纳米锌对杀鲑气单胞菌的灭活效果及其细胞毒性
|
摘要
为探究纳米锌在制备水产动物致病菌灭活疫苗应用中的可行性。本研究利用不同浓度的纳米锌水溶液在不同温度条件下对杀鲑气单胞菌(Aeromonas salmonicida)进行不同时长的孵育处理。研究显示,纳米锌对杀鲑气单胞菌的灭活效果与浓度、温度及孵育时间均呈现正相关性,在高浓度与高温条件下灭活菌体所需的时间短。检测了不同灭活条件下纳米锌对杀鲑气单胞菌菌体蛋白及免疫原性的影响,SDS-PAGE结果显示不同条件下纳米锌灭活的杀鲑气单胞菌菌体蛋白组成及丰度与正常菌体没有明显差异;而扫描电镜结果显示,在37℃条件下纳米锌会造成菌体结构破损并出现细菌碎片;ELISA检测结果4℃条件下各纳米锌浓度灭活组菌体抗原性均未受到显著性影响,然而在28℃条件下,200和400mg/L纳米锌灭活组菌体抗原性显著低于对照组,而在37℃条件下除50mg/L浓度组外,其它3个浓度灭活组抗原性均显著低于对照组。比较研究了100mg/L-28℃灭活组与甲醛灭活的全菌疫苗的免疫保护效果,结果显示纳米锌与甲醛灭活菌体免疫组鱼体的免疫保护率分别为86.2%与79.3%。利用鲤上皮瘤细胞对纳米锌的细胞毒性进行了检测,结果显示400mg/L浓度以下纳米锌对细胞的贴壁、生长及代谢均无明显影响。本研究结果说明纳米锌对杀鲑气单胞菌具有明显的灭活效果,且灭活的全菌疫苗表现出良好的免疫保护效果,而其对菌体的抑杀能力及损伤程度与温度和时间密切相关,本研究为纳米锌在水产全菌灭活疫苗的制备提供了基础。
In order to explore the applicability of nanometer zinc(Nano-Zn)in the preparation of inactivated vaccine of aquatic animals,nano-Zn aqueous solution was used to inactivate Aeromonas salmonicida at different temperatures and for different durations.The results showed that the inactivation effect of nano-Zn on A.salmonicida was positively correlated with concentration,temperature and incubation time,and a shorter time was needed to inactivate the bacterium at higher concentrations of nano-Zn and temperatures.Meanwhile,the effects of nano-Zn on the protein and immunogenicity of A.salmonicida were also detected under different inactivation conditions.SDS-PAGE showed that the composition and abundance of the nano-Zn inactivated A.salmonicidacells were not significantly different under different inactivation conditions.However,scanning electron microscopy showed that the nano-Zn broke bacteria at 37℃.ELISA showed that the antigenicity of inactivated bacterium at different concentrations of nano-Zn and 4℃ was similar to that of the normal while the antigenicity of 200 and 400 mg/L nano-Zn inactivated bacterium at 28℃and 100,200 and 400 mg/L nano-Zn inactivated bacterium at 37 ℃ was significantly lower than that of the normal.In addiiton,the immune protective effect of 100 mg/L-28℃inactivated bacterium and formalin killed bacterium was compared.The results showed that the relative percentage of survival rate of nano-Zn and formalin killed cells was 86.2% and 79.3%,respectively.The cytotoxicity of nano-Zn was detected using EPC cell line.When the concentration of nano-Zn was less than 400 mg/L,there was no significant effect on cell growth and metabolism.Our findings indicated that nano-zinc has a significant inactivation effect on A.salmonicida,and the nano-Zn inactivated bacterium showed a good immune protective effect.The inactivation effect and cytotoxicity of nano-Zn were closely related to temperature and incubation time.These results provided a potential application for the preparation of whole bacterium inactivated vaccine using nano-Zn in aquaculture.
