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Veterinary Microbiology: Subuntith Nimrat, Sunisa Suksawat, Traimat Boonthai, Verapong Vuthiphandchai
Veterinary Microbiology: Subuntith Nimrat, Sunisa Suksawat, Traimat Boonthai, Verapong Vuthiphandchai
Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic
Department of Microbiology and Environmental Science Program, Faculty of Science, Burapha University, Chon Buri, 20131, Thailand
Environmental Science Program, Faculty of Science, Burapha University, Chon Buri, 20131, Thailand
Biological Science Program, Faculty of Science, Burapha University, Chon Buri, 20131, Thailand
d
Department of Aquatic Science, Faculty of Science, Burapha University, Chon Buri, 20131, Thailand
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 15 January 2012
Received in revised form 18 April 2012
Accepted 20 April 2012
Keywords:
Bacillus
Probiotics
Litopenaeus vannamei
Growth
Water quality
1. Introduction
White shrimp (Litopenaeus vannamei) are commercially
cultivated in Thailand since 2001 to replace Penaeus
monodon destroyed by viral disease. Production of white
shrimp reached nearly 400,000 tons in 2006 and represented over 98% of total shrimp production in Thailand
(Wyban, 2007). Marine shrimp aquaculture in Thailand has
suffered also from opportunistic and pathogenic bacteria
* Corresponding author. Tel.: +66 38 103 120; fax: +66 38 393 490.
E-mail address: subunti@buu.ac.th (S. Nimrat).
0378-1135/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2012.04.029
444
445
2.6.1. Experiment 1
This experiment evaluated the effect of Bacillus
probiotics on larval stages. The experimental design was
completely randomized with 7 treatments in triplicate.
Each treatment was randomly assigned to triplicate glass
tanks (width length height = 0.2 m 0.4 m 0.25 m)
wrapped with black plastic to reduce light intensity. Each
tank was stocked with zoea stage 3 of shrimp (n = 1000;
100 zoea l 1). Water (10 l) was ltered through the cotton
lter at about 1 l min 1. An equal amount of each probiotic
species (109 CFU g 1) in forms of freeze-dried Bacillus (FB) or
microencapsulated Bacillus (MB) (T1T2 for probiotics A and
T4T5 for probiotics B) was introduced daily into culture
water to maintain a nal concentration of 109 CFU ml 1.
Shrimp larvae were fed with enriched Artemia (T3 and T6) or
nonenriched Artemia (T1T2, T4T5 and control) at a rate of
34 nauplii per shrimp larvae four-times daily at 6:00,
12:00, 18:00 and 24:00 h. Culture water was not discharged
during the experiment and Chaetoceros was added daily into
culture water at a xed 106 cells ml 1 in all treatments. The
salinity and temperature of culture water were maintained
at a range of 3031% and 3132 8C, respectively. Shrimp
survivals were recorded daily. At the 4th day after beginning
of the experiment, larval shrimp were randomly measured
for weight and length and bacterial numbers were
enumerated.
The enumeration of bacteria was performed in triplicate in shrimp at the same interval of growth and survival
determination. At each sampling, twenty shrimp larvae or
postlarvae were sampled from each tank, immersed
individually in 0.05 g l 1 formalin solution for 5 min to
eliminate the external bacteria and washed thoroughly
using sterilized water for 1 min to remove the disinfectant.
Whole shrimp were separately homogenized in 1 g l 1
Peptone Water and diluted serially by 10 fold-dilution
method with the same buffer solution. One hundred
milliliter of each dilution was plated onto PCA supplemented with 10 g l 1 NaCl for determination of total
heterotrophic bacteria (THB) and total Bacillus numbers by
spread plate standard method (Boonthai et al., 2011;
Nimrat et al., 2011). Water samples (50 ml per tank) were
collected from each of the four edges and the center of each
tank and evaluated for THB and total Bacillus numbers as
previously described. All petri dishes were incubated at
30 8C, 2448 h (Nimrat et al., 2008, 2011). All colonies of
Bacillus were identied using Gram staining, spore
staining, catalase test and other selected biochemical tests
described by Krieg and Holt (1984). All bacterial colonies
and Bacillus colonies were calculated as a colony forming
unit of THB and total Bacillus numbers, respectively.
2.9. Water quality analysis
2.6.2. Experiment 2
The effect of Bacillus probiotic on postlarval (PL) stages
was examined in Experiment 2. Mysis 3 obtained from the
Experiment 1 were reared until reaching PL 1 stage. PL 1
shrimp were reared in the same glass tanks at a density of
500 per tank (50 postlarvae l 1). The experiment was
divided into 7 treatments as previously described. The
treatments and control were repeated in triplicate. One to
fourteen-day-old and fteen to twenty two-day-old PL
shrimp were fed nonenriched Artemia at 810 and 1520
nauplii per postlarvae, respectively, six-time daily at 6:00,
446
Table 1
Growth and survival of treated and untreated PL shrimp.
