bioassay was conducted for 21 days with shrimp weighing 12.8 1.8 g.
During the first seven days of experimental condition, animals were fed two
treatments: I) shrimp fed with commercial feed (control group); II) shrimp fed with
Bacillus stratosphericus (AMET1601) + Arthrobacter sp (AMET1852) +
Lactobacillus sp (AMET1506) incorporated feed (Table 2). At day eight, animals
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R Karthik, Angelin C Pushpam, and M. C. Vanitha and D. Yuvaraj

from all treatments were fed with only 1 g per tank of muscle shrimp paste positive
for WSSV (one-step PCR). From day nine to 21 animals were fed as the first seven
days. At the end of the bioassay, shrimp survival was determined as a percentage and
shrimp were weighed.
Table 2 Experimental setup for probiotics administration and wssv prevalence (Bioassay II)
Treatments
T-1
T-2

Feed incorporated with mixture of

multi probiotic isolates @ ratio 1:1
Commercial feed
Bacillus stratosphericus (AMET1601) +
Lactobacillus sp (AMET1506) +
Arthrobacter sp (AMET1852)

Experiments
Control
E -10
E - 11
E - 12

Dose cfu
mL-1
1x106
3106
5106

2.7. Water quality parameters

Water parameters were measured and maintained during both bioassays remained
within the optimum ranges for shrimp using standard protocols (Boyd and Tucker,
1998; Golterman and Clymo (1969); Wetzel and Likens (1979); APHA (1999).

2.8. Microbiological analysis

Shrimps and the bottom water samples were taken from from all the control and
experimental tanks. Total heterotrophic bacteria (THB) and Vibrio sp load in the
shrimp intestine and culture water was enumerated by growth on Zobell Marine agar
and TCBS agar (Himedia, India) respectively (Sivakumar et al., 2012; Karthik et al.,
2013; Akponah et al., 2014).
3. RESULTS AND DISCUSSION
In shrimp farming, the passage and permanent existence of large amounts of
antibiotics in the environment of water and sediments also have the potential to affect
the presence of the normal flora and plankton in those niches, resulting in shifts in the
diversity of the microbiota. In this view, the development of non antibiotic agents for
health management in shrimp farming is felt necessary. To date, probiotics can be
considered a valid alternative to the use of antibiotics in aquaculture, to prevent high
mortality and to improve welfare and promote growth and survival (Talpur et al.,
2012; Bestha Lakshmi et al., 2013). In this, study all the three potentially selected
probiotic bacterial strains viz., Bacillus stratosphericus (AMET1601) Arthrobacter sp
(AMET1852) and Lactobacillus sp (AMET1506) have shown a strongest antibacterial
activity towards all the tested bacterial pathogens such as, E.coli, V. cholerae, V.
parahaemolyticus, V. harveyi, V. alginolyticus, V. fischeri, Salmonella sp. and
Shigella sp. Far et al., 2009 as also suggested that the use of Bacillus sp as a probiotic
in shrimp culture will colonize both the culture water and the shrimp digestive tract
and also replace Vibrio spp. in the gut of the shrimp, thereby increasing shrimp
survival. Abrashev et al. (1998), have reported that, some Arthrobacter species have
the ability to produce a number of valuable substances like amino acids, vitamins,
enzymes, specific growth factors, and polysaccharides and its possess many
advantageous properties and nutritional benefits to be probiotics in aquaculture (Li et
al., 2006). Recently, Amnah, (2013) have reported that the Arthrobacter sp. can be
regarded as a probiotic bacterium for the culture of shrimp while -1,3 glucan and,
Moringa oleifera leaf were considered as immunostimulants for cultured of shrimp
Penaeus indicus Juvenile against pathogenic vibrios Juvenile. Natesan et al., 2012
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Development of Marine Derived Probiotic Bacterial Consortium For The Sustainable

