哈维氏弧菌引起的条纹海马表皮溃疡综合征的研究
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摘要
近年来,条纹海马的养殖在我国发展迅速,但由于多种养殖疾病的频繁暴发,严重制约了条纹海马养殖业的健康发展。自2017年以来,我国北方地区在条纹海马的养殖过程中,多次发生表皮溃疡综合征,患病海马游泳能力减弱,皮肤大面积溃疡,同时伴随烂尾症状,解剖后发现其肠道发白,肝脏及鳃颜色暗淡,死亡率较高,给海马养殖业造成巨大的经济损失。本研究从患病条纹海马的溃烂肌肉及肠道中分离出13株优势菌株,经回归感染实验证实菌株HDM-2可引起条纹海马出现表皮溃疡综合征的类似症状,且半数致死量(LD_(50))约为2.89×10~8 CFU/mL。为确定菌株HDM-2的分类地位,采用生理生化及基于16S rDNA的分子生物学方法对其进行鉴定,结果显示,HDM-2为哈维氏弧菌;为了解菌株HDM-2的菌体特征,采用透射电镜(TEM)对菌株HDM-2进行观察,结果显示,HDM-2为周生鞭毛,有荚膜,大小约2.20μm×4.75μm;为获知HDM-2的抗生素敏感性,采用纸片扩散法进行药敏试验,结果显示,菌株HDM-2对氟苯尼考和头孢他啶等较敏感;患病条纹海马的组织病理学研究表明,菌株HDM-2对条纹海马的皮肤、肝脏以及鳃组织损伤较严重。本文可为条纹海马表皮溃疡综合征的防治及深入研究提供基础资料。
Hippocampus spp. is unique marine fish with special body morphology and life history traits. Compared with other marine fishes, the most special part of Hippocampus spp. is male breeding and strict monogamy. In addition, its unique body morphology, including curvaceous torso and horse-shaped head, has given them higher ornamental value. The trade volume of Hippocampus spp. is huge in the world, and the majority of the trade in Hippocampus spp. is for traditional Chinese medicine. In recent years, the destruction of wild habitats and overfishing lead to a sharp decline of the wild populations. Aquaculture of Hippocampus spp. has been considered as an effective way to solve the problem of decline of the wild populations and the unsustainable trade for traditional Chinese medicine. Therefore, the development of the Hippocampus spp. aquaculture is essential.However, the threat of various diseases has seriously affected the healthy development of the aquaculture industry.Since 2017, in the breeding process of the H. erectus in northern China, epidermis ulcer syndrome has frequently occurred, the morbidity is rapid and the mortalily is high. Epidermis ulcer syndrome has become a high incidence disease in the aquaculture process. Diseased H. erectus showed obvious symptoms, such as large rot area in body surface, darker and lackluster of the gills and liver, diseased H. erectus would die within 3 days. If the diseased H.erectus were not removed in time, a large number of seahorses would be infected. Although most fish diseases can be treated with antibiotics, the overuse of antibiotics may produce many potential threats, which are not conducive to the sustainable and healthy development of the aquaculture industry. Therefore, it is essential to further study the pathogens of various fish diseases and then we can develop biological control on this basis. Accordingly, the priority of research should be disease treatment and health management, especially disease treatment. Specific vaccines can be developed from isolated pathogenic bacteria, which provide effective way for disease treatment. In this study, dominant strains were isolated from the lesion tissue and intestine of H. erectus with epidermis ulcer syndrome. In order to determine the pathogenic bacteria, we carried out artificial infection, the infection results suggested that strain HDM-2 was the pathogen causing epidermis ulcer syndrome, and the LD_(50) of strain HDM-2 was about 2.89×10~8 CFU/mL. We identified strain HDM-2 with physiological and biochemical identification and molecular biological identification methods, and we observed strain HDM-2 under transmission electron microscope. 16 S rDNA gene sequence analysis by BLAST in GenBank suggested that the similarity between strain HDM-2 and V. harveyi reached 100%. The observation results showed that HDM-2 was about 2.2 μm×4.75 μm,with peritrichous flagella and capsule. In order to study antibiotic susceptibility of strain HDM-2, we implemented the antibiotic sensitivity test by using Kirby-Bauerdiffusion method, and results showed that strain HDM-2 was highly sensitive to florfenicol. In order to study the pathogenicity of the pathogen to the host, we carried out histopathological observation of the diseased H. erectus. Observation results suggested that skin, liver and gills were severely damaged by HDM-2. This study would lay a foundation for the prevention and further study of the epidermis ulcer syndrome of H. erectus.
