- Email: [email protected]
Effects of dietary probiotic, Lactococcus lactis subsp. lactis I2, supplementation on the growth and immune response of olive flounder (Paralichthys olivaceus)
Aquaculture 376–379 (2013) 20–24
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Effects of dietary probiotic, Lactococcus lactis subsp. lactis I2, supplementation on the growth and immune response of olive flounder (Paralichthys olivaceus) Won-Seok Heo a, 1, Yu-Ri Kim a, 1, Eun-Young Kim a, Sungchul C. Bai b, In-Soo Kong a,⁎ a b
Department of Biotechnology, Pukyong National University, Busan 608-737, Korea Department of Marine Bio-materials and Aquaculture, Pukyong National University, Busan 608-737, Korea
a r t i c l e
i n f o
Article history: Received 31 October 2012 Received in revised form 7 November 2012 Accepted 8 November 2012 Available online 16 November 2012 Keywords: Probiotic Lactococcus lactis Immune response Olive flounder
a b s t r a c t We investigated the effects of a potential probiotic strain Lactococcus lactis subsp. lactis I2 on the immune response and growth of olive flounder (Paralichthys olivaceus), and their capacity to prevent streptococcosis after Streptococcus iniae challenge. The L. lactis subsp. lactis I2 strain, isolated from olive flounder intestine, was supplemented orally as a feed additive (~10 8 CFU g−1) to fish for 5 weeks. Compared with the untreated group, the rate of growth was increased in the I2-diet group. The administration of I2 to olive flounder enhanced non-specific immune parameters, such as lysozyme, antiprotease, serum peroxidase and blood respiratory burst activities. At 9 days after challenge with S. iniae (10 8 CFU), the untreated control group experienced a 90% mortality rate, whereas all of the I2-cell-supplemented fish survived. These results show that L. lactis subsp. lactis I2 exerted beneficial effects as a probiotic and has potential as an alternative to antibiotics for the prevention of streptococcosis in aquaculture. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The olive flounder (Paralichthys olivaceus) is a farmed fish frequently consumed in Korea, for which demand is increasing. In 2011, the output of olive flounder was more than 50% (~40,805 tons) of the total output of fish by Fisheries Information Service (http://www.fips.go.kr/). The increased productivity, high-density culture and poor water quality have weakened disease-resistance, resulting in frequent outbreaks of fish diseases, and ultimately causes economic losses (MIFAFF, 2003). Fish diseases are caused by a number of pathogens, such as parasites, bacteria and viruses. Of these, bacteria are the leading pathogen in commercial aquaculture. Antibiotics or formalin-inactivated antigen vaccines have been used to treat bacterial infections in aquaculture systems. However, the development of antibiotic-resistant strains due to overuse of antibiotics and the reduced efficacy of the remaining antibiotics in fish have given rise to important problems (Karunasagar et al., 1994; Smith et al., 1994). The stress associated with vaccine administration to fish places a limitation on vaccination. To overcome these problems and to protect fish from bacterial diseases, many studies have evaluated the application of lactic acid bacteria (LAB) as feed additives instead of antibiotics or vaccine treatments (Brunt and Austin, 2005; Choi and Yoon, 2008; Gildberg and Mikkelsen, 1998; Pirarat et al., 2006; Sakai et al., 1995; Sharifuzzaman and Austin, 2009; Vendrell et al., 2008). LAB are recognized as an alternative to ⁎ Corresponding author. Tel.: +82 51 629 5865; fax: +82 51 629 5863. E-mail address: [email protected] (I.-S. Kong). 1 These authors contributed equally to this paper. 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2012.11.009
antibiotics or vaccines because they are safe and induce an immune response against pathogenic bacteria (Gatesoupe, 1999). Because LAB produce antimicrobial compounds as metabolites (e.g., lactic acid, diacetyl, carbon dioxide, hydrogen peroxide and bacteriocins) and to inhibit the increment of harmful intestinal bacteria (Calo-Mata et al., 2008; Gatesoupe, 1999; Perdigon et al., 1990), traditional fermented foods, such as soy sauce, and dairy products containing LAB have been used in the food industry. In addition, LAB cell wall components (capsular polysaccharides, peptidoglycans, lipoteichoic acids) induce cytokine production by macrophagocytes (Haza et al., 2004) and complement activation (Kim et al., 2002). The method of attachment and metabolite-producing stages of probiotic bacteria in host bodies are not well-understood (Gatesoupe, 1999). A probiotic strain suitable for treatment in marine aquaculture should be identified. According to a report by the Fisheries Information Service of Korea, 32% of total fish infections are caused by bacteria, of which 19% are due to Streptococcus spp. (http://www.fips.go.kr/). Streptococcus iniae, which is a marine pathogen and the causal agent of streptococcosis, is a gram-positive bacterium that infects at least 27 cultured fish species, including olive flounder, causing a greater than 50% mortality rate and a marked economic burden (Agnew and Barnes, 2007; Kim et al., 2011). Probiotic bacteria isolated from marine sources may be effective against pathogenic bacteria in fish (Kesarcodi-Warson et al., 2008). Previously, we isolated Lactococcus lactis subsp. lactis I2, a nisin Z-producing LAB, from the intestine of healthy olive flounder; this bacterium inhibited the growth of pathogenic bacteria, including S. iniae (Heo et al., 2012). The nisin Z produced by L. lactis subsp. lactis
W.-S. Heo et al. / Aquaculture 376–379 (2013) 20–24
has been approved as a safe food additive by both the Food and Drug Administration (FDA) and the World Health Organization (WHO). In this study, to determine the applicability of the nisin Z-producing strain in aquaculture, we investigated the effect of L. lactis subsp. lactis I2 as a food additive in terms of growth of flounder and induction of an immune response against S. iniae infection. 2. Materials and methods 2.1. Bacterial cultivation Nisin-Z-producing L. lactis subsp. lactis I2, isolated in our laboratory from flounder intestine, was cultured in MRS (Difco, Detroit, MI, USA) (Heo et al., 2012). S. iniae (KCTC 3657) was cultured in brain–heart infusion (BHI; Becton Dickinson, Heidelberg, Germany) broth at 37 °C up to 108 CFU/ml and used in a challenge test (Heo et al., 2012). Bacterial stocks were stored at −80 °C in their respective broths with 20% glycerol until use. 2.2. Fish A total of 300 juvenile olive flounder (P. olivaceus; average weight = 5.72 ± 0.17 g) were obtained from Geojedo, Korea. The fish were divided into two equal groups, an I2 group and a control group, and reared in continuously aerated free-flowing seawater, which was maintained at ~ 18 °C, for at least 1 week. Of the fish in each group, 10 were used to investigate the effects of the food additive on growth, 12 were used in a challenge test and 40 were used to investigate the immune response. Each experiment was repeated three times. All fish were fed the appropriate diet at 1.5% of their body weight daily. The health of the fish was checked immediately upon arrival in the laboratory and at 14-day intervals thereafter (Kim and Austin, 2006).
21
(Sigma, St. Louis, MO, USA) at a concentration of 0.2 mg ml −1 (w/v) in phosphate-buffered saline (PBS, pH 6.2). The optical density (OD) was recorded at 530 nm after 1 and 20 min at 22 °C. One unit of lysozyme activity was defined as the amount of serum that caused a decrease in the OD of 0.001 units min −1. For antiprotease activity, 10 μl of serum was incubated with 10 μl of 5 mg/ml trypsin solution (Sigma, St. Louis, MO, USA) for 10 min at 22 °C. Then, 100 μl of 0.1 M PBS (pH 7.0) and 125 μl of 2% (w/v) azocasein (Sigma, St. Louis, MO, USA) were added and the solution was incubated for 1 h at 22 °C. The reaction was stopped by addition of 500 μl of 10% (v/v) trichloroacetic acid (TCA) and then centrifuged at 6000 rpm for 5 min. The supernatants (400 μl) were transferred to new tubes containing 1 N sodium hydroxide (400 μl) and the OD at 450 nm was determined using a spectrophotometer (Ultrospec 3000, Pharmacia Biotech, USA). PBS was used as the control. The inhibitory activity of antiprotease was expressed in terms of the percentage of trypsin inhibition [(Control OD − Sample OD) / Control OD × 100] (Zuo and Woo, 1997). The peroxidase content of serum was measured using the method described by Sitjà-Bobadilla et al. (2008), with some modifications. In brief, 15 μl of serum was mixed with 85 μl of Hank's Buffered salt solution (HBSS) and then added to peroxidase substrate [100 μl of 5 mM tetramethylbenzidine hydrochloride (TMB; Sigma, St. Louis, MO, USA) and 100 μl of 2.5 mM H2O2]. The serum mixture (300 μl) was incubated for 2 min. The reaction was stopped with 100 μl of 2 M H2SO4 and the OD at 450 nm was determined. PBS was used as a blank instead of serum. The production of oxygen radicals by leukocytes was assessed by the reduction of NBT according to Taoka et al. (2006). Blood was added to an identical volume of NBT solution (2 mg ml −1) and incubated for 30 min. Then, 100 μl of N,N-dimethylformamide was added. The solution was centrifuged at 3000 rpm for 10 min. Supernatant (100 μl) was diluted with 900-μl PBS (pH 7.0) and then the OD at 550 nm was determined using a spectrophotometer.
