一株虾源蜡样芽胞杆菌D7作为潜在益生菌的毒理学研究
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摘要
为评价一株虾源潜在益生菌蜡样芽胞杆菌D7的毒理安全性,分别从溶血试验、抗菌药物敏感试验、斑点叉尾鮰急性毒性试验、凡纳滨对虾急性毒性试验、破损皮肤刺激性试验、急性眼刺激试验和急性腹腔注射试验等7个指标进行了测试。采用血琼脂平板法测定溶血性,纸片扩散法测试药物的敏感性。采用浸浴和注射两种方式在斑点叉尾鮰上进行急性毒性试验,采用注射方式在凡纳滨对虾进行急性毒性试验。破损皮肤刺激性试验、急性眼刺激试验用家兔,急性腹腔注射性试验用小鼠。结果表明,D7菌株具有明显溶血性,对13种抗菌药物中的9种敏感,104CFU/mL～108CFU/mL浓度下浸浴斑点叉尾鮰,96h内未出现不良症状及死亡,96hLC50>108 CFU/mL。以1×109CFU/mL注射斑点叉尾鮰,24h后出现感染死亡,14d内部分试验组出现感染症状,死亡9尾,肝脏是该菌株感染的靶器官。以1×106CFU/mL注射凡纳滨对虾,96h累积死亡率同对照组差异不显著(P>0.05)。对家兔急性眼刺激中,该菌株属于无刺激性级别,皮肤刺激试验中,该菌株属于轻刺激性级别,急性腹腔注射,发现该菌株属于强致病性级别。说明虾源蜡样芽胞杆菌D7对养殖鱼类具有一定的毒性,对凡纳滨对虾较为安全。
In order to evaluate the toxicology safety of Bcillus cereus D7,the hemolysis test,antimicrobial susceptibility test,acute toxicity tests in Ictalurus punctat and Penaeus vanmamei,damaged skin irritation test,acute eye irritation test,acute abdominal injection test were conducted.The blood argar plate method was used to test hemolysis,and disc diffusion method was used to test drug sensitivity.Acute toxicity was tested in I.punctatus using bath immersion and intraperitoneal injection methods,and acute toxicity in P.vanmamei was tested using injection method.Acute eye and skin irritating tests were conducted in Oryctolagus cuniculus,and acute intraperitoneal injection was tested in Mus musculus.The results showed that the D7 strain had obvious hemolysis,and was sensitive to 9 kinds of antimicrobials.I.punctatus were bathed in the contrations of 104 CFU/mL-108 CFU/mL,there was no symptoms or death in 96 h,and 96 hLC50>108 CFU/mL.The I.punctatus were intraperitoneally injected with the concentration of 1×109 CFU/mL,death fish happened after 24 hbecause of infection,parts of experiment group fishes appeared symptoms and there were 9 fishes died in 14 d,and the target organ was liver.P.vanmamei were intraperitoneally injected with the concentration of 1×106 CFU/mL,there was no significant difference with the control group(P>0.05)in cumulative mortality of 96 h.The strain belongs to the nonirritating grade and light stimulus level in the acute eye irritating test and skin irritating test,respectively,and belongs to highly pathogenic grade in acute intraperitoneal injection test.In conclusion,B.cereus D7 strain has a certain degree of toxicity to fish,but is safe to shrimp.