Hippocampus spp. is unique marine fish with special body morphology and life history traits. Compared with other marine fishes, the most special part of Hippocampus spp. is male breeding and strict monogamy. In addition, its unique body morphology, including curvaceous torso and horse-shaped head, has given them higher ornamental value. The trade volume of Hippocampus spp. is huge in the world, and the majority of the trade in Hippocampus spp. is for traditional Chinese medicine. In recent years, the destruction of wild habitats and overfishing lead to a sharp decline of the wild populations. Aquaculture of Hippocampus spp. has been considered as an effective way to solve the problem of decline of the wild populations and the unsustainable trade for traditional Chinese medicine. Therefore, the development of the Hippocampus spp. aquaculture is essential.However, the threat of various diseases has seriously affected the healthy development of the aquaculture industry.Since 2017, in the breeding process of the H. erectus in northern China, epidermis ulcer syndrome has frequently occurred, the morbidity is rapid and the mortalily is high. Epidermis ulcer syndrome has become a high incidence disease in the aquaculture process. Diseased H. erectus showed obvious symptoms, such as large rot area in body surface, darker and lackluster of the gills and liver, diseased H. erectus would die within 3 days. If the diseased H.erectus were not removed in time, a large number of seahorses would be infected. Although most fish diseases can be treated with antibiotics, the overuse of antibiotics may produce many potential threats, which are not conducive to the sustainable and healthy development of the aquaculture industry. Therefore, it is essential to further study the pathogens of various fish diseases and then we can develop biological control on this basis. Accordingly, the priority of research should be disease treatment and health management, especially disease treatment. Specific vaccines can be developed from isolated pathogenic bacteria, which provide effective way for disease treatment. In this study, dominant strains were isolated from the lesion tissue and intestine of H. erectus with epidermis ulcer syndrome. In order to determine the pathogenic bacteria, we carried out artificial infection, the infection results suggested that strain HDM-2 was the pathogen causing epidermis ulcer syndrome, and the LD_(50) of strain HDM-2 was about 2.89×10~8 CFU/mL. We identified strain HDM-2 with physiological and biochemical identification and molecular biological identification methods, and we observed strain HDM-2 under transmission electron microscope. 16 S rDNA gene sequence analysis by BLAST in GenBank suggested that the similarity between strain HDM-2 and V. harveyi reached 100%. The observation results showed that HDM-2 was about 2.2 μm×4.75 μm,with peritrichous flagella and capsule. In order to study antibiotic susceptibility of strain HDM-2, we implemented the antibiotic sensitivity test by using Kirby-Bauerdiffusion method, and results showed that strain HDM-2 was highly sensitive to florfenicol. In order to study the pathogenicity of the pathogen to the host, we carried out histopathological observation of the diseased H. erectus. Observation results suggested that skin, liver and gills were severely damaged by HDM-2. This study would lay a foundation for the prevention and further study of the epidermis ulcer syndrome of H. erectus.
引文
[1] Lin Q,Lin J D,Zhang D. Breeding and juvenile culture of the lined seahorse, Hippocampus erectus Perry, 1810[J].Aquaculture, 2008, 277(3-4):287-292.
[2] Zhang D,Yin F,Lin J D. Criteria for assessing juvenile quality of the lined seahorse Hippocampus erectus[J].Aquaculture, 2011, 322-323:255-258.
[3] Li H D, Sun H S, Bai X F, et al. HC2 of Pseudomonas sp. induced enteritis in Hippocampus japonicus[J]. Aquaculture Research, 2016, 47(6):2027-2030.
