鳗弧菌O1/O2二价灭活疫苗免疫大菱鲆的抗体持续期和免疫保护期
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摘要
本研究分析了鳗弧菌(Vibrio anguillarum)O1/O2血清型二价灭活疫苗免疫大菱鲆后的抗体持续期和免疫保护期。以鳗弧菌O1血清型VAM003株和O2血清型VAM007株为抗原制备了福尔马林灭活二价疫苗,将疫苗按照三种剂量(10~7 cells/尾、10~8 cells/尾、10~9 cells/尾)以腹腔注射途径免疫大菱鲆,在免疫后3 d、7 d、14 d、30 d、60 d、90 d、120 d、150 d,用血清凝集实验检测了免疫鱼血清的VAM003和VAM007抗体效价,用攻毒实验检测了疫苗的免疫保护率(RPS)。结果显示,在免疫后7 d三个剂量组的大菱鲆均产生了特异抗体,并获得27%～60%的RPS。三个剂量组大菱鲆的O1血清型抗体持续期分别>90 d (10~7 cells/尾组)、>150 d (10~8 cells/尾组)、>150 d (10~9cells/尾组),而三个剂量组大菱鲆的O2血清型抗体持续期均>150 d。三个剂量组的大菱鲆获得的免疫保护持续期均>150 d;以RPS>75%为有效免疫保护,各剂量组大菱鲆抵抗O1血清型病原感染的有效免疫保护期为:14～120d(10~7 cells组)、14～120 d (10~8 cells/尾)、14～150 d (10~9 cells/尾),抵抗O2血清型病原感染的有效免疫保护期为:14～60 d (10~7 cells组)、14～120 d (10~8 cells/尾)、14～120 d (10~9 cells/尾)。研究结果表明鳗弧菌二价灭活疫苗可为大菱鲆提供有效而稳定的免疫保护,获得的抗体持续期和免疫保护期为该疫苗的临床中试研究提供了基础。
Vibrio anguillarum is an important bacterial fish pathogen that can cause vibriosis extensively in economically-important fish including Scophthalmus maximus, Paralichthys olivaceus, Anguilla anguilla, and Lateolabrax japonicus in China. In our previous studies, we have carried out the epidemiological work of V. anguillarum in China, and demonstrated that V. anguillarum O1, O2, and O3 serotype strains are prevalent in marine fish farms. Vaccination is proven to be a safe and efficient route to prevent and control vibriosis. However, no commercial V. anguillarum vaccine is available in China. In this study, the antibody persistence and immune protection duration of a V. anguillarum inactivated bivalent vaccine based on O1 and O2 serotype strains were evaluated in turbot(Scophthalmus maximus). The V. anguillarum formalin-inactivated bivalent vaccine was prepared using O1 serotype VAM003 and O2 serotype VAM007 strains. Groups of turbot were injected intraperitoneally with three dosages of vaccine preparation, i.e, 10~7 cells/ind, 10~8 cells/ind, and 10~9 cells/ind. At 3 d, 7 d, 14 d, 30 d, 60 d, 90 d,120 d, and 150 d after injection, each immunized fish group was evaluated for their serum-antibody titers against VAM003 and VAM007 using a serum-agglutination test, and the relative percent survival(RPS) was evaluated by challenge with a 10 LD50(50% lethal dose) of VAM003 or VAM007. The results showed that specific antibodies against VAM003 and VAM007 were noticeably induced in the immunized fish as early as 7 d post-immunization,and an RPS of 27%–60% was detected in these fish. Specifically, the induced VAM003 antibody persisted in three dosage fish groups for at least 90 d for 10~7 cells/ind, 150 d for 10~8 cells/ind, and 150 d for 10~9 cells/ind, respectively, while anti-VAM007 persisted in each of three dosage fish groups for more than 150 d. The immune protection duration for each of the three dosage groups all exceeded 150 d. With an RPS of more than 75% as an effective immune protection, the effective immune protection duration against O2 serotype V. anguillarum was 14–120 d for 10~7 cells/ind fish group, 14–120 d for 10~8 cells/ind group, and 14–150 d for 10~9 cells/ind group, respectively, while the effective immune protection duration against O2 serotype were 14 d–60 d for 10~7 cells/ind group, 14–120 d for 10~8 cells/ind group, and 14–120 d for 10~9 cells/ind group, respectively. We also analyzed the relationship between serum antibody titer and RPS of bivalent vaccine. When the serum antibody titer was over 1 : 40 for the O1 serotype and 1 : 160 for the O2 serotype, the corresponsive RPS to V. anguillarum O1 or O2 serotype were all over 75%. These results suggest that the V. anguillarum inactivated bivalent vaccine could provide effective immune protection for turbot, and the data of antibody persistence and immune protection duration obtained in this study will provide support for clinical trials of this vaccine.
