Dietary administration of the probiotic, Saccharomyces cerevisiae P13, enhanced the growth, innate immune responses, and disease resistance of the grouper, Epinephelus coioides

Fish & Shellfish Immunology 29 (2010) 1053e1059

Contents lists available at ScienceDirect

Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Dietary administration of the probiotic, Saccharomyces cerevisiae P13, enhanced the growth, innate immune responses, and disease resistance of the grouper, Epinephelus coioides Chiu-Hsia Chiu a, **, Chih-Hsin Cheng b, Wen-Ren Gua b, Yuan-Kuang Guu a, Winton Cheng b, * a b

Department of Food Science, National Pingtung University of Science and Technology, Pingtung, Taiwan 91201, ROC Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan 91201, ROC

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 June 2010 Received in revised form 2 August 2010 Accepted 24 August 2010 Available online 8 September 2010

The percent weight gain (PWG) and feeding efficiency (FE) of Epinephelus coioides were calculated. The survival of Saccharomyces cerevisiae P13 in the posterior intestines using a specific primer pair of YMR245w-F/YMR245w-R, non-specific immune parameters of grouper, and its susceptibility to Streptococcus sp. and an iridovirus were determined when the fish were fed diets containing S. cerevisiae at 0 (control), 103, 105, or 107 colony-forming units (cfu) kg
1 for 4 weeks. Results showed that grouper fed a diet containing S. cerevisiae at the levels of 103, 105, and 107 cfu kg
1 had significantly increased PGW and FE especially in the 107 cfu kg
1 group which were 211.6% and 1.2, respectively. S. cerevisiae was able to survive in the fish posterior intestines during the S. cerevisiae feeding period. Fish fed a diet containing S. cerevisiae at 107 cfu kg
1 had significantly higher survival rates than those fed the 103 cfu kg
1 S. cerevisiae diet and the control diet after challenge with Streptococcus sp. and an iridovirus, with increased survival rates of 26.6% and 36.6%, respectively, compared to the challenge control group. The phagocytic activity, respiratory burst and superoxide dismutase (SOD) level of head kidney leucocytes as well as serum lysozyme activity and serum alternative complement activity (ACH50) of fish fed diets containing S. cerevisiae at 105 and 107 cfu kg
1 were significantly higher than those of fish fed the 103 cfu kg
1 S. cerevisiae-contained diet and the control diets after 4 weeks of feeding, and had increased by 20% and 20%, 27.6% and 19.7%, 30.5% and 36.2%, 205.8% and 169.6%, and 90.8% and 80.3%, respectively, compared to the control group. We therefore recommend dietary S. cerevisiae administration of 105 and 107 cfu kg
1 to E. coioides to promote growth and enhance immunity and resistance against Streptococcus sp. and an iridovirus especially in the 107 cfu kg
1 group. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Dietary administration Saccharomyces cerevisiae Epinephelus coioides Streptococcus sp. Iridovirus Growth Innate immunity

1. Introduction Groupers, Epinephelus spp., are one of the most important commercial mariculture fish species in Asia and around the world. Because of their hardiness in crowded environments and rapid growth, the intensive culture of grouper has dramatically developed in Taiwan. It is known that the rapid degradation of environments in intensive culture ponds may result in increased incidences of diseases that can lead to culture failure of cultured organisms. During the past few years, commercial fish farming has been severely hit by epidemics associated with viruses and bacteria,

* Corresponding author. Tel.: þ886 8 770 3202; fax: þ886 8 774 0225. ** Corresponding author. Tel.: þ886 8 770 3202; fax: þ886 8 774 0378. E-mail addresses: [email protected] (C.-H. Chiu), [email protected]. edu.tw (W. Cheng). 1050-4648/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2010.08.019

which have caused serious economic losses. Therefore, the health of fish and enhancement of their immunity are of primary concern. Probiotics are defined as live microbial or cultured product feed supplements, which beneficially affect the host by producing inhibitory compounds, competing for chemicals and adhesion sites, modulating and stimulating the immune function, and improving the microbial balance [1e3]. They have been used in aquaculture as a means of controlling disease, enhancing the immune response, supplementing or even in some cases replacing the use of antimicrobial compounds, providing nutrients and enzymatic contributions, and improving water quality [4]. A wide range of microalgae (Tetraselmis), yeasts (Debaryomyces, Phaffia, and Saccharomyces), and gram-positive (Bacillus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Streptococcus, and Weissella) and -negative bacteria (Aeromonas, Alteromonas, Photorbodobacterium, Pseudomonas, and Vibrio) have been applied as probiotics to improve aquatic animal growth, survival, health, and disease prevention [3e6].

1054

C.-H. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 1053e1059

Saccharomyces cerevisiae cell walls are constructed almost entirely of b-1,3-D-glucan, b-1,6-D-glucan, mannoproteins and chitin, bound together by covalent linkages [7]. In vertebrates, each one of these purified compounds is known to promote innate defense mechanisms and/or disease resistance [8e11]. The whole cells of S. cerevisiae, as well as its single cell wall components, can stimulate fish immune systems [12e14]. In addition, supplementation of a dietary S. cerevisiae fermentation product (DVAQUAÒ) showed no effects on growth performance, although potentially beneficial bacteria in the gut and non-specific immunity were stimulated in hybrid tilapia (Oreochromis niloticus \  Oreochromis aureus _) [15]. Non-specific immune systems are very important in the defense mechanisms of fish against pathogens and microorganisms. The objective of this study was to examine innate immune parameters such as serum lysozyme activity, serum alternative complement activity, respiratory burst activity, phagocytic activity, and superoxide dismutase (SOD) of head kidney leukocytes in grouper, Epinephelus coioides, and its resistance against Streptococcus sp. and a grouper iridovirus (GIV) following dietary administration of S. cerevisiae. In addition, the growth performance of fish and their intestinal S. cerevisiae survival were also examined.