In order to explore the applicability of nanometer zinc(Nano-Zn)in the preparation of inactivated vaccine of aquatic animals,nano-Zn aqueous solution was used to inactivate Aeromonas salmonicida at different temperatures and for different durations.The results showed that the inactivation effect of nano-Zn on A.salmonicida was positively correlated with concentration,temperature and incubation time,and a shorter time was needed to inactivate the bacterium at higher concentrations of nano-Zn and temperatures.Meanwhile,the effects of nano-Zn on the protein and immunogenicity of A.salmonicida were also detected under different inactivation conditions.SDS-PAGE showed that the composition and abundance of the nano-Zn inactivated A.salmonicidacells were not significantly different under different inactivation conditions.However,scanning electron microscopy showed that the nano-Zn broke bacteria at 37℃.ELISA showed that the antigenicity of inactivated bacterium at different concentrations of nano-Zn and 4℃ was similar to that of the normal while the antigenicity of 200 and 400 mg/L nano-Zn inactivated bacterium at 28℃and 100,200 and 400 mg/L nano-Zn inactivated bacterium at 37 ℃ was significantly lower than that of the normal.In addiiton,the immune protective effect of 100 mg/L-28℃inactivated bacterium and formalin killed bacterium was compared.The results showed that the relative percentage of survival rate of nano-Zn and formalin killed cells was 86.2% and 79.3%,respectively.The cytotoxicity of nano-Zn was detected using EPC cell line.When the concentration of nano-Zn was less than 400 mg/L,there was no significant effect on cell growth and metabolism.Our findings indicated that nano-zinc has a significant inactivation effect on A.salmonicida,and the nano-Zn inactivated bacterium showed a good immune protective effect.The inactivation effect and cytotoxicity of nano-Zn were closely related to temperature and incubation time.These results provided a potential application for the preparation of whole bacterium inactivated vaccine using nano-Zn in aquaculture.
引文
[1]Wiklund T,Dalsgaard I.Occurrence and significance of atypical Aeromonas salmonicidain non-salmonid and salmonid fish species:A review[J].Disease of Aquatic Organisms,1998,32:49-69.
[2] Reith M E,Singh R K,Curtis B,et al.The genome of Aeromonas salmonicidasubsp.salmonicida A449:Insights into the evolution of a fish pathogen[J].BMC Genomics,2008,9(427):1-15.
[3] Garrity G M.Bergey’s Manual of Systematic Bacteriology[M].9th Edition.New York:Springer,2004.
[4]高冬梅,李健,王群.鳗弧菌灭活疫苗对牙鲆免疫效果的研究[J].渔业科学进展,2014,25(1):34-40.Gao D M,Li J,Wang Q.Study on immune efficacy induced by killed Vibrio anguillarumvaccine on flounder(Paralichthys olivaceus)[J].Marine Fisheries Research,2014,25(1):34-40.
[5]孙金辉,王庆奎,陈成勋,等.嗜水气单胞菌灭活疫苗对虹鳟免疫力和抗病力的影响[J].淡水渔业,2013,43(1):44-49.Sun J H,Wang Q K,Chen C X,et al.Effects of inactivated Aeromonas hydrophila vaccine on immunity and disease resistance of Oncorhynchus mykiss[J].Freshwater Fisheries,2013,43(1):44-49.
[6] Zeng W,Wang Q,Wang Y,et al.Immunogenicity of a cell culture-derived inactivated vaccine against a common virulent isolate of grass carp reovirus[J].Fish and Shellfish Immunology,2016,54:473-480.
[7]徐守振,尹燕博,王新.动物疫苗中常用抗原灭活剂的研究进展[J].中国畜牧兽医,2010,37(9):162-167.Xu S Z,Yin Y B,Wang X.Research progress on routine inactivants in veterinary vaccines[J].Chinese Animal Husbandry and Veterinary Medicine 2010,37(9):162-167.
[8]张爱凤,邹明畅.锌对成骨细胞增殖、分化、矿化活性的影响[J].微量元素与健康研究,2018,35(1):6-8.Zhang A F,Zou M C.The effect of zinc on the propagation,differentiation and mineralization activity of osteoblast[J].Studies of Trace elements and Health,2018,35(1):6-8.
[9]鲍若虹.纳米微量元素在畜牧业中应用[J].福建畜牧兽医,2004,26(2):16-17.Bao R H.The application of nano-micro elements in animal husbandry[J].Fujian Journal of Animal Husbandry and Veterinary Medicine,2004,26(2):16-17.
