6 : 1359 KocuiiaSM cells/g,,, 10% 28%, (73% 92%), 3 [13] Wang Yan-Bo [14] Enterococcus faecium ZJ CFU/ ml, 40 d (DWG), [15], [5, 16 17

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1 , 18(6): Journal of Fishery Sciences of China 研究论文 DOI: /SP.J 李桂英 1,2, 宋晓玲 2, 孙艳 1,2, 麦康森 1, 谢国驷 3, 黄倢 1., ; 2., ; 3., 摘要 : (Bacillus firmus) ( CFU/g)+ (Photobacterium damsela) (1%) ( CFU/g)+ (Vibrio alginolyticus) (1%) 3 3, (3.2±0.26) g (Litopenaeus vannamei) 30 d 5 d, (ACP) (AKP) (NOS) (SOD) (UL) ; 16, 0.9 g/10, (WSSV), : SOD ACP AKP NOS (P<0.05), WSSV ( CFU/g) (1%), WSSV 14 d 10.71%; 64.28%,, : ; ; ; ; WSSV 中图分类号 : S941 文献标志码 : A 文章编号 : (2011) (Litopenaeus vannamei),,,, [1], [2],, [3] [4], [5 6], [7 8] [9],, Kozasa [10], Olsson [11] Irene Salinas [12],,,, Sharifuzzaman 收稿日期 : ; 修订日期 : 基金项目 : ( ). 作者简介 : (1985 ),,,. lgytaqf@163.com 通信作者 :. songxl@ysfri.ac.cn

2 6 : 1359 KocuiiaSM cells/g,,, 10% 28%, (73% 92%), 3 [13] Wang Yan-Bo [14] Enterococcus faecium ZJ CFU/ ml, 40 d (DWG), [15], [5, 16 17] Garriques [18],,, Rengpipat [5,7] (Penaeus monodon) Sll, ( ) 3 (Vibrio harveyi), Sll 100%, 26%, 3 Evelyne Bache`re [19],, 100% [20 21], 3,,,, (Litopenaeus vannamei), (3.2±0.26) g,, WSSV 7 d, 720, 4, 3, 60, 1, 1/3; 3,, 10% 15%( ), 23 28, (30±1), ph 8.0±0.2, 3 (4 d, 3 d, 7 d 1 ), (Bacillus firmus) (Photobacterium damsela) (Vibrio alginolyticus) 2006, 80,,, CFU/mL, 1% [21] ( 250 g/kg, 250 g/kg, 300 g/kg, 100 g/kg, 30 g/kg, 10 g/kg, C 2 g/kg, NaH 2 PO 4 2H 2 O 2 g/kg, Na 2 HPO 4 12H 2 O 3 g/kg, 10 g/kg, 20 g/kg), 3, (B. firmus) ( CFU/g), 1 ( CFU/g) (P. damsela) (1%) 2 ( CFU/g) (V. alginolyticus) (1%) 3 80,,, 37, WSSV

1360 18 WSSV,, 80 1.2 5 d 10 15, (1mL), 1.5 ml, 4,, [18] 1.3 (SOD) (AKP) (ACP) (NOS) (UL), 1.4 WSSV 0.9 g/10 16, 30, 3 1d,, 80,,,, 14 d 1.5 SPSS16.0 (one-way ANOVE), (P<0.05) Duncan 2 2.

3 WSSV,, d 10 15, (1mL), 1.5 ml, 4,, [18] 1.3 (SOD) (AKP) (ACP) (NOS) (UL), 1.4 WSSV 0.9 g/10 16, 30, 3 1d,, 80,,,, 14 d 1.5 SPSS16.0 (one-way ANOVE), (P<0.05) Duncan (SOD) 1, 15 d 1 2 SOD (P<0.05), 15 d, 1 2 SOD (P<0.05), (P>0.05) 15 d (P<0.05), 15 d 5 d, SOD (P<0.05), SOD, SOD, 5 d,, 1 ( CFU/g) ( 1%) ; : ( CFU/g) ; 2: ( CFU/g) ( 1%). Fig.1 Serum superoxide dismutase (SOD) activity of Litopenaeus vannamei in test groups Values with different letters mean significant difference (P<0.05) between groups. Control: normal diet; Compound group one: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated P. damsela; single group: immune group with addition of live B. firmus ( CFU/g); Compound group two: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated V. alginolyticus.

6 : 1361 SOD, 2.2 (AKP) 2, 1 2 AKP 25 d (P<0.05), 20 d 15 d, AKP (P<0.05), 10 d,, AKP, AKP (P<0.05), AKP, 2.3 (ACP) 3, 1 (P<0.05), 10 d 2 (AKP) (1.0 10 8 CFU/g) ( 1%) ; : (1.0 10 8 CFU/g) ; 2: (1.