2. Materials and methods 2.1. Preparation of the S. cerevisiae mixture and diet S. cerevisiae P13 isolated from fermented peaches were used in this study. Yeast was cultured in a sterilized 2-L flask with yeast malt (YM) broth for 24 h at 30  1  C, and then centrifuged at 8000g for 15 min at 4  C. The pellet was collected and mixed with skim milk at a ratio of 1:4, and then freeze-dried. The yeast mixture was stored at 4  C until used. The viability of the yeast mixture was determined by plate counting on YM agar. Four diets containing different doses of the S. cerevisiae P13 mixture were prepared as described in Table 1. The proximate analysis of the basal diet according to the AOAC method [16] was 48.2% crude protein, 7.0% crude lipid, 12.9% ash, and 6.2% moisture. A S. cerevisiae mixture (5.3  106 cfu g
1) was added to the basal diet at different amounts of 0.001, 0.1, and 10.0 g, resulting in 103, 105, and 107 colony-forming units (cfu) (kg diet)
1 as the respective test diets, with corresponding decreases in the amount of skim milk. The measured population levels of S. cerevisiae in the test diets were (4.9  0.5)  103, (5.1  0.3)  105, and (5.2  0.6)  107 cfu (kg diet)
1 for respective treatments with 103, 105, and 107 cfu (kg diet)
1 diets. The ingredients were ground in a Hammer mill so as to pass through an 80-mesh screen. Experimental diets were prepared as described previously [17].

Table 1 Composition of the basal diet (g kg
1) for grouper, Epinephelus coioides. Saccharomyces cerevisiae in diet (g kg
1) 0

103

105

107

Fish meal a-starch Squid cream Defat soy meal Shrimp shell meal Mineral mixturea Vitamin mixtureb Skim milk Probiotic

640 133 27 91 54 47 8 10 0

640 133 27 91 54 47 8 9.999 0.001

640 133 27 91 54 47 8 9.9 0.1

640 133 27 91 54 47 8 0 10

a

Mineral. Vitamin mix: provided by Shinta Feed Company, Pingtung, Taiwan.

In this study, a bacterial pathogen, Streptococcus sp. was provided by Dr. J. P. Shu, chairman of the Animal Health Inspection and Quarantine Institute, Pingtung County, Taiwan. The bacterium was cultured and collected by a previously described method [18] except that saline was used to adjust the bacterial suspension to 2.5  108 cfu ml
1 for the susceptibility study in E. coioides. In this study, a GIV was provided by Dr. C. Y Chang, Institute of Zoology, Academia Sinica, Nankang, Taipei, Taiwan. It was isolated and prepared according to a previously described approach [19]. The virus titer was determined using a method described by Reed and Muench [20] at 107 TCID50 ml
1 as the stock viral suspension for the susceptibility study in E. coioides. 2.3. Experimental design Grouper, E. coioides, juveniles purchased from a private farm in Pingtung, Taiwan, were shipped to our lab. The fish were acclimated indoors in a 2-ton tank with recirculating aerated seawater (25&) at 28  1  C, and fed the control diet (without S. cerevisiae) for 2 weeks before the experiment. Four studies were conducted. For the growing-out study of grouper, four diet groups (one control and three tests) were comprised of 30 fish each in triplicate, and were conducted for 4 weeks. For studies of the resistance of grouper to both Streptococcus sp. and the GIV, test and control groups were comprised of ten fish each in triplicate, and were conducted on grouper following 4 weeks of feeding of S. cerevisiaecontaining and control diets. For non-specific immune parameter examination of grouper, 24 0.5-ton FRP tanks containing 0.4 ton of aerated seawater were used for this study. Each tank was used to rear 10 fish. Four different groups containing S. cerevisiae at 0, 103, 105, and 107 cfu (kg diet)
1 were categorized, and each group consisted of six tanks (six replicate). Each replicate consisted of one fish randomly sampled from a tank at the beginning, and after 7, 14, and 28 days of feeding the S. cerevisiae-containing or control diets. In all tests, fish were fed the test diet twice daily. In general, the growing-out is more apparent and sensitive to GIV, but the immune system is rather immature and uneasy to excise the head kidney in fry and fingerling than that of juvenile fish. For handy sampling, manipulation and obtaining a more notable positive effect in the experiment, the fish average weight of 1.7  0.4 g were used for the growing-out and challenge trails and 25.8  1.5 g for the immune parameter analyses, respectively. No significant differences in weight were observed among treatments. During the experiments, 30% of the seawater was exchanged daily to maintain the water quality, and the water temperature was maintained at 27  1  C, the pH at 7.8e8.5, and the salinity at 25&. For the microbiological analysis, S. cerevisiae levels in the intestines of fish were determined after 4 weeks of feeding (at the end of the experiment). 2.4. Effect of S. cerevisiae on the growing-out of grouper

Ingredients

b

2.2. Culture of pathogens

At the beginning of the growing-out trial, grouper juveniles were randomly sampled and placed in a 60-l glass aquaria with 40 l of seawater at 25&. All aquaria were equipped with an air filter. During the growing-out trial, fish were fed the experimental diets which contained S. cerevisiae at 0, 103, 105, and 107 cfu (kg diet)
1. Fish were fed the respective diets at a rate of 5e7% body weight, and were bulk-weighed from each aquarium once every week until the end of the trial. The percent weight gain (PWG) and feed efficiency (FE) were calculated as follows: PWG ¼ [100  (final body weight
initial body weight) (initial body weight)
1] and FE ¼ [(final body weight
initial body weight) (feed intake)
1].

