环形泰勒虫喀什株感染牛淋巴细胞系的建立及其生物学特征的鉴定
|
摘要
建立1株环形泰勒虫(Theileria annulata)感染的牛淋巴细胞系,分析其生物学特征,为环形泰勒虫病疫苗研发提供新的细胞系。用环形泰勒虫喀什株感染的小亚璃眼蜱成蜱叮咬健康黄牛,当动物体温升高,体表淋巴结肿大时,用显微镜检查肩前和股前淋巴结穿刺物涂片,出现裂殖体感染的淋巴细胞时,手术摘取淋巴结,无菌条件下进行淋巴细胞的分离培养;选择合适的培养条件对该细胞进行适应性传代培养,可稳定传代培养后,绘制细胞生长曲线,测定细胞倍增时间和细胞周期,测定细胞系环形泰勒虫18S rRNA基因序列,通过序列比对分析其亲缘关系。结果显示,建立了1株可传代培养的环形泰勒虫喀什株裂殖体感染的淋巴细胞系(TaKashi),接种5.0×10~5 mL~(-1)的细胞,经72 h培养周期,细胞浓度可达到(2.8～3.0)×10~6mL~(-1),细胞存活率维持在90%以上;细胞群体倍增时间为33.4 h。细胞周期分析显示,G1期占46.2%,G2期占10.4%,S1期占43.4%,TaKashi传50代后仍然保持着淋巴细胞的形态学特征。18S rRNA基因序列比对及抗原基因Tams1同源性分析显示,该虫株与我国不同省份流行的环形泰勒虫虫株的相似性在91.9%以上,并处于同一个分支。
To establish a Theileria annulata infected bovine lymphocyte cell line and to analyze its biological characteristics that provides the opportunity for the development of vaccine against tropical theileriosis,adult Hyalomma anatolicum anatolicum ticks infected with T.annulata Kashi strain were feed on yellow cattle.Meanwhile,the physical and microscopic parameters of animal like body temperature,lymph node enlargement,microscopic examination of blood smear,and pre-scapular lymph node biopsy smear were performed for the appearance of schizonts in the lymphocytes.After confirmation of schizonts in lymphocytes,lymph nodes of infected animal were dissected aseptically for cell separation.The 18 S r RNA gene sequence of the cytoplasmic T.annulata was determined for its confirmation.A lymphocyte cell line(Ta Kashi) infected by the Kashi strain of the T.annulata schizont was established.After the stable in-vitro cell culture,the growth curve was drawn for the calculation of the cell doubling time and cell cycle.Cells density of 5.0×105mL-1reached to(2.8-3.0)×106mL-1after 72 h after in vitro culture of transformed cells in complete cell culture medium with the cell survival rate of 90% as determined.Recorded cell population doubling time was 33.4 h,and cell cycle analysis showed that G1,G2 and S1 stages were 46.2%,10.4%,and 43.4%,respectively.The morphological characteristics of Ta Kashi transformed lymphocytes remained unchanged until the 50 th generation.The comparative analysis of 18 S r RNA gene sequences and homogenous comparison of Tams1 gene of Ta Kashi strain showed that the similarity index between this strain and the other strains prevalent in different provinces of China were more than 91.9% and phylogenetic relationships were shown in the T.annulata branch.
To establish a Theileria annulata infected bovine lymphocyte cell line and to analyze its biological characteristics that provides the opportunity for the development of vaccine against tropical theileriosis,adult Hyalomma anatolicum anatolicum ticks infected with T.annulata Kashi strain were feed on yellow cattle.Meanwhile,the physical and microscopic parameters of animal like body temperature,lymph node enlargement,microscopic examination of blood smear,and pre-scapular lymph node biopsy smear were performed for the appearance of schizonts in the lymphocytes.After confirmation of schizonts in lymphocytes,lymph nodes of infected animal were dissected aseptically for cell separation.The 18 S r RNA gene sequence of the cytoplasmic T.annulata was determined for its confirmation.A lymphocyte cell line(Ta Kashi) infected by the Kashi strain of the T.annulata schizont was established.After the stable in-vitro cell culture,the growth curve was drawn for the calculation of the cell doubling time and cell cycle.Cells density of 5.0×105mL-1reached to(2.8-3.0)×106mL-1after 72 h after in vitro culture of transformed cells in complete cell culture medium with the cell survival rate of 90% as determined.Recorded cell population doubling time was 33.4 h,and cell cycle analysis showed that G1,G2 and S1 stages were 46.2%,10.4%,and 43.4%,respectively.The morphological characteristics of Ta Kashi transformed lymphocytes remained unchanged until the 50 th generation.The comparative analysis of 18 S r RNA gene sequences and homogenous comparison of Tams1 gene of Ta Kashi strain showed that the similarity index between this strain and the other strains prevalent in different provinces of China were more than 91.9% and phylogenetic relationships were shown in the T.annulata branch.
引文
[1] SERVICE M W.Theileriosis[M]//WALLINGFORD M W.Encyclopedia of Arthropod-Transmitted Infections of Man and Domesticated Animals.UK:CABI Publishing,2001:487-502.
