南美白对虾肝肠胞虫的分离及形态学观察
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摘要
为了研究江苏地区感染南美白对虾(Penaeus vannamei)的肝肠胞虫(Enterocytozoon hepatopenaei,EHP)的形态学特点,分析与国外报道的差异性,本研究以江苏地区感染肝肠胞虫的南美白对虾肝胰腺组织为研究材料,通过蔗糖密度梯度离心等方法进行肝肠胞虫初步分离纯化,构建一种肝肠胞虫荧光染色检测方法,显微观察肝肠胞虫的孢子形态结构。结果发现感染江苏地区南美白对虾的肝肠胞虫孢子主要分布在35%~40%浓度的蔗糖层面,可推算其密度为1.15~1.17 g/m L。荧光显微镜下可见孢子椭圆或圆形,呈亮蓝色,孢子微小但可计数,显微观察可见肝肠胞虫孢子大小约为1.7μm×0.9μm,外壁上有大量白色瘤状物,疑似胞壁蛋白,孢子表面布满细小褶皱,孢子具有一个细胞核,极丝4~6圈,细胞壁由一相对薄的电子透亮的孢子内壁和电子致密的孢子外壁组成。结论认为江苏地区南美白对虾肝肠胞虫的形态结构与国外报道的吻合,本研究建立的肝肠胞虫分离纯化、荧光染色方法以及取得的肝肠胞虫形态学资料可为其检测和研究提供参考。
Enterocytozoon hepatopenaei is a new species of microsporidia found to be affecting shrimp culture. Previous studies have shown that shrimps infected with Enterocytozoon hepatopenaei grow slowly. In recent years, slow growth has been observed in Penaeus vannamei cultured in Jiangsu Province, and severe Enterocytozoon hepatopenaei infection has been reported. To study the morphological characteristics of Enterocytozoon hepatopenaei in Jiangsu Province and interpret the differences in context of the reports from other studies conducted abroad, this study used Enterocytozoon hepatopenaei-infected hepatopancreas of Penaeus vannamei cultured in Jiangsu Province as the research material. Preliminary isolation and purification of Enterocytozoon hepatopenaei through sucrose density gradient centrifugation was performed. A method of diagnosing Enterocytozoon hepatopenaei using Fluorescent Brightener 28 staining was established and the spore morphology was observed under a microscope. The results showed that the spores of Enterocytozoon hepatopenaei were mainly distributed along the sucrose gradient of 35%–40%, and their buoyant density was 1.15 g/m L–1.17 g/m L. The spores were elliptical or round, bright blue, and small, but countable under the fluorescence microscope. The spores were approximately 1.7 μm×0.9 μm. The spore surface is covered with small folds and there is a large amount of white nodules present on their outer wall, which may be wall protein; the cell wall is composed of a relatively thin, electron-transparent inner wall and an electron dense spore wall. The spores have a nucleus, and 4–6 coils of the pole filament. The morphological structure of Enterocytozoon hepatopenaei in Jiangsu was consistent with the reports abroad. The methods of isolation, purification, and fluorescent staining developed, as well as the morphological data of Enterocytozoon hepatopenaei obtained in this study provide reference for diagnosis and research.
Enterocytozoon hepatopenaei is a new species of microsporidia found to be affecting shrimp culture. Previous studies have shown that shrimps infected with Enterocytozoon hepatopenaei grow slowly. In recent years, slow growth has been observed in Penaeus vannamei cultured in Jiangsu Province, and severe Enterocytozoon hepatopenaei infection has been reported. To study the morphological characteristics of Enterocytozoon hepatopenaei in Jiangsu Province and interpret the differences in context of the reports from other studies conducted abroad, this study used Enterocytozoon hepatopenaei-infected hepatopancreas of Penaeus vannamei cultured in Jiangsu Province as the research material. Preliminary isolation and purification of Enterocytozoon hepatopenaei through sucrose density gradient centrifugation was performed. A method of diagnosing Enterocytozoon hepatopenaei using Fluorescent Brightener 28 staining was established and the spore morphology was observed under a microscope. The results showed that the spores of Enterocytozoon hepatopenaei were mainly distributed along the sucrose gradient of 35%–40%, and their buoyant density was 1.15 g/m L–1.17 g/m L. The spores were elliptical or round, bright blue, and small, but countable under the fluorescence microscope. The spores were approximately 1.7 μm×0.9 μm. The spore surface is covered with small folds and there is a large amount of white nodules present on their outer wall, which may be wall protein; the cell wall is composed of a relatively thin, electron-transparent inner wall and an electron dense spore wall. The spores have a nucleus, and 4–6 coils of the pole filament. The morphological structure of Enterocytozoon hepatopenaei in Jiangsu was consistent with the reports abroad. The methods of isolation, purification, and fluorescent staining developed, as well as the morphological data of Enterocytozoon hepatopenaei obtained in this study provide reference for diagnosis and research.