4 6 : 1361 SOD, 2.2 (AKP) 2, 1 2 AKP 25 d (P<0.05), 20 d 15 d, AKP (P<0.05), 10 d,, AKP, AKP (P<0.05), AKP, 2.3 (ACP) 3, 1 (P<0.05), 10 d 2 (AKP) ( CFU/g) ( 1%) ; : ( CFU/g) ; 2: ( CFU/g) ( 1%). Fig.2 Serum alkaline phosphatase (AKP) activity of Litopenaeus vannamei in test groups Values with different letters mean significant difference (P<0.05) between groups. Control: normal diet; compound group 1: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated P. damsela; single group: immune group with addition of live B. firmus ( CFU/g); compound group 2: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated V. alginolyticus. 3 (ACP) ( CFU/g) (1%) ; : ( CFU/g) ; 2: ( CFU/g) ( 1%). Fig.3 Serum acid phosphatase(acp) activity of Litopenaeus vannamei in test groups Values with different letters mean significant difference (P<0.05) between groups. Control: normal diet; compound group 1: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated P. damsela; single group: immune group with addition of live B. firmus ( CFU/g); compound group 2: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated V. alginolyticus.

5 d 10 d (P>0.05), (P< 0.05), 10 d 5 d (P>0.05), (P<0.05), 15 d ACP, (P<0.05), ACP (P<0.05), ACP 2.4 (TNOS) 4, 1 TNOS 10 d (P<0.05), 5 d 15 d (P>0.05), 10 d 5 d, 2 10 d 5 d (P>0.05), (P<0.05), 5 d 10 d 5 d 5 d (P>0.05), (P<0.05), 5 d,,, 5 d TNOS (P>0.05) TNOS, 2.5 (inos) 5, 1 2, 5 d, 10 d,, 10 d inos, 5 d,, inos, (P<0.05), inos, 4 (TNOS) ( CFU/g) ( 1%) ; : ( CFU/g) ; 2: ( CFU/g) ( 1%). Fig. 4 Serum total nitric oxide synthase (TNOS) activity of Litopenaeus vannamei in test groups Values with different letters mean significant difference (P<0.05) between groups. Control: normal diet; compound group 1: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated P. damsela; single group: immune group with addition of live B. firmus ( CFU/g); compound group 2: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated V. alginolyticus.

6 6 : (UL) 6,, UL (P>0.05), 5 d UL, UL 2.7 7, 4 d (P>0.05), 5 6, (P<0.05), 9 d (64.28±12.8)% 1 (10.71± 7.1)%, (39.28±12.9)% 2 (26.19±5.45)% (P<0.05), WSSV, 5 (inos) ( CFU/g) ( 1%) ; : ( CFU/g) ; 2: ( CFU/g) ( 1%). Fig.5 Serum inducible nitric oxide synthase (inos) activity of Litopenaeus vannamei in test groups Values with different letters mean significant difference (P<0.05) between groups. Control: normal diet; compound group 1: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated P. damsela; single group: immune group with addition of live B. firmus ( CFU/g); compound group 2: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated V. alginolyticus. 6 (UL) ( CFU/g) ( 1%) ; : ( CFU/g) ; 2: ( CFU/g) ( 1%). Fig.6 Serum lysozyme (UL) activity of Litopenaeus vannamei in test groups Values with different letters mean significant difference (P<0.05) between groups. Control: normal diet; compound group 1: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated P. damsela; single group: immune group with addition of live B. firmus ( CFU/g); compound group 2: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated V. alginolyticus.

7 WSSV ( CFU/g) ( 1%) ; : ( CFU/g) ; 2: ( CFU/g) ( 1%). Fig.7 Cumulative mortality of L. vannamei after challenged with WSSV Values with different letters mean significant difference (P<0.05) between groups. Control: normal diet; compound group 1: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated P. damsela; single group: immune group with addition of live B. firmus ( CFU/g); compound group 2: immune group with addition of live B. firmus ( CFU/g) and 1% inactivated V. alginolyticus. 3,, [22] [23 26],,,, [23, 27 28], 1992,, 35% Austin [29],, 21 Garriques [18] Gatesoupe [30],,, [31 32] [21] [12, 33],,

