饲料中添加复合芽孢杆菌对凡纳滨对虾抗病毒感染能力及抗病基因表达的影响
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摘要
自健康凡纳滨对虾(Litopenaeus vannamei)分离到枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(B. licheniformis)和短小芽孢杆菌(B. pumilus),将上述芽孢杆菌以单一和3株复合的方式包裹在基础饲料表面,制成益生菌饲料;每日投喂对虾,3周后进行白斑综合征病毒(WSSV)人工感染。统计实验组和对照组的累积死亡率,测定对虾鳃组织内WSSV拷贝数,分析对虾肠道组织含半胱氨酸的天冬氨酸蛋白酶基因(Caspase)和硫氧还原蛋白基因(Trx)的相对表达量。结果显示,感染实验结束时,A组(枯草芽孢杆菌)、B组(地衣芽孢杆菌)、C组(短小芽孢杆菌)和D组(枯草芽孢杆菌+地衣芽孢杆菌+短小芽孢杆菌复合益生菌)的对虾累积死亡率分别为(73.3±7.0)%、(63.3±5.5)%、(75.0±7.9)%和(50.0±5.3)%,显著低于对照组(PBS组)(100%);在整个感染阶段,各实验组的病毒拷贝数呈先上升后下降的趋势,但对照组呈现一直上升趋势,且显著高于实验组。抗病基因表达结果显示,WSSV感染后,各组对虾肠道Caspase相对表达量随感染时间的延长呈先上调再下调的趋势,且在18h各组对虾肠道Caspase表达量达到最大值;益生菌摄取和WSSV感染都能刺激Trx的表达,益生菌的刺激相对平缓,且各实验组对虾肠道Trx相对表达量在WSSV感染后的18h时陡升到最大值,极显著高于对照组,且以D组的激活能力最强。研究证实,枯草芽孢杆菌、地衣芽孢杆菌和短小芽孢杆菌均可提高对虾抗WSSV感染能力,复合芽孢杆菌抗病毒能力最突出。对虾抗病力的提高可能与芽孢杆菌减缓了病毒在靶组织的增殖速率、提高了Caspase和Trx基因表达水平相关。
Bacillus subtilis, B. licheniformis, and B. pumilus, were isolated from healthy Litopenaeus vannamei, and were added to the base feed surface in single and mixed treatments. Probiotic feed was fed to the shrimp daily, and the shrimp were infected by white spot syndrome virus(WSSV) three weeks later.During the virus infection, the cumulative mortality and the number of WSSV copies in the gill tissue were statistically analyzed for each group. The relative expression of cysteinyl aspartate specific proteinase gene(Caspase) and Thioredoxin gene(Trx) in the intestinal tissue of the shrimp was measured by real-time PCR during feeding of probiotic feed and virus infection. The results showed that the cumulative mortality of B. subtilis(Group A), B. licheniformis(Group B), B. pumilus(Group C) and B.subtilis+B. licheniformis+B. pumilus(Group D), were(73.3±7.0)%,(63.3±5.5)%,(75.0±7.9)% and(50.0±5.3)% respectively, significantly lower than that of the control group(Group PBS), where the cumulative mortality of shrimp was 100%. The number of WSSV copies in each experimental group firstly increased, and then declined in the whole infection stage, but the number of WSSV copies of Bacillus groups was significantly higher than that of the control group every time(P<0.05 or P<0.01).The number of WSSV copies in the gill tissue of the mixed Bacillus group was extremely significantly lower than that of the control group, in the 8 d after virus infection. The relative expression of the Caspase gene was not significant in the 21 d of feeding with probiotic feed. After WSSV infection, the relative expression of Caspase in the intestinal tissue of each group firstly increased and then decreased, with time,reaching the maximum at the 18 th h. The relative expression of Caspase in the intestinal tissue of the mixed Bacillus group was the highest, with the expression level at the 96 th h still significantly higher than that of the control group. Feeding with Bacillus spp. and infection of WSSV, both could stimulate expression of the Trx gene, and the stimulation of feeding with Bacillus spp. was relatively gentle. After WSSV infection, relative expression of the Trx gene in the intestinal tissue of each Bacillus group grew quickly to its maximum at the 18 th h, which was extremely significantly higher than that of the control group, and the activation on Trx gene expression from the mixed Bacillus group was the strongest. It can be surmised that the enhancement of the anti WSSV infection ability of shrimp may be related to the reduction of the speed of virus amplification in the target tissue, and the increase of the expression level of the anti-infection genes, such as Caspase and Trx, that were brought by feeding Bacillus spp.