引文
[1]Flegel T W,Nelson L,Thamavit V,et al.Presence of multiple viruses in non-diseased,cultivated shrimp at harvest[J].Aquaculture,2004,240:55-68.
[2]Chayaburakul K,Nash G,Pratanpipat P,et al.Multiple pathogens found in growth-retarded black tiger shrimpPenaeus monodon cultivated in Thailand[J].Diseases of Aquatic Organisms,2004,60(2):89-96.
[3]Anantasomboon G,Sriurairatana S,Flegel T W,et al.Unique lesions and viral-like particles found in growth retarded black tiger shrimp Penaeus monodon from East Africa[J].Aquaculture,2006,253:197-203.
[4]Sritunyalucksana K,Apisawetakan S,Boon-nat A,et al.A new RNA virus found in black tiger shrimp Penaeus monodon from Thailand[J].Virus Research,2006,118(1-2):31-38.
[5]Tourtip S.Histology,ultrastructure and molecular biology of a new microsporidium infecting the black tiger shrimp Penaeus monodon[R].Department of Anatomy,Faculty of Science.Bangkok:Mahidol University,2005.
[6]Tourtip S,Wongtripop S,Stentiford G D,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationships[J].Journal of Invertebrate Pathology,2009,102(1):21-29.
[7]Tangprasittipap A,Srisala J,Chouwdee S,et al.The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus(Litopenaeus)vannamei[J].BMC Veterinary Research,2013,9:139-148.
[8]Liu Y M,Qiu L,Cheng D Y,et al.The relationship of body length and weight in the Litopenaeus vannamei populations detected Enterocytozoon hepatopenaei[J].Progress in Fishery Sciences,2017,38(4):96-103.[刘雅梅,邱亮,程东远,等.检出虾肝肠胞虫(Enterocytozoon hepatopenaei)的凡纳滨对虾(Litopenaeus vannamei)群体的体长和体重关系[J].渔业科学进展,2017,38(4):96-103.]
[9]Chen L Z,Yu X Y,Hu Y C,et al.Preliminary investigation of Enterocytozoon hepatopenaei(EHP)and infectious hypodermal and hematopoietic necrosis virus(IHHNV)from Litopenaeus vannamei in west Guangdong Province[J].Journal of Fisheries Research,2016,38(4):273-280.[陈禄芝,余霞艳,胡一丞,等.粤西地区凡纳滨对虾虾肝肠胞虫、传染性皮下和造血组织坏死病毒感染情况的初步调查[J].渔业研究,2016,38(4):273-280.]
[10]Biju N,Sathiyaraj G,Raj M,et al.High prevalence of Enterocytozoon hepatopenaei in shrimps Penaeus monodon and Litopenaeus vannamei sampled from slow growth ponds in India[J].Diseases of Aquatic Organisms,2016,120(3):225-230.
[11]Tang K F J,Pantoja C R,Redman R M,et al.Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei(EHP)a microsporidian parasite infecting penaeid shrimp[J].Journal of Invertebrate Pathology,2015,130:37-41.
[12]Tang K F J,Han J E,Aranguren L F,et al.Dense populations of the microsporidian Enterocytozoon hepatopenaei(EHP)in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp[J].Journal of Invertebrate Pathology,2016,140:1-7.
[13]Aranguren L F,Han J E,Tang K F J.Enterocytozoon hepatopenaei(EHP)is a risk factor for acute hepatopancreatic necrosis disease(AHPND)and septic hepatopancreatic necrosis(SHPN)in the Pacific white shrimp Penaeus vannamei[J].Aquaculture,2017,471:37-42.
[14]HàN T,HàD T,Thùy N T,et al.Enterocytozoon hepatopenaei parasitizing on tiger shrimp(Penaeus monodon)infected by white feces culture in Vietnam,has been detected[J].Journal of Agricultural Science and Technology,2010,12:45-50.
[15]Flegel T W.Historic emergence,impact and current status of shrimp pathogens in Asia[J].Journal of Invertebrate Pathology,2012,110(2):166-173.
[16]Rajendran K V,Shivam S,Praveena P E,et al.Emergence of Enterocytozoon hepatopenaei(EHP)in farmed penaeus(Litopenaeus)vannamei in India[J].Aquaculture,2016,454:272-280.