In order to evaluate the toxicology safety of Bcillus cereus D7,the hemolysis test,antimicrobial susceptibility test,acute toxicity tests in Ictalurus punctat and Penaeus vanmamei,damaged skin irritation test,acute eye irritation test,acute abdominal injection test were conducted.The blood argar plate method was used to test hemolysis,and disc diffusion method was used to test drug sensitivity.Acute toxicity was tested in I.punctatus using bath immersion and intraperitoneal injection methods,and acute toxicity in P.vanmamei was tested using injection method.Acute eye and skin irritating tests were conducted in Oryctolagus cuniculus,and acute intraperitoneal injection was tested in Mus musculus.The results showed that the D7 strain had obvious hemolysis,and was sensitive to 9 kinds of antimicrobials.I.punctatus were bathed in the contrations of 104 CFU/mL-108 CFU/mL,there was no symptoms or death in 96 h,and 96 hLC50>108 CFU/mL.The I.punctatus were intraperitoneally injected with the concentration of 1×109 CFU/mL,death fish happened after 24 hbecause of infection,parts of experiment group fishes appeared symptoms and there were 9 fishes died in 14 d,and the target organ was liver.P.vanmamei were intraperitoneally injected with the concentration of 1×106 CFU/mL,there was no significant difference with the control group(P>0.05)in cumulative mortality of 96 h.The strain belongs to the nonirritating grade and light stimulus level in the acute eye irritating test and skin irritating test,respectively,and belongs to highly pathogenic grade in acute intraperitoneal injection test.In conclusion,B.cereus D7 strain has a certain degree of toxicity to fish,but is safe to shrimp.
引文
[1]Stenfors A L,Fagerlund A,Granum P E.From soil to gut:Bacillus cereus and its food poisoning toxins[J].FEMS Microbiol Rev,2008,32(4):579-606.
[2]刘源.饲用微生物添加剂的研究与应用(综述)[J].中国实验动物学杂志,1997(3):59-63.
[3]中国兽药委员会.兽药质量标准:生物制品卷[M].北京:中国农业出版社,2017:184-187.
[4]Kirchgessner M,Roth F X,Eidelsburger U,et al.The nutritive efficiency of Bacillus cereus as a probiotic in the raising of piglets.1.Effect on the growth parameters and gastrointestinal environment[J].Arch Animal Nutr,1993,44(2):111-121.
[5]Niu D D,Wang C J,Guo Y H,et al.The plant growth-promoting rhizobacterium Bacillus cereus AR156induces resistance in tomato with induction and priming of defence response[J].Biocont Sci Technol,2012,22(9):991-1004.
[6]Navinchandran M,Iyapparaj P,Moovendhan S,et al.Influence of probiotic bacteriumBacillus cereus isolated from the gut of wild shrimp Penaeus monodonin turn as a potent growth promoter and immune enhancer in P.monodon[J].Fish Shellfish Immun,2014,36(1):38-45.
[7]王春迪,宋晓玲,张晓静,等.养殖水体中添加蜡样芽孢杆菌PC465对凡纳滨对虾抗病力的影响[J].中国水产科学,2016(1):146-155.
[8]刘文亮,许华,唐杨,等.饲料中补充蜡样芽孢杆菌(Bacillus cereus)生物膜对凡纳滨对虾(Litopenaeus vannamei)生长、抗病力及其肠道微生物组成的影响[J].渔业科学进展,2017,38(4):87-95.
[9]Hao K,Liu J Y,Ling F,et al.Effect of dietary administration of Shewanella haliotis D4,Bacillus cereus D7and Aeromonas bivalvium D15,single or combined,on the growth,innate immunity and disease resistance of shrimp,Litopenaeus vannamei[J].Aquaculture,2014,428-429(3):141-149.
[10]夏磊,赵明军,张洪玉,等.不同比例复合益生菌对凡纳滨对虾生长、免疫及抗氨氮能力的影响[J].中国水产科学,2015,22(6):1299-1307.
[11]Ramarao N,Sanchis V.The pore-forming haemolysins of Bacillus cereus:A review[J].Toxins,2013,5(6):1119-1139.
[12]Bennett S D,Walsh K A,Gould L H.Foodborne disease outbreaks caused by Bacillus cereus,Clostridium perfringens,and Staphylococcus aureus-United States,1998-2008[J].Clin Infect Dis,2013,57(3):425-433.
[13]Glasset B,Herbin S,Guillier L,et al.Bacillus cereus-induced food-borne outbreaks in France,2007to 2014:epidemiology and genetic characterisation[J].Eurosurveillance,2016,21(48):30413.doi:10.2807/1560-7917.ES.2016.21.48.30413.
[14]Chou Y L,Cheng S N,Hsieh K H,et al.Bacillus cereus septicemia in a patient with acute lymphoblastic leukemia:A case report and review of the literature[J].J Microbiol Immunol Infect,2016,49(3):448-451.