C.-H. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 1053e1059

2.6. Effects of S. cerevisiae on the non-specific immune parameters of grouper At the beginning, and after 7, 14 and 28 days of feeding, one fish from each tank was sampled to determine the immune parameters. In total, 78 fish were used in this study. Blood was individually withdrawn and serum was collected, and the head kidneys of fish were then excised, and the leucocytes were harvested following the method of Yeh et al. [18]. Harvested cells were adjusted to 2  106 cell ml
1 for the assay. In addition, the intestines were excised for microbiological analyses. 2.6.1. Alternative complement activity (ACH50) assay ACH50 was determined and calculated using the method of Sunyer and Tort [22] with previously described modifications [18]. The volume of serum complement producing 50% hemolysis (ACH50) was determined, and the number of ACH50 units ml
1 was calculated for each experimental fish. 2.6.2. Lysozyme activity assay Serum lysozyme activity was modified as described by Ellis [23] and Obach et al. [24]. The detailed procedures were previously described [18]. 2.6.3. Respiratory burst assay Respiratory burst activity produced by leucocytes of the head kidneys was assayed according to the method of Cook et al. [25] as previously described [18] except that the leucocyte suspension was adjusted to 2  106 cells ml
1. 2.6.4. SOD assay Leucocytes of the head kidneys were homogenized in PBS (Sigma, St. Louis, MO, USA) and centrifuged at 10,000g for 10 min at 4  C. The supernatant was transferred to a new tube previously placed on ice and immediately used for the SOD analysis. SOD activity was measured by its ability to inhibit superoxide radical-dependent

2.6.5. Phagocytic activity assays The method of phagocytic activity analysis was described previously [18]. Three hundred phagocytes were counted. Phagocytic activity, defined as the phagocytic rate (PR) or phagocytic index (PI), was expressed as: PR ¼ [100  (phagocytic leucocytes) (total leucocytes)
1] or PI ¼ [(phagocytic number of particle beads) (phagocytic leucocytes)
1]. 2.7. Survival analysis of S. cerevisiae in the intestines of grouper For the microbiological analysis, S. cerevisiae levels in the intestines of fish were determined after 4 weeks of feeding by reverse-transcription polymerase chain reaction (RT-PCR) using a specific primer pair of YMR245w-F (50 -TCTGTATATTCTGTATCTATGTTCCTGC-30 ) and YMR245w-R (50 -AAATGGCCTATTGTATTGTCAGGTC-30 ) from a specific open reading frame of the S. cerevisiae genome [27]. One centimeter of intestines was shatter in 15 ml of YM broth and cultured at 30  C for 16 h, and then centrifuged at 7155g for 15 min at 4  C. The pellet was collected and total DNA was extracted and further purified using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) following the manufacturer’s instructions.

300 Percent weight gain (%)

To confirm the effect of received a similar dose of pathogens in the treated fish, the injected challenge test was chose to evaluate the susceptibility of E. coioides to pathogens. Grouper were fed S. cerevisiae-containing diets at the concentrations of 0, 103, 105, and 107 cfu (kg diet)
1 for 4 weeks, before the challenge tests were conducted. During the challenge period, fish continued to be fed their respective diets. The bacterial and viral challenge tests were individually conducted in triplicate by respective intramuscular and intraperitoneal injections of 20 ml of a stock bacterial suspension or 60 ml of viral suspension resulting in 5.0  105 cfu (g fish)
1 and 1.0  105 TCID50 (g fish)
1. Fish that were fed the 107 cfu kg
1 S. cerevisiae-containing diet, and then received 20 ml saline and 60 ml L-15 medium served as the unchallenged control for the bacterial and viral challenge trials, respectively. Experimental fish (10 fish aquarium
1) were kept in 60-l glass aquaria containing 40 l of seawater at 25& and 27  1  C. There were five treatments (unchallenged control, and those containing S. cerevisiae at 0, 103, 105, and 107 cfu (kg diet)
1) for each challenge test. Each treatment was conducted with 30 grouper (with 10 fish in an aquarium serving as one replicate). Seawater was renewed and mortalities were counted daily for a total experimental period of 7 days. The relative percentage survival (RPS) of fish was calculated in the end of experiment according to Amend [21] using the following formula: RPS ¼ 1
[(percentage mortality in treatment) (percentage mortality in control)
1]  100

reactions using a Ransod kit (Randox, Crumlin, UK). Details of the procedures were described previously [18]. The specific activity was expressed as units of (mg protein)
1. The concentration of protein in the leucocyte suspension was determined by the Bradford method [26] using bovine serum albumin as a standard and the Bio-Rad Protein assay reagent (Bio-Rad Laboratories, Mississauga, ON, Canada).

A

Saccharomyces cerevisiae in diet (cfu kg-1) 105

103

0

107 c

200

100 c

b a b

0

c

b

7

a a

c

a a

b

14

b

a

d

21

28

Time elapsed (days) 2

B

Saccharomyces cerevisiae in diet (cfu kg-1) 0

Feed efficiency

2.5. Susceptibility of E. coioides to Streptococcus sp. and the GIV

1055

105

103

107

1.5

1

c

a a

b

c

b

ab

a c

a a

b

c

b

a a

0.5

0

7

14

21

28

Time elapsed (days) Fig. 1. Percent weight gain (%) (A) and feed efficiency (B) of Epinephelus coioides fed a control diet and Saccharomyces cerevisiae-containing diets at 103, 105, and 107 colonyforming units (cfu) (kg diet)
1. Each bar represents the mean value from six determinations with the standard error (S.E.). Data at the same sampling time with different letters significantly differ (p < 0.05) among treatments.