[17]Suebsing R,Prombun P,Srisala J,et al.Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp[J].Journal of Applied Microbiology,2013,114(5):1254-1263.
[18]Liu Z,Zhang Q L,Wan X Y,et al.Development of Real-Time PCR assay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates[J].Progress In Fishery Sciences,2016,37(2):119-126.[刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测[J].渔业科学进展,2016,37(2):119-126.]
[19]Luo Y H,Shi J,Fang L,et al.Development and application of a Taq Man real-time PCR assay for the detection of Enterocytozoon hepatopenaei[J].Chinese Veterinary Science,2016,46(7):847-852.[骆云慧,石坚,方磊,等.虾肝肠胞虫Taq Man实时荧光定量PCR检测方法的建立及应用[J].中国兽医科学,2016,46(7):847-852.]
[20]Jiang Y R,Deng Z H,Wang B Y,et al.Study on the method of separation and purification in Nosema pernyi[J].Chinese Journal of Applied Entomology,2011,48(2):452-456.[姜义仁,邓真华,王伯阳,等.柞蚕微孢子虫孢子分离纯化方法[J].应用昆虫学报,2011,48(2):452-456.]
[21]Harrington B J,Haneage G J.Calcofluor white:A review of its uses and application in clinical mycology and parasitology[J].Laboratory Medicine,2003,34:361-367.
[22]Rasconi S,Jobard M,Jouve L,et al.Use of Calcofluor white for detection,identification and quantification of phytoplanktonic fungal parasites[J].Applied and Environmental Microbiology,2009,75:2545-2553.
[23]Yang T,He L T,Chen T,et al.Application of calcofluor white method for detection of fungi in biopsy tissues[J].Journal of Molecular Diagnosis and Therapy,2014,6(5):307-311.[杨通,何炼图,陈涛,等.荧光染色剂Calcofluor White在活检组织中真菌染色的应用[J].分子诊断与治疗杂志,2014,6(5):307-311.]
[24]Liu J P,Zeng L.Staining and discrimination of Nosema bombycis spores with Calcofluor White M2R[J].Acta Entomologica Sinica,2007,50(11):1185-1186.[刘吉平,曾玲.Calcofluor White M2R荧光染色法识别家蚕微孢子虫[J].昆虫学报,2007,50(11):1185-1186.]
[25]Qin H R,Li J L,He S Y,et al.Detection and identification of Nosema ceranae by dual fluorescent staining with Cal-cofluor White M2R and Sytox Green[J].Chinese Journal of Applied Entomology,2012,49(5):1392-1396.[秦浩然,李继莲,和绍禹,等.Calcofluor White M2R与Sytox Green双重染色法鉴别蜜蜂微孢子虫[J].应用昆虫学报,2012,49(5):1392-1396.]
[26]Wang T C,Nai Y S,Wang C Y,et al.A new microsporidium,Triwangia caridinae gen.nov.,parasitizing fresh water shrimp,Cariding formosae(Decapoda:Atyidae)in Taiwan[J].Journal of Invertebrate Pathology,2013,112(3):281-293.
[27]Ramasamy P,Jayakumar R,Brennan G P.Muscle degeneration associated with cotton shrimp disease of Penaeus indicus[J].Journal of Fish diseases,2000,23(1):77-81.
[28]Li Z,Pan G,Li T,et al.SWP5,a spore wall protein,interacts with polar tube proteins in the parasitic microsporidian Nosema bombycis[J].Eukaryot Cell,2011,11(2):229-237.
[29]Wu Z,Li Y,Pan G,et al.Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis(Microsporidia)[J].Proteomics,2008,8(12):2447-2461.
[30]Yan Y D,Dang X,Peng P,et al.Nb HSWP11,a microsporidia Nosema bombycis protein,localizing in the spore wall and membranes,reduces spore adherence to host cell BME[J].Journal of Parasitology,2014,100(5):623-632.
[31]Liu R H,Wan Y J,Liu Q B,et al.Infectious experiment of microsporidium,Vairimorpha ceracessp.nov.from Cerace stipatana to silkworm(Bombyxmori)and fish(Red Carp:Cyprinus carpio L.red Var.)[J].Science of Sericulture,2006,32(1):58-62.[刘仁华,万永继,刘强波,等.裳卷蛾变形孢虫(Vairimorpha ceraces sp.nov.)对家蚕、鱼类的感染性试验[J].蚕业科学,2006,32(1):58-62.]