[10] Mirhosseini M,Firouzabadi F B.Antibacterial activity of zinc oxide nanoparticle suspensions on food-borne pathogens[J].International Journal of Dairy Technology,2013,66(2):291-295.
[11]肖月,康梁,吕庆,等.纳米载银抗菌剂与四针状氧化锌抗白色念珠菌的性能[J].中国组织工程研究,2013,17(25):4609-4615.Xiao Y,Liang K,Lv Q,et al.Properties of nanometer silver antibacterial agent and tetrapod-like zinc oxide antibacterial agent against Candida albicans[J].Chinese Journal of Tissue Engineering Research,2013,17(25):4609-4615.
[12]张铭伟,唐小千,绳秀珍,等.利用鱼抗血清及鼠抗血清分析鱼肠道弧菌全菌蛋白抗原性[J].中国海洋大学学报(自然科学版),2016,46(10):24-31.Zhang M W,Tang X Q,Sheng X Z,et al.Antigenic analysis of total protein of Vibrio ichthyoenteri using mice and flounder antisera[J].Periodical of Ocean University of China,2016,46(10):24-31.
[13]孙千月,罗雯静,梁丹,等.纳米锌抗菌机理的研究进展[J].口腔医学研究,2015,31(2):195-197.Sun Q Y,Luo W J,Liang D,et al.Research progress on the antibacterial mechanism of nanometer zinc[J].Journal of Oral Science Research,2015,31(2):195-197.
[14] Raghupathi K R,Koodali R T,Manna A C.Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles[J].Langmuir,2011,27(7):4020-4028.
[15] Xie Y,He Y,Irwin Y L,et al.Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni[J].Applied and Environmental Microbiology,2011,77(7):2325-2331.
[16]高艳玲,刘熙,王宗贤,等.纳米金属氧化物对食品污染菌的杀、抑能力研究[J].食品科学,2005,26(4):45-48.Gao Y L,Liu X,Wang Z X,et al.Antibacterial effects nanostructural metal oxide's on bacterium contaminated Food[J].Journal of Food Science,2005,26(4):45-48.
[17] Jones N,Ray B,Ranjit K T.Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms[J].FEMS Microbiology Letters,2008,279(1):71-76.
[18] Padmavathy N,Vijayaraghavan R.Enhanced bioactivity of ZnO nanoparticles-an antimicrobial study[J].Science and Technology of Advanced Materials,2008,9(3):035004.
[19] Zhang L,Jiang Y,Ding Y,et al.Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles(ZnO nanofluids)[J].Journal of Nanoparticle Research,2007,9(3):479-489.
[20] Sirelkhatim A,Mahmud S,Seeni A,et al.Review on zinc oxide nanoparticles:Antibacterial activity and toxicity mechanism[J].Nano-Micro Letters,2015,7(3):219-242.
[21] Pace J L,Rossi H A,Esposito V M,et al.Inactivated whole-cell bacterial vaccines:current status and novel strategies[J].Vaccine,1998,16(16):1563-1574.
[22]麻晓霞,马玉龙.氧化锌型复合抗菌材料抗菌活性研究进展[J].功能材料,2018,49(9):9061-9066.Ma X X,Ma Y L.Research progress on antibacterial activity of zinc oxide type composite antibacterial materials[J].Journal of Functional Materials,2018,49(9):9061-9066.
[23]朱玲英,郭大伟,顾宁.纳米银细胞毒性体外检测方法研究进展[J].中国科学,2014,59(22):2145-2152.Zhu L Y,Guo D W,Gu N.Advances in in vitro detection of cytotoxicity induced by silver nanoparticles[J].Science China,2014,59(22):2145-2152.
[24]余文娟,王向晖,史琴,等.纳米银对体外培养细胞附着形态及膜功能的影响[J].华东师范大学学报(自然科学版),2010,2:102-110.Yu W J,Wang X H,Shi Q,et al.In vitro study of the effect of silver nanoparticles on the adhesion and membrane function of cells[J].Journal of East China Normal University(Natural Science),2010,2:102-110.