1056

C.-H. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 1053e1059

The primer pair, YMR245w-F/YMR245w-R, was used to amplify an S. cerevisiae DNA fragment. Amplification was performed using 1 ml of 2.5 mM dNTP mix, 2.5 ml Taq 10 buffer, 0.5 ml Taq DNA polymerase (5 U ml
1), 1 ml of each primer, 3 ml of DNA, and 40 ml DEPC-H2O in a final volume of 50 ml. The PCR was carried out in a Perkin-Elmer 9700 thermocycler (Applied Biosystems, Lincoln, NE, USA) with a denaturing step of 94  C for 5 min followed by 30 cycles of 95  C for 20 s, annealing at 55  C for 20 s, elongation at 72  C for 30 s, and extension at 72  C for 7 min, with final cooling to 4  C. The PCR fragments were subjected to electrophoresis on a 1.5% agarose gel to determine length differences. 2.8. Statistical analysis One-way analysis of variance (ANOVA) was used to analyze the data. When ANOVA identified differences among groups, a multiple comparisons (Tukey’s) test was conducted to examine significant differences among treatments using the SAS computer software (SAS Institute, Cary, NC, USA). Before the analysis, the percent data (from the growing-out, susceptibility, and phagocytic activity assays) were normalized by arcsine-transformation. Statistically significant differences required that p < 0.05. 3. Results 3.1. Growth measurements The PWG of groupers fed S. cerevisiae-supplemented diets was significantly higher than that of groupers fed the control diet from 1 to 4 weeks of feeding. After 4 weeks of feeding, the PWG of fish supplemented with concentrations of S. cerevisiae in the diets of 103e107 cfu kg
1 increased. Values of the PWG of fish fed the control diet, and 103, 105, and 107 cfu kg
1 S. cerevisiae-containing diets were 166.6%, 177.7%, 199.8%, and 211.6%, respectively (Fig. 1A). FEs of fish fed the 103, 105, and 107 cfu kg
1 S. cerevisiae-supplemented diets were significantly higher than those of fish fed the control diet at 1e4 weeks of feeding. After 4 weeks of feeding, the FEs of fish fed the 105 and 107 cfu kg
1 S. cerevisiae-containing diet were significantly higher than those of fish fed the 103 cfu kg
1 S. cerevisiae diet. The FEs of fish fed the control diet, and 103, 105, and 107 cfu kg
1 S. cerevisiae-containing diets were 0.97, 1.04, 1.16, and 1.20, respectively (Fig. 1B). 3.2. Challenge tests All unchallenged fish (control) group survived in the Streptococcus spp. and iridovirus challenge tests. For the Streptococcus sp. challenge test, death occurred after 24 h for fish fed the control diet and 103 cfu kg
1 S. cerevisiae diet (Table 2). After 6 days of challenge, the survival rate of fish fed the 107 cfu kg
1 S. cerevisiaecontaining diets was significantly higher than that of fish fed the

control diet, and was 26.6% higher compared to the control group. The relative percentage survival of fish fed the 103, 105 and 107 cfu kg
1 S. cerevisiae-containing diets are 0, 25.0 and 45.8, respectively. However, no significant differences in survival rates were observed between fish fed the 103 cfu kg
1 S. cerevisiae diet and the control diet (Table 2). For the iridovirus challenge test, the survival rate of fish fed the 107 cfu kg
1 S. cerevisiae-supplemented diet was significantly higher than that of fish fed 103 cfu kg
1 S. cerevisiae-supplemented diet and the control diet after 7 days of challenge, with a 36.6% increased survival rate compared to the control group. The relative percentage survival of fish fed the 103, 105 and 107 cfu kg
1 S. cerevisiae-containing diets are 0, 24.0 and 68.1, respectively. However, no significant differences in survival rates were observed between fish fed the 103 cfu kg
1 S. cerevisiae diet and the control diet (Table 3).

3.3. Immune parameters ACH50 levels of fish fed the diet containing S. cerevisiae at 105 and 107 cfu kg
1 at 2e4 weeks were significantly higher than those of fish fed 103 cfu kg
1 S. cerevisiae diet and the control diet, and had increased by 1.9- and 1.8-fold, respectively, compared to the control group after 4 weeks of feeding (Fig. 2A). The serum lysozyme activities of fish fed the 105 and 107 cfu kg
1 S. cerevisiae diets were significantly higher than those of fish fed the control diet at 1e4 weeks, and had increased by 3.1- and 2.7-fold, respectively, compared to the control diet after 4 weeks of feeding (Fig. 2B). The PAs of head kidney leucocytes of fish fed 105 and 107 cfu kg
1 S. cerevisiae-containing diets were significantly higher than those of fish fed the 103 cfu kg
1 S. cerevisiae-supplemented diet and the control diet at 1e4 weeks (Fig. 3A). However, no significant differences in PAs were observed between fish fed the 105 and 107 cfu kg
1 S. cerevisiae-supplemented diets after 4 weeks of feeding. After 4 weeks of feeding, fish fed a diet containing S. cerevisiae at 103 cfu kg
1 had a significantly higher PA than those fed the control diet. The relative PAs (compared to the control group) of fish fed the 103, 105, and 107 cfu kg
1 S. cerevisiae-supplemented diets for 4 weeks respectively increased by 1.1-, 1.2-, and 1.2-fold (Fig. 3A). Respiratory bursts of fish fed the 103, 105, and 107 cfu kg
1 S. cerevisiae diets were significantly higher than those of fish fed the control diet after 2 and 4 weeks of feeding, and had respectively increased by 19.8%, 27.6% and 37.9%, and 10.3%, 26.1% and 19.7% compared to the control group (Fig. 3B). SOD activities of fish fed the 103, 105, and 107 cfu kg
1 S. cerevisiae-supplemented diets were significantly lower than those of the control group after 1 and 2 weeks of feeding. After 4 weeks of feeding, SOD activities of fish fed a diet containing S. cerevisiae at 105 and 107 cfu kg
1 were significantly higher than those of fish fed the 103 cfu kg
1 S. cerevisiae-supplemented diet and the

