DEVELOPMENT OF MARINE DERIVED PROBIOTIC BACTERIAL ...€¦ · culture period is significantly...

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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

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ISSN Print: 0976-6480 and ISSN Online: 0976-6499

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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 5×106

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 didn’t eliminate the WSSV in

cultured shrimps, but increase the survival rate and decrease the vibrio load

in the culture systems and water.

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

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 Bergey’s

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 30°C 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 μm) sea water (34 to 35 g/l)




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 mL−1

, 5.0×106 cfu mL

−1 and 1.0×10

7 cfu mL

−1) 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 Feed incorporated with mixture of

multi probiotic isolates @ ratio 1:1 Experiments

Dose cfu

mL-1

T - 1 Commercial feed Control -

T – 2 Bacillus stratosphericus (AMET1601) +

Arthrobacter sp (AMET1852)

E - 1 1x106

E - 2 3×106

E - 3 5×106

T – 3 Bacillus stratosphericus (AMET1601) +

Lactobacillus sp (AMET1506)

E - 4 1x106

E -5 3×106

E - 6 5×106

T – 4 Lactobacillus sp (AMET1506) +


E - 7 1x106

E - 8 3×106

E - 9 5×106

T - 5 Bacillus stratosphericus (AMET1601) +

Lactobacillus sp (AMET1506) +


E -10 1x106

E - 11 3×106

E - 12 5×106

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



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 Feed incorporated with mixture of

multi probiotic isolates @ ratio 1:1 Experiments

Dose cfu

mL-1

T - 1 Commercial feed Control -

T - 2 Bacillus stratosphericus (AMET1601) +

Lactobacillus sp (AMET1506) +


E -10 1x106

E - 11 3×106

E - 12 5×106

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 β-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




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 (Chabrillón 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 ± 0.03°C), 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 (10.2±0.20 g), Mean weight

gain (6.17±17 g), FCR (2.65±0.07), DWG (0.68±0.05 g/days), Yield (265.31±15.11

g) and Survival rate (86.1±1.18) 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 5×106

cfu mL) followed by T-3, T-4, T-2 and control groups (Table 3 &

4).



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

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

Dose

cfu

mL-1

FCR

(g)

DWG

(g/days)

Yield of

Shrimps (g)

Survival

(%)

T – 1 Control - 3.80±0.06 0.42±0.12 42.12±11.13 36.5±5.92

T – 2 E - 1 1x106 3.84±0.03 0.50±0.02 114.88±12.22 64.2±1.74

E - 2 3×106 3.52±0.07 0.56±0.04 152.32±18.12 68.5±2.66

E - 3 5×106 3.08±0.05 0.54±0.13 150.84±14.11 72.3±1.52

T – 3 E - 4 1x106 3.88±0.09 0.54±0.11 134.97±12.10 66.2±3.88

E -5 3×106 3.11±0.02 0.54±0.02 146.3±18.12 70.3±4.11

E - 6 5×106 2.85±0.05 0.61±0.05 191.29±16.19 74.5±1.88

T – 4 E - 7 1x106 3.94±0.02 0.50±0.05 108.19±12.14 62.3±2.66

E - 8 3×106 3.71±0.04 0.54±0.11 138.27±13.16 66.8±2.11

E - 9 5×106 3.58±0.08 0.58±0.16 163.45±14.16 70.6±1.88

T – 5 E -10 1x106 2.88±0.02 0.56±0.07 171.21±18.14 78.3±21

E - 11 3×106 2.77±0.05 0.62±0.09 215.6±16.12 80.5±3.5

E - 12 5×106 2.65±0.07 0.68±0.05 265.31±15.11 86.1±1.18

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 5×106

cfu mL-1

Treatments Experiments

Dose

cfu mL-

1

Initial mean

weight (g)

Final mean

weight (g)

Mean

weight gain

(g)

Mean weight

gain (%)

T – 1 Control - 4.01±0.13 6.4±0.1 2.34±11 59.62±22

T – 2 E - 1 1x106 4.01±0.14 7.6±0.2 3.59±22 89.52±11

E - 2 3×106 4.02±0.11 8.5±0.2 4.48±10 111.44±08

E - 3 5×106 4.02±0.14 8.2±0.07 4.19±33 104.22±16

T – 3 E - 4 1x106 4.01±0.11 8.1±0.11 4.09±18 101.99±31

E -5 3×106 4.02±0.34 8.2±0.21 4.18±26 103.98±17

E - 6 5×106 4.02±0.21 9.19±0.11 5.17±33 128.60±33

T – 4 E - 7 1x106 4.01±0.26 7.5±0.15 3.49±28 87.03±11

E - 8 3×106 4.01±0.22 8.2±0.25 4.19±11 104.48±23

E - 9 5×106 4.03±0.33 8.7±0.1 4.67±0.8 115.88±44

T – 5 E -10 1x106 4.01±0.13 8.4±0.1 4.39±15 109.47±28

E - 11 3×106 4.01±0.12 9.4±0.3 5.39±0.7 134.41±32

E - 12 5×106 4.03±0.22 10.2±0.20 6.17±17 153.10±18




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

Note: W- Water; L- Larvae

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).

Treatments

Experiments

Dose

cfu mL-

1

15th

Day

30th

Day

45th

Day

60th

Day

W L W L W L W L

T - 1 Control - + + + + + + + +

T - 2 E - 1 1x106 + + + + - - - -

E - 2 3×106 + + + + - - - -

E - 3 5×106 + + - - - - - -

T - 3 E - 4 1x106 + + + + - - - -

E -5 3×106 + + - - - - - -

E - 6 5×106 + + - - - - - -

T - 4

E - 7 1x106 + + + + - - - -

E - 8 3×106 + + + + - - - -

E - 9 5×106 + + - - - - - -

T - 5 E -10 1x106 + + - - - - - -

E - 11 3×106 + + - - - - - -

E - 12 5×106 - - - - - - - -



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 ± 0.01°C, 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




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

(AMET1506) + Arthrobacter sp (AMET1852)) incorporated feed (in the range of

5×106

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



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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