Vibrio anguillarum is an important bacterial fish pathogen that can cause vibriosis extensively in economically-important fish including Scophthalmus maximus, Paralichthys olivaceus, Anguilla anguilla, and Lateolabrax japonicus in China. In our previous studies, we have carried out the epidemiological work of V. anguillarum in China, and demonstrated that V. anguillarum O1, O2, and O3 serotype strains are prevalent in marine fish farms. Vaccination is proven to be a safe and efficient route to prevent and control vibriosis. However, no commercial V. anguillarum vaccine is available in China. In this study, the antibody persistence and immune protection duration of a V. anguillarum inactivated bivalent vaccine based on O1 and O2 serotype strains were evaluated in turbot(Scophthalmus maximus). The V. anguillarum formalin-inactivated bivalent vaccine was prepared using O1 serotype VAM003 and O2 serotype VAM007 strains. Groups of turbot were injected intraperitoneally with three dosages of vaccine preparation, i.e, 10~7 cells/ind, 10~8 cells/ind, and 10~9 cells/ind. At 3 d, 7 d, 14 d, 30 d, 60 d, 90 d,120 d, and 150 d after injection, each immunized fish group was evaluated for their serum-antibody titers against VAM003 and VAM007 using a serum-agglutination test, and the relative percent survival(RPS) was evaluated by challenge with a 10 LD50(50% lethal dose) of VAM003 or VAM007. The results showed that specific antibodies against VAM003 and VAM007 were noticeably induced in the immunized fish as early as 7 d post-immunization,and an RPS of 27%–60% was detected in these fish. Specifically, the induced VAM003 antibody persisted in three dosage fish groups for at least 90 d for 10~7 cells/ind, 150 d for 10~8 cells/ind, and 150 d for 10~9 cells/ind, respectively, while anti-VAM007 persisted in each of three dosage fish groups for more than 150 d. The immune protection duration for each of the three dosage groups all exceeded 150 d. With an RPS of more than 75% as an effective immune protection, the effective immune protection duration against O2 serotype V. anguillarum was 14–120 d for 10~7 cells/ind fish group, 14–120 d for 10~8 cells/ind group, and 14–150 d for 10~9 cells/ind group, respectively, while the effective immune protection duration against O2 serotype were 14 d–60 d for 10~7 cells/ind group, 14–120 d for 10~8 cells/ind group, and 14–120 d for 10~9 cells/ind group, respectively. We also analyzed the relationship between serum antibody titer and RPS of bivalent vaccine. When the serum antibody titer was over 1 : 40 for the O1 serotype and 1 : 160 for the O2 serotype, the corresponsive RPS to V. anguillarum O1 or O2 serotype were all over 75%. These results suggest that the V. anguillarum inactivated bivalent vaccine could provide effective immune protection for turbot, and the data of antibody persistence and immune protection duration obtained in this study will provide support for clinical trials of this vaccine.
引文
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[2]Xiao H,Li J,Wang X H,et al.Studies on pathogens of rotted gill and rotted caudal fins of seaperch(Lateolabrax japonicus)fry[J].Journal of Ocean University of Qingdao,1999,29(1):87-93.[肖慧,李军,王祥红,等.鲈鱼苗烂鳃、烂尾病病原菌的研究[J].青岛海洋大学学报,1999,29(1):87-93.]