2.3. Experimental diet 2.7. Challenge test A pure culture of L. lactis subsp. lactis I2 was incubated at 37 °C for 16 h and used to prepare the experimental diet. The diet of the I2 group was mixed with L. lactis subsp. lactis I2 to ~ 10 8 cells per gram commercial diet (Su-Hyup No.4, Korea); this dose was determined in preliminary experiments. The viability of L. lactis subsp. lactis I2 in the feed was assessed by plate counts on MRS medium. Fish in the control group were supplied with commercial feed only.
Juvenile olive flounders from each group were fed twice per day for up to 5 weeks with each experimental diet. After two weeks, 12 fish from each sub-group were intraperitoneally challenged with 100-μl S. iniae suspension (1 × 10 8 CFU), S. iniae was injected. Mortality was recorded daily for 2 weeks post-challenge; the results are presented as cumulative survival rates.
2.4. Sample collection
2.8. Statistical analysis
lood was collected from all fish from the caudal vein and Alserver's solution was used to perform a nitroblue tetrazolium (NBT; Sigma, St. Louis, MO, USA) reduction activity test. Serum was separated and subjected to lysozyme, antiprotease, peroxidase and bacterial killing assays.
All experimental data were analyzed by one-way analysis of variance (ANOVA), least significant difference (LSD), and Duncan's comparison tests as appropriate. All data were analyzed using the software Statistical Package for the Social Sciences (SPSS ver. 17.0, SPSS, Chicago, IL, USA). Differences were considered to be significant at values of P b 0.05.
2.5. Growth effects of experimental diets on fish populations 3. Results Ten fish from each sub-group were fed the experimental diet for 5 weeks; body weights and lengths were measured during this period. Growth effects were determined by calculating weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER), condition factor (CF), hepatosomatic index (HSI), and visceral somatic index (VSI). 2.6. Immunological assays Lysozyme and antiprotease activities were determined using the ethods of Ellis (1990). To determine lysozyme activity, 50 μl of serum was added to 1 ml lyophilized Micrococcus lysodeikticus
3.1. Effects of experimental diets on the growth of flounder After 5 weeks of feeding, the body weights of olive flounder fed probiotic-containing feed was higher than that of the control group (Table 1). Growth performance parameters for fish in the I2-diet and control groups were 0.97 ± 0.03% and 0.89 ± 0.01% for CF, and 1.80 ± 0.20% and 1.28 ± 0.15% for HIS, respectively. Especially, CF and HIS were significantly higher (P b 0.05) in the I2-diet group compared to the control group. Other parameter were 46.94 ± 0.70 % and 44.52 ± 0.34 % for WG, 78.62 ± 0.81% and 77.97 ± 0.32% for FE, 1.12 ± 0.01% and 1.06 ± 0.01% for SGR, 0.94 ± 0.01% and 0.77 ± 0.01% for FER,
W.-S. Heo et al. / Aquaculture 376–379 (2013) 20–24
Pooled SEMa
Diet
WGb FEc SGRd PERe CFf HISg VSIh
Probiotic (I2)
Control
46.94 ± 0.7 78.62 ± 0.81 1.12 ± 0.01 0.94 ± 0.01 0.97 ± 0.03⁎ 1.80 ± 0.20⁎
44.52 ± 0.34 77.97 ± 0.32 1.06 ± 0.01 0.77 ± 0.01 0.89 ± 0.01 1.28 ± 0.15 2.04 ± 0.20
2.30 ± 0.14
1.21 0.32 0.03 0.09 0.04 0.26 0.13
⁎ Significantly different (P b 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (P b 0.05) between the feeding regimes. a Pooled standard error of the mean. b Weight gain (%) = (final weight − initial weight) × 100 / initial weight. c Feed efficiency (%) = (wet weight gain / dry feed intake ) × 100. d Specific growth rate = (ln final weight − ln initial weight) × 100/days. e Protein efficiency ratio = wet weight gain / protein fed. f Condition factor = body weight(g) /total body length(cm)3 × 100. g Hepatosomatic Index = liver weight / body weight × 100. h Visceralsomatic Index = visceral weight /body weight × 100.
and 2.30 ± 0.14% and 2.04 ± 0.20% for VSI in the I2-diet and control groups, respectively. These findings indicated that the probiotic L. lactis subsp. lactis I2 has good effects on the growth performance of olive flounder. 3.2. Challenge test The survival rates of olive flounder in the I2-diet and control groups after S. iniae challenge were determined. Olive flounder mortality was first observed in the control group on day 3 postchallenge. The mortality rate was 20% on day 5 and > 80% on day 8 post-challenge. In contrast, fish mortality was observed starting on day 9 in the probiotic-supplemented group and >80% mortality was observed after day 11 post-challenge. This indicates that L. lactis subsp. lactis I2 delayed the occurrence of a > 80% mortality rate by 3 days (Fig. 5). 3.3. Immune parameters
Lysozyme activity (unit ml-1)
3.3.1. Lysozyme assay Lysozyme activity increased from the 2nd to 4th weeks, and decreased by the 5th week in both groups. However, significant differences (P b 0.05) in lysozyme activity between the two groups were observed at the 3rd (247 ± 47.7 unit ml −1) and 4th weeks
a *
400
Probiotic (I2) Control
a *
300
ab
200 b 100
85
Trypsin inhibition (%)
Table 1 Growth performance of juvenile olive flounder after probiotic supplementation for 5 weeks.
83
Probiotic (I2) Control
a * b
b
81
b
79 77 75 2
3
4
5
Week Fig. 2. Serum total antiprotease activity levels of juvenile olive flounder over 5 weeks. I2: commercial diet containing L. lactis subsp. lactis I2 (~108 cells g−1); Control: commercial diet. Data represent means ± SD of a triplicate set of nine fish. *Significantly different (P b 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (P b 0.05) between the feeding regimes.
(310 ± 50 unit ml −1). Lysozyme activities during the 2nd (110 ± 30 unit ml −1) and 5th weeks (170 ± 10 unit ml −1) were not significantly different between the groups (P > 0.05), but were higher in the I2-diet group (Fig. 1). 3.3.2. Antiprotease activity Trypsin inhibition activities in the I2-diet group from the 2nd to 5th weeks were 82.79 ± 0.09%, 81.30 ± 0.09%, 80.69 ± 0.55% and 79.72 ± 0.54%, respectively. The antiprotease activity of the I2-diet group was significantly different from the control group at the 2nd week (Fig. 2), and decreased with time in both groups. In the I2-diet group, antiprotease activities during the 4th and 5th weeks were slightly higher than those of the control group. Activity levels during the 3rd to 5th weeks were similar both groups (Fig. 2). 3.3.3. Serum peroxidase activity Serum peroxidase activity levels of the I2-diet group during the 2nd to 5th weeks were 0.71 ± 0.22, 0.96 ± 0.10, 1.23 ± 0.15 and 1.50 ± 0.02, respectively, and were higher than those of the control group. The activity level increased with time throughout the 5 weeks, and during the 5th week were more than twofold greater than during the 2nd week in both groups. Peroxidase activity in the I2-diet group was significantly different from the control group during the 2nd week (P b 0.05), and slightly higher in the I2-diet group compared to the control group during the 3rd to 5th weeks (Fig. 3).
