BALB/c小鼠用于评价尼罗罗非鱼无乳链球菌毒力的研究
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摘要
实验采用BALB/c小鼠作为实验动物,旨在建立尼罗罗非鱼无乳链球菌毒力测定的BALB/c小鼠模型。BALB/c小鼠经腹腔注射尼罗罗非鱼源无乳链球菌建立感染模型,比较了尼罗罗非鱼源无乳链球菌分别感染尼罗罗非鱼和小鼠的LD_(50)差异,分别测定了不同毒力尼罗罗非鱼无乳链球菌对尼罗罗非鱼和小鼠的毒力。结果显示,小鼠经腹腔注射无乳链球菌,在24 h内出现死亡现象,且对小鼠脑、肝脏、脾脏、肾脏等组织造成损伤。3次测定尼罗罗非鱼无乳链球菌TFJ0901对尼罗罗非鱼和小鼠LD_(50)分别为7.7×10~7、2.2×10~8、3.5×10~9 CFU/mL和405、361、419 CFU/只。将无乳链球菌TFJ0901和THN0901感染尼罗罗非鱼(1.0×10~7 CFU/mL)和小鼠(100 CFU/只),尼罗罗非鱼和小鼠存活率分别为100%、6.7%±5.8%和100%、0,其存活率都具有显著性差异。将无乳链球菌TFJ0901和TFJ-F感染尼罗罗非鱼(3.0×10~8 CFU/mL)和小鼠(2 500 CFU/只),尼罗罗非鱼的存活率分别为73.3%±11.5%和80.0%±10.0%,存活率差异不显著,小鼠存活率分别为13.3%±11.5%和100.0%,存活率具有显著性差异。研究表明,本实验成功建立了BALB/c小鼠作为尼罗罗非鱼源无乳链球菌毒力测定的稳定模型,测定不同毒力的尼罗罗非鱼源无乳链球菌对小鼠毒力与对尼罗罗非鱼毒力一致,且该模型能够区分尼罗罗非鱼模型难以区分的毒力相近的无乳链球菌。
Streptococcus agalactiae is one of the major pathogens of tilapia(Oreochromis niloticus). At present,the virulence of strains was evaluated mainly by comparing the median lethal dose(LD_(50)) to tilapia. However,tilapia as experimental animals for evaluation of the virulence of S. agalactiae was often unstable. In this experiment, BALB/c mice were used as experimental animals, in order to establish a method for the determination of virulence of S. agalactiae. BALB/c mice were injected intraperitoneally with tilapia Streptococcus agalactiae to establish an infection model. The LD_(50) of Streptococcus agalactiae to tilapia and mice were tested 3 times and the virulence of different Streptococcus agalactiae to tilapia and mice was determined. Results show that: by intraperitoneal injection, S. agalactiae can cause the death of mice within 24 hours. S. agalactiae could damage brain, liver, spleen, kidney and other tissues in mice. The LD_(50) of S. agalactiae to tilapia and mice 3 times was7.7×10~7, 2.2×10~8, 3.5×10~9 CFU and 405, 361, 419 CFU, respectively. When S. agalactiae TFJ0901 and THN0901 infected tilapia(1.0×10~7 CFU) and mice(100 CFU), the survival rates of tilapia and mouse were 100.0%,6.7%±5.8%, and 100.0%, 0%, respectively, both of which were significantly(P<0.05) different. When S.agalactiae TFJ0901 and TFJ-F infected tilapia(3.0×10~8 CFU) and mice(2 500 CFU), the survival rates of tilapia were 73.3%±11.5% and 80.0%±10.0%, respectively, which were not significantly(P>0.05) different from each other, while the survival rates of mice were 13.3%±11.5% and 100.0%, respectively, which were of significant(P<0.05) difference. Taken together, BALB/c mice were successfully established as a stable model for virulence determination of S. agalactiae in tilapia. The determination of the virulence of different S. agalactiae in mice is consistent with tilapia, and this model was able to distinguish S. agalactiae with similar virulence that was difficult to be distinguished by using tilapia model.