Bacillus subtilis, B. licheniformis, and B. pumilus, were isolated from healthy Litopenaeus vannamei, and were added to the base feed surface in single and mixed treatments. Probiotic feed was fed to the shrimp daily, and the shrimp were infected by white spot syndrome virus(WSSV) three weeks later.During the virus infection, the cumulative mortality and the number of WSSV copies in the gill tissue were statistically analyzed for each group. The relative expression of cysteinyl aspartate specific proteinase gene(Caspase) and Thioredoxin gene(Trx) in the intestinal tissue of the shrimp was measured by real-time PCR during feeding of probiotic feed and virus infection. The results showed that the cumulative mortality of B. subtilis(Group A), B. licheniformis(Group B), B. pumilus(Group C) and B.subtilis+B. licheniformis+B. pumilus(Group D), were(73.3±7.0)%,(63.3±5.5)%,(75.0±7.9)% and(50.0±5.3)% respectively, significantly lower than that of the control group(Group PBS), where the cumulative mortality of shrimp was 100%. The number of WSSV copies in each experimental group firstly increased, and then declined in the whole infection stage, but the number of WSSV copies of Bacillus groups was significantly higher than that of the control group every time(P<0.05 or P<0.01).The number of WSSV copies in the gill tissue of the mixed Bacillus group was extremely significantly lower than that of the control group, in the 8 d after virus infection. The relative expression of the Caspase gene was not significant in the 21 d of feeding with probiotic feed. After WSSV infection, the relative expression of Caspase in the intestinal tissue of each group firstly increased and then decreased, with time,reaching the maximum at the 18 th h. The relative expression of Caspase in the intestinal tissue of the mixed Bacillus group was the highest, with the expression level at the 96 th h still significantly higher than that of the control group. Feeding with Bacillus spp. and infection of WSSV, both could stimulate expression of the Trx gene, and the stimulation of feeding with Bacillus spp. was relatively gentle. After WSSV infection, relative expression of the Trx gene in the intestinal tissue of each Bacillus group grew quickly to its maximum at the 18 th h, which was extremely significantly higher than that of the control group, and the activation on Trx gene expression from the mixed Bacillus group was the strongest. It can be surmised that the enhancement of the anti WSSV infection ability of shrimp may be related to the reduction of the speed of virus amplification in the target tissue, and the increase of the expression level of the anti-infection genes, such as Caspase and Trx, that were brought by feeding Bacillus spp.
引文
Chai PC,Song XL,Chen GF,et al.Dietary supplementation of probiotic Bacillus PC465 isolated from the gut of Fenneropenaeus chinensis improves the health status and resistance of Litopenaeus vannamei against white spot syndrome virus.Fish and Shellfish Immunology,2016,54:602-611
Cheng XY,Liu QH,Huang J.Establishment of real-time fluorescent quantitative PCR assay for detecting white spot syndrome virus.Journal of Anhui Agricultural Sciences,2010,38(26):14265-14267[程晓艳,刘庆慧,黄倢.实时荧光定量PCR检测对虾白斑综合症病毒方法的建立.安徽农业科学,2010,38(26):14265-14267]
Du T,Li B,Wang YG,et al.Screening and characteristic analysis of potential probiotics from large water ponds used for sea cucumber(Apostichopus japonicus)farming.Progress in Fishery Sciences,2017,38(3):180-186[杜佗,李彬,王印庚等.刺参(Apostichopus japonicus)大水面养殖池塘环境中优势益生菌筛选及其特性分析.渔业科学进展,2017,38(3):180-186]