1.8
Probiotic (I2) Control
1.6
Absorbance at 550nm
22
b
1.4
ab
1.2 1
ab
a *
0.8 0.6 0.4 0.2
0
0 2
3
4
5
Week Fig. 1. Serum lysozyme activity levels of juvenile olive flounder over 5 weeks. I2: commercial diet containing L. lactis subsp. lactis I2 (~108 cells g−1); Control: commercial diet. Data represent means ± SD of a triplicate set of nine fish. *Significantly different (P b 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (Pb 0.05) between the feeding regimes.
2
3
Week
4
5
Fig. 3. Serum peroxidase activity levels of juvenile olive flounder over five weeks. I2: commercial diet containing L. lactis subsp. lactis I2 (~108 cells g−1); Control: commercial diet. Data represent means±SD of a triplicate set of nine fish. *Significantly different (Pb 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (Pb 0.05) between the feeding regimes.
W.-S. Heo et al. / Aquaculture 376–379 (2013) 20–24
0.8
Probiotic (I2) Control a
a
a
a
0.6 0.4 0.2 0 2
3
4
5
Week Fig. 4. Respiratory burst activity of blood collected from juvenile olive flounders. I2: commercial diet containing L. lactis subsp. lactis I2 (~108 cells g−1); Control: commercial diet. Data represent means±SD of a triplicate set of nine fish. *Significantly different (Pb 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (Pb 0.05) between the feeding regimes.
3.3.4. Respiratory burst activity In terms of oxygen radical production, there was no significant difference between the I2-diet group (0.55 ± 0.08 to 0.65 ± 0.03) and the control group (0.41 ± 0.05 to 0.55 ± 0.05) (P > 0.05). However, the I2-diet group showed slightly greater NBT reduction compared to the control group throughout the 5 weeks. NBT reduction activity decreased gradually with time in both groups (Fig. 4). 4. Discussion Probiotic bacteria are considered an alternative method for control and prevention of the diverse diseases caused by pathogenic bacteria in aquaculture. There are many reports of protection against various diseases by probiotic-containing food. Lactobacillus rhamnosus was successfully used as a feed additive in tilapia (Pirarat et al., 2006) and rainbow trout (Choi and Yoon, 2008) to prevent edwardsiellosis. Similarly, C. divergens (Gildberg and Mikkelsen, 1998), Clostridium butyricum (Sakai et al., 1995) and Kocuria species (Sharifuzzaman and Austin, 2009) were used against vibriosis, A. sobria (Brunt and Austin, 2005), L. plantarum (Vendrell et al., 2008) and Enterococcus faecium (Kim et al., 2012) to prevent lactococcosis and A. sobria (Brunt and Austin, 2005), Bacillus subtilis (Kamgar and Ghane, 2012) to prevent streptococcosis. Streptococcosis causes greater damage to farmed fishes, such as flounder, when seawater temperatures exceed 17 °C (Agnew and Barnes, 2007). The methods that can be used for the application of probiotics in aquaculture include baths, suspensions and feeding. However, supplementation of food with probiotics is the most effective and simplest method to ensure successful colonization of and establishment in the gut (Nayak, 2010). In a previous study, we isolated L. lactis subsp. lactis I2 from the intestines of healthy olive flounder. We expected that the isolated LAB would retain its activity in the presence of salt and found that it effectively inhibited the growth of S. iniae, a potent pathogen associated with streptococcosis (Heo et al., 2012). To determine whether the isolated strain could be used to protect against streptococcosis, we supplemented feed with L. lactis subsp. lactis I2 (I2-diet) as a probiotic and determined its effects on the growth and immune response of olive flounder. The load of L. lactis subsp. lactis I2 in the intestine of olive flounder was 1.4× 107 CFU/ml after supplementation with an I2-diet for 2 weeks (data not shown). The growth of fish was monitored by means of a variety of physiological factors, such as digestion, energy absorption, metabolism and discharge ability (Brett, 1979). The growth rate was higher in the I2-diet group than in the control group (Table 1). We also investigated a number of innate immune factors, such as lysozyme, antiprotease, and peroxidase activities and oxygen radical production, in olive flounder from the 2nd to 5th weeks (at 1-week
intervals) following the commencement of probiotic-supplemented feeding. To determine the effects of a probiotic, it should be supplied as a feed additive for a minimum of 2 weeks to allow colonization of the gut (Desriac et al., 2010; Nayak, 2010). Levels of all of the above immune factors were similar to the control group during the 1st week (data not shown). Lysozyme is a component of the innate immune system and plays an important role in defense mechanisms due to its anticancer, antiviral and opsonization properties (Jollès and Jollès, 1984). Panigrahi et al. (2004) noted that supplementation of L. rhamnosus (109 CFU g −1) as a feed additive for 30 days enhanced lysozyme activity in rainbow trout. Stimulation of lysozyme activity in rainbow trout was observed after 2 weeks of feeding with Kocuria sp. SM1 (Sharifuzzaman and Austin, 2009) and Carnobacterium divergens B33 (Kim and Austin, 2006). Lysozyme activity was significantly enhanced during the 3rd and 4th weeks in the I2-diet group (Fig. 2). These findings suggest that high lysozyme activity may contribute to defense against bacterial diseases in probiotic-supplemented fish. Antiprotease activity in serum, which contains α1-antiprotease, α2-antiprotease and α2-macroglobulin, inhibits the action of the proteases used by some bacteria to invade the host. This activity is generally high and unaffected by immunization or infection (Ellis, 2001; Magnadottir et al., 2006). In this study, total antiprotease activity, in terms of mean serum antitrypsin activity, increased in the I2-diet group relative to the control group during the 2nd week, but the overall antiprotease regime in the I2-diet (79.72 ± 0.34 to 81.6 ±0.15) and control (79.66 ± 0.11 to 81.6± 0.11) groups was similar. Phagocytic cells, such as neutrophils and macrophages, play an important role in antibacterial defense. These cells engulf and kill bacteria by producing reactive oxygen species (ROS), comprising superoxide anions (O2−), hydrogen peroxide (H2O2) and hydroxyl free radicals (OH −) during the respiratory burst (Ellis, 1999). Taoka et al. (2006) reported that NBT reduction activity increased slightly on the 15th day after probiotic feed supplementation in tilapia. In rainbow trout, enhanced activity was identified relative to controls after 4 weeks (Sharifuzzaman and Austin, 2009). Moreover, the greatest activity against V. anguillarum was observed in rainbow trout 2 weeks after the start of a probiotic feeding regime. When we determined blood ROS production by NBT assay, the result was very similar to those in other fish species (Fig. 4). Cross (2002) defined probiotics as beneficial microorganisms that can colonize and multiply in the gut of a host and exert various beneficial effects, including immunomodulation, by influencing diverse host biological systems. Previously, we reported that L. lactis subsp. lactis I2 effectively inhibits the growth of S. iniae in vitro. This effect was confirmed by the increased survival rate of olive flounder infected with S. iniae (Fig. 5). When normal assorted feed
100
Cumulative survival rate (%)
Absorbance at 550nm
1
23
80 60 40 20
Probiotics (I2) Control
0 0
2
4
6
8
10
12
14
Day after challenge Fig. 5. Cumulative survival rates of juvenile olive flounder after challenge with S. iniae.