[2] SCHEIN E,BUSCHER G,FRIEDHOFF K T.Light microscopic studies on the development of Theileria annulata(Dschunkowsky and Luhs,1904)in Hyalomma anatolicum excavatum(Koch,1844)[J].Z Parasitenkd,1975,48(2):123-136.
[3] MEHLHORN H,SCHEIN E.The piroplasms:life cycle and sexual stages[J].Advances in Parasitology,1984,23(23):37-103.
[4]孔繁瑶.家畜寄生虫学[M].2版.北京:中国农业大学出版社,2008.KONG Fan-yao.Parasitology of Domestic Animals[M].2nd ed.Beijing:China Agricultural University Press,2008.(in Chinese)
[5]兰州兽医研究所.环形泰勒焦虫(Theileria annulata)裂殖体细胞培养的研究[J].畜牧兽医科技资料,1973,3(3):1-9.Lanzhou Veterinary Research Institute.Study on cell culture of Theileria annulata schizont[J].Chinese Journal of Animal Husbandry and Veterinary,1973,3(3):1-9.(in Chinese)
[6] GILL B S,BHATTACHARYULU Y,KAUR D,et al.Vaccination against bovine tropical theileriasis(Theileria annulata)[J].Nature,1976,264(25):355-356.
[7] TSUR I,ADLERS H,PIPANO E.The cultivation of lymphoid cells and Theileria annulata schizonts from infected bovine blood[J].Refuah Vet,1965,22:60-62.
[8] PIPANO E,TSUR I.Experimental immunization against Theileria annulata with a tissue culture vaccine[J].Refuah Vet,1966,23:133-136.
[9]罗建勋.我国第一个兽医寄生虫病疫苗——环形泰勒虫裂殖体胶冻细胞苗[J].中国兽医寄生虫病,2003,11(4):62.LUO Jian-xun.Chinese first veterinary parasitosis vaccine—gel cellular vaccine of Theileria annulata schizont[J].Chinese Journal of Veterinary Parasitology,2003,11(4):62.(in Chinese)
[10]宋世荣,杨佐文,李良荣,等.牛环形泰勒虫裂殖体胶冻细胞苗的应用效果[J].中国兽医杂志,2001,37(3):18-19.SONG Shi-rong,YANG Zuo-wen,LI Liang-rong,et al.Application effect of gel cellular vaccine of Theileria annulata schizont[J].Chinese Journal of Veterinary Medicine,2001,37(3):18-19.(in Chinese)
[11]黄国顺.奶牛环形泰勒焦虫病的诊断与防治[J].贵州畜牧兽医,1994,18(4):38-40.HUANG Guo-shun.Diagnosis and treatment of cow Theileria annulata[J].Guizhou Animal Science and Veterinary Medicine,1994,18(4):38-40.(in Chinese)
[12] YIN F,LIU Z,LIU J,et al.Population genetic analysis of Theileria annulata from six geographical regions in China,determined on the basis of micro-and mini-satellite markers[J].Frontiers in Genetics,2018,9:50.
[13] LIU J,LI Y,LIU A,et al.Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle[J].Parasitology Research,2015,114(7):2715-2721.
[14]罗建勋,殷宏,关贵全,等.小亚璃眼蜱和亚洲璃眼蜱对环形泰勒虫传播能力的研究[J].中国兽医科技,2003,33(2):22-23.LUO Jian-xun,YIN Hong,GUAN Gui-quan,et al.Studies on ability to transmit Theileria annulata with Hyalomma anatolicum anatolicum and Hyalomma asiaticum asiaticum[J].Chinese Journal of Veterinary Science and Technology,2003,33(2):22-23.(in Chinese)
[15] HULLIGER L,BROWN C G,WILDE J K.Transition of developmental stages of Theileria parva in vitro at high temperature[J].Nature,1966,16(7):328-329.
[16]兰州兽医研究所、宁夏回族自治区农科所兽医系.牛泰勒焦虫病人工免疫的研究[J].畜牧兽医科技资料,1972,2(2):43-65.Lanzhou Veterinary Research Institute,Ningxia Veterinary Medicine of Agriculture Institute.Study on artificial immunity of theileriosis[J].Chinese Journal of Animal Husbandry and Veterinary,1972,2(2):43-65.(in Chinese)
[17] HULLIGER L.Cultivation of three species of Theileria in lymphoid cells in vitro[J].The Journal of Protozoology,1965,12(4):649-655.
[18]罗金,刘光远,田占成,等.基于18S rRNA基因序列的我国马梨形虫分类学地位分析[J].动物分类学报,2011,36(1):99-103.LUO Jin,LIU Guang-yuan,TIAN Zhan-cheng,et al.Taxonomic status of equine piroplasmid assays based on 18S rRNA gene sequencing in China[J].Acta Zootaxonomica Sinica,2011,36(1):99-103.(in Chinese)
[19] ALLSOPP M T,CAVALIER-SMITH T,DE WAAL D T,et al.Phylogeny and evolution of the piroplasms[J].Parasitology,1994,108(2):147-152.