Flegel TW.Major viral diseases of the black tiger prawn(Penaeus monodon)in Thailand.World Journal of Microbiology and Biotechnology,1997,13(4):433-442
Garcia-Orozco KD,Sanchez-Paz A,Aispuro-Hernandez E,et al.Gene expression and protein levels of thioredoxin in the gills from the whiteleg shrimp(Litopenaeus vannamei)infected with two different viruses:The WSSV or IHHNV.Fish and Shellfish Immunology,2012,32(6):1141-1147
Jin DY,Chae HZ,Rhee SG,et al.Regulatory role for a novel human thioredoxin peroxidase in NF-κB activation.Journal of Biological Chemistry,1997,272(49):30952-30961
Lakshmi B,Viswanath B,Sai Gopal DV.Probiotics as antiviral agents in shrimp aquaculture.Journal of Pathogens,2013,424123
Liu SL,Chen J,Li J,et al.Effects of complex-probioticpreparation on the growth,digestive enzymes and the nonspecific immune indices of Atlantic salmon(Salmo salar).Progress in Fishery Sciences,2017,38(5):100-106[刘淑兰,陈娟,李杰,等.复合益生菌制剂对大西洋鲑(Salmo salar)生长、消化酶和非特异性免疫指标的影响.渔业科学进展,2017,38(5):100-106]
Livak KJ,Schmittgen TD.Analysis of relative gene expression data using rea-time quantitative PCR and 2-CT method.Methods,2001,25:402-408
Ninawe AS,Selvin J.Probiotics in shrimp aquaculture:Avenues and challenges.Critical Reviews in Microbiology,2009,35(1):43-66
Phongdara A,Wanna W,Chotigeat W.Molecular cloning and expression of caspase from white shrimp Penaeus merguiensis.Aquaculture,2006,252(2-4):114-120
Ravi AV,Musthafa KS,Jegathammbal G,et al.Screening and evaluation of probiotics as a biocontrol agent against pathogenic Vibrios in marine aquaculture.Letters in Applied Microbiology,2007,45(2):219-223
Rengpipat S,Rukpratanporn S,Piyatiratitivorakul S,et al.Immunity enhancement on black tiger shrimp(Penaeus monodon)by a probiont bacterium(Bacillus S11).Aquaculture2000,191(4):271-288
Salinas I,Cueste A,Esteban Má,et al.Dietary administration of Lactobacillus delbreeckii and Bacillus subtilis,single or combined,on gilthead seabream cellular innate immune responses.Fish and Shellfish Immunology,2005,19(1):67-77
Sánchez-Ortiz AC,Angulo C,Luna-González A,et al.Effect of mixed-Bacillus spp isolated from pustulose ark Anadara tuberculosa,on growth,survival,viral prevalence and immune-related gene expression in shrimp Litopenaeus vannamei.Fish and Shellfish Immunology,2016,59:95-102
Sha Y,Liu M,Wang B,et al.Bacterial population in intestines of Litopenaeus vannamei fed different probiotics or probiotic supernatant.Journal of Microbiology and Biotechnology,2016,26(10):1736-1745
Song GN,Jin SJ,Zhang JQ,et al.Cloning and expression of a caspase gene in Chinese shrimp Fenneropenaeus chinensis.Marine Sciences,2010,34(12):1-7[宋光年,金松君,张继泉,等.中国明对虾caspase基因的克隆与表达分析.海洋科学,2010,34(12):1-7]
Song XL,Huang J,Tang KFL,et al.The construction and analysis for cDNA clones of white spot syndrome virus of shrimp.Acta Hydrobiologica Sinica,2002,26(5):444-451[宋晓玲,黄倢,Tang KFL,等.对虾白斑综合症病毒重组c DNA克隆的构建与分析.水生生物学报,2002,26(5):444-451]
Sruthy KS,Chaithanya ER,Sathyan N,et al.Molecular characterization and phylogenetic analysis of novel isoform of anti-lipopolysaccharide factor from the mantis shrimp,Miyakea nepa.Probiotics and Antimicrobial Proteins,2015,7(4):275-283
Sun Y,Liu F,Song XL,et al.Effects of adding probiotics in the feed on non-specific immune gene expression and disease resistance of Litopenaeus vannamei.Oceanologia et Limnologia Sinica,2012,43(4):845-851[孙艳,刘飞,宋晓玲,等.饲料中添加益生菌对凡纳滨对虾(Litopenaeus vannamei)非特异免疫基因表达量和抗病力的影响.海洋与湖沼,2012,43(4):845-851]
Tannock GW.Studies of the intestinal microflora:A prerequisite for the development of probiotics.International Dairy Journal,1998,8(5-6):527-533