24
W.-S. Heo et al. / Aquaculture 376–379 (2013) 20–24
supplemented with the isolated LAB was supplied to olive flounder, activity against S. iniae was 2.5-fold higher than in the control. This may have been due to enhancement of the gut mucosal barrier, ‘colonization’ of the gastrointestinal tract of olive flounder, or enhanced induction of immune factors. Also, a role for nisin Z, a bacteriocin produced by L. lactis subsp. lactis I2, cannot be excluded. However, the enhanced immune factors in the I2-supplemented group certainly contributed to the improved growth rate in olive flounder. The synergistic effects of these diverse factors can also be considered the final influence on the high survival rate of flounder. Therefore, the enhancement of immune activity by the probiotics themselves or their production of nisin Z prevented infection. In conclusion, L. lactis subsp. lactis I2 isolated from intestines of healthy olive flounder enhanced the immune response and effectively controlled S. iniae infection of olive flounder. This strain is a good candidate feed additive to improve fish growth. However, further extensive investigations, including field tests and a full commercial cost-benefit analysis, are needed prior to its widespread application in aquaculture. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant 2010-0006364). References Agnew, W., Barnes, A.C., 2007. Streptococcus iniae: an aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination. Veterinary Microbiology 122, 1–15. Brett, J.R., 1979. Environmental factors and growth. In: Hoar, W.S., Randall, D.J., Brett, J.R. (Eds.), Fish Physiology. Bioenergetics and Growth, vol. VIII. Academic Press, New York, U.S.A., pp. 599–675. Brunt, J., Austin, B., 2005. Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 28, 693–701. Calo-Mata, P., Arlindo, S., Boehme, K., de Miguel, T., Pascoal, A., Barros-Velazquez, J., 2008. Current applications and future trends of lactic acid bacteria and their bacteriocins for the biopreservation of aquatic food products. Food Bioprocess Technology 1, 43–63. Choi, S.H., Yoon, T.J., 2008. Non-specific immune response of rainbow trout (Oncorhynchus mykiss) by dietary heat-inactivated poteintial probiotics. Immune Network 8 (3), 67–74. Cross, M.L., 2002. Microbes versus microbes: immune signals generated by probiotic lactobacilli and their role in protection against microbial pathogens. FEMS Immunology and Medical Microbiology 34, 245–253. Desriac, F., Defer, D., Bourgougnon, N., Brillet, B., Chevalier, P.L., Fleury, Y., 2010. Bacteriocin as weapons in the marine animal-associated bacteria warfare: inventory and potential applications as an aquaculture probiotic. Marine Drugs 8, 1153–1177. Ellis, A.E., 1990. Serum antiproteases in fish. In: Stolen, J.S., Fletcher, T.C., Anderson, D.P., Roberson, W.B., Van Muiswinkel, W.B. (Eds.), Techniques in fish Immunology. SOS Publication, Fair Haven, U.S.A., pp. 95–103. Ellis, A.E., 1999. Immunity to bacteria in fish. Fish & Shellfish Immunology 9, 291–308. Ellis, A.E., 2001. Innate host defense mechanisms of fish against viruses and bacteria. Development & Comparative Immunology 25, 827–839. Gatesoupe, F.J., 1999. The use of probiotics in aquaculture. Aquaculture 180, 147–165. Gildberg, A., Mikkelsen, H., 1998. Effect of supplementing the diet to Atlantic cod (Gadus morhua) fry with lactic acid bacteria and immuno-stimulating peptides during a challenge trial with Vibrio anguillarum. Aquaculture 167, 103–113. Haza, A.I., Zabala, A., Morales, P., 2004. Protective effect and cytokine production of a Lactobacillus plantarum strain isolated from ewes' milk cheese. International Dairy Journal 14, 29–38.
Heo, W.S., Kim, E.Y., Kim, Y.R., Hossain, M.T., Kong, I.S., 2012. Salt effect of nisin Z isolated from a marine fish on the growth inhibition of Streptococcus iniae, a pathogen of streptococcosis. Biotechnology Letters 34, 315–320. Jollès, P., Jollès, J., 1984. What is new in lysozyme research? Always a model system, todays as yesterday. Molecular and Cell Biochemistry 63, 156–189. Kamgar, M., Ghane, M., 2012. Evaluation of Bacillus subtilis effect as probiotic on hematological parameters of rainbow trout, Oncorhynchus mykiss (Walbaum) following experimental infection with Streptococcus iniae. Journal of Fisheries and Aquatic Science 7 (6), 422–430. Karunasagar, I., Pai, R., Malathi, G.R., Karunasagar, I., 1994. Mass mortality of Pecaeus monodon larvae due to antibiotic resistant Vibrio harveyi infection. Aquaculture 128, 203–209. Kesarcodi-Warson, A., Kaspar, H., Lategan, M.J., Gilson, L., 2008. Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 274, 1–14. Kim, D.H., Austin, B., 2006. Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish & Shellfish Immunology 21, 513–524. Kim, K.W., Bai, S.C., Koo, J.W., Wang, X., Kim, S.K., 2002. Effect of dietary Chlorella ellipsoidea supplementation on growth, blood characteristics, and whole-body composition in juvenile Japanese flounder Paralichthys olivaceus. Journal of the World Aquaculture Society 33 (4), 425–431. Kim, K.W., Kim, S.S., Jeong, J.B., Jeon, Y.J., Kim, K.D., An, C.M., Lee, K.J., 2011. Effects of dietary supplementation of fermented garlic powder and fluid on growth performance, immune responses, blood components, and disease resistance against Edwardsiella tarda and Streptococcus iniae in olive flounder Paralichthys olivaceus. Korean Journal of Fisheries and Aquatic Sciences 44 (6), 644–652. Kim, Y.R., Kim, E.Y., Choi, S.Y., Hossain, M.T., Oh, R., Heo, W.S., Lee, J.M., Cho, Y.C., Kong, I.S., 2012. Effect of a probiotic strain, Enterococcus faecium, on the immune responses of olive flounder (Paralichthys olivaceus). Journal of Microbiology and Biotechnology 22 (4), 526–529. Magnadottir, B., Gudmundsdottir, B.K., Lange, S., Steinarsson, A., Oddgeirsson, M., Bowden, T., et al., 2006. Immunostimulation of larvae and juveniles of cod, Gadus morhua L. Journal of Fish Disease 29 (3), 147–155. MIFAFF, Ministry for Food Agriculture Forestry and Fisheries, 2003. Statistical year book of maritime affairs and fisheries. Nayak, S.K., 2010. Probiotics and immunity: a fish perspective. Fish & Shellfish Immunology 29, 2–14. Panigrahi, A., Kiron, V., Kobayashi, T., Puangkaew, J., Satoh, S., Sugita, H., 2004. Immune response in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136. Veterinary Immunology and Immunopatholpogy 102, 379–388. Perdigon, G., Alvarez, S., Narder de Macas, M.E., Roux, M.E., de Ruiz, Pesce, Holgado, A., 1990. The oral administration of lactic acid bacteria increases the mucosal intestinal immunity in response to enterocpathogens. Journal of Food Protection 53, 404–410. Pirarat, N., Kobayashi, T., Katagiri, T., Maita, M., Endo, M., 2006. Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Veterinary Immunology and Immunopatholpogy 113, 339–347. Sakai, M., Yoshida, T., Astuta, S., Kobayashi, M., Endo, M., 1995. Enhancement of resistance to vibriosis in rainbow trout, Oncorhynchus mykiss (Walbaum) by oral administration of Clostridium butyricum bacteria. Journal of Fish Diseases 18, 187–190. Sharifuzzaman, S.M., Austin, B., 2009. Influence of probiotic feeding duration on disease resistance and immune parameters in rainbow trout. Fish & Shellfish Immunology 27, 440–445. Sitjà-Bobadilla, A., Palenzuela, O., Alvarez-Pellitero, P., 2008. Immune response of turbot, Psetta maxima (L.) (Pisces: Teleostei), to formalin-killed scuticociliates (Ciliophora) and adjuvanted formulations. Fish & Shellfish Immunology 24, 1–10. Smith, P., Hiney, M.P., Samuelsen, O.B., 1994. Bacterial resistance to antimicrobial agents used in fish farming: a critical evaluation of method and meaning. Annual Review of Fish Diseases 4, 273–313. Taoka, Y., Maeda, H., Jo, J.Y., Kim, S.M., Park, S.I., Yoshikawa, T., Sakata, T., 2006. Use of live and dead probiotic cells in tilapia Oreochromis niloticus. Fisheries Science 72, 755–766. Vendrell, D., Balcárzar, J.L., de Blas, I., Ruiz-Zarzuela, I., Girones, O., Muzquiz, J.L., 2008. Protection of rainbow trout (Oncorhynchus mykiss) from lactococcosis by probiotic bacteria. Comparative Immunology, Microbiology and Infectious Diseases 31, 337–345. Zuo, X., Woo, P.T.K., 1997. Natural anti-proteases in rainbow trout, Oncorhynchus mykiss and brook charr, Salvelinus fontinalis and the in vitro neutralization of fish α2-macroglobulin by the metalloprotease from the pathogenic haemoflagellate, Crytobia salmositica. Parasitology 114, 375–381.