Table 2 The survival rate and relative percentage survival (RPS) of grouper Epinephelus coioides challenged with Streptococcus sp., when the grouper were fed different doses of Saccharomyces cerevisiae (0, 103, 105, 107 cfu kg
1) containing diets after 28 days. Challeng dose (cfu g fish
1)

S. cerevisiae in diet (cfu kg
1)

Survival ratio and RPS (%), time after challenge (hrs) 24

48

72

96

120

144

Saline 5  105 5  105 5  105 5  105

0 0 5.3  103 5.3  105 5.3  107

100 100a 96.73.3a 100a 100a

100 86.7 90.0 93.3 96.7

100 70.0 63.3 90.0 90.0

100 43.3 43.3 66.7 76.7

100 26.7 33.3 43.3 63.3

100 20.0 20.0 40.0 56.6

   

3.3a 5.7a 3.3a 3.3a

   

5.7a 3.3a 5.7a 5.7a

   

8.8b 3.3b 3.3a 6.6a

Data in the same column with different letters are significant differ (p < 0.05) among different treatments. Values are mean  S.E. a Values in parentheses are relative percentage survival (RPS).

   

3.3c 3.3bc 6.6b 3.3a

   

5.7c 5.7c (0)a 5.7b (25.0)a 3.3a (45.8)a

C.-H. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 1053e1059

1057

Table 3 The survival rate and relative percentage survival (RPS) of grouper Epinephelus coioides challenged with grouper iridovirus, when the grouper were fed different doses of Saccharomyces cerevisiae (0, 103, 105, 107 cfu kg
1) containing diets after 28 days. Challeng dose (TCID50 g fish
1)

S. cerevisiae in diet (cfu kg
1)

L15 1 1 1 1

0 0 5.3  103 5.3  105 5.3  107

105 105 105 105

Survival ratio and RPS (%), time after challenge (hrs) 24

48

72

96

120

144

168

100 93.3  3.3a 96.7  3.3a 100a 100a

100 80  5.7b 76.7  3.3b 86.7  3.3b 96.7  3.3a

100 56.7 63.3 80.0 90.0

100 33.3 50.3 63.3 70.0

100 26.7 33.3 56.7 60.0

100 16.7 20.0 43.3 46.7

100 16.7 16.7 36.7 43.3

   

6.6b 3.3b 5.7ab 5.7a

   

8.8b 5.7ab 8.8a 5.7a

   

6.6b 3.3b 6.6a 5.7a

   

3.3c 5.7bc 8.8ab 8.8a

   

3.3b 6.6b (0)a 6.6ab (24.0)a 6.6a (68.1)a

Data in the same column with different letters are significant differ (p < 0.05) among different treatments. Values are mean  S.E. a Values in parentheses are relative percentage survival (RPS).

control diet. The relative SOD levels (compared to the control group) of fish fed the 105 and 107 cfu kg
1 S. cerevisiae-supplemented diets increased by 1.3- and 1.4-fold, respectively, compared to the control group (Fig. 3C). 3.4. Detection of S. cerevisiae in fish intestines Before the trial and after control diet feeding for 4 weeks, no contamination by S. cerevisiae in the intestines of fish was observed by PCR. A 300-bp fragment was observed in all S. cerevisiae-containing diets at 4 weeks (Fig. 4). 4. Discussion

300

103

0

107

105 a

a a a a

a a

ab

b b

a

b b

b

a

b

150

0

103

a a a a b

7 14 Time elapsed (days)

B

Saccharomyces cerevisiae in diet (cfu kg ) 103

0

107

105

a

0.6 a 0.3 a a a a

b b

a

a a a

b b

b b

0

a a c

b

a a

b

5

0.6

7 14 Time elapsed (days)

28

B

Saccharomyces cerevisiae in diet (cfu kg-1) 103

0

107

105

a

0.4 b b b a a a a 0.2

b

a a a

c

b

a a

0 7 14 Time elapsed (days)

28

-1

b

b

0

SOD activity (unit mg protein-1)

Lysozyme activity (µg ml-1)

0.9

0

a

a

0

0

107

105

15 10

A

Saccharomyces cerevisiae in diet (cfu kg-1)

0

Respiratory burst (O.D. 630 nm)

ACH50 (units ml-1)

450

A

Saccharomyces cerevisiae in diet (cfu kg-1)

Phagocytic activity (%)

20

In the present study, grouper E. coioides fed diets containing S. cerevisiae at 103e107 cfu kg
1 had significantly increased PWG levels and FEs, and these increased with the supplemented concentration of S. cerevisiae in the diets. In our previous study, grouper E. coioides fed a diet containing L. plantarum at the levels of 106, 108, and 1010 cfu kg
1 had significantly increased PWG levels and FEs especially at 108 cfu kg
1, which indicated that the optimal L. plantarum concentration for supplementing diets may promote the growth rate and FE which may have resulted from increased protein turnover in grouper [28]. This result is in agreement with 600

the use of higher concentrations of probiotics not always leading to better growth performances [29]. Supplementation with a dietary S. cerevisiae fermentation product (DAVAQUAÒ) at concentrations of 0.125e2.0 g kg
1 showed no effects on growth performance, diet conversion, or survival rates of hybrid tilapia (O. niloticus \  O. aureus _) [15]. Dietary DAVAQUA (1.25 g kg
1) improved weight gain by 27.8% in rainbow trout, Oncorhynchus mykiss, reared in tanks [30]. However, they did not report the concentration of S. cerevisiae in the diets. Differences in the growth performances and FEs might have been due to differences in the type of basal ingredients in the diets, fish species, and feeding dosages.