[15]艾海新,于晶晶,郑方亮,等.刺参“腐皮综合症”致病菌LNUB415的分离及防治的初步研究[J].微生物学杂志.2012,32(2):68-72.
[16]中华人民共和国农业部.微生物肥料生物安全通用技术准则:NY 1109-2006[S].北京:中国农业出版社,2006:1-10.
[17]钟妮娜,王文龙,吴宥析,等.人工感染西伯利亚鲟体内嗜水气单胞菌分布的免疫组织化学检测[J].中国兽医科学,2010,40(8):797-800.
[18]王晓琳.两株潜在益生菌安全性评价及其对凡纳滨对虾生长、免疫相关酶活力、抗WSSV能力的影响[D].山东青岛:中国海洋大学,2015.
[19]EFSA.Opinion of the Scientific Committee on a request from EFSA on the introduction of a Qualified Presumption of Safety(QPS)approach for assessment of selected microorganisms referred to EFSA[J].EFSA J,2017,5871-5916:2007.
[20]刘勇,张勇,张和平.世界益生菌安全性评价方法[J].中国食品学报,2011,11(6):141-151.
[21]国家环境保护总局.环保用微生物菌剂环境安全评价导则:HJ/T 415-2008[S].北京:中国环境科学出版社,2008:1-6.
[22]农业部渔业渔政管理局.中国渔业统计年鉴[M].北京:中国农业出版社,2017:1-2.
[23]Andreas B,Kueffer M,Simmen A,et al.Relatedness of Lactobacillus rhamnosus strains isolated from clinical specimens and such from food-stuffs,humans and technology[J].LWT-Food Sci Technol,1998,31(5):489-494.
[24]Ike Y,Hashimoto H,Clewell D B.Hemolysin of Streptococcus faecalis subspecies zymogenes contributes to virulence in mice[J].Infect Immun,1984,45(2):528-530.
[25]胡永红,陈卫,欧阳平凯.高效有益微生态制剂开发与利用:蜡样芽孢杆菌[M].北京:化学工业出版社,2013:2.
[26]Decker H.Recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins[J].Dev Comp Immunol,2004,28(7-8):673-687.
[27]宋增福,陈彪,郭婧,等.具有群体感应抑制作用的地衣芽孢杆菌T-1的动物安全及生态评价[J].水产学报,2015,39(9):1395-1404.
[28]曹海鹏,卫若鹏,何珊,等.水产养殖用解淀粉芽孢杆菌微胶囊的安全性评价[J].中国生物工程杂志,2012,32(5):58-65.
[2]刘源.饲用微生物添加剂的研究与应用(综述)[J].中国实验动物学杂志,1997(3):59-63.
[3]中国兽药委员会.兽药质量标准:生物制品卷[M].北京:中国农业出版社,2017:184-187.
[4]Kirchgessner M,Roth F X,Eidelsburger U,et al.The nutritive efficiency of Bacillus cereus as a probiotic in the raising of piglets.1.Effect on the growth parameters and gastrointestinal environment[J].Arch Animal Nutr,1993,44(2):111-121.
[5]Niu D D,Wang C J,Guo Y H,et al.The plant growth-promoting rhizobacterium Bacillus cereus AR156induces resistance in tomato with induction and priming of defence response[J].Biocont Sci Technol,2012,22(9):991-1004.
[6]Navinchandran M,Iyapparaj P,Moovendhan S,et al.Influence of probiotic bacteriumBacillus cereus isolated from the gut of wild shrimp Penaeus monodonin turn as a potent growth promoter and immune enhancer in P.monodon[J].Fish Shellfish Immun,2014,36(1):38-45.
[7]王春迪,宋晓玲,张晓静,等.养殖水体中添加蜡样芽孢杆菌PC465对凡纳滨对虾抗病力的影响[J].中国水产科学,2016(1):146-155.
[8]刘文亮,许华,唐杨,等.饲料中补充蜡样芽孢杆菌(Bacillus cereus)生物膜对凡纳滨对虾(Litopenaeus vannamei)生长、抗病力及其肠道微生物组成的影响[J].渔业科学进展,2017,38(4):87-95.