Streptococcus agalactiae is one of the major pathogens of tilapia(Oreochromis niloticus). At present,the virulence of strains was evaluated mainly by comparing the median lethal dose(LD_(50)) to tilapia. However,tilapia as experimental animals for evaluation of the virulence of S. agalactiae was often unstable. In this experiment, BALB/c mice were used as experimental animals, in order to establish a method for the determination of virulence of S. agalactiae. BALB/c mice were injected intraperitoneally with tilapia Streptococcus agalactiae to establish an infection model. The LD_(50) of Streptococcus agalactiae to tilapia and mice were tested 3 times and the virulence of different Streptococcus agalactiae to tilapia and mice was determined. Results show that: by intraperitoneal injection, S. agalactiae can cause the death of mice within 24 hours. S. agalactiae could damage brain, liver, spleen, kidney and other tissues in mice. The LD_(50) of S. agalactiae to tilapia and mice 3 times was7.7×10~7, 2.2×10~8, 3.5×10~9 CFU and 405, 361, 419 CFU, respectively. When S. agalactiae TFJ0901 and THN0901 infected tilapia(1.0×10~7 CFU) and mice(100 CFU), the survival rates of tilapia and mouse were 100.0%,6.7%±5.8%, and 100.0%, 0%, respectively, both of which were significantly(P<0.05) different. When S.agalactiae TFJ0901 and TFJ-F infected tilapia(3.0×10~8 CFU) and mice(2 500 CFU), the survival rates of tilapia were 73.3%±11.5% and 80.0%±10.0%, respectively, which were not significantly(P>0.05) different from each other, while the survival rates of mice were 13.3%±11.5% and 100.0%, respectively, which were of significant(P<0.05) difference. Taken together, BALB/c mice were successfully established as a stable model for virulence determination of S. agalactiae in tilapia. The determination of the virulence of different S. agalactiae in mice is consistent with tilapia, and this model was able to distinguish S. agalactiae with similar virulence that was difficult to be distinguished by using tilapia model.
引文
[1]刘志刚,可小丽,卢迈新,等.温度对尼罗罗非鱼无乳链球菌毒力的影响[J].水产学报,2013, 37(11):1733-1741.Liu Z G, Ke X L, Lu M X, et al. Effect of temperature on the virulence of Streptococcus agalactiae from Nile tilapia(Oreochromis niloticus)[J]. Journal of Fisheries of China, 2013, 37(11):1733-1741(in Chinese).
[2]祝璟琳,杨弘,邹芝英,等.海南养殖罗非鱼(Oreochromis niloticus)致病链球菌的分离、鉴定及其药敏试验[J].海洋与湖沼,2010,41(4):590-596.Zhu J L, Yang H, Zou Z Y, et al. Isolation, identification and drug sensitivity test of pathogenic streptococcus from tilapias Oreochromis niloticus cultured in Hainan[J].Oceanologia et Limnologia Sinica,2010, 41(4):590-596(in Chinese).
[3]Naccari C, Niutta P P, Trombetta D, et al. Pharmacokinetics and efficacy of teicoplanin against intramammary infections in sheep[J]. Veterinary Record, 2009,165(1):19-22.
[4]Amundson N R, Flores A E, Hillier S L, et al. DNA macrorestriction analysis of nontypeable group B streptococcal isolates:clonal evolution of nontypeable and type V isolates[J]. Journal of Clinical Microbiology,2005,43(2):572-576.
[5]汪开毓,付希,肖丹,等.罗非鱼源无乳链球菌cpsE基因的克隆和分子特性分析[J].水产学报,2011,35(5):660-667.Wang K Y, Fu X, Xiao D, et al. Cloning and molecular characterization of cpsE gene of Streptococcus agalactiae isolated from tilapia[J]. Journal of Fisheries of China,2011, 35(5):660-667(in Chinese).
[6] Pridgeon J W, Klesius P H. Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin[J]. Vaccine,2013,31(24):2705-2712.
[7] Li L P, Wang R, Liang W W, et al. Development of live attenuated Streptococcus agalactiae vaccine for tilapia via continuous passage in vitro[J]. Fish&Shellfish Immunology, 2015,45(2):955-963.
[8] Mereghetti L, Sitkiewicz I, Green N M, et al. Remodeling of the Streptococcus agalactiae transcriptome in response to growth temperature[J]. PLoS One,2008,3(7):e2785.
[9] Wang L, Liu P, Wan Z Y, et al. RNA-Seq revealed the impairment of immune defence of tilapia against the infection of Streptococcus agalactiae with simulated climate warming[J]. Fish&Shellfish Immunology,2016, 55:679-689.
[10] Seo H S, Mu R, Kim B J, et al. Binding of glycoprotein Srrl of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis[J]. PLoS Pathogens, 2012, 8(10):e1002947.
[11]郭玉娟,张德锋,樊海平,等.中国南方地区罗非鱼无茵乳链球菌的分子流行病学研究[J].水产学报,2012,36(3):399-406.Guo Y J, Zhang D F, Fan H P, et al. Molecular epidemiology of Streptococcus agalactiae isolated from tilapia in Southern China[J]. Journal of Fisheries of China,2012, 36(3):399-406(in Chinese).