1.5

C

Saccharomyces cerevisiae in diet (cfu kg-1) 103

0 1.0

28

107

105 a

a a

a a a a b b

a

b 0.5

b

b b

b

b

0

0

7 14 Time elapsed (days)

28

Fig. 2. Alternative complement activity (ACH50) (A) and lysozyme activity (B) of. Epinephelus coioides fed the control diet and Saccharomyces cerevisiae-containing diets at 103, 105, and 107 colony-forming units (cfu) (kg diet)
1. See Fig. 1 for statistical information.

0

7

14

28

Time elapsed (days) Fig. 3. Phagocytic activity (A), respiratory bursts (B) and superoxide dismutase activity (C) of Epinephelus coioides fed the control diet and Saccharomyces cerevisiae-containing diets at 103, 105, and 107 colony-forming units (cfu) (kg diet)
1. See Fig. 1 for statistical information.

1058

C.-H. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 1053e1059

Fig. 4. Detection of Saccharomyces cerevisiae (P13) in the intestines of Epinephelus coioides fed the control diet and S. cerevisiae-containing diets at 103, 105, and 107 colony-forming units (cfu) (kg diet)
1 by gel electrophoresis of polymerase chain reaction products amplified using the specific primer pair, YMR245w-F (50 -TCTGTATATTCTGTATCTATGTTCCTGC-30 ) and YMR245w-R (50 -AAATGGCCTATTGTATTGTCAGGTC-30 ), of a specific open reading frame from the S. cerevisiae genome.

E. coioides fed a diet containing S. cerevisiae at 107 cfu kg
1 had the best resistance against Streptococcus sp. and GIV in the present study. Dietary administration of lactic acid bacteria significantly increased the survival rate in rainbow trout, O. mykiss, challenged with Aeromonas salmonicida [31,32], in orange grouper, E. coioides, challenged with Streptococcus sp. and GIV [28], and white shrimp, L. vannamei, challenged with V. alginolyticus [17]. Dietary fermented S. cerevisiae (XP Yeast CultureÒ) administration at 1% for 4 weeks revealed the best resistance against Vibrio sp. in L. vannamei [33]. Nile tilapia, O. niloticus, fed a S. cerevisiae-containing diet for 21 days showed an increased survival rate following challenge with A. hydrophila [14]. The facts suggest that S. cerevisiae administration can enhance shrimp and fish resistance to bacterial and viral pathogens. Differences in the probiotic effects against bacterial and viral pathogens possibly result from differences in defense mechanisms of fish to different pathogens and different pathogenic mechanisms of various pathogens [28]. Macrophages play a central role in non-specific cellular defense. The present study indicated that dietary S. cerevisiae administration at 103, 105, and 107 cfu kg
1 for 4 weeks significantly increased the head kidney macrophage phagocytic activity, respiratory burst activity, and SOD regulation in E. coioides. The results are in agreement with studies on hybrid tilapia [14,15] and gilthead seabream [12,13], in which dietary supplementation with whole yeast cells (S. cerevisiae) improved the head kidney macrophage phagocytic, respiratory burst, and myeloperoxidase activities in fish. The alternative pathway of complement activity has emerged as a powerful nonspecific defense mechanism for protecting fish against a wide range of potentially invasive organisms, such as bacteria, fungi, viruses, and parasites [34]. Lysozymes are also one of the defensive factors against invasive microorganisms in vertebrates [35]. They lyse gram-positive bacteria, and kill gramnegative bacteria after a complement and other enzymes have disrupted the outer cell walls [36e38]. In addition, lysozymes promote phagocytosis as an opsonin, or by directly activating polymorphonuclear leukocytes and macrophages [39,40]. Our results showed that E. coioides fed S. cerevisiae-supplemented diets at 105 and 107 cfu kg
1 exhibited significant increases in both serum lysozyme and alternative complement pathway activities (ACH50). The same result was observed in hybrid tilapia fed diets containing DVAQUAÒ at 0.125e2.0 g kg
1 diet [15]. A significant increase in lysozyme activity was also observed in Nile tilapia,

O. niloticus, fed diets containing S. cerevisiae at a concentration of 10 g kg
1 (BiosalÒ, KW Alternative Feeds) for 21 days [14]. The facts suggest that resistance against bacterial and viral pathogens is correlated with increases in ACH50 and lysozyme activities of grouper fed S. cerevisiae-supplemented diets. Compounds of the S. cerevisiae cell wall are known to promote innate defense mechanisms and/or disease resistance in fish [8e11]. A S. cerevisiae fermentation product (DVAQUAÒ) consisting of yeast cell walls (b-glucan and mannan-oligosaccharides) and soluble materials (vitamins, amino acid, proteins, peptides, nucleotides, oligosaccharides, etc.) of cells, seldom contains living cells [33], which might enhance the nonspecific immunity of fish. He et al. [15] concluded that the components of DVAQUAÒ may be responsible for the improved nonspecific immune response, including b-glucans that are recognized by receptors expressed on fish monocytes/ macrophages and are able to stimulate the innate immune system in fish [41e43]. Dietary nucleotides may be involved as they are reported to increase resistance to infections as a consequence of increasing phagocytic activity of peritoneal macrophages [44], Tcell-dependent antibody production [45], natural killer cell activity, interleukin-2 production [46], and bone marrow cell and peripheral neutrophil numbers [47]. Mannan-oligosaccharides, as the prebiotic-ingredient of DVAQUA may also alter the immune response due to the presence of mannose receptors on many cells in the immune system [48]. In addition to those components, dietary oligosaccharides can also indirectly improve the nonspecific immunity of hybrid tilapia by selectively stimulating potentially beneficial intestinal bacteria [15,49]. Much research has demonstrated that probiotics are able to survive in the digestive tract of fish fed probiotic-supplemented diets. The same result was observed in E. coioides fed S. cerevisiae-supplemented diets in the present study. The facts suggested that dietary S. cerevisiae might be able to survive in digestive tract of grouper and improve the immunity and disease resistance via the above mechanisms. Using plate counting on a selective medium, the viable lactobacilli in the posterior intestines of fish fed the 106, 108, and 1010 cfu kg
1 L. plantarum-containing diets had dominantly increased from (2.2  0.3)  107, (1.9  0.4)  108 and (2.2  0.1)  108 cfu (g gut)
1, respectively, and the amount of L. plantarum exhibited a relation between diet and intestines of fish after 4 weeks of feeding. Using PCR by a specific primer pair, the S. cerevisiae amount had no relation between diet and intestines of fish in the present study, which possibly resulting from the