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[4]Zou Y X,Zhang P J,Mo Z L,et al.Isolation and identification of a pathogenic bacterium from haemorrhagic turbot(Scophthalmus maximus)[J].Chinese High Technology Letters,2004,14(4):89-93.[邹玉霞,张培军,莫照兰,等.大菱鲆出血症病原菌的分离和鉴定[J].高技术通讯,2004,14(4):89-93.]
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[6]Larsen J L,Pedersen K,Dalsgaard I.Vibrio anguillarum serovars associated with vibriosis in fish[J].Journal of Fish Disease,1994,7:259-267.
[7]Zhao L N,Li G Y,Li J,et al.Serotyping and antibiotics sensitivity of Vibrio anguillarum strains isolated from marine farmed fish[J].Oceanologia et Limnologia Sinica,2015,46(5):1109-1118.[赵鲁宁,李贵阳,李杰,等.海水养殖鱼类鳗弧菌(Vibrio anguillarum)临床分离株的血清型和抗生素耐药谱[J].海洋与湖沼,2015,46(5):1109-1118.]
[8]Xiao H,Li J,Wang X H,et al.Preparation of vaccine against vibriosis of sea perch(Lateolabrax japonicus)and its immune efficiency evaluation[J].Journal of Ocean University of Qingdao,2003,33(2):226-232.[肖慧,李军,王祥红,等.鲈鱼鳗弧菌病疫苗的制备及免疫防治效果[J].青岛海洋大学学报,2003,33(2):226-232.]
[9]Mo Z L,Xu Y L,Zhang P J.Vaccination against Vibrio anguillarum on cultured flounder,Paralichthys olivaceus[J].Marine Sciences,2002,26(4):63-67.[莫照兰,徐永立,张培军.养殖牙鲆鳗弧菌疫苗的研究[J].海洋科学,2002,26(4):63-67.]
[10]Zhu K L,Chen J X,Li Y,et al.Study on the vaccination against V.anguillarum in farmed marine turbot[J].Chinese High Technology Letters,2004,14(2):76-80.[朱开玲,陈吉祥,李筠,等.鳗弧菌灭活疫苗对海水养殖大菱鲆的免疫预防研究[J].高技术通讯,2004,14(2):76-80.]
[11]Xiao P,Mo Z L,Zou Y X,et al.Immune responses and efficacy of an oral-emulsified bivalent vaccine against Vibrio anguillarum in turbot(Scophthalmus maximus)[J].Chinese High Technology Letters,2007,17(9):979-985.[肖鹏,莫照兰,邹玉霞,等.鳗弧菌油乳化二价口服疫苗免疫养殖大菱鲆的免疫应答及免疫效果的研究[J].高技术通讯,2007,17(9):979-985.]
[12]Liu R,Zhao M J,Yang H,et al.Construction and protection evaluation of a bivalent DNA vaccine containing tdh2 of Vibrio parahaemolyticus and ompU of V.anguillarum on turbot on turbot Scophthalmus maximus[J].Oceanologia et Limnologia Sinica,2011,42(4):580-586.[刘瑞,赵明君,杨慧,等.副溶血弧菌(Vibrio parahaemolyticus)tdh2基因和鳗弧菌(V.anguillarum)ompU基因二联DNA疫苗制备及其对大菱鲆(Scophthalmus maximus)免疫保护作用[J].海洋与湖沼,2011,42(4):580-586.]
[13]Pan Y H,Ma Y,Zhao D L,et al.Study on immune response of Paralichthys olivaceus induced by live attenuated V.anguillarum vaccine[J].Food and Drug,2009,11(5):12-15.[潘燕华,马悦,赵东玲,等.鳗弧菌减毒活疫苗对牙鲆免疫效果的研究[J].食品与药品,2009,11(5):12-15.]
[14]Shen P,Fan X R,Li G W.Experiment of Microbiology[M].Ver.3.Beijing:Higher Education Press,1999.[沈萍,范秀容,李广武.微生物学实验[M].第三版.北京:高等教育出版社,1999.]