Treatment
Control
T1
T2
T3
T4
T5
T6
Survival
rate (%)
Initial
length
(mm)
Final
length
(mm)
Length
gain (%)
Initial
weight
(mg)
Final
weight
(mg)
Weight
gain (%)
ADG
(mg day
70.8 0.8b
80.7 0.6a
80.9 1.0a
81.1 0.8a
83.9 1.5a
84.3 3.1a
84.7 3.5a
4.5 0.1a
4.4 0.1a
4.5 0.1a
4.5 0.1a
4.5 0.0a
4.5 0.1a
4.5 0.0a
19.9 0.8c
22.2 0.5b
21.9 0.6b
22.2 0.6b
23.7 0.7a
23.6 0.7a
23.5 0.7a
347.8 16.9c
398.5 10.4b
393.2 13.9b
396.1 15.8b
431.8 12.5a
431.3 12.6a
432.3 12.8a
0.2 0.0a
0.2 0.0a
0.2 0.0a
0.2 0.0a
0.2 0.0a
0.2 0.0a
0.2 0.0a
20.1 0.7c
24.4 0.8b
24.3 0.8b
24.6 0.8b
25.9 0.4a
25.6 0.6a
25.4 1.1a
8,852.7 447.3c
10,710.4 345.8b
10,658.9 335.4b
10,795.6 318.0b
10,987.0 248.8a
11,256.7 462.7a
11,032.7 283.7a
0.8 0.0c
1.0 0.0b
1.0 0.1b
1.0 0.0b
1.1 0.0a
1.1 0.0a
1.1 0.0a
SGR (%)
1
)
18.6 0.1c
19.4 0.0b
19.4 0.1b
19.3 0.1b
19.6 0.1a
19.6 0.1a
19.5 0.0a
Data were expressed as mean S.D. Means with different superscript indicate signicant difference (P < 0.05). T1: addition of mixed freeze-dried Bacillus (FB) of
probiotic A, T2: addition of mixed microencapsulated Bacillus (MB) of probiotic A, T3: addition of artemia enriched with mixed MB of probiotic A, T4: addition of
mixed FB of probiotic B, T5: addition of mixed MB of probiotic B, T6: addition of artemia enriched with mixed MB of probiotic B and control: no addition of
probiotics, ADG: average daily weight gain, SRG: specic growth rate.
Control
Zoea 3 (at beginning
of Experiment 1)
Data were expressed as mean S.D. Means with different superscript at the same shrimp stage indicate signicant difference (P < 0.05). T1: addition of mixed freeze-dried Bacillus (FB) of probiotic A, T2: addition of mixed
microencapsulated Bacillus (MB) of probiotic A, T3: addition of artemia enriched with mixed MB of probiotic A, T4: addition of mixed FB of probiotic B, T5: addition of mixed MB of probiotic B, T6: addition of artemia
enriched with mixed MB of probiotic B and control: no addition of probiotics.
0(c)
10.9 1.3(a)
10.5 1.5(a)
0.9 0.1(b)
11.4 2.9(a)
10.2 1.6(a)
1.0 0.2(b)
0(c)
9.2 0.9 104(a)
8.0 1.0 104(a)
6.3 1.1 102(b)
1.3 0.6 105(a)
7.9 1.3 104(a)
6.7 1.6 102(b)
4.2 0.9 104(c)
8.7 1.1 105(a)
2.3 1.2 106(a)
8.8 0.9 104(b)
9.2 1.1 105(a)
7.9 0.1 105(a)
9.7 1.1 104(b)
0(b)
7.8 1.0 105(a)
9.0 1.7 105(a)
2.3 0.1 106(a)
8.7 0.9 105(a)
1.5 0.1 106(a)
8.9 0.2 105(a)
5.7 1.5 105(b)
3.4 1.6 106(a)
4.5 1.3 106(a)
6.0 0.8 106(a)
3.9 1.1 106(a)
4.7 1.1 106(a)
5.3 1.0 106(a)
Control
T1
T2
T3
T4
T5
T6
0(b)
26.8 2.3(a)
27.5 3.1(a)
21.7 2.0(a)
32.8 3.5(a)
23.0 4.6(a)
25.8 3.6(a)
13.3 1.3(a)
14.3 3.8(a)
0.4 0.1(b)
15.2 4.1(a)
14.8 0.4(a)
0.5 0.1(b)
7.0 1.6 104(a)
8.2 1.6 104(a)
2.8 0.6 102(b)
8.7 1.0 104(a)
6.9 1.2 104(a)
4.3 1.2 102(b)
5.3 1.3 105(a)
6.2 1.3 105(a)
7.4 1.1 104(b)
6.0 1.3 105(a)
4.9 1.2 105(a)
6.0 1.1 104(b)
5.4 0.8 103(a)
3.9 0.7 103(a)
4.0 0.7 103(a)
6.2 0.9 103(a)
5.9 0.9 103(a)
4.8 0.9 103(a)
2.0 1.0 105(a)
4.2 0.9 105(a)
3.0 1.1 105(a)
3.7 1.1 105(a)
1.6 0.9 105(a)
4.5 0.8 105(a)
T1
T2
T3
T4
T5
T6
0.8 0.1(a)
0.2 0.0(a)
0.5 0.0(a)
0.6 0.0(a)
0.8 0.1(a)
0.4 0.1(a)
0(c)
0(c)
3.7 0.0 104(c)
0(b)
0(b)
2.1 0.1 104(b)
Culture water
Larval shrimp
Treatments
Duration (days)
Table 2
THB and Bacillus numbers of treated and untreated larval shrimp and culture water.