Management of Litopenaeus Vannamei Culture

also observed the maximum zone of inhibition (16mm) against V. alginolyticus using
their strain L. acidophilus 04. The previous authors also described that, the
antibacterial activity of Lactobacillus sp against the pathogenic microbes may be due
to the production of its metabolites such as, organic acids (lactic and acetic acid),
hydrogen peroxide, diacetyl and bacteriocins (Valenzuela et al., 2010).
In the two last decades, many studies reported promising results using a single
beneficial bacterial strain in the culture of many finfish species (Avella et al., 2010a)
Looking for novel approach, during present study the was aimed to determine the
probiotic effectiveness three different bacterial mixtures of probiotic bacteria such as,
of Bacillus stratosphericus (AMET1601) Arthrobacter defluvii sp (AMET1852) and
Lactobacillus sp (AMET1506) on Litopenaeus vannamei culture, under laboratory
scale experimental conditions. The use of probiotics in aquaculture might represent a
valuable mechanism to increase shrimp growth and survival rate. In general, the
gastro intestinal tract (GIT) of the aquatic animal is mainly composed of gram
negative bacteria (Vine et al., 2006). Hence, the incorporation of beneficial gram
positive (probiotic) bacteria in feed can modify the gastro intestinal tract (Vieira et al.,
2007). Lactic acid bacteria and other probiotics have been shown to be beneficial for
aquaculture in terms of growth when compared with normal controls (Ten-Doeschate
and Coyne, 2008) However, wide range research on probiotics has been done on
health benefits of organisms against pathogens (Chabrilln et al., 2006; Lategan et al.,
2004) and some of their research results has not shown any positive effects on the
growth parameters or survival rate (Jeevan Kumar et al., 2013). Moreover, there are
no much reports on a mixture of probiotic bacteria against pathogens in aquaculture.
Thus, in this study, all the three probiotic bacterial strains were incorporated into the
feeds at four different combinations in the range of three different concentrations and
fed to the shrimps in the experimental tanks and the control diet was fed to the
shrimps in control tanks.
The first bioassay was carried out for 60 days. During the culture period, the water
temperature (26.02 0.03C), oxygen (5.82 0.19 mg/l), pH (8.15 0.42), salinity
(35.10 0.1), total ammonium concentration (0.77 0.01), nitrites (0.06 0.00) and
nitrates (0.68 0.05 mg/l) were maintained at optimum. After 60 days of culture,
there were significance differences in initial mean weight (10.20.20 g), Mean weight
gain (6.1717 g), FCR (2.650.07), DWG (0.680.05 g/days), Yield (265.3115.11
g) and Survival rate (86.11.18) in Treatment T-5, where the shrimps fed with all the
three different probiotic strains (Bacillus stratosphericus (AMET1601) +
Lactobacillus sp (AMET1506) + Arthrobacter sp (AMET1852)) incorporated feed (in
the range of 5106 cfu mL) followed by T-3, T-4, T-2 and control groups (Table 3 &
4).

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R Karthik, Angelin C Pushpam, and M. C. Vanitha and D. Yuvaraj

Table 3 Initial mean weight (g), Final mean weight (g), Mean weight gain (g) and Mean
weight gain (%) of L. vannamei larvae fed with different feeds incorporated with mixture of
multi probiotic isolates at various doses

Treatments Experiments

Dose
cfu mL-

Initial mean
weight (g)

T1
T2

Control
E-1
E-2
E-3
E-4
E -5
E-6
E-7
E-8
E-9
E -10
E - 11
E - 12

T3

T4

T5

1x106
3106
5106
1x106
3106
5106
1x106
3106
5106
1x106
3106
5106

4.010.13
4.010.14
4.020.11
4.020.14
4.010.11
4.020.34
4.020.21
4.010.26
4.010.22
4.030.33
4.010.13
4.010.12
4.030.22

Final mean
weight (g)
6.40.1
7.60.2
8.50.2
8.20.07
8.10.11
8.20.21
9.190.11
7.50.15
8.20.25
8.70.1
8.40.1
9.40.3
10.20.20

Mean
weight gain
(g)
2.3411
3.5922
4.4810
4.1933
4.0918
4.1826
5.1733
3.4928
4.1911
4.670.8
4.3915
5.390.7
6.1717

Mean weight
gain (%)
59.6222
89.5211
111.4408
104.2216
101.9931
103.9817
128.6033
87.0311
104.4823
115.8844
109.4728
134.4132
153.1018

Table 4 FCR (g), DWG (g/days), yield of shrimps (g) and survival (%) of L. vannamei larvae
fed with different feeds incorporated with mixture of multi probiotic isolates at various doses
Treatments