8 6 : 1365,,,, [21 22] [34 35], Salinas [12], (WSSV),,,, (IHNV) [36], [37] NICA 1030 NICA 1031 IHNV (OMV) [38] WSSV,,,,,,, [21], WSSV,, :,,, WSSV, 3, WSSV,, 参考文献 : [1] Li J Q, Tan B P, Mai K S. Comparative study between probiotic bacterium Arthrobacter XE-7 and chloramphenicol on protection of Penaeus chinensis post-larvae from pathogenic vibrios [J]. Aquaculture, 2006, 253(1 4): [2] Nomoto K. Prevention of infections by probiotics [J]. J Biosci Bioengin, 2005, 100(6): [3] Suzer C, Coban D, Kamaci H,et al. Lactobacillus spp. bacteria as probiotics in gilthead sea bream (Sparus aurata, L.) larvae: Effects on growth performance and digestive enzyme activities [J]. Aquaculture, 2008, 280(1 4): [4] Vaseeharan B, Ramasamy P. Control of pathogenic Vibrio spp. by Bacillus subtilis BT23, a possible probiotic treatment for black tiger shrimp Penaeus monodon[j]. Lett Appl Microbiol, 2003, 36(2): [5] Rengpipat S, Phianphak W, Piyatiratitivorakul S. Effects of a probiotic bacterium on black tiger shrimp Penaeus monodon

9 survival and growth [J]. Aquaculture, 1998, 167(3 4): [6] Liu K F, Chiu C H, Shiu Y L. Effects of the probiotic, Bacillus subtilis E20, on the survival, development, stress tolerance, and immune status of white shrimp, Litopenaeus vannamei larvae[j]. Fish Shellf Immunol, 2010, 28(5 6): [7] Rengpipat S, Rukpratanporn S, Piyatiratitivorakul S. Immunity enhancement in black tiger shrimp (Penaeus monodon) by a probiont bacterium (Bacillus S11)[J]. Aquaculture, 2000, 191(4): [8] Wang Y, Zha L, Xu Z. Effects of probiotics on Penaeus vannamei pond sediments[j]., 2006, 17(9): [9] Wang Y B, Li J R, Lin J. Probiotics in aquaculture: Challenges and outlook[j]. Aquaculture, 2008, 281(1 4): 1 4. [10] Kozasa M. Toyocerin (Bacillu stoyoi) as growth promoter for animal reeding[j]. Microbiol Aliment Nutr, 1986, 4: [11] Ollson J C, Westerdahl A, Conway pl et al. Intestinal colonization potential of turbot (Scophthalmus maximus) and dab (Limandali manda) associated bacteria with inhibitory effects against Vibrio anguillarum[j]. Appland Environ Micro, 2005, 58 (2): [12] Salinas I, Abelli L, Bertoni F. Monospecies and multispecies probiotic formulations produce different systemic and local immunostimulatory effects in the gilthead seabream (Sparus aurata L.)[J]. Fish Shellf Immunol, 2008, 25(1 2): [13] Sharifuzzaman S M, Austin B. Development of protection in rainbow trout (Oncorhynchus mykiss, Walbaum) to Vibrio anguillarum following use of the probiotic Kocuria SM1[J]. Fish Shellf Immunol, 2010, 29(2): [14] Wang Y B, Tian Z Q, Yao J T. Effect of probiotics, Enteroccus faecium, on tilapia (Oreochromis niloticus) growth performance and immune response[j]. Aquaculture, 2008, 277(3 4): [15] Dalmin G., Kathiresan K, Purushothaman A. Effect of probiotics on bacterial population and health status of shrimp in culture pond ecosystem[j]. Indian J Exp Biol, 2001, 39(9): [16]. [D]. :, [17] Gullian M, Thompson F, Rodriguez J. Selection of probiotic bacteria and study of their immunostimulatory effect in Penaeus vannamei[j]. Aquaculture, 2004, 233(1 4): [18] Garriques D, Arevalo G. An evaluation of the production and use of a live bacterial isolate to manipulate the microbial flora in commercial production of Penaeus vannamei postlarvae in Ecuador[J]. In: Browdy C L, Hopkins J S (Eds.), Swimming through Troubled Water. Proceedings of the Special Session on Prawn Farming, Aquaculture '95. World Aquaculture Society, 1995: [19] Evelyne Bache`re. Anti-infectious immune effectors in marine: potential tools for disease control in larviculture[j]. Aquaculture, 2003, 227: [20],,,. 4 [J]., 2008(4): [21],,. [J]., 2010(1): [22]. [D]. :, [23] Gomez G B, Roque A, Turnbull J F. The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms [J]. Aquaculture, 2000, 191(1 3): [24],,. [J]., 2007(4): [25] Li K, Zheng T, Tian Y. Beneficial effects of Bacillus licheniformis on the intestinal microflora and immunity of the white shrimp, Litopenaeus vannamei[j]. Biotechnol Lett, 2007, 29(4): [26] Ziaei N S, Rezaei M H, Takami G A. The effect of Bacillus spp. bacteria used as probiotics on digestive enzyme activity, survival and growth in the Indian white shrimp Fenneropenaeus indicus[j]. Aquaculture, 2006, 252(2 4): [27] Gatesoupe F J. The use of probiotics in aquaculture[j]. Aquaculture, 1999, 180(1 2): [28] Ninawe A S, Selvin J. Probiotics in shrimp aquaculture: avenues and challenges[j]. Crit Rev Microbiol, 2009, 35(1): [29] Austin B, Stuckey L F, Robertson P A W, et al. A probiotic strain of Vibrio alginolyticus effective in reducing diseases caused by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalii [J]. Fish Dis, 1995, 18: [30] Gatesoupe F J. Siderophore production and probiotic effect of Vibriosp. associated with turbot larvae, Scophthalmus maximus[j]. Aquat Living Resour, 1997, 10: [31] Krupesha Sharma S R, Shankar K M, Sathyanarayana M L.