[4] Hora M dos S C da, Joyeux J C. Closing the reproductive cycle:growth of the seahorse Hippocampus reidi(Teleostei, Syngnathidae)from birth to adulthood under ex-perimental conditions[J]. Aquaculture, 2009, 292(1-2):37-41.
[5] Job S D, Do H H, Meeuwig J J, et al. Culturing the oceanic seahorse, Hippocampus kuda[J]. Aquaculture, 2002,214(1-4):333-341.
[6] Koldewey H J,Martin-Smith K M. A global review of seahorse aquaculture[J]. Aquaculture, 2010, 302(3-4):131-152.
[7] Payne M F,Rippingale R J. Rearing West Australian Seahorse, Hippocampus subelongatus, juveniles on copepod nauplii and enriched Artemia[J]. Aquaculture, 2000,188(3-4):353-361.
[8] Balcazar J L, Lee N M, Pintado J, et al. Phylogeneticcharacterization and in situ detection of bacterial communities associated with seahorses(Hippocampus guttulatus)in captivity[J]. Systematic and Applied Microbiology,2010, 33(2):71-77.
[9]Lin T T, Zhang D, Liu X, et al. Variations of immune parameters in the lined seahorse Hippocampus erectus after infection with enteritis pathogen of Vibrio parahaemolyticus[J]. Fish&Shellfish Immunology, 2016, 50:247-254.
[10]徐晓丽,邵蓬,李灏,等.豹纹鳃棘鲈致病性哈维氏弧菌的分离鉴定与系统发育分析[J].华中农业大学学报,2014, 33(4):112-118.Xu X L, Shao P, Li H, et al. Identification and phylogenetic analyses of Vibrio harveyi islated from Plectropomus leopardus[J]. Journal of Huazhong Agricultural University,2014, 33(4):112-118(in Chinese).
[11] Li J, Xu H S. Isolation and biological characteristics of Vibrio harveyi affecting hatchery-reared Penaeus chinensis larvae[J]. Oceanologia et Limnologia Sinica, 1998,29(4):353-361.
[12]胡亮,赵凤梅,于兰萍,等.副溶血弧菌对养殖大菱鲆致病性研究[J].水产科学,2008, 27(7):340-343.Hu L, Zhao F M, Yu L P, et al. Identification of pathogenic bacteria in turbot Scophthalmus maximus[J]. Fisheries Science, 2008, 27(7):340-343(in Chinese).
[13]靳晓敏,葛慕湘,张艳英,等.大菱鲆源鳗弧菌耐药表型及耐药基因检测[J].水产科学,2015, 34(8):510-514.Jin X M, Ge M X, Zhang Y Y, et al. Detection of drug resistance phenotypes and genes in Vibrio anguillarum from turbot Scophthalmus maximus[J]. Fisheries Science, 2015, 34(8):510-514(in Chinese).
[14]栾林林,张永刚,王凤军,等.大菱鲆源鳗弧菌的分离鉴定及药敏分析[J].饲料工业,2018, 39(22):60-64.Luan L L, Zhang Y G, Wang F J, et al. Isolation and identification of Vibrio anguillarum from Scophthalmus maximus and its drug sensitivity analysis[J]. Feed Industry Magazine, 2018, 39(22):60-64(in Chinese).
[15]薛淑霞,冯守明,孙金生.海水工厂化养殖大菱鲆(Scophthalmus maximus)和褐牙鲆(Paralichthys olivaceus)腹水病病原菌的分离与鉴定[J].海洋与湖沼,2006,37(6):548-554.Xue S X, Feng S M, Sun J S. Isolation and identification of pathogenic bacteria in swollen abdomen of cultured turbot(Scophthalmus maximus)and flounder(Parali-chthys olivaceus)[J]. Oceanologia et Limnologia Sinica,2006, 37(6):548-554(in Chinese).
[16]范文辉,黄捷,王秀华,等.养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析[J].微生物学报,2005,45(5):665-670.Fan W H, Huang J, Wang X H, et al. Identification and phylogenetic study of pathogenic bacteria causing ulcer disease of cultured turbot(Scophthalmus maximus)[J].Acta Microbiologica Sinica,2005, 45(5):665-670(in Chinese).