Experiments

T1
T2

Control
E-1
E-2
E-3
E-4
E -5
E-6
E-7
E-8
E-9
E -10
E - 11
E - 12

T3

T4

T5

Dose
cfu
mL-1
1x106
3106
5106
1x106
3106
5106
1x106
3106
5106
1x106
3106
5106

FCR
(g)

DWG
(g/days)

Yield of
Shrimps (g)

Survival
(%)

3.800.06
3.840.03
3.520.07
3.080.05
3.880.09
3.110.02
2.850.05
3.940.02
3.710.04
3.580.08
2.880.02
2.770.05
2.650.07

0.420.12
0.500.02
0.560.04
0.540.13
0.540.11
0.540.02
0.610.05
0.500.05
0.540.11
0.580.16
0.560.07
0.620.09
0.680.05

42.1211.13
114.8812.22
152.3218.12
150.8414.11
134.9712.10
146.318.12
191.2916.19
108.1912.14
138.2713.16
163.4514.16
171.2118.14
215.616.12
265.3115.11

36.55.92
64.21.74
68.52.66
72.31.52
66.23.88
70.34.11
74.51.88
62.32.66
66.82.11
70.61.88
78.321
80.53.5
86.11.18

In general, among the aquatic pathogens vibrio species are highly dangerous and it
will detached with shrimp epithelium and affect highly by eliminating the two layers
which protects the shrimp from infections and finally end with high mortality (Martin
et al. 2004). Normally, probiotics may prevent the pathogens from the shrimp gut by
production of antimicrobial compounds (Balcazar et al., 2006a). In this study, while
checking the Vibrio sp load in all the experimental and control groups, it was totally
reduced in the T-4 treatment at all the three different dose cfu mL-1, and not even a
single colony was observed in 15th day while fed with concentration of 5106 cfu mL-1
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Development of Marine Derived Probiotic Bacterial Consortium For The Sustainable

Management of Litopenaeus Vannamei Culture

probiotic mixtures incorporated feed (Table 5). The previous study by several authors
also stated that the aapplication of probiotics in aquaculture has yielded to positive
effects, mainly in survival and growth rates (Avella et al., 2010b; Carnevali et al.,
2006; 2004; Gatesoupe, 2008; Wang et al., 2008).
The previous study showed that supplementation of the commercial lactic acid
producing Bacillus probiotic significantly increased the survival rate of Indian white
shrimp (Fenneropenaeus indicus) in the treatments over the controls (Ziaei-Nejad et
al., 2006). In Penaues monodon, a probiotic Bacillus, was able to colonize in both the
culture water and the shrimp digestive tract, thereby increasing the black tiger shrimp
survival (Rengpipat et al., 1998). However, Shariff et al. ((2001) found that treatment
of P. monodon with a commercial Bacillus probiotic did not significantly increase
survival. Furthermore, the probiotic, Bacillus coagulans SC8168, supplemented as
water additive could significantly increased survival rate of shrimp Penaeus vannamei
larvae (Zhou et al., 2009).
Table 5 Detection of Vibrio sp in culture water and larvae during probiotic treatments

Treatments

Experiments

T-1
T-2

Control
E-1
E-2
E-3
T-3
E-4
E -5
E-6
T-4
E-7
E-8
E-9
T-5
E -10
E - 11
E - 12
Note: W- Water; L- Larvae

15th
Day

Dose
cfu mL1x106
3106
5106
1x106
3106
5106
1x106
3106
5106
1x106
3106
5106

W
+
+
+
+
+
+
+
+
+
+
+
+
-

L
+
+
+
+
+
+
+
+
+
+
+
+
-

30th
Day
W
+
+
+
+
+
+
-

L
+
+
+
+
+
+
-

45th
Day
W
+
-

L
+
-

60th
Day
W
+
-

L
+
-

The first bioassay results indicates that, incorporating a mixture (consortium) of

Bacillus stratosphericus (AMET1601),
Lactobacillus sp (AMET1506) and
Arthrobacter sp (AMET1852) as probiotics in shrimp feed will definitely provide
supplementary support against the unfavorable conditions or pathogen attacks during
the culture and increased significant survival and yield. Other studies previously
demonstrated enhanced protection with multi-species probiotics (Timmerman et al.,
2007; Zoppi et al., 2001), based on the theory that multiple species-specific benefits
possessed broaden spectrum of probiotic effect. Indeed, three LAB probiotics were
effective against Vibrio harveyi, V. parahaemolyticus and Pseudoalteromonas
piscicida in an in vitro assay (Talpur et al., 2012). The higher survival of shrimp fed
with probiotic supplemented feed might be related to an immune reactive effect of
probiotics on the host immune system, by producing extracellular compounds to
stimulate the non specific immune response in vertebrates (Marteau et al., 2002; Gill,
2001).