Contents lists available at SciVerse ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Effects of dietary probiotic, Lactococcus lactis subsp. lactis I2, supplementation on the growth and immune response of olive flounder (Paralichthys olivaceus) Won-Seok Heo a, 1, Yu-Ri Kim a, 1, Eun-Young Kim a, Sungchul C. Bai b, In-Soo Kong a,⁎ a b
Department of Biotechnology, Pukyong National University, Busan 608-737, Korea Department of Marine Bio-materials and Aquaculture, Pukyong National University, Busan 608-737, Korea
a r t i c l e
i n f o
Article history: Received 31 October 2012 Received in revised form 7 November 2012 Accepted 8 November 2012 Available online 16 November 2012 Keywords: Probiotic Lactococcus lactis Immune response Olive flounder
a b s t r a c t We investigated the effects of a potential probiotic strain Lactococcus lactis subsp. lactis I2 on the immune response and growth of olive flounder (Paralichthys olivaceus), and their capacity to prevent streptococcosis after Streptococcus iniae challenge. The L. lactis subsp. lactis I2 strain, isolated from olive flounder intestine, was supplemented orally as a feed additive (~10 8 CFU g−1) to fish for 5 weeks. Compared with the untreated group, the rate of growth was increased in the I2-diet group. The administration of I2 to olive flounder enhanced non-specific immune parameters, such as lysozyme, antiprotease, serum peroxidase and blood respiratory burst activities. At 9 days after challenge with S. iniae (10 8 CFU), the untreated control group experienced a 90% mortality rate, whereas all of the I2-cell-supplemented fish survived. These results show that L. lactis subsp. lactis I2 exerted beneficial effects as a probiotic and has potential as an alternative to antibiotics for the prevention of streptococcosis in aquaculture. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The olive flounder (Paralichthys olivaceus) is a farmed fish frequently consumed in Korea, for which demand is increasing. In 2011, the output of olive flounder was more than 50% (~40,805 tons) of the total output of fish by Fisheries Information Service (http://www.fips.go.kr/). The increased productivity, high-density culture and poor water quality have weakened disease-resistance, resulting in frequent outbreaks of fish diseases, and ultimately causes economic losses (MIFAFF, 2003). Fish diseases are caused by a number of pathogens, such as parasites, bacteria and viruses. Of these, bacteria are the leading pathogen in commercial aquaculture. Antibiotics or formalin-inactivated antigen vaccines have been used to treat bacterial infections in aquaculture systems. However, the development of antibiotic-resistant strains due to overuse of antibiotics and the reduced efficacy of the remaining antibiotics in fish have given rise to important problems (Karunasagar et al., 1994; Smith et al., 1994). The stress associated with vaccine administration to fish places a limitation on vaccination. To overcome these problems and to protect fish from bacterial diseases, many studies have evaluated the application of lactic acid bacteria (LAB) as feed additives instead of antibiotics or vaccine treatments (Brunt and Austin, 2005; Choi and Yoon, 2008; Gildberg and Mikkelsen, 1998; Pirarat et al., 2006; Sakai et al., 1995; Sharifuzzaman and Austin, 2009; Vendrell et al., 2008). LAB are recognized as an alternative to ⁎ Corresponding author. Tel.: +82 51 629 5865; fax: +82 51 629 5863. E-mail address: [email protected] (I.-S. Kong). 1 These authors contributed equally to this paper. 0044-8486/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2012.11.009
antibiotics or vaccines because they are safe and induce an immune response against pathogenic bacteria (Gatesoupe, 1999). Because LAB produce antimicrobial compounds as metabolites (e.g., lactic acid, diacetyl, carbon dioxide, hydrogen peroxide and bacteriocins) and to inhibit the increment of harmful intestinal bacteria (Calo-Mata et al., 2008; Gatesoupe, 1999; Perdigon et al., 1990), traditional fermented foods, such as soy sauce, and dairy products containing LAB have been used in the food industry. In addition, LAB cell wall components (capsular polysaccharides, peptidoglycans, lipoteichoic acids) induce cytokine production by macrophagocytes (Haza et al., 2004) and complement activation (Kim et al., 2002). The method of attachment and metabolite-producing stages of probiotic bacteria in host bodies are not well-understood (Gatesoupe, 1999). A probiotic strain suitable for treatment in marine aquaculture should be identified. According to a report by the Fisheries Information Service of Korea, 32% of total fish infections are caused by bacteria, of which 19% are due to Streptococcus spp. (http://www.fips.go.kr/). Streptococcus iniae, which is a marine pathogen and the causal agent of streptococcosis, is a gram-positive bacterium that infects at least 27 cultured fish species, including olive flounder, causing a greater than 50% mortality rate and a marked economic burden (Agnew and Barnes, 2007; Kim et al., 2011). Probiotic bacteria isolated from marine sources may be effective against pathogenic bacteria in fish (Kesarcodi-Warson et al., 2008). Previously, we isolated Lactococcus lactis subsp. lactis I2, a nisin Z-producing LAB, from the intestine of healthy olive flounder; this bacterium inhibited the growth of pathogenic bacteria, including S. iniae (Heo et al., 2012). The nisin Z produced by L. lactis subsp. lactis
W.-S. Heo et al. / Aquaculture 376–379 (2013) 20–24
has been approved as a safe food additive by both the Food and Drug Administration (FDA) and the World Health Organization (WHO). In this study, to determine the applicability of the nisin Z-producing strain in aquaculture, we investigated the effect of L. lactis subsp. lactis I2 as a food additive in terms of growth of flounder and induction of an immune response against S. iniae infection. 2. Materials and methods 2.1. Bacterial cultivation Nisin-Z-producing L. lactis subsp. lactis I2, isolated in our laboratory from flounder intestine, was cultured in MRS (Difco, Detroit, MI, USA) (Heo et al., 2012). S. iniae (KCTC 3657) was cultured in brain–heart infusion (BHI; Becton Dickinson, Heidelberg, Germany) broth at 37 °C up to 108 CFU/ml and used in a challenge test (Heo et al., 2012). Bacterial stocks were stored at −80 °C in their respective broths with 20% glycerol until use. 2.2. Fish A total of 300 juvenile olive flounder (P. olivaceus; average weight = 5.72 ± 0.17 g) were obtained from Geojedo, Korea. The fish were divided into two equal groups, an I2 group and a control group, and reared in continuously aerated free-flowing seawater, which was maintained at ~ 18 °C, for at least 1 week. Of the fish in each group, 10 were used to investigate the effects of the food additive on growth, 12 were used in a challenge test and 40 were used to investigate the immune response. Each experiment was repeated three times. All fish were fed the appropriate diet at 1.5% of their body weight daily. The health of the fish was checked immediately upon arrival in the laboratory and at 14-day intervals thereafter (Kim and Austin, 2006).
21
(Sigma, St. Louis, MO, USA) at a concentration of 0.2 mg ml −1 (w/v) in phosphate-buffered saline (PBS, pH 6.2). The optical density (OD) was recorded at 530 nm after 1 and 20 min at 22 °C. One unit of lysozyme activity was defined as the amount of serum that caused a decrease in the OD of 0.001 units min −1. For antiprotease activity, 10 μl of serum was incubated with 10 μl of 5 mg/ml trypsin solution (Sigma, St. Louis, MO, USA) for 10 min at 22 °C. Then, 100 μl of 0.1 M PBS (pH 7.0) and 125 μl of 2% (w/v) azocasein (Sigma, St. Louis, MO, USA) were added and the solution was incubated for 1 h at 22 °C. The reaction was stopped by addition of 500 μl of 10% (v/v) trichloroacetic acid (TCA) and then centrifuged at 6000 rpm for 5 min. The supernatants (400 μl) were transferred to new tubes containing 1 N sodium hydroxide (400 μl) and the OD at 450 nm was determined using a spectrophotometer (Ultrospec 3000, Pharmacia Biotech, USA). PBS was used as the control. The inhibitory activity of antiprotease was expressed in terms of the percentage of trypsin inhibition [(Control OD − Sample OD) / Control OD × 100] (Zuo and Woo, 1997). The peroxidase content of serum was measured using the method described by Sitjà-Bobadilla et al. (2008), with some modifications. In brief, 15 μl of serum was mixed with 85 μl of Hank's Buffered salt solution (HBSS) and then added to peroxidase substrate [100 μl of 5 mM tetramethylbenzidine hydrochloride (TMB; Sigma, St. Louis, MO, USA) and 100 μl of 2.5 mM H2O2]. The serum mixture (300 μl) was incubated for 2 min. The reaction was stopped with 100 μl of 2 M H2SO4 and the OD at 450 nm was determined. PBS was used as a blank instead of serum. The production of oxygen radicals by leukocytes was assessed by the reduction of NBT according to Taoka et al. (2006). Blood was added to an identical volume of NBT solution (2 mg ml −1) and incubated for 30 min. Then, 100 μl of N,N-dimethylformamide was added. The solution was centrifuged at 3000 rpm for 10 min. Supernatant (100 μl) was diluted with 900-μl PBS (pH 7.0) and then the OD at 550 nm was determined using a spectrophotometer.