Wakiyama M,Kaitsu Y,Yokoyama S.Cell-free translation system from Drosophila S2 cells that recapitulates RNAi.Biochemical and Biophysical Research Communications,2006,343(4):1067-1071
Wen CQ,Xue M,Liang HF,et al.Beneficial effects of Ectothiorhodospira shaposhnikovii WF on larval cultivation of Litopenaeus vannamei.Beneficial Microbes,2015;6(4):525-533
Wu J,Muroga K.Apoptosis does not play an important role in the resistance of‘immune’Penaeus japonicus against white spot syndrome virus.Journal of Fish Diseases,2004,27(1):15-21
Zhang SJ,Zhao XJ,Song XL,et al.Analysis of the culturable bacteria’s quantity and composition in the intestinal tract of cultivation shrimp.Journal of Shanghai Ocean University,2015,24(2):211-218[张盛静,赵小金,宋晓玲,等.人工养殖对虾肠道内可培养细菌数量及组成分析.上海海洋大学学报,2015,24(2):211-218]
Zhang XJ,Song XL,Huang J.Impact of Vibrio parahaemolyticus and white spot syndrome virus(WSSV)co-infection on survival of penaeid shrimp Litopenaeus vannamei.Chinese Journal of Oceanology and Limnology,2016,34(6):1278-1286
Zhou J,Song XL,Huang J,et al.Effects of dietary supplementation of A3α-peptidoglycan on innate immune responses and defense activity of Japanese flounder(Paralichthys olivaceus).Aquaculture,2006,251(1-4):172-181
Zokaeifar H,Babaei N,Saad CR,et al.Administration of Bacillus subtilis strains in the rearing water enhances the water quality,growth performance,immune response,and resistance against Vibrio harveyi infection in juvenile white shrimp,Litopenaeus vannamei.Fish and Shellfish Immunology,2014,36(1):68-74
Cheng XY,Liu QH,Huang J.Establishment of real-time fluorescent quantitative PCR assay for detecting white spot syndrome virus.Journal of Anhui Agricultural Sciences,2010,38(26):14265-14267[程晓艳,刘庆慧,黄倢.实时荧光定量PCR检测对虾白斑综合症病毒方法的建立.安徽农业科学,2010,38(26):14265-14267]
Du T,Li B,Wang YG,et al.Screening and characteristic analysis of potential probiotics from large water ponds used for sea cucumber(Apostichopus japonicus)farming.Progress in Fishery Sciences,2017,38(3):180-186[杜佗,李彬,王印庚等.刺参(Apostichopus japonicus)大水面养殖池塘环境中优势益生菌筛选及其特性分析.渔业科学进展,2017,38(3):180-186]
Flegel TW.Major viral diseases of the black tiger prawn(Penaeus monodon)in Thailand.World Journal of Microbiology and Biotechnology,1997,13(4):433-442
Garcia-Orozco KD,Sanchez-Paz A,Aispuro-Hernandez E,et al.Gene expression and protein levels of thioredoxin in the gills from the whiteleg shrimp(Litopenaeus vannamei)infected with two different viruses:The WSSV or IHHNV.Fish and Shellfish Immunology,2012,32(6):1141-1147
Jin DY,Chae HZ,Rhee SG,et al.Regulatory role for a novel human thioredoxin peroxidase in NF-κB activation.Journal of Biological Chemistry,1997,272(49):30952-30961
Lakshmi B,Viswanath B,Sai Gopal DV.Probiotics as antiviral agents in shrimp aquaculture.Journal of Pathogens,2013,424123
Liu SL,Chen J,Li J,et al.Effects of complex-probioticpreparation on the growth,digestive enzymes and the nonspecific immune indices of Atlantic salmon(Salmo salar).Progress in Fishery Sciences,2017,38(5):100-106[刘淑兰,陈娟,李杰,等.复合益生菌制剂对大西洋鲑(Salmo salar)生长、消化酶和非特异性免疫指标的影响.渔业科学进展,2017,38(5):100-106]
Livak KJ,Schmittgen TD.Analysis of relative gene expression data using rea-time quantitative PCR and 2-CT method.Methods,2001,25:402-408
Ninawe AS,Selvin J.Probiotics in shrimp aquaculture:Avenues and challenges.Critical Reviews in Microbiology,2009,35(1):43-66
Phongdara A,Wanna W,Chotigeat W.Molecular cloning and expression of caspase from white shrimp Penaeus merguiensis.Aquaculture,2006,252(2-4):114-120
Ravi AV,Musthafa KS,Jegathammbal G,et al.Screening and evaluation of probiotics as a biocontrol agent against pathogenic Vibrios in marine aquaculture.Letters in Applied Microbiology,2007,45(2):219-223