[15]Song X Q,Xing J,Zhan W B.Variations of seven immune related genes expression in flounder(Paralichthys olivaceus)injected and immersed with inactivated Vibrio anguillarum[J].Journal of Fishery Sciences of China,2014,21(4):747-758.[宋晓青,邢婧,战文斌.牙鲆经注射和浸泡免疫鳗弧菌灭活疫苗后7种免疫相关基因表达的变化[J].中国水产科学,2014,21(4):747-758.]
[16]Xing J,Wang Y,Song X Q,et al.Variations in expression of eleven immune related genes in flounder(Paralichthys olivaceus)injected with inactivated Vibrio anguillarum[J].Oceanologia et Limnologia Sinica,2015,46(5):1024-1033.[邢婧,王洋,宋晓青,等.牙鲆(Paralichthys olivaceus)浸泡免疫鳗弧菌(Vibrio anguillarum)灭活疫苗后11种免疫相关基因的表达变化[J].海洋与湖沼,2015,46(5):1024-1033.]
[17]Xue J,Song X Q,Xing J,et al.Expression analysis of thirteen immune related genes of flounder(Paralichthys olivaceus)vaccinated with formalin-inactivated Vibrio anguillarum[J].Periodical of Ocean University of China,2016,46(2):42-53.[薛洁,宋晓青,邢婧,等.牙鲆注射免疫鳗弧菌灭活疫苗后13种免疫相关基因表达的变化[J].中国海洋大学学报,2016,46(2):42-53.]
[2]Xiao H,Li J,Wang X H,et al.Studies on pathogens of rotted gill and rotted caudal fins of seaperch(Lateolabrax japonicus)fry[J].Journal of Ocean University of Qingdao,1999,29(1):87-93.[肖慧,李军,王祥红,等.鲈鱼苗烂鳃、烂尾病病原菌的研究[J].青岛海洋大学学报,1999,29(1):87-93.]
[3]Mo Z L,Mao Y X,Chen S Y,et al.Classification for one pathogenic Vibrio anguillarum strain isolated from skinulcer flounder[J].Acta Microbiologica Sinica,2002,42(3):263-269.[莫照兰,茅云翔,陈师勇,等.一株牙鲆皮肤溃烂症病原菌的鉴定[J].微生物学报,2002,42(3):263-269.]
[4]Zou Y X,Zhang P J,Mo Z L,et al.Isolation and identification of a pathogenic bacterium from haemorrhagic turbot(Scophthalmus maximus)[J].Chinese High Technology Letters,2004,14(4):89-93.[邹玉霞,张培军,莫照兰,等.大菱鲆出血症病原菌的分离和鉴定[J].高技术通讯,2004,14(4):89-93.]
[5]Chen J X,Li C F,Yan X H,et al.Studies on biological characteristics of five pathogenic Vibrio anguillarum strains isolated from diseased turbot(Scophthalmus maximus)in Shandong province of China[J].Chinese High Technology Letters,2005,15(6):92-96.[陈吉祥,李彩风,颜显辉,等.大菱鲆病原鳗弧菌生物学及分子特征研究.高技术通讯,2005,15(6):92-96.]
[6]Larsen J L,Pedersen K,Dalsgaard I.Vibrio anguillarum serovars associated with vibriosis in fish[J].Journal of Fish Disease,1994,7:259-267.
[7]Zhao L N,Li G Y,Li J,et al.Serotyping and antibiotics sensitivity of Vibrio anguillarum strains isolated from marine farmed fish[J].Oceanologia et Limnologia Sinica,2015,46(5):1109-1118.[赵鲁宁,李贵阳,李杰,等.海水养殖鱼类鳗弧菌(Vibrio anguillarum)临床分离株的血清型和抗生素耐药谱[J].海洋与湖沼,2015,46(5):1109-1118.]
[8]Xiao H,Li J,Wang X H,et al.Preparation of vaccine against vibriosis of sea perch(Lateolabrax japonicus)and its immune efficiency evaluation[J].Journal of Ocean University of Qingdao,2003,33(2):226-232.[肖慧,李军,王祥红,等.鲈鱼鳗弧菌病疫苗的制备及免疫防治效果[J].青岛海洋大学学报,2003,33(2):226-232.]