2.3. Experimental diet 2.7. Challenge test A pure culture of L. lactis subsp. lactis I2 was incubated at 37 °C for 16 h and used to prepare the experimental diet. The diet of the I2 group was mixed with L. lactis subsp. lactis I2 to ~ 10 8 cells per gram commercial diet (Su-Hyup No.4, Korea); this dose was determined in preliminary experiments. The viability of L. lactis subsp. lactis I2 in the feed was assessed by plate counts on MRS medium. Fish in the control group were supplied with commercial feed only.
Juvenile olive flounders from each group were fed twice per day for up to 5 weeks with each experimental diet. After two weeks, 12 fish from each sub-group were intraperitoneally challenged with 100-μl S. iniae suspension (1 × 10 8 CFU), S. iniae was injected. Mortality was recorded daily for 2 weeks post-challenge; the results are presented as cumulative survival rates.
2.4. Sample collection
2.8. Statistical analysis
lood was collected from all fish from the caudal vein and Alserver's solution was used to perform a nitroblue tetrazolium (NBT; Sigma, St. Louis, MO, USA) reduction activity test. Serum was separated and subjected to lysozyme, antiprotease, peroxidase and bacterial killing assays.
All experimental data were analyzed by one-way analysis of variance (ANOVA), least significant difference (LSD), and Duncan's comparison tests as appropriate. All data were analyzed using the software Statistical Package for the Social Sciences (SPSS ver. 17.0, SPSS, Chicago, IL, USA). Differences were considered to be significant at values of P b 0.05.
2.5. Growth effects of experimental diets on fish populations 3. Results Ten fish from each sub-group were fed the experimental diet for 5 weeks; body weights and lengths were measured during this period. Growth effects were determined by calculating weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER), condition factor (CF), hepatosomatic index (HSI), and visceral somatic index (VSI). 2.6. Immunological assays Lysozyme and antiprotease activities were determined using the ethods of Ellis (1990). To determine lysozyme activity, 50 μl of serum was added to 1 ml lyophilized Micrococcus lysodeikticus
3.1. Effects of experimental diets on the growth of flounder After 5 weeks of feeding, the body weights of olive flounder fed probiotic-containing feed was higher than that of the control group (Table 1). Growth performance parameters for fish in the I2-diet and control groups were 0.97 ± 0.03% and 0.89 ± 0.01% for CF, and 1.80 ± 0.20% and 1.28 ± 0.15% for HIS, respectively. Especially, CF and HIS were significantly higher (P b 0.05) in the I2-diet group compared to the control group. Other parameter were 46.94 ± 0.70 % and 44.52 ± 0.34 % for WG, 78.62 ± 0.81% and 77.97 ± 0.32% for FE, 1.12 ± 0.01% and 1.06 ± 0.01% for SGR, 0.94 ± 0.01% and 0.77 ± 0.01% for FER,
W.-S. Heo et al. / Aquaculture 376–379 (2013) 20–24
Pooled SEMa
Diet
WGb FEc SGRd PERe CFf HISg VSIh
Probiotic (I2)
Control
46.94 ± 0.7 78.62 ± 0.81 1.12 ± 0.01 0.94 ± 0.01 0.97 ± 0.03⁎ 1.80 ± 0.20⁎
44.52 ± 0.34 77.97 ± 0.32 1.06 ± 0.01 0.77 ± 0.01 0.89 ± 0.01 1.28 ± 0.15 2.04 ± 0.20
2.30 ± 0.14
1.21 0.32 0.03 0.09 0.04 0.26 0.13
⁎ Significantly different (P b 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (P b 0.05) between the feeding regimes. a Pooled standard error of the mean. b Weight gain (%) = (final weight − initial weight) × 100 / initial weight. c Feed efficiency (%) = (wet weight gain / dry feed intake ) × 100. d Specific growth rate = (ln final weight − ln initial weight) × 100/days. e Protein efficiency ratio = wet weight gain / protein fed. f Condition factor = body weight(g) /total body length(cm)3 × 100. g Hepatosomatic Index = liver weight / body weight × 100. h Visceralsomatic Index = visceral weight /body weight × 100.
and 2.30 ± 0.14% and 2.04 ± 0.20% for VSI in the I2-diet and control groups, respectively. These findings indicated that the probiotic L. lactis subsp. lactis I2 has good effects on the growth performance of olive flounder. 3.2. Challenge test The survival rates of olive flounder in the I2-diet and control groups after S. iniae challenge were determined. Olive flounder mortality was first observed in the control group on day 3 postchallenge. The mortality rate was 20% on day 5 and > 80% on day 8 post-challenge. In contrast, fish mortality was observed starting on day 9 in the probiotic-supplemented group and >80% mortality was observed after day 11 post-challenge. This indicates that L. lactis subsp. lactis I2 delayed the occurrence of a > 80% mortality rate by 3 days (Fig. 5). 3.3. Immune parameters
Lysozyme activity (unit ml-1)
3.3.1. Lysozyme assay Lysozyme activity increased from the 2nd to 4th weeks, and decreased by the 5th week in both groups. However, significant differences (P b 0.05) in lysozyme activity between the two groups were observed at the 3rd (247 ± 47.7 unit ml −1) and 4th weeks
a *
400
Probiotic (I2) Control
a *
300
ab
200 b 100
85
Trypsin inhibition (%)
Table 1 Growth performance of juvenile olive flounder after probiotic supplementation for 5 weeks.
83
Probiotic (I2) Control
a * b
b
81
b
79 77 75 2
3
4
5
Week Fig. 2. Serum total antiprotease activity levels of juvenile olive flounder over 5 weeks. I2: commercial diet containing L. lactis subsp. lactis I2 (~108 cells g−1); Control: commercial diet. Data represent means ± SD of a triplicate set of nine fish. *Significantly different (P b 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (P b 0.05) between the feeding regimes.
(310 ± 50 unit ml −1). Lysozyme activities during the 2nd (110 ± 30 unit ml −1) and 5th weeks (170 ± 10 unit ml −1) were not significantly different between the groups (P > 0.05), but were higher in the I2-diet group (Fig. 1). 3.3.2. Antiprotease activity Trypsin inhibition activities in the I2-diet group from the 2nd to 5th weeks were 82.79 ± 0.09%, 81.30 ± 0.09%, 80.69 ± 0.55% and 79.72 ± 0.54%, respectively. The antiprotease activity of the I2-diet group was significantly different from the control group at the 2nd week (Fig. 2), and decreased with time in both groups. In the I2-diet group, antiprotease activities during the 4th and 5th weeks were slightly higher than those of the control group. Activity levels during the 3rd to 5th weeks were similar both groups (Fig. 2). 3.3.3. Serum peroxidase activity Serum peroxidase activity levels of the I2-diet group during the 2nd to 5th weeks were 0.71 ± 0.22, 0.96 ± 0.10, 1.23 ± 0.15 and 1.50 ± 0.02, respectively, and were higher than those of the control group. The activity level increased with time throughout the 5 weeks, and during the 5th week were more than twofold greater than during the 2nd week in both groups. Peroxidase activity in the I2-diet group was significantly different from the control group during the 2nd week (P b 0.05), and slightly higher in the I2-diet group compared to the control group during the 3rd to 5th weeks (Fig. 3).
1.8
Probiotic (I2) Control
1.6
Absorbance at 550nm
22
b
1.4
ab
1.2 1
ab
a *
0.8 0.6 0.4 0.2
0
0 2
3
4
5
Week Fig. 1. Serum lysozyme activity levels of juvenile olive flounder over 5 weeks. I2: commercial diet containing L. lactis subsp. lactis I2 (~108 cells g−1); Control: commercial diet. Data represent means ± SD of a triplicate set of nine fish. *Significantly different (P b 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (Pb 0.05) between the feeding regimes.