[9]Hao K,Liu J Y,Ling F,et al.Effect of dietary administration of Shewanella haliotis D4,Bacillus cereus D7and Aeromonas bivalvium D15,single or combined,on the growth,innate immunity and disease resistance of shrimp,Litopenaeus vannamei[J].Aquaculture,2014,428-429(3):141-149.
[10]夏磊,赵明军,张洪玉,等.不同比例复合益生菌对凡纳滨对虾生长、免疫及抗氨氮能力的影响[J].中国水产科学,2015,22(6):1299-1307.
[11]Ramarao N,Sanchis V.The pore-forming haemolysins of Bacillus cereus:A review[J].Toxins,2013,5(6):1119-1139.
[12]Bennett S D,Walsh K A,Gould L H.Foodborne disease outbreaks caused by Bacillus cereus,Clostridium perfringens,and Staphylococcus aureus-United States,1998-2008[J].Clin Infect Dis,2013,57(3):425-433.
[13]Glasset B,Herbin S,Guillier L,et al.Bacillus cereus-induced food-borne outbreaks in France,2007to 2014:epidemiology and genetic characterisation[J].Eurosurveillance,2016,21(48):30413.doi:10.2807/1560-7917.ES.2016.21.48.30413.
[14]Chou Y L,Cheng S N,Hsieh K H,et al.Bacillus cereus septicemia in a patient with acute lymphoblastic leukemia:A case report and review of the literature[J].J Microbiol Immunol Infect,2016,49(3):448-451.
[15]艾海新,于晶晶,郑方亮,等.刺参“腐皮综合症”致病菌LNUB415的分离及防治的初步研究[J].微生物学杂志.2012,32(2):68-72.
[16]中华人民共和国农业部.微生物肥料生物安全通用技术准则:NY 1109-2006[S].北京:中国农业出版社,2006:1-10.
[17]钟妮娜,王文龙,吴宥析,等.人工感染西伯利亚鲟体内嗜水气单胞菌分布的免疫组织化学检测[J].中国兽医科学,2010,40(8):797-800.
[18]王晓琳.两株潜在益生菌安全性评价及其对凡纳滨对虾生长、免疫相关酶活力、抗WSSV能力的影响[D].山东青岛:中国海洋大学,2015.
[19]EFSA.Opinion of the Scientific Committee on a request from EFSA on the introduction of a Qualified Presumption of Safety(QPS)approach for assessment of selected microorganisms referred to EFSA[J].EFSA J,2017,5871-5916:2007.
[20]刘勇,张勇,张和平.世界益生菌安全性评价方法[J].中国食品学报,2011,11(6):141-151.
[21]国家环境保护总局.环保用微生物菌剂环境安全评价导则:HJ/T 415-2008[S].北京:中国环境科学出版社,2008:1-6.
[22]农业部渔业渔政管理局.中国渔业统计年鉴[M].北京:中国农业出版社,2017:1-2.
[23]Andreas B,Kueffer M,Simmen A,et al.Relatedness of Lactobacillus rhamnosus strains isolated from clinical specimens and such from food-stuffs,humans and technology[J].LWT-Food Sci Technol,1998,31(5):489-494.
[24]Ike Y,Hashimoto H,Clewell D B.Hemolysin of Streptococcus faecalis subspecies zymogenes contributes to virulence in mice[J].Infect Immun,1984,45(2):528-530.
[25]胡永红,陈卫,欧阳平凯.高效有益微生态制剂开发与利用:蜡样芽孢杆菌[M].北京:化学工业出版社,2013:2.
[26]Decker H.Recent findings on phenoloxidase activity and antimicrobial activity of hemocyanins[J].Dev Comp Immunol,2004,28(7-8):673-687.
[27]宋增福,陈彪,郭婧,等.具有群体感应抑制作用的地衣芽孢杆菌T-1的动物安全及生态评价[J].水产学报,2015,39(9):1395-1404.
[28]曹海鹏,卫若鹏,何珊,等.水产养殖用解淀粉芽孢杆菌微胶囊的安全性评价[J].中国生物工程杂志,2012,32(5):58-65.