[32]Burges H D,Canning E U,Hulls I K.Ultrastructure of Nosema oryzaephili and the taxonomic value of the polar filament[J].Journal of Invertebrate Pathology,1974,23(2):135-139.
[2]Chayaburakul K,Nash G,Pratanpipat P,et al.Multiple pathogens found in growth-retarded black tiger shrimpPenaeus monodon cultivated in Thailand[J].Diseases of Aquatic Organisms,2004,60(2):89-96.
[3]Anantasomboon G,Sriurairatana S,Flegel T W,et al.Unique lesions and viral-like particles found in growth retarded black tiger shrimp Penaeus monodon from East Africa[J].Aquaculture,2006,253:197-203.
[4]Sritunyalucksana K,Apisawetakan S,Boon-nat A,et al.A new RNA virus found in black tiger shrimp Penaeus monodon from Thailand[J].Virus Research,2006,118(1-2):31-38.
[5]Tourtip S.Histology,ultrastructure and molecular biology of a new microsporidium infecting the black tiger shrimp Penaeus monodon[R].Department of Anatomy,Faculty of Science.Bangkok:Mahidol University,2005.
[6]Tourtip S,Wongtripop S,Stentiford G D,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationships[J].Journal of Invertebrate Pathology,2009,102(1):21-29.
[7]Tangprasittipap A,Srisala J,Chouwdee S,et al.The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus(Litopenaeus)vannamei[J].BMC Veterinary Research,2013,9:139-148.
[8]Liu Y M,Qiu L,Cheng D Y,et al.The relationship of body length and weight in the Litopenaeus vannamei populations detected Enterocytozoon hepatopenaei[J].Progress in Fishery Sciences,2017,38(4):96-103.[刘雅梅,邱亮,程东远,等.检出虾肝肠胞虫(Enterocytozoon hepatopenaei)的凡纳滨对虾(Litopenaeus vannamei)群体的体长和体重关系[J].渔业科学进展,2017,38(4):96-103.]
[9]Chen L Z,Yu X Y,Hu Y C,et al.Preliminary investigation of Enterocytozoon hepatopenaei(EHP)and infectious hypodermal and hematopoietic necrosis virus(IHHNV)from Litopenaeus vannamei in west Guangdong Province[J].Journal of Fisheries Research,2016,38(4):273-280.[陈禄芝,余霞艳,胡一丞,等.粤西地区凡纳滨对虾虾肝肠胞虫、传染性皮下和造血组织坏死病毒感染情况的初步调查[J].渔业研究,2016,38(4):273-280.]
[10]Biju N,Sathiyaraj G,Raj M,et al.High prevalence of Enterocytozoon hepatopenaei in shrimps Penaeus monodon and Litopenaeus vannamei sampled from slow growth ponds in India[J].Diseases of Aquatic Organisms,2016,120(3):225-230.
[11]Tang K F J,Pantoja C R,Redman R M,et al.Development of in situ hybridization and PCR assays for the detection of Enterocytozoon hepatopenaei(EHP)a microsporidian parasite infecting penaeid shrimp[J].Journal of Invertebrate Pathology,2015,130:37-41.
[12]Tang K F J,Han J E,Aranguren L F,et al.Dense populations of the microsporidian Enterocytozoon hepatopenaei(EHP)in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp[J].Journal of Invertebrate Pathology,2016,140:1-7.
[13]Aranguren L F,Han J E,Tang K F J.Enterocytozoon hepatopenaei(EHP)is a risk factor for acute hepatopancreatic necrosis disease(AHPND)and septic hepatopancreatic necrosis(SHPN)in the Pacific white shrimp Penaeus vannamei[J].Aquaculture,2017,471:37-42.
[14]HàN T,HàD T,Thùy N T,et al.Enterocytozoon hepatopenaei parasitizing on tiger shrimp(Penaeus monodon)infected by white feces culture in Vietnam,has been detected[J].Journal of Agricultural Science and Technology,2010,12:45-50.
[15]Flegel T W.Historic emergence,impact and current status of shrimp pathogens in Asia[J].Journal of Invertebrate Pathology,2012,110(2):166-173.
[16]Rajendran K V,Shivam S,Praveena P E,et al.Emergence of Enterocytozoon hepatopenaei(EHP)in farmed penaeus(Litopenaeus)vannamei in India[J].Aquaculture,2016,454:272-280.
[17]Suebsing R,Prombun P,Srisala J,et al.Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp[J].Journal of Applied Microbiology,2013,114(5):1254-1263.