2
3
Week
4
5
Fig. 3. Serum peroxidase activity levels of juvenile olive flounder over five weeks. I2: commercial diet containing L. lactis subsp. lactis I2 (~108 cells g−1); Control: commercial diet. Data represent means±SD of a triplicate set of nine fish. *Significantly different (Pb 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (Pb 0.05) between the feeding regimes.
W.-S. Heo et al. / Aquaculture 376–379 (2013) 20–24
0.8
Probiotic (I2) Control a
a
a
a
0.6 0.4 0.2 0 2
3
4
5
Week Fig. 4. Respiratory burst activity of blood collected from juvenile olive flounders. I2: commercial diet containing L. lactis subsp. lactis I2 (~108 cells g−1); Control: commercial diet. Data represent means±SD of a triplicate set of nine fish. *Significantly different (Pb 0.05) from the control group within the same sampling week. Means without a common letter differed significantly (Pb 0.05) between the feeding regimes.
3.3.4. Respiratory burst activity In terms of oxygen radical production, there was no significant difference between the I2-diet group (0.55 ± 0.08 to 0.65 ± 0.03) and the control group (0.41 ± 0.05 to 0.55 ± 0.05) (P > 0.05). However, the I2-diet group showed slightly greater NBT reduction compared to the control group throughout the 5 weeks. NBT reduction activity decreased gradually with time in both groups (Fig. 4). 4. Discussion Probiotic bacteria are considered an alternative method for control and prevention of the diverse diseases caused by pathogenic bacteria in aquaculture. There are many reports of protection against various diseases by probiotic-containing food. Lactobacillus rhamnosus was successfully used as a feed additive in tilapia (Pirarat et al., 2006) and rainbow trout (Choi and Yoon, 2008) to prevent edwardsiellosis. Similarly, C. divergens (Gildberg and Mikkelsen, 1998), Clostridium butyricum (Sakai et al., 1995) and Kocuria species (Sharifuzzaman and Austin, 2009) were used against vibriosis, A. sobria (Brunt and Austin, 2005), L. plantarum (Vendrell et al., 2008) and Enterococcus faecium (Kim et al., 2012) to prevent lactococcosis and A. sobria (Brunt and Austin, 2005), Bacillus subtilis (Kamgar and Ghane, 2012) to prevent streptococcosis. Streptococcosis causes greater damage to farmed fishes, such as flounder, when seawater temperatures exceed 17 °C (Agnew and Barnes, 2007). The methods that can be used for the application of probiotics in aquaculture include baths, suspensions and feeding. However, supplementation of food with probiotics is the most effective and simplest method to ensure successful colonization of and establishment in the gut (Nayak, 2010). In a previous study, we isolated L. lactis subsp. lactis I2 from the intestines of healthy olive flounder. We expected that the isolated LAB would retain its activity in the presence of salt and found that it effectively inhibited the growth of S. iniae, a potent pathogen associated with streptococcosis (Heo et al., 2012). To determine whether the isolated strain could be used to protect against streptococcosis, we supplemented feed with L. lactis subsp. lactis I2 (I2-diet) as a probiotic and determined its effects on the growth and immune response of olive flounder. The load of L. lactis subsp. lactis I2 in the intestine of olive flounder was 1.4× 107 CFU/ml after supplementation with an I2-diet for 2 weeks (data not shown). The growth of fish was monitored by means of a variety of physiological factors, such as digestion, energy absorption, metabolism and discharge ability (Brett, 1979). The growth rate was higher in the I2-diet group than in the control group (Table 1). We also investigated a number of innate immune factors, such as lysozyme, antiprotease, and peroxidase activities and oxygen radical production, in olive flounder from the 2nd to 5th weeks (at 1-week
intervals) following the commencement of probiotic-supplemented feeding. To determine the effects of a probiotic, it should be supplied as a feed additive for a minimum of 2 weeks to allow colonization of the gut (Desriac et al., 2010; Nayak, 2010). Levels of all of the above immune factors were similar to the control group during the 1st week (data not shown). Lysozyme is a component of the innate immune system and plays an important role in defense mechanisms due to its anticancer, antiviral and opsonization properties (Jollès and Jollès, 1984). Panigrahi et al. (2004) noted that supplementation of L. rhamnosus (109 CFU g −1) as a feed additive for 30 days enhanced lysozyme activity in rainbow trout. Stimulation of lysozyme activity in rainbow trout was observed after 2 weeks of feeding with Kocuria sp. SM1 (Sharifuzzaman and Austin, 2009) and Carnobacterium divergens B33 (Kim and Austin, 2006). Lysozyme activity was significantly enhanced during the 3rd and 4th weeks in the I2-diet group (Fig. 2). These findings suggest that high lysozyme activity may contribute to defense against bacterial diseases in probiotic-supplemented fish. Antiprotease activity in serum, which contains α1-antiprotease, α2-antiprotease and α2-macroglobulin, inhibits the action of the proteases used by some bacteria to invade the host. This activity is generally high and unaffected by immunization or infection (Ellis, 2001; Magnadottir et al., 2006). In this study, total antiprotease activity, in terms of mean serum antitrypsin activity, increased in the I2-diet group relative to the control group during the 2nd week, but the overall antiprotease regime in the I2-diet (79.72 ± 0.34 to 81.6 ±0.15) and control (79.66 ± 0.11 to 81.6± 0.11) groups was similar. Phagocytic cells, such as neutrophils and macrophages, play an important role in antibacterial defense. These cells engulf and kill bacteria by producing reactive oxygen species (ROS), comprising superoxide anions (O2−), hydrogen peroxide (H2O2) and hydroxyl free radicals (OH −) during the respiratory burst (Ellis, 1999). Taoka et al. (2006) reported that NBT reduction activity increased slightly on the 15th day after probiotic feed supplementation in tilapia. In rainbow trout, enhanced activity was identified relative to controls after 4 weeks (Sharifuzzaman and Austin, 2009). Moreover, the greatest activity against V. anguillarum was observed in rainbow trout 2 weeks after the start of a probiotic feeding regime. When we determined blood ROS production by NBT assay, the result was very similar to those in other fish species (Fig. 4). Cross (2002) defined probiotics as beneficial microorganisms that can colonize and multiply in the gut of a host and exert various beneficial effects, including immunomodulation, by influencing diverse host biological systems. Previously, we reported that L. lactis subsp. lactis I2 effectively inhibits the growth of S. iniae in vitro. This effect was confirmed by the increased survival rate of olive flounder infected with S. iniae (Fig. 5). When normal assorted feed
100
Cumulative survival rate (%)
Absorbance at 550nm
1
23
80 60 40 20
Probiotics (I2) Control
0 0
2
4
6
8
10
12
14
Day after challenge Fig. 5. Cumulative survival rates of juvenile olive flounder after challenge with S. iniae.