C.-H. Chiu et al. / Fish & Shellfish Immunology 29 (2010) 1053e1059

intestines microflora were beforehand proliferated in the YM broth and/or the too high cycles used in PCR. In conclusion, S. cerevisiae colonized the intestines of the grouper E. coioides fed S. cerevisiae-supplemented diets, which improved the feed efficiency and growth rate, and induced upregulation of innate cellular and humoral immune responses together with increasing the resistance to challenge by Streptococcus sp. and a grouper iridovirus. To elevate the growth and immune resistance ability of E. coioides, dietary S. cerevisiae administration at 107 cfu kg
1 is an optimal dose. Acknowledgements This study was supported by grants from the Council of Agriculture (98AS-5.3.3-PT-f2) and National Science Council (NSC962313-B-020-009), ROC. We appreciate Dr. J. P. Shu and Dr. C. Y Chang for providing bacterial and viral pathogens, respectively. References [1] Fuller R. A review: probiotics in man and animals. J Appl Bacteriol 1989;66:365e78. [2] McCracken VJ, Gaskins HR. Probiotics and the immune systems. In: Tannock GW, editor. Probiotics: a critical review. Wymondham, UK: Horizon Scientific Press; 1999. p. 85e112. [3] Verschuere L, Rombaut G, Sorgeloos P, Verstraete W. Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 2000;64:655e71. [4] Balcázar JL, Id Blas, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Muzquiz JL. The role of probiotics in aquaculture. Vet Microbiol 2006;114:173e86. [5] Gatesoupe FJ. The use of probiotics in aquaculture. Aquaculture 1999;180:147e65. [6] Irianto A, Austin B. Probiotics in aquaculture. J Fish Dis 2002;25:633e42. [7] Cabib E, Roberts R, Bowers B. Synthesis of the yeast cell wall and its regulation. Annu Rev Biochem 1982;51:763e93. [8] Czop JK, Austen KF. A beta-glucan inhabitable receptor on human monocytes: its identity with the phagocytic receptor for particulate activators of the alternative complement pathway. J Immunol 1985;134:2588e93. [9] Engstad RE, Robertsen B. Recognition of yeast cell wall glucan by Atlantic salmon (Salmo salar L.) macrophages. Dev Comp Immunol 1993;17:319e30. [10] Esteban MA, Mulero V, Cuesta A, Ortuño J, Meseguer J. Immunomodulatory effects of dietary intake of chitin in gilthead seabream (Sparus aurata L.) innate immune response. Fish Shellfish Immunol 2001;11:303e15. [11] Pietrella D, Cherniak R, Strappini C, Perito S, Mosci P, Bistoni F, et al. Role of mannoprotein in induction and regulation of immunity to Cryptococcus neoformans. Infect Immun 2001;69:2808e14. [12] Ortuño J, Cuesta A, Rodríguez A, Esteban MA, Meseguer J. Oral administration of yeast, Saccharomyces cerevisiae, enhances the cellular innate immune response of gilthead seabream (Sparus aurata L.). Vet Immunol Immunopathol 2002;85:41e50. [13] Rodríguez A, Cuesta A, Ortuño J, Esteban MA, Meseguer J. Immunostimulant properties of a cell wall-modified whole Saccharomyces cerevisiae strain administratered by diet to seabream (Sparus aurata L.). Vet Immunol Immunopathol 2003;96:183e92. [14] El-Boshy ME, El-Ashram AM, AbdelHamid FM, Gadalla HA. Immunomodulatory effect of dietary Saccharomyces cerevisiae, ß-glucan and laminaran in mercuric chloride treated Nile tilapia (Oreochromis niloticus) and experimentally infected with Aeromonas hydrophila. Fish Shellfish Immunol 2010;28:802e8. [15] He S, Zhou Z, Liu Y, Shi P, Yao B, Ringø E, et al. Effects of dietary Saccharomyces cerevisiae fermentation product (DVAQUAÒ) on growth performance, intestinal autochthonous bacterial community and non-specific immunity of hybrid tilapia (Oreochromis niloticus \  Oreochromis aureus _) cultured in cages. Aquaculture 2009;294:99e107. [16] AOAC. Official methods of analyses. 16th ed. Washington, DC: Association of Official Analytical Chemists (AOAC); 1997. [17] Chiu CH, Guu YK, Liu CH, Pan TM, Cheng W. Immune responses and gene expression in white shrimp, Litopenaeus vannamei, induced by Lactobacillus plantarum. Fish Shellfish Immunol 2007;23:364e77. [18] Yeh SP, Chang CA, Chang CY, Liu CH, Cheng W. Dietary sodium alginate administration affects the fingerling growth and resistance to Streptococcus sp. and iridovirus, and juvenile non-specific immune responses of the orangespotted grouper, Epinephelus coioides. Fish Shellfish Immunol 2008;25:19e27. [19] Lai YS, John JAC, Lin CH, Guo IC, Chen SC, Fanf K, et al. Establishment of cell lines from a tropical grouper, Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses. J Fish Dis 2003;26:31e42. [20] Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. Am J Hyg 1938;27:493e7.