[12] Tort L. Stress and immune modulation in fish[J].Developmental&Comparative Immunology, 2011,35(12):1366-1375.
[13] Chen W H, Sun L T, Tsai C L, et al. Cold-stress induced the modulation of catecholamines, cortisol, immunoglobulin M, and leukocyte phagocytosis in tilapia[J]. General and Comparative Endocrinology, 2002, 126(1):90-100.
[14] Dominguez M, Takemura A, Tsuchiya M, et al. Impact of different environmental factors on the circulating immunoglobulin levels in the Nile tilapia, Oreochromisniloticus[J]. Aquaculture, 2004, 241(1-4):491-500.
[15] Varsamos S, Flik G, Pepin J F, et al. Husbandry stress during early life stages affects the stress response and health status of juvenile sea bass, Dicentrarchus labrax[J]. Fish&Shellfish Immunology, 2006, 20(1):83-96.
[16] Huang Z H, Ma A J, Wang X A. The immune response of turbot, Scophthalmus maximus(L.), skin to high water temperature[J]. Journal of Fish Diseases, 2011, 34(8):619-627.
[17] Gallage S, Katagiri T, Endo M, et al. Influence of moderate hypoxia on vaccine efficacy against Vibrio anguillarum in Oreochromis niloticus(Nile tilapia)[J].Fish&Shellfish Immunology, 2016, 51:271-281.
[18]祝璟琳,邹芝英,李大宇,等.四个罗非鱼选育品种抗链球菌病能力差异研究[J].水生生物学报,2017,41(6):1232-1241.Zhu J L, Zou Z Y, Li D Y, et al. Study on resistant difference among four bleeding species of tilapia Oreochromis spp. following Streptococcus agalactiae challenge[J]. Acta Hydrobiologica Sinica, 2017, 41(6):1232-1241(in Chinese).
[19] van Sorge N M, Quach D, Gurney M A, et al. The group B streptococcal serine-rich repeat 1 glycoprotein mediates penetration of the blood-brain barrier[J]. The Journal of Infectious Diseases, 2009, 199(10):1479-1487.
[20]刘一,隋丽华,曾林,等.BABL/c小鼠感染大肠杆菌0127模型的建立及TGF-β1因子的荧光定量检测[J].中国实验动物学报,2013, 21(2):13-16.Liu Y, Sui L H, Zeng L, et al. Establishment of a mouse model of Escherichia coli O127 infection and real-time PCR detection of TGF-β1 expression[J]. Acta Laboratorium Animalis Scientia Sinica,2013, 21(2):13-16(in Chinese).
[21] von Kockritz-Blickwede M, Rohde M, Oehmcke S, et al.Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model[J]. The American Journal of Pathology,2008, 173(6):1657-1668.
[22] Buscetta M, Papasergi S, Firon A, et al. FbsC, a novel fibrinogen-binding protein, promotes Streptococcus agalactiae-host cell interactions[J]. The Journal of Biological Chemistry, 2014, 289(30):21003-21015.
[2]祝璟琳,杨弘,邹芝英,等.海南养殖罗非鱼(Oreochromis niloticus)致病链球菌的分离、鉴定及其药敏试验[J].海洋与湖沼,2010,41(4):590-596.Zhu J L, Yang H, Zou Z Y, et al. Isolation, identification and drug sensitivity test of pathogenic streptococcus from tilapias Oreochromis niloticus cultured in Hainan[J].Oceanologia et Limnologia Sinica,2010, 41(4):590-596(in Chinese).
[3]Naccari C, Niutta P P, Trombetta D, et al. Pharmacokinetics and efficacy of teicoplanin against intramammary infections in sheep[J]. Veterinary Record, 2009,165(1):19-22.
[4]Amundson N R, Flores A E, Hillier S L, et al. DNA macrorestriction analysis of nontypeable group B streptococcal isolates:clonal evolution of nontypeable and type V isolates[J]. Journal of Clinical Microbiology,2005,43(2):572-576.
[5]汪开毓,付希,肖丹,等.罗非鱼源无乳链球菌cpsE基因的克隆和分子特性分析[J].水产学报,2011,35(5):660-667.Wang K Y, Fu X, Xiao D, et al. Cloning and molecular characterization of cpsE gene of Streptococcus agalactiae isolated from tilapia[J]. Journal of Fisheries of China,2011, 35(5):660-667(in Chinese).