10 6 : 1367 Evaluation of immune response and resistance to diseases in tiger shrimp, Penaeus monodon fed with biofilm of Vibrio alginolyticus [J]. Fish Shellf Immunol, 2010, 29(5): [32] Xiong X P, Zhang B W, Yang M J. Identification of vaccine candidates from differentially expressed outer membrane proteins of Vibrio alginolyticus in response to NaCl and iron limitation [J]. Fish Shellf Immunol, 2010, 29(5): [33],,. [J]., 2008, 02: [34] 樑,,. [J]., 2009(2): [35],, 倢. [J]., 2004, 11(1): [36] Kamei Y, Yoshimizu M, Ezura Y, et al. Screening of bacteria with antiviral activity from fresh water salmonid hatcheries[j]. Microbiol Immunol, 1988, 32: [37] Girones R, Jofre J T, Bosch A. Isolation of marine bacteria with antiviral properties [J]. Can J Microbiol, 1989, 35: [38] Direkbusarakom S, Yoshimizu M, Ezura Y, et al. Vibrio spp. the dominant flora in shrimp hatchery against some fish pathogenic viruses [J]. Mar Biotechnol, 1998, 6: Effects of probiotics from the shrimp intestine on the non-specific immunity and antiviral capacity of Litopenaeus vannamei LI Guiying 1, 2, SONG Xiaoling 2, SUN Yan 1, 2, MAI Kangsen 1, XIE Guosi 3, HUANG Jie 2 1. The Key Laboratory of Mariculture, Ministry of Education; Ocean University of China, Qingdao , China; 2. Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao , China; 3. Shanghai Ocean University, Shanghai , China Abstract: We evaluated the effects of probiotics on the non-specific immunity and antiviral capacity of Litopenaeus vannamei. We fed groups of shrimp (body weight 3.2 g±0.26 g) a diet supplemented with either CFU/g Bacillus firmus (live) plus 1% Photobacterium damsela (inactivated), CFU/g Bacillus firmus (live), or CFU/g Bacillus firmus (live) plus 1% Vibrio alginolyticus (inactivated). The control groups were fed a non-supplemented diet. Each diet was fed to triplicate groups of 60 shrimp that were reared in aquaria. We randomly selected 10 shrimp per group every 5 d and measured a range of humoral immune parameters (superoxide dismutase SOD, acidic phosphatase ACP, alkaline phosphatase AKP, nitric oxide synthase NOS and lysozyme UL) in each individual. The shrimp were challenged with white spot syndrome virus (WSSV) after two weeks. We then calculated the cumulative mortality of each experimental group. Supplementation with bacteria significantly increased serum SOD, ACP, AKP, and NOS compared with the controls. There was significantly lower cumulative mortality in shrimp that were fed the probiotics diet (10.7%, 39.3% and 26.2%, respectively) compared to the control (64.28%). Our results suggest that supplementing the diet with shrimp intestine probiotics improves immunity and the antiviral capacity of Litopenaeus vannamei. Key words: intestinal probiotics; Litopenaeus vannamei; non-specific immunity; WSSV Corresponding author: SONG Xiaoling. songxl@ysfri.ac.cn

29期

29期中国农学通报 01,8(9):109-114 Chinese Agricultural Science Bulletin 复方中草药对凡纳滨对虾生长及非特异性免疫功能的影响 1, 王芸, 李健, 陈萍 1,, 张喆, 何玉英, 李吉涛, 刘德月 ( 1 农业部水产品加工重点实验室 / 中国水产