24
W.-S. Heo et al. / Aquaculture 376–379 (2013) 20–24
supplemented with the isolated LAB was supplied to olive flounder, activity against S. iniae was 2.5-fold higher than in the control. This may have been due to enhancement of the gut mucosal barrier, ‘colonization’ of the gastrointestinal tract of olive flounder, or enhanced induction of immune factors. Also, a role for nisin Z, a bacteriocin produced by L. lactis subsp. lactis I2, cannot be excluded. However, the enhanced immune factors in the I2-supplemented group certainly contributed to the improved growth rate in olive flounder. The synergistic effects of these diverse factors can also be considered the final influence on the high survival rate of flounder. Therefore, the enhancement of immune activity by the probiotics themselves or their production of nisin Z prevented infection. In conclusion, L. lactis subsp. lactis I2 isolated from intestines of healthy olive flounder enhanced the immune response and effectively controlled S. iniae infection of olive flounder. This strain is a good candidate feed additive to improve fish growth. However, further extensive investigations, including field tests and a full commercial cost-benefit analysis, are needed prior to its widespread application in aquaculture. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (grant 2010-0006364). References Agnew, W., Barnes, A.C., 2007. Streptococcus iniae: an aquatic pathogen of global veterinary significance and a challenging candidate for reliable vaccination. Veterinary Microbiology 122, 1–15. Brett, J.R., 1979. Environmental factors and growth. In: Hoar, W.S., Randall, D.J., Brett, J.R. (Eds.), Fish Physiology. Bioenergetics and Growth, vol. VIII. Academic Press, New York, U.S.A., pp. 599–675. Brunt, J., Austin, B., 2005. Use of a probiotic to control lactococcosis and streptococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum). Journal of Fish Diseases 28, 693–701. Calo-Mata, P., Arlindo, S., Boehme, K., de Miguel, T., Pascoal, A., Barros-Velazquez, J., 2008. Current applications and future trends of lactic acid bacteria and their bacteriocins for the biopreservation of aquatic food products. Food Bioprocess Technology 1, 43–63. Choi, S.H., Yoon, T.J., 2008. Non-specific immune response of rainbow trout (Oncorhynchus mykiss) by dietary heat-inactivated poteintial probiotics. Immune Network 8 (3), 67–74. Cross, M.L., 2002. Microbes versus microbes: immune signals generated by probiotic lactobacilli and their role in protection against microbial pathogens. FEMS Immunology and Medical Microbiology 34, 245–253. Desriac, F., Defer, D., Bourgougnon, N., Brillet, B., Chevalier, P.L., Fleury, Y., 2010. Bacteriocin as weapons in the marine animal-associated bacteria warfare: inventory and potential applications as an aquaculture probiotic. Marine Drugs 8, 1153–1177. Ellis, A.E., 1990. Serum antiproteases in fish. In: Stolen, J.S., Fletcher, T.C., Anderson, D.P., Roberson, W.B., Van Muiswinkel, W.B. (Eds.), Techniques in fish Immunology. SOS Publication, Fair Haven, U.S.A., pp. 95–103. Ellis, A.E., 1999. Immunity to bacteria in fish. Fish & Shellfish Immunology 9, 291–308. Ellis, A.E., 2001. Innate host defense mechanisms of fish against viruses and bacteria. Development & Comparative Immunology 25, 827–839. Gatesoupe, F.J., 1999. The use of probiotics in aquaculture. Aquaculture 180, 147–165. Gildberg, A., Mikkelsen, H., 1998. Effect of supplementing the diet to Atlantic cod (Gadus morhua) fry with lactic acid bacteria and immuno-stimulating peptides during a challenge trial with Vibrio anguillarum. Aquaculture 167, 103–113. Haza, A.I., Zabala, A., Morales, P., 2004. Protective effect and cytokine production of a Lactobacillus plantarum strain isolated from ewes' milk cheese. International Dairy Journal 14, 29–38.
Heo, W.S., Kim, E.Y., Kim, Y.R., Hossain, M.T., Kong, I.S., 2012. Salt effect of nisin Z isolated from a marine fish on the growth inhibition of Streptococcus iniae, a pathogen of streptococcosis. Biotechnology Letters 34, 315–320. Jollès, P., Jollès, J., 1984. What is new in lysozyme research? Always a model system, todays as yesterday. Molecular and Cell Biochemistry 63, 156–189. Kamgar, M., Ghane, M., 2012. Evaluation of Bacillus subtilis effect as probiotic on hematological parameters of rainbow trout, Oncorhynchus mykiss (Walbaum) following experimental infection with Streptococcus iniae. Journal of Fisheries and Aquatic Science 7 (6), 422–430. Karunasagar, I., Pai, R., Malathi, G.R., Karunasagar, I., 1994. Mass mortality of Pecaeus monodon larvae due to antibiotic resistant Vibrio harveyi infection. Aquaculture 128, 203–209. Kesarcodi-Warson, A., Kaspar, H., Lategan, M.J., Gilson, L., 2008. Probiotics in aquaculture: the need, principles and mechanisms of action and screening processes. Aquaculture 274, 1–14. Kim, D.H., Austin, B., 2006. Innate immune responses in rainbow trout (Oncorhynchus mykiss, Walbaum) induced by probiotics. Fish & Shellfish Immunology 21, 513–524. Kim, K.W., Bai, S.C., Koo, J.W., Wang, X., Kim, S.K., 2002. Effect of dietary Chlorella ellipsoidea supplementation on growth, blood characteristics, and whole-body composition in juvenile Japanese flounder Paralichthys olivaceus. Journal of the World Aquaculture Society 33 (4), 425–431. Kim, K.W., Kim, S.S., Jeong, J.B., Jeon, Y.J., Kim, K.D., An, C.M., Lee, K.J., 2011. Effects of dietary supplementation of fermented garlic powder and fluid on growth performance, immune responses, blood components, and disease resistance against Edwardsiella tarda and Streptococcus iniae in olive flounder Paralichthys olivaceus. Korean Journal of Fisheries and Aquatic Sciences 44 (6), 644–652. Kim, Y.R., Kim, E.Y., Choi, S.Y., Hossain, M.T., Oh, R., Heo, W.S., Lee, J.M., Cho, Y.C., Kong, I.S., 2012. Effect of a probiotic strain, Enterococcus faecium, on the immune responses of olive flounder (Paralichthys olivaceus). Journal of Microbiology and Biotechnology 22 (4), 526–529. Magnadottir, B., Gudmundsdottir, B.K., Lange, S., Steinarsson, A., Oddgeirsson, M., Bowden, T., et al., 2006. Immunostimulation of larvae and juveniles of cod, Gadus morhua L. Journal of Fish Disease 29 (3), 147–155. MIFAFF, Ministry for Food Agriculture Forestry and Fisheries, 2003. Statistical year book of maritime affairs and fisheries. Nayak, S.K., 2010. Probiotics and immunity: a fish perspective. Fish & Shellfish Immunology 29, 2–14. Panigrahi, A., Kiron, V., Kobayashi, T., Puangkaew, J., Satoh, S., Sugita, H., 2004. Immune response in rainbow trout Oncorhynchus mykiss induced by a potential probiotic bacteria Lactobacillus rhamnosus JCM 1136. Veterinary Immunology and Immunopatholpogy 102, 379–388. Perdigon, G., Alvarez, S., Narder de Macas, M.E., Roux, M.E., de Ruiz, Pesce, Holgado, A., 1990. The oral administration of lactic acid bacteria increases the mucosal intestinal immunity in response to enterocpathogens. Journal of Food Protection 53, 404–410. Pirarat, N., Kobayashi, T., Katagiri, T., Maita, M., Endo, M., 2006. Protective effects and mechanisms of a probiotic bacterium Lactobacillus rhamnosus against experimental Edwardsiella tarda infection in tilapia (Oreochromis niloticus). Veterinary Immunology and Immunopatholpogy 113, 339–347. Sakai, M., Yoshida, T., Astuta, S., Kobayashi, M., Endo, M., 1995. Enhancement of resistance to vibriosis in rainbow trout, Oncorhynchus mykiss (Walbaum) by oral administration of Clostridium butyricum bacteria. Journal of Fish Diseases 18, 187–190. Sharifuzzaman, S.M., Austin, B., 2009. Influence of probiotic feeding duration on disease resistance and immune parameters in rainbow trout. Fish & Shellfish Immunology 27, 440–445. Sitjà-Bobadilla, A., Palenzuela, O., Alvarez-Pellitero, P., 2008. Immune response of turbot, Psetta maxima (L.) (Pisces: Teleostei), to formalin-killed scuticociliates (Ciliophora) and adjuvanted formulations. Fish & Shellfish Immunology 24, 1–10. Smith, P., Hiney, M.P., Samuelsen, O.B., 1994. Bacterial resistance to antimicrobial agents used in fish farming: a critical evaluation of method and meaning. Annual Review of Fish Diseases 4, 273–313. Taoka, Y., Maeda, H., Jo, J.Y., Kim, S.M., Park, S.I., Yoshikawa, T., Sakata, T., 2006. Use of live and dead probiotic cells in tilapia Oreochromis niloticus. Fisheries Science 72, 755–766. Vendrell, D., Balcárzar, J.L., de Blas, I., Ruiz-Zarzuela, I., Girones, O., Muzquiz, J.L., 2008. Protection of rainbow trout (Oncorhynchus mykiss) from lactococcosis by probiotic bacteria. Comparative Immunology, Microbiology and Infectious Diseases 31, 337–345. Zuo, X., Woo, P.T.K., 1997. Natural anti-proteases in rainbow trout, Oncorhynchus mykiss and brook charr, Salvelinus fontinalis and the in vitro neutralization of fish α2-macroglobulin by the metalloprotease from the pathogenic haemoflagellate, Crytobia salmositica. Parasitology 114, 375–381.