1059

[21] Amend DF. Potency testing of fish vaccines. Dev Biol Stand 1981;49:447e54. International symposium on fish biologics: serodiagnostics and vaccines. [22] Sunyer JO, Tort L. Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are effected by the alternative complement pathway. Vet Immunol Immunopathol 1995;45:333e45. [23] Ellis AE. Lysozyme assay. In: Stolen JS, Fletcher DP, Anderson BS, Robertson BS, editors. Techniques in fish immunology. Fair Haven, NJ: SOS Publication; 1990. p. 101e3. [24] Obach A, Quentel C, Bandin LF. Effects of alpha-tocopherol and dietary oxidized fish oil on the immune response of sea bass Dicentrarchus labrax. Dis Aquat Org 1993;15:175e85. [25] Cook MT, Hayball PJ, Hutchinson W, Nowak B, Hayball JD. The efficacy of a commercial beta-glucan preparation, EcoActiva, on stimulating respiratory burst activity of head-kidney macrophages from pink snapper (Pagrus auratus) Sparidae. Fish Shellfish Immunol 2001;11:661e72. [26] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 1976;72:248e54. [27] Havilio M, Levanon EY, Lerman G, Kupiec M, Eisenberg E. Evidence for abundant transcription of non-coding regions in the Saccharomyces cerevisiae genome. BMC Genomics 2005;6:93. [28] Vo MS, Chang CC, Wu MC, Guu YK, Chiu CH, Cheng W. Dietary administration of the probiotic, Lactobacillus plantarum, enhanced the growth, innate immune responses, and disease resistance of the grouper Epinephelus coioides. Fish Shellfish Immunol 2009;26:691e8. [29] Ghosh S, Sinha A, Sahu C. Dietary probiotic supplementation in growth and health of live-bearing ornamental fishes. Aquacult Nutr 2007;13:1e11. [30] Barnes ME, Durben DJ, Reeves SG, Sanders R. Dietary yeast culture supplementation improves initial rearing of McConaughy strain rainbow trout. Aquacult Nutr 2006;12:388e94. [31] Balcázar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Girones O, Muzquiz JL. Enhancement of the immune response and protection induced by probiotic lactic acid bacteria against furunculosis in rainbow trout (Oncorhynchus mykiss). FEMS Immunol Med Microbiol 2007;51:185e93. [32] Nikoskelainen S, Ouwehand A, Salminen S, Bylund G. Protection of rainbow trout (Oncorhynchus mykiss) from furunculosis by Lactobacillus rhamnosus. Aquaculture 2001;198:229e36. [33] Burgents JE, Burnett KG, Burnett LE. Disease resistance of Pacific white shrimp, Litopenaeus vannamei, following the dietary administration of a yeast culture food supplement. Aquaculture 2004;231:1e8. [34] Müller-Eberhard HJ. Molecular organization and function of the complement system. Annu Rev Biochem 1988;57:321e47. [35] Osserman EF, Canfield RE, Beychok S, editors. Lysozyme. New York: Academic Press; 1974. 641 p. [36] Salton MRJ, Ghuysen JM. The structure of di- and tetra-saccharides released from cell walls by lysozyme and Streptomyces F1 enzyme and the b(1 / 4) Nacetylhexosaminidase activity of these enzymes. Biochim Biophys Acta 1959;36:552e4. [37] Glynn AA. The complement lysozyme sequence in immune bacteriolysis. Immunology 1969;16:463e71. [38] Hjelmeland K, Christie M, Raa J. Skin mucus protease from rainbow trout, Salmo gairdneri Richardson, and its biological significance. J Fish Biol 1983;23:13e22. [39] Klockar M, Roberts P. Stimulation of phagocytosis by human lysozyme. Acta Haematol 1976;55:289e95. [40] Jollès P, Jollès J. What’s new in lysozyme research? Always a model system, today as yesterday. Mol Cell Biochem 1984;63:165e89. [41] Toranzo AE, Devesa S, Romalde JL, Lamas J, Riaza A, Leiro J, et al. Efficacy of intraperitoneal and immersion vaccination against Enterococcus sp. infection in turbot. Aquaculture 1995;134:17e27. [42] Yoshida T, Kruger R, Inglis V. Augmentation of nonspecific protection in African catfish, Claris gariepinus (Burchell), by the long-term oral-administration of immunostimulants. J Fish Dis 1995;18:195e8. [43] Siwicki AK, Anderson DP, Rumsey G. Dietary intake of immunostimulants by rainbow trout affects non-specific immunity and protection against furunculosis. Vet Immunol Immunopathol 1994;41:125e39. [44] Kulkarni AD, Fanslow WC, Rudolph FB, van Buren CT. Effect of dietary nucleotides on response to bacterial infections. J Parenter Enteral Nutr 1986;10:169e71. [45] Jyonouchi H. Nucleotide actions on humoral immune responses. J Nutr 1994;124:138Se43S. [46] Carver JD. Dietary nucleotides: cellular immune, intestinal and hepatic system effects. J Nutr 1994;124(Suppl):144Se8S. [47] Matsumoto Y, Adje AA, Yamauchi K, Kise M, Nakasone Y, Shinegawa Y, et al. Mixture of nucleosides and nucleotides increases bone marrow cell and peripheral neutrophil number in mice infected with methicillin-resistant Staphylococcus aureus. Biochemical and molecular roles of nutrients. J Nutr 1995;125:815e22. [48] Djeraba A, Quere P. In vivo macrophage activation in chickens with acemannan, a complex carbohydrate extracted from Aloe vera. Int Immunopharmacol 2000;22:365e72. [49] Lv H, Zhou Z, Rudeaux F, Respondek F. Effects of dietary short chain fructooligosaccharides on intestinal microflora, mortality and growth performance of O. nilotica \  O. aurea _. Chin J Anim Nutr 2007;19:691e7.