[25]石慧,邢克智,王庆奎,等.饲料中不同纳米锌含量对半滑舌鳎肝胰脏、肠道消化酶的影响[J].饲料工业,2016,37(12):21-25.Shi H,Xing K Z,Wang Q K,et al.Effects of dietary nano-zinc on digestive enzyme of hepatopancreas and intestine in Cynoglossus semilaevis[J].Feed Industry,2016,37(12):21-25.
[26]孙学亮,王庆奎,陈成勋,等.饲料中添加纳米锌对半滑舌鳎生长、肌肉营养成分及部分血液生化指标的影响[J].饲料工业,2017,38(10):9-14.Sun X L,Wang Q K,Chen C X,et al.Effects of dietary nanozinc on growth,muscle nutrition and some blood biochemical indices of Cynoglossus semilaevis Günther[J].Feed Industry,2017,38(10):9-14.
[2] Reith M E,Singh R K,Curtis B,et al.The genome of Aeromonas salmonicidasubsp.salmonicida A449:Insights into the evolution of a fish pathogen[J].BMC Genomics,2008,9(427):1-15.
[3] Garrity G M.Bergey’s Manual of Systematic Bacteriology[M].9th Edition.New York:Springer,2004.
[4]高冬梅,李健,王群.鳗弧菌灭活疫苗对牙鲆免疫效果的研究[J].渔业科学进展,2014,25(1):34-40.Gao D M,Li J,Wang Q.Study on immune efficacy induced by killed Vibrio anguillarumvaccine on flounder(Paralichthys olivaceus)[J].Marine Fisheries Research,2014,25(1):34-40.
[5]孙金辉,王庆奎,陈成勋,等.嗜水气单胞菌灭活疫苗对虹鳟免疫力和抗病力的影响[J].淡水渔业,2013,43(1):44-49.Sun J H,Wang Q K,Chen C X,et al.Effects of inactivated Aeromonas hydrophila vaccine on immunity and disease resistance of Oncorhynchus mykiss[J].Freshwater Fisheries,2013,43(1):44-49.
[6] Zeng W,Wang Q,Wang Y,et al.Immunogenicity of a cell culture-derived inactivated vaccine against a common virulent isolate of grass carp reovirus[J].Fish and Shellfish Immunology,2016,54:473-480.
[7]徐守振,尹燕博,王新.动物疫苗中常用抗原灭活剂的研究进展[J].中国畜牧兽医,2010,37(9):162-167.Xu S Z,Yin Y B,Wang X.Research progress on routine inactivants in veterinary vaccines[J].Chinese Animal Husbandry and Veterinary Medicine 2010,37(9):162-167.
[8]张爱凤,邹明畅.锌对成骨细胞增殖、分化、矿化活性的影响[J].微量元素与健康研究,2018,35(1):6-8.Zhang A F,Zou M C.The effect of zinc on the propagation,differentiation and mineralization activity of osteoblast[J].Studies of Trace elements and Health,2018,35(1):6-8.
[9]鲍若虹.纳米微量元素在畜牧业中应用[J].福建畜牧兽医,2004,26(2):16-17.Bao R H.The application of nano-micro elements in animal husbandry[J].Fujian Journal of Animal Husbandry and Veterinary Medicine,2004,26(2):16-17.
[10] Mirhosseini M,Firouzabadi F B.Antibacterial activity of zinc oxide nanoparticle suspensions on food-borne pathogens[J].International Journal of Dairy Technology,2013,66(2):291-295.
[11]肖月,康梁,吕庆,等.纳米载银抗菌剂与四针状氧化锌抗白色念珠菌的性能[J].中国组织工程研究,2013,17(25):4609-4615.Xiao Y,Liang K,Lv Q,et al.Properties of nanometer silver antibacterial agent and tetrapod-like zinc oxide antibacterial agent against Candida albicans[J].Chinese Journal of Tissue Engineering Research,2013,17(25):4609-4615.
[12]张铭伟,唐小千,绳秀珍,等.利用鱼抗血清及鼠抗血清分析鱼肠道弧菌全菌蛋白抗原性[J].中国海洋大学学报(自然科学版),2016,46(10):24-31.Zhang M W,Tang X Q,Sheng X Z,et al.Antigenic analysis of total protein of Vibrio ichthyoenteri using mice and flounder antisera[J].Periodical of Ocean University of China,2016,46(10):24-31.