[17]张晓君,毕可然,阎斌伦,等.矛尾复鰕虎鱼病原哈氏弧菌的鉴定及特异性检测方法的建立[J].水产科学,2011, 30(12):758-763.Zhang X J, Bi K R, Yan B L, et al. Identification and PCR detection of pathogenic Vibrio harveyi isolated from Gobby Synechogobius hasta[J]. Fisheries Science,2011, 30(12):758-763(in Chinese).
[18] Won K M, Park S I. Pathogenicity of Vibrio harveyi to cultured marine fishes in Korea[J]. Aquaculture, 2008,285(1-4):8-13.
[19] Kraxberger-Beatty T, Mcgarey D J, Grier H J, et al.Vibrio harveyi, an opportunistic pathogen of common snook, Centropomus undecimalis(Bloch), held in captivity[J]. Journal of Fish Diseases, 1990, 13(6):557-560.
[20] Pujalte M J, Sitja-Bobadilla A, Macian M C, et al.Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex, gilthead sea bream and European sea bass[J]. Systematic and Applied Microbiology, 2003,26(2):284-292.
[21] Zhang X H, Austin B. Pathogenicity of Vibrio harveyi to salmonids[J]. Journal of Fish Diseases, 2000, 23(2):93-102.
[22] Lee K K, Liu P C, Chuang W H. Pathogenesis of gastroenteritis caused by Vibrio carchariae in cultured marine fish[J]. Marine Biotechnology, 2002,4(3):267-277.
[23]李海东.日本海马白斑状皮肤溃烂病病原学及组织病理学研究[D].烟台:鲁东大学,2015.Li H D. Etiology and histopathology study for white porphyritic ulcer disease of Hippocampus japonicus[D].Yantai:Ludong University,2015(in Chinese).
[24] Balcazar J L, Gallo-Bueno A, Planas M, et al. Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms[J]. Antonie Van Leeuwenhoek,2010, 97(2):207-210.
[25] Alcaide E, Gil-Sanz C, Sanjuan E, et al. Vibrio harveyi causes disease in seahorse, Hippocampus sp.[J]. Journal of Fish Diseases, 2001, 24(5):311-313.
[26] Tendencia E A. The first report of Vibrio harveyi infection in the sea horse Hippocampus kuda Bleekers 1852in the Philippines[J]. Aquaculture Research, 2004,35(13):1292-1294.
[27] Esteban M A. An overview of the immunological defenses in fish skin[J]. ISRN Immunology, 2012, 2012:853470.
[28]杨桂文,安利国.鱼类粘液细胞研究进展[J].水产学报,1999, 23(4):403-408.Yang G W, An L G. A review on the researches of fish mucous cells[J]. Journal of Fisheries of China,1999,23(4):403-408(in Chinese).
[29] Fast M D, Sims D E, Burka J F, et al. Skin morphologyand humoral non-specific defence parameters of mucus and plasma in rainbow trout, coho and Atlantic salmon[J]. Comparative Biochemistry and Physiology-Part A:Molecular&Integrative Physiology, 2002, 132(3):645-657.
[30] Guardiola F A, Cuesta A, Abellan E, et al. Comparative analysis of the humoral immunity of skin mucus from several marine teleost fish[J]. Fish&Shellfish Immunology, 2014, 40(1):24-31.
[31] Palaksha K J, Shin G W, Kim Y R, et al. Evaluation of non-specific immune components from the skin mucus of olive flounder(Paralichthys olivaceus)[J]. Fish&Shellfish Immunology, 2008, 24(4):479-488.
[32] Baquero F, Martinez J L, Canton R. Antibiotics and antibiotic resistance in water environments[J]. Current Opinion in Biotechnology, 2008, 19(3):260-265.
[2] Zhang D,Yin F,Lin J D. Criteria for assessing juvenile quality of the lined seahorse Hippocampus erectus[J].Aquaculture, 2011, 322-323:255-258.
[3] Li H D, Sun H S, Bai X F, et al. HC2 of Pseudomonas sp. induced enteritis in Hippocampus japonicus[J]. Aquaculture Research, 2016, 47(6):2027-2030.