[18]Liu Z,Zhang Q L,Wan X Y,et al.Development of Real-Time PCR assay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates[J].Progress In Fishery Sciences,2016,37(2):119-126.[刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测[J].渔业科学进展,2016,37(2):119-126.]
[19]Luo Y H,Shi J,Fang L,et al.Development and application of a Taq Man real-time PCR assay for the detection of Enterocytozoon hepatopenaei[J].Chinese Veterinary Science,2016,46(7):847-852.[骆云慧,石坚,方磊,等.虾肝肠胞虫Taq Man实时荧光定量PCR检测方法的建立及应用[J].中国兽医科学,2016,46(7):847-852.]
[20]Jiang Y R,Deng Z H,Wang B Y,et al.Study on the method of separation and purification in Nosema pernyi[J].Chinese Journal of Applied Entomology,2011,48(2):452-456.[姜义仁,邓真华,王伯阳,等.柞蚕微孢子虫孢子分离纯化方法[J].应用昆虫学报,2011,48(2):452-456.]
[21]Harrington B J,Haneage G J.Calcofluor white:A review of its uses and application in clinical mycology and parasitology[J].Laboratory Medicine,2003,34:361-367.
[22]Rasconi S,Jobard M,Jouve L,et al.Use of Calcofluor white for detection,identification and quantification of phytoplanktonic fungal parasites[J].Applied and Environmental Microbiology,2009,75:2545-2553.
[23]Yang T,He L T,Chen T,et al.Application of calcofluor white method for detection of fungi in biopsy tissues[J].Journal of Molecular Diagnosis and Therapy,2014,6(5):307-311.[杨通,何炼图,陈涛,等.荧光染色剂Calcofluor White在活检组织中真菌染色的应用[J].分子诊断与治疗杂志,2014,6(5):307-311.]
[24]Liu J P,Zeng L.Staining and discrimination of Nosema bombycis spores with Calcofluor White M2R[J].Acta Entomologica Sinica,2007,50(11):1185-1186.[刘吉平,曾玲.Calcofluor White M2R荧光染色法识别家蚕微孢子虫[J].昆虫学报,2007,50(11):1185-1186.]
[25]Qin H R,Li J L,He S Y,et al.Detection and identification of Nosema ceranae by dual fluorescent staining with Cal-cofluor White M2R and Sytox Green[J].Chinese Journal of Applied Entomology,2012,49(5):1392-1396.[秦浩然,李继莲,和绍禹,等.Calcofluor White M2R与Sytox Green双重染色法鉴别蜜蜂微孢子虫[J].应用昆虫学报,2012,49(5):1392-1396.]
[26]Wang T C,Nai Y S,Wang C Y,et al.A new microsporidium,Triwangia caridinae gen.nov.,parasitizing fresh water shrimp,Cariding formosae(Decapoda:Atyidae)in Taiwan[J].Journal of Invertebrate Pathology,2013,112(3):281-293.
[27]Ramasamy P,Jayakumar R,Brennan G P.Muscle degeneration associated with cotton shrimp disease of Penaeus indicus[J].Journal of Fish diseases,2000,23(1):77-81.
[28]Li Z,Pan G,Li T,et al.SWP5,a spore wall protein,interacts with polar tube proteins in the parasitic microsporidian Nosema bombycis[J].Eukaryot Cell,2011,11(2):229-237.
[29]Wu Z,Li Y,Pan G,et al.Proteomic analysis of spore wall proteins and identification of two spore wall proteins from Nosema bombycis(Microsporidia)[J].Proteomics,2008,8(12):2447-2461.
[30]Yan Y D,Dang X,Peng P,et al.Nb HSWP11,a microsporidia Nosema bombycis protein,localizing in the spore wall and membranes,reduces spore adherence to host cell BME[J].Journal of Parasitology,2014,100(5):623-632.
[31]Liu R H,Wan Y J,Liu Q B,et al.Infectious experiment of microsporidium,Vairimorpha ceracessp.nov.from Cerace stipatana to silkworm(Bombyxmori)and fish(Red Carp:Cyprinus carpio L.red Var.)[J].Science of Sericulture,2006,32(1):58-62.[刘仁华,万永继,刘强波,等.裳卷蛾变形孢虫(Vairimorpha ceraces sp.nov.)对家蚕、鱼类的感染性试验[J].蚕业科学,2006,32(1):58-62.]
[32]Burges H D,Canning E U,Hulls I K.Ultrastructure of Nosema oryzaephili and the taxonomic value of the polar filament[J].Journal of Invertebrate Pathology,1974,23(2):135-139.