Rengpipat S,Rukpratanporn S,Piyatiratitivorakul S,et al.Immunity enhancement on black tiger shrimp(Penaeus monodon)by a probiont bacterium(Bacillus S11).Aquaculture2000,191(4):271-288
Salinas I,Cueste A,Esteban Má,et al.Dietary administration of Lactobacillus delbreeckii and Bacillus subtilis,single or combined,on gilthead seabream cellular innate immune responses.Fish and Shellfish Immunology,2005,19(1):67-77
Sánchez-Ortiz AC,Angulo C,Luna-González A,et al.Effect of mixed-Bacillus spp isolated from pustulose ark Anadara tuberculosa,on growth,survival,viral prevalence and immune-related gene expression in shrimp Litopenaeus vannamei.Fish and Shellfish Immunology,2016,59:95-102
Sha Y,Liu M,Wang B,et al.Bacterial population in intestines of Litopenaeus vannamei fed different probiotics or probiotic supernatant.Journal of Microbiology and Biotechnology,2016,26(10):1736-1745
Song GN,Jin SJ,Zhang JQ,et al.Cloning and expression of a caspase gene in Chinese shrimp Fenneropenaeus chinensis.Marine Sciences,2010,34(12):1-7[宋光年,金松君,张继泉,等.中国明对虾caspase基因的克隆与表达分析.海洋科学,2010,34(12):1-7]
Song XL,Huang J,Tang KFL,et al.The construction and analysis for cDNA clones of white spot syndrome virus of shrimp.Acta Hydrobiologica Sinica,2002,26(5):444-451[宋晓玲,黄倢,Tang KFL,等.对虾白斑综合症病毒重组c DNA克隆的构建与分析.水生生物学报,2002,26(5):444-451]
Sruthy KS,Chaithanya ER,Sathyan N,et al.Molecular characterization and phylogenetic analysis of novel isoform of anti-lipopolysaccharide factor from the mantis shrimp,Miyakea nepa.Probiotics and Antimicrobial Proteins,2015,7(4):275-283
Sun Y,Liu F,Song XL,et al.Effects of adding probiotics in the feed on non-specific immune gene expression and disease resistance of Litopenaeus vannamei.Oceanologia et Limnologia Sinica,2012,43(4):845-851[孙艳,刘飞,宋晓玲,等.饲料中添加益生菌对凡纳滨对虾(Litopenaeus vannamei)非特异免疫基因表达量和抗病力的影响.海洋与湖沼,2012,43(4):845-851]
Tannock GW.Studies of the intestinal microflora:A prerequisite for the development of probiotics.International Dairy Journal,1998,8(5-6):527-533
Wakiyama M,Kaitsu Y,Yokoyama S.Cell-free translation system from Drosophila S2 cells that recapitulates RNAi.Biochemical and Biophysical Research Communications,2006,343(4):1067-1071
Wen CQ,Xue M,Liang HF,et al.Beneficial effects of Ectothiorhodospira shaposhnikovii WF on larval cultivation of Litopenaeus vannamei.Beneficial Microbes,2015;6(4):525-533
Wu J,Muroga K.Apoptosis does not play an important role in the resistance of‘immune’Penaeus japonicus against white spot syndrome virus.Journal of Fish Diseases,2004,27(1):15-21
Zhang SJ,Zhao XJ,Song XL,et al.Analysis of the culturable bacteria’s quantity and composition in the intestinal tract of cultivation shrimp.Journal of Shanghai Ocean University,2015,24(2):211-218[张盛静,赵小金,宋晓玲,等.人工养殖对虾肠道内可培养细菌数量及组成分析.上海海洋大学学报,2015,24(2):211-218]
Zhang XJ,Song XL,Huang J.Impact of Vibrio parahaemolyticus and white spot syndrome virus(WSSV)co-infection on survival of penaeid shrimp Litopenaeus vannamei.Chinese Journal of Oceanology and Limnology,2016,34(6):1278-1286
Zhou J,Song XL,Huang J,et al.Effects of dietary supplementation of A3α-peptidoglycan on innate immune responses and defense activity of Japanese flounder(Paralichthys olivaceus).Aquaculture,2006,251(1-4):172-181
Zokaeifar H,Babaei N,Saad CR,et al.Administration of Bacillus subtilis strains in the rearing water enhances the water quality,growth performance,immune response,and resistance against Vibrio harveyi infection in juvenile white shrimp,Litopenaeus vannamei.Fish and Shellfish Immunology,2014,36(1):68-74