[13]孙千月,罗雯静,梁丹,等.纳米锌抗菌机理的研究进展[J].口腔医学研究,2015,31(2):195-197.Sun Q Y,Luo W J,Liang D,et al.Research progress on the antibacterial mechanism of nanometer zinc[J].Journal of Oral Science Research,2015,31(2):195-197.
[14] Raghupathi K R,Koodali R T,Manna A C.Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles[J].Langmuir,2011,27(7):4020-4028.
[15] Xie Y,He Y,Irwin Y L,et al.Antibacterial activity and mechanism of action of zinc oxide nanoparticles against Campylobacter jejuni[J].Applied and Environmental Microbiology,2011,77(7):2325-2331.
[16]高艳玲,刘熙,王宗贤,等.纳米金属氧化物对食品污染菌的杀、抑能力研究[J].食品科学,2005,26(4):45-48.Gao Y L,Liu X,Wang Z X,et al.Antibacterial effects nanostructural metal oxide's on bacterium contaminated Food[J].Journal of Food Science,2005,26(4):45-48.
[17] Jones N,Ray B,Ranjit K T.Antibacterial activity of ZnO nanoparticle suspensions on a broad spectrum of microorganisms[J].FEMS Microbiology Letters,2008,279(1):71-76.
[18] Padmavathy N,Vijayaraghavan R.Enhanced bioactivity of ZnO nanoparticles-an antimicrobial study[J].Science and Technology of Advanced Materials,2008,9(3):035004.
[19] Zhang L,Jiang Y,Ding Y,et al.Investigation into the antibacterial behaviour of suspensions of ZnO nanoparticles(ZnO nanofluids)[J].Journal of Nanoparticle Research,2007,9(3):479-489.
[20] Sirelkhatim A,Mahmud S,Seeni A,et al.Review on zinc oxide nanoparticles:Antibacterial activity and toxicity mechanism[J].Nano-Micro Letters,2015,7(3):219-242.
[21] Pace J L,Rossi H A,Esposito V M,et al.Inactivated whole-cell bacterial vaccines:current status and novel strategies[J].Vaccine,1998,16(16):1563-1574.
[22]麻晓霞,马玉龙.氧化锌型复合抗菌材料抗菌活性研究进展[J].功能材料,2018,49(9):9061-9066.Ma X X,Ma Y L.Research progress on antibacterial activity of zinc oxide type composite antibacterial materials[J].Journal of Functional Materials,2018,49(9):9061-9066.
[23]朱玲英,郭大伟,顾宁.纳米银细胞毒性体外检测方法研究进展[J].中国科学,2014,59(22):2145-2152.Zhu L Y,Guo D W,Gu N.Advances in in vitro detection of cytotoxicity induced by silver nanoparticles[J].Science China,2014,59(22):2145-2152.
[24]余文娟,王向晖,史琴,等.纳米银对体外培养细胞附着形态及膜功能的影响[J].华东师范大学学报(自然科学版),2010,2:102-110.Yu W J,Wang X H,Shi Q,et al.In vitro study of the effect of silver nanoparticles on the adhesion and membrane function of cells[J].Journal of East China Normal University(Natural Science),2010,2:102-110.
[25]石慧,邢克智,王庆奎,等.饲料中不同纳米锌含量对半滑舌鳎肝胰脏、肠道消化酶的影响[J].饲料工业,2016,37(12):21-25.Shi H,Xing K Z,Wang Q K,et al.Effects of dietary nano-zinc on digestive enzyme of hepatopancreas and intestine in Cynoglossus semilaevis[J].Feed Industry,2016,37(12):21-25.
[26]孙学亮,王庆奎,陈成勋,等.饲料中添加纳米锌对半滑舌鳎生长、肌肉营养成分及部分血液生化指标的影响[J].饲料工业,2017,38(10):9-14.Sun X L,Wang Q K,Chen C X,et al.Effects of dietary nanozinc on growth,muscle nutrition and some blood biochemical indices of Cynoglossus semilaevis Günther[J].Feed Industry,2017,38(10):9-14.