[4] Hora M dos S C da, Joyeux J C. Closing the reproductive cycle:growth of the seahorse Hippocampus reidi(Teleostei, Syngnathidae)from birth to adulthood under ex-perimental conditions[J]. Aquaculture, 2009, 292(1-2):37-41.
[5] Job S D, Do H H, Meeuwig J J, et al. Culturing the oceanic seahorse, Hippocampus kuda[J]. Aquaculture, 2002,214(1-4):333-341.
[6] Koldewey H J,Martin-Smith K M. A global review of seahorse aquaculture[J]. Aquaculture, 2010, 302(3-4):131-152.
[7] Payne M F,Rippingale R J. Rearing West Australian Seahorse, Hippocampus subelongatus, juveniles on copepod nauplii and enriched Artemia[J]. Aquaculture, 2000,188(3-4):353-361.
[8] Balcazar J L, Lee N M, Pintado J, et al. Phylogeneticcharacterization and in situ detection of bacterial communities associated with seahorses(Hippocampus guttulatus)in captivity[J]. Systematic and Applied Microbiology,2010, 33(2):71-77.
[9]Lin T T, Zhang D, Liu X, et al. Variations of immune parameters in the lined seahorse Hippocampus erectus after infection with enteritis pathogen of Vibrio parahaemolyticus[J]. Fish&Shellfish Immunology, 2016, 50:247-254.
[10]徐晓丽,邵蓬,李灏,等.豹纹鳃棘鲈致病性哈维氏弧菌的分离鉴定与系统发育分析[J].华中农业大学学报,2014, 33(4):112-118.Xu X L, Shao P, Li H, et al. Identification and phylogenetic analyses of Vibrio harveyi islated from Plectropomus leopardus[J]. Journal of Huazhong Agricultural University,2014, 33(4):112-118(in Chinese).
[11] Li J, Xu H S. Isolation and biological characteristics of Vibrio harveyi affecting hatchery-reared Penaeus chinensis larvae[J]. Oceanologia et Limnologia Sinica, 1998,29(4):353-361.
[12]胡亮,赵凤梅,于兰萍,等.副溶血弧菌对养殖大菱鲆致病性研究[J].水产科学,2008, 27(7):340-343.Hu L, Zhao F M, Yu L P, et al. Identification of pathogenic bacteria in turbot Scophthalmus maximus[J]. Fisheries Science, 2008, 27(7):340-343(in Chinese).
[13]靳晓敏,葛慕湘,张艳英,等.大菱鲆源鳗弧菌耐药表型及耐药基因检测[J].水产科学,2015, 34(8):510-514.Jin X M, Ge M X, Zhang Y Y, et al. Detection of drug resistance phenotypes and genes in Vibrio anguillarum from turbot Scophthalmus maximus[J]. Fisheries Science, 2015, 34(8):510-514(in Chinese).
[14]栾林林,张永刚,王凤军,等.大菱鲆源鳗弧菌的分离鉴定及药敏分析[J].饲料工业,2018, 39(22):60-64.Luan L L, Zhang Y G, Wang F J, et al. Isolation and identification of Vibrio anguillarum from Scophthalmus maximus and its drug sensitivity analysis[J]. Feed Industry Magazine, 2018, 39(22):60-64(in Chinese).
[15]薛淑霞,冯守明,孙金生.海水工厂化养殖大菱鲆(Scophthalmus maximus)和褐牙鲆(Paralichthys olivaceus)腹水病病原菌的分离与鉴定[J].海洋与湖沼,2006,37(6):548-554.Xue S X, Feng S M, Sun J S. Isolation and identification of pathogenic bacteria in swollen abdomen of cultured turbot(Scophthalmus maximus)and flounder(Parali-chthys olivaceus)[J]. Oceanologia et Limnologia Sinica,2006, 37(6):548-554(in Chinese).