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R Karthik, Angelin C Pushpam, and M. C. Vanitha and D. Yuvaraj

WSSV is a large dsDNA virus infecting crustaceans and is the most important
viral pathogen of cultured penaeid shrimp worldwide. In cultured shrimp, WSSV
causes a cumulative mortality of up to 100% within 3-10 days. Due to its rapid spread
and high associated mortality rates, WSSV is an extremely virulent pathogen in
shrimp culture (James et al., 2010). Hence, in this study, the second bioassay was
conducted for 21 days with shrimp weighing 12.8 1.8 g. After acclimation of eight
days, animals from all treatments were fed with only 1 g per tank of muscle shrimp
paste positive for WSSV and confirmed positive for the White Spot Syndrome Virus
(Fig: 1 & 2). During the second bioassay, the water temperature was found between
28.91 0.01C, oxygen between 5.02 0.10mg/l, pH between 8.10 0.53, and
salinity between 35.10 0.1%. The total ammonium concentration was 0.76
0.01mg/l, nitrites 0.18 0.01mg/l, and nitrates between 0.62 0.07mg/l.

Figure 1 Amplification plot showing WSSV negative

Figure 2 Melt Cure showing WSSV negative

While checking the vibrio load in the culture water and shrimp (in both control
and experimental groups) on 7th, 14th and 21st day, the higher Vibrio load was
observed in shrimp intestine and culture water where the shrimps fed with control
diet, and it was decreased in treatment (T- II), where the shrimps were fed with all

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Development of Marine Derived Probiotic Bacterial Consortium For The Sustainable

Management of Litopenaeus Vannamei Culture

the three different probiotic strains (Bacillus sp (AMET1601) + Lactobacillus sp

(AMET1506) + Arthrobacter sp (AMET1852)) incorporated feed (in the range of
5106 cfu mL). After 21 days of culture, the maximum shrimp survival (46%) was
observed in treatment II comparatively than control (28%), and the shrimps from both
control and experiments were confirmed with WSSV positive (Fig 3 & 4). From the
results, it has been concluded that the, the consortium of these three potential
beneficial probiotic bacterial consortium have not eliminated the WSSV infection in
cultured shrimps, but increased the survival rate and decreased the vibrio load in the
culture systems and water. A perusal of literature revealed thus far no reports on the
effect of incorporating a mixture (consortium) of Bacillus sp (AMET1601),
Lactobacillus sp (AMET1506) and Arthrobacter sp (AMET1852) as probiotics in
shrimp feed and WSSV prevalence in L. vannamei or other penaeid shrimp.

Figure 3 Amplification plot showing WSSV positive

Figure 4 Melt Cure showing WSSV positive

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R Karthik, Angelin C Pushpam, and M. C. Vanitha and D. Yuvaraj

The employment of Bacillus stratosphericus (AMET1601) in the present study is
novel in the sense that it is distributed in two environmental extremes viz the
stratospherical layer of air (Shivaji et al., 2006) and in the deep region of seas (Lima
et al., 2013). Hence, this is the first report on the probiotic effect of these three
marine derived bacterial strains on L. vannamei culture. Similarly, Partida Arangure et
al., 2013, reported that, inulin and probiotic bacteria increased the shrimp survival of
100% and a decrease in the prevalence of WSSV (22.2%) in shrimp fed inulin (8.0
g/kg feed) and bacteria (1 x 105 CFU/g feed) compared with control (44.4 and
51.8%). However, study of the antiviral activity of the probiotics is an upcoming
research which needs a deep insight. Further research works should be needed to
adapt the WSSV to the BHK cell lines, to study their antiviral efficacy and cytopathic
effect (CPE) in the BHK cell lines for the qualitative assessment of antiviral efficacy
of the probiotic bacteria strains used in this study.

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