Bacillus number
(CFU larvae 1)
THB number
(CFU ml 1)
Bacillus number
(CFU ml 1)
447
448
Table 3
THB and Bacillus numbers of treated and untreated PL shrimp and culture water.
Duration (days)
Treatments
Postlarval shrimp
THB number
(CFU postlarvae
Culture water
)
Bacillus number
(CFU postlarvae 1)
Bacillus number
(CFU ml 1)
Control
T1
T2
T3
T4
T5
T6
0(b)
2.1 0.6 106(a)
4.3 1.3 106(a)
1.9 0.8 106(a)
3.8 1.1 106(a)
4.0 1.0 106(a)
3.6 0.8 106(a)
0(b)
53.7 5.5(a)
62.0 6.1(a)
51.5 4.3(a)
55.9 5.3(a)
58.3 2.7(a)
54.4 3.9(a)
0(c)
8.3 1.1 104(a)
1.1 0.5 105(a)
9.7 1.4 102(b)
9.0 1.0 104(a)
7.7 1.1 104(a)
7.8 1.3 102(b)
0(c)
16.4 1.8(a)
18.4 2.0(a)
5.2 0.6(b)
16.5 2.8(a)
17.2 3.6(a)
4.5 0.8(b)
14
Control
T1
T2
T3
T4
T5
T6
0(b)
7.1 0.9 106(a)
6.6 1.7 106(a)
5.0 1.2 106(a)
4.9 0.9 106(a)
9.7 1.0 106(a)
8.4 1.1 106(a)
0(b)
82.0 6.2(a)
80.2 5.2(a)
74.7 4.2(a)
76.8 3.8(a)
73.1 5.1(a)
73.8 4.5(a)
0(b)
9.2 1.2 106(a)
8.8 0.8 106(a)
7.2 1.0 106(a)
6.7 1.9 106(a)
7.7 1.2 106(a)
6.3 0.8 106(a)
0(b)
42.3 2.5(a)
54.7 4.5(a)
37.8 3.5(a)
43.0 6.5(a)
47.8 4.6(a)
34.3 3.9(a)
22
Control
T1
T2
T3
T4
T5
T6
0(b)
7.3 0.8 106(a)
6.1 1.2 106(a)
7.8 0.9 106(a)
6.9 1.1 106(a)
8.7 1.3 106(a)
9.2 0.9 106(a)
0(b)
76.0 7.2(a)
85.7 3.2(a)
81.1 5.6(a)
76.6 4.3(a)
80.2 6.4(a)
79.6 7.8(a)
0(b)
7.0 1.2 106(a)
4.1 1.3 106(a)
7.7 0.7 106(a)
7.3 1.0 106(a)
6.5 1.7 106(a)
5.4 0.5 106(a)
0(b)
49.9 2.8(a)
51.9 0.6(a)
45.0 1.6(a)
50.8 1.2(a)
50.6 3.6(a)
42.5 2.3(a)
Data were expressed as mean S.D. Means with different superscript at the same shrimp stage indicate signicant difference (P < 0.05). T1: addition of mixed freeze-dried Bacillus (FB) of probiotic A, T2: addition of mixed
microencapsulated Bacillus (MB) of probiotic A, T3: addition of artemia enriched with mixed MB of probiotic A, T4: addition of mixed FB of probiotic B, T5: addition of mixed MB of probiotic B, T6: addition of artemia
enriched with mixed MB of probiotic B and control: no addition of probiotics.
449
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