[16]范文辉,黄捷,王秀华,等.养殖大菱鲆溃疡症病原菌的分离鉴定及系统发育分析[J].微生物学报,2005,45(5):665-670.Fan W H, Huang J, Wang X H, et al. Identification and phylogenetic study of pathogenic bacteria causing ulcer disease of cultured turbot(Scophthalmus maximus)[J].Acta Microbiologica Sinica,2005, 45(5):665-670(in Chinese).
[17]张晓君,毕可然,阎斌伦,等.矛尾复鰕虎鱼病原哈氏弧菌的鉴定及特异性检测方法的建立[J].水产科学,2011, 30(12):758-763.Zhang X J, Bi K R, Yan B L, et al. Identification and PCR detection of pathogenic Vibrio harveyi isolated from Gobby Synechogobius hasta[J]. Fisheries Science,2011, 30(12):758-763(in Chinese).
[18] Won K M, Park S I. Pathogenicity of Vibrio harveyi to cultured marine fishes in Korea[J]. Aquaculture, 2008,285(1-4):8-13.
[19] Kraxberger-Beatty T, Mcgarey D J, Grier H J, et al.Vibrio harveyi, an opportunistic pathogen of common snook, Centropomus undecimalis(Bloch), held in captivity[J]. Journal of Fish Diseases, 1990, 13(6):557-560.
[20] Pujalte M J, Sitja-Bobadilla A, Macian M C, et al.Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex, gilthead sea bream and European sea bass[J]. Systematic and Applied Microbiology, 2003,26(2):284-292.
[21] Zhang X H, Austin B. Pathogenicity of Vibrio harveyi to salmonids[J]. Journal of Fish Diseases, 2000, 23(2):93-102.
[22] Lee K K, Liu P C, Chuang W H. Pathogenesis of gastroenteritis caused by Vibrio carchariae in cultured marine fish[J]. Marine Biotechnology, 2002,4(3):267-277.
[23]李海东.日本海马白斑状皮肤溃烂病病原学及组织病理学研究[D].烟台:鲁东大学,2015.Li H D. Etiology and histopathology study for white porphyritic ulcer disease of Hippocampus japonicus[D].Yantai:Ludong University,2015(in Chinese).
[24] Balcazar J L, Gallo-Bueno A, Planas M, et al. Isolation of Vibrio alginolyticus and Vibrio splendidus from captive-bred seahorses with disease symptoms[J]. Antonie Van Leeuwenhoek,2010, 97(2):207-210.
[25] Alcaide E, Gil-Sanz C, Sanjuan E, et al. Vibrio harveyi causes disease in seahorse, Hippocampus sp.[J]. Journal of Fish Diseases, 2001, 24(5):311-313.
[26] Tendencia E A. The first report of Vibrio harveyi infection in the sea horse Hippocampus kuda Bleekers 1852in the Philippines[J]. Aquaculture Research, 2004,35(13):1292-1294.
[27] Esteban M A. An overview of the immunological defenses in fish skin[J]. ISRN Immunology, 2012, 2012:853470.
[28]杨桂文,安利国.鱼类粘液细胞研究进展[J].水产学报,1999, 23(4):403-408.Yang G W, An L G. A review on the researches of fish mucous cells[J]. Journal of Fisheries of China,1999,23(4):403-408(in Chinese).
[29] Fast M D, Sims D E, Burka J F, et al. Skin morphologyand humoral non-specific defence parameters of mucus and plasma in rainbow trout, coho and Atlantic salmon[J]. Comparative Biochemistry and Physiology-Part A:Molecular&Integrative Physiology, 2002, 132(3):645-657.
[30] Guardiola F A, Cuesta A, Abellan E, et al. Comparative analysis of the humoral immunity of skin mucus from several marine teleost fish[J]. Fish&Shellfish Immunology, 2014, 40(1):24-31.
[31] Palaksha K J, Shin G W, Kim Y R, et al. Evaluation of non-specific immune components from the skin mucus of olive flounder(Paralichthys olivaceus)[J]. Fish&Shellfish Immunology, 2008, 24(4):479-488.
[32] Baquero F, Martinez J L, Canton R. Antibiotics and antibiotic resistance in water environments[J]. Current Opinion in Biotechnology, 2008, 19(3):260-265.