[9]Mo Z L,Xu Y L,Zhang P J.Vaccination against Vibrio anguillarum on cultured flounder,Paralichthys olivaceus[J].Marine Sciences,2002,26(4):63-67.[莫照兰,徐永立,张培军.养殖牙鲆鳗弧菌疫苗的研究[J].海洋科学,2002,26(4):63-67.]
[10]Zhu K L,Chen J X,Li Y,et al.Study on the vaccination against V.anguillarum in farmed marine turbot[J].Chinese High Technology Letters,2004,14(2):76-80.[朱开玲,陈吉祥,李筠,等.鳗弧菌灭活疫苗对海水养殖大菱鲆的免疫预防研究[J].高技术通讯,2004,14(2):76-80.]
[11]Xiao P,Mo Z L,Zou Y X,et al.Immune responses and efficacy of an oral-emulsified bivalent vaccine against Vibrio anguillarum in turbot(Scophthalmus maximus)[J].Chinese High Technology Letters,2007,17(9):979-985.[肖鹏,莫照兰,邹玉霞,等.鳗弧菌油乳化二价口服疫苗免疫养殖大菱鲆的免疫应答及免疫效果的研究[J].高技术通讯,2007,17(9):979-985.]
[12]Liu R,Zhao M J,Yang H,et al.Construction and protection evaluation of a bivalent DNA vaccine containing tdh2 of Vibrio parahaemolyticus and ompU of V.anguillarum on turbot on turbot Scophthalmus maximus[J].Oceanologia et Limnologia Sinica,2011,42(4):580-586.[刘瑞,赵明君,杨慧,等.副溶血弧菌(Vibrio parahaemolyticus)tdh2基因和鳗弧菌(V.anguillarum)ompU基因二联DNA疫苗制备及其对大菱鲆(Scophthalmus maximus)免疫保护作用[J].海洋与湖沼,2011,42(4):580-586.]
[13]Pan Y H,Ma Y,Zhao D L,et al.Study on immune response of Paralichthys olivaceus induced by live attenuated V.anguillarum vaccine[J].Food and Drug,2009,11(5):12-15.[潘燕华,马悦,赵东玲,等.鳗弧菌减毒活疫苗对牙鲆免疫效果的研究[J].食品与药品,2009,11(5):12-15.]
[14]Shen P,Fan X R,Li G W.Experiment of Microbiology[M].Ver.3.Beijing:Higher Education Press,1999.[沈萍,范秀容,李广武.微生物学实验[M].第三版.北京:高等教育出版社,1999.]
[15]Song X Q,Xing J,Zhan W B.Variations of seven immune related genes expression in flounder(Paralichthys olivaceus)injected and immersed with inactivated Vibrio anguillarum[J].Journal of Fishery Sciences of China,2014,21(4):747-758.[宋晓青,邢婧,战文斌.牙鲆经注射和浸泡免疫鳗弧菌灭活疫苗后7种免疫相关基因表达的变化[J].中国水产科学,2014,21(4):747-758.]
[16]Xing J,Wang Y,Song X Q,et al.Variations in expression of eleven immune related genes in flounder(Paralichthys olivaceus)injected with inactivated Vibrio anguillarum[J].Oceanologia et Limnologia Sinica,2015,46(5):1024-1033.[邢婧,王洋,宋晓青,等.牙鲆(Paralichthys olivaceus)浸泡免疫鳗弧菌(Vibrio anguillarum)灭活疫苗后11种免疫相关基因的表达变化[J].海洋与湖沼,2015,46(5):1024-1033.]
[17]Xue J,Song X Q,Xing J,et al.Expression analysis of thirteen immune related genes of flounder(Paralichthys olivaceus)vaccinated with formalin-inactivated Vibrio anguillarum[J].Periodical of Ocean University of China,2016,46(2):42-53.[薛洁,宋晓青,邢婧,等.牙鲆注射免疫鳗弧菌灭活疫苗后13种免疫相关基因表达的变化[J].中国海洋大学学报,2016,46(2):42-53.]