[6] Pridgeon J W, Klesius P H. Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin[J]. Vaccine,2013,31(24):2705-2712.
[7] Li L P, Wang R, Liang W W, et al. Development of live attenuated Streptococcus agalactiae vaccine for tilapia via continuous passage in vitro[J]. Fish&Shellfish Immunology, 2015,45(2):955-963.
[8] Mereghetti L, Sitkiewicz I, Green N M, et al. Remodeling of the Streptococcus agalactiae transcriptome in response to growth temperature[J]. PLoS One,2008,3(7):e2785.
[9] Wang L, Liu P, Wan Z Y, et al. RNA-Seq revealed the impairment of immune defence of tilapia against the infection of Streptococcus agalactiae with simulated climate warming[J]. Fish&Shellfish Immunology,2016, 55:679-689.
[10] Seo H S, Mu R, Kim B J, et al. Binding of glycoprotein Srrl of Streptococcus agalactiae to fibrinogen promotes attachment to brain endothelium and the development of meningitis[J]. PLoS Pathogens, 2012, 8(10):e1002947.
[11]郭玉娟,张德锋,樊海平,等.中国南方地区罗非鱼无茵乳链球菌的分子流行病学研究[J].水产学报,2012,36(3):399-406.Guo Y J, Zhang D F, Fan H P, et al. Molecular epidemiology of Streptococcus agalactiae isolated from tilapia in Southern China[J]. Journal of Fisheries of China,2012, 36(3):399-406(in Chinese).
[12] Tort L. Stress and immune modulation in fish[J].Developmental&Comparative Immunology, 2011,35(12):1366-1375.
[13] Chen W H, Sun L T, Tsai C L, et al. Cold-stress induced the modulation of catecholamines, cortisol, immunoglobulin M, and leukocyte phagocytosis in tilapia[J]. General and Comparative Endocrinology, 2002, 126(1):90-100.
[14] Dominguez M, Takemura A, Tsuchiya M, et al. Impact of different environmental factors on the circulating immunoglobulin levels in the Nile tilapia, Oreochromisniloticus[J]. Aquaculture, 2004, 241(1-4):491-500.
[15] Varsamos S, Flik G, Pepin J F, et al. Husbandry stress during early life stages affects the stress response and health status of juvenile sea bass, Dicentrarchus labrax[J]. Fish&Shellfish Immunology, 2006, 20(1):83-96.
[16] Huang Z H, Ma A J, Wang X A. The immune response of turbot, Scophthalmus maximus(L.), skin to high water temperature[J]. Journal of Fish Diseases, 2011, 34(8):619-627.
[17] Gallage S, Katagiri T, Endo M, et al. Influence of moderate hypoxia on vaccine efficacy against Vibrio anguillarum in Oreochromis niloticus(Nile tilapia)[J].Fish&Shellfish Immunology, 2016, 51:271-281.
[18]祝璟琳,邹芝英,李大宇,等.四个罗非鱼选育品种抗链球菌病能力差异研究[J].水生生物学报,2017,41(6):1232-1241.Zhu J L, Zou Z Y, Li D Y, et al. Study on resistant difference among four bleeding species of tilapia Oreochromis spp. following Streptococcus agalactiae challenge[J]. Acta Hydrobiologica Sinica, 2017, 41(6):1232-1241(in Chinese).
[19] van Sorge N M, Quach D, Gurney M A, et al. The group B streptococcal serine-rich repeat 1 glycoprotein mediates penetration of the blood-brain barrier[J]. The Journal of Infectious Diseases, 2009, 199(10):1479-1487.
[20]刘一,隋丽华,曾林,等.BABL/c小鼠感染大肠杆菌0127模型的建立及TGF-β1因子的荧光定量检测[J].中国实验动物学报,2013, 21(2):13-16.Liu Y, Sui L H, Zeng L, et al. Establishment of a mouse model of Escherichia coli O127 infection and real-time PCR detection of TGF-β1 expression[J]. Acta Laboratorium Animalis Scientia Sinica,2013, 21(2):13-16(in Chinese).
[21] von Kockritz-Blickwede M, Rohde M, Oehmcke S, et al.Immunological mechanisms underlying the genetic predisposition to severe Staphylococcus aureus infection in the mouse model[J]. The American Journal of Pathology,2008, 173(6):1657-1668.
[22] Buscetta M, Papasergi S, Firon A, et al. FbsC, a novel fibrinogen-binding protein, promotes Streptococcus agalactiae-host cell interactions[J]. The Journal of Biological Chemistry, 2014, 289(30):21003-21015.