[20]罗金,刘光远,田占成,等.马泰勒虫EMA1基因的克隆及生物学特性分析[J].中国兽医科学,2012,42(4):352-356.LUO Jin,LIU Guang-yuan,TIAN Zhan-cheng,et al.Cloning and phylogenetic analysis of EMA1 gene of Theileria equi[J].Chinese Veterinary Science,2012,42(4):352-356.(in Chinese)
[21] GLASCODINE J,TETLEY L,TAIT A,et al.Developmental expression of a Theileria annulata merozoite surface antigen[J].Molecular and Biochemical Parasitology,1990,40(1):105-112.
[22] TANAKA M,ONOE S,MATSUBA T,et al.Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA[J].Journal of Clinical Microbiology,1993,31(10):2565-2569.
[23] D’OLIVEIRA C,VAN DER WEIDE M,JACQUIET P,et al.Detection of Theileria annulata by the PCR in ticks(Acari:Ixodidae)collected from cattle in Mauritania[J].Experimental&Applied Acarology,1997,21(5):279-291.
[24] D’OLIVEIRA C,FEENSTRA A,VOS H,et al.Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems[J].Vaccine,1997,15(16):1796-1804.
[25] GUBBELS M J,D’OLIVEIRA C,JONGEJAN F.Development of an indirect Tams1 enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle[J].Clinical and Vaccine Immunology,2000,7(3):404-411.
[26]吕文祥,吕文顺,张其才,等.我国牛蜱传性血液原虫的虫种调查和分布特征的研究[J].中国兽医科技,1995,25(8):13-16.LUWen-xiang,LUWen-shun,ZHANG Qi-cai,et al.Species investigation and distribution characteristics of bovine tickborne protozoa in China[J].Chinese Journal of Veterinary Science and Technology,1995,25(8):13-16.(in Chinese)
[2] SCHEIN E,BUSCHER G,FRIEDHOFF K T.Light microscopic studies on the development of Theileria annulata(Dschunkowsky and Luhs,1904)in Hyalomma anatolicum excavatum(Koch,1844)[J].Z Parasitenkd,1975,48(2):123-136.
[3] MEHLHORN H,SCHEIN E.The piroplasms:life cycle and sexual stages[J].Advances in Parasitology,1984,23(23):37-103.
[4]孔繁瑶.家畜寄生虫学[M].2版.北京:中国农业大学出版社,2008.KONG Fan-yao.Parasitology of Domestic Animals[M].2nd ed.Beijing:China Agricultural University Press,2008.(in Chinese)
[5]兰州兽医研究所.环形泰勒焦虫(Theileria annulata)裂殖体细胞培养的研究[J].畜牧兽医科技资料,1973,3(3):1-9.Lanzhou Veterinary Research Institute.Study on cell culture of Theileria annulata schizont[J].Chinese Journal of Animal Husbandry and Veterinary,1973,3(3):1-9.(in Chinese)
[6] GILL B S,BHATTACHARYULU Y,KAUR D,et al.Vaccination against bovine tropical theileriasis(Theileria annulata)[J].Nature,1976,264(25):355-356.
[7] TSUR I,ADLERS H,PIPANO E.The cultivation of lymphoid cells and Theileria annulata schizonts from infected bovine blood[J].Refuah Vet,1965,22:60-62.
[8] PIPANO E,TSUR I.Experimental immunization against Theileria annulata with a tissue culture vaccine[J].Refuah Vet,1966,23:133-136.
[9]罗建勋.我国第一个兽医寄生虫病疫苗——环形泰勒虫裂殖体胶冻细胞苗[J].中国兽医寄生虫病,2003,11(4):62.LUO Jian-xun.Chinese first veterinary parasitosis vaccine—gel cellular vaccine of Theileria annulata schizont[J].Chinese Journal of Veterinary Parasitology,2003,11(4):62.(in Chinese)
[10]宋世荣,杨佐文,李良荣,等.牛环形泰勒虫裂殖体胶冻细胞苗的应用效果[J].中国兽医杂志,2001,37(3):18-19.SONG Shi-rong,YANG Zuo-wen,LI Liang-rong,et al.Application effect of gel cellular vaccine of Theileria annulata schizont[J].Chinese Journal of Veterinary Medicine,2001,37(3):18-19.(in Chinese)
[11]黄国顺.奶牛环形泰勒焦虫病的诊断与防治[J].贵州畜牧兽医,1994,18(4):38-40.HUANG Guo-shun.Diagnosis and treatment of cow Theileria annulata[J].Guizhou Animal Science and Veterinary Medicine,1994,18(4):38-40.(in Chinese)
[12] YIN F,LIU Z,LIU J,et al.Population genetic analysis of Theileria annulata from six geographical regions in China,determined on the basis of micro-and mini-satellite markers[J].Frontiers in Genetics,2018,9:50.
[13] LIU J,LI Y,LIU A,et al.Development of a multiplex PCR assay for detection and discrimination of Theileria annulata and Theileria sergenti in cattle[J].Parasitology Research,2015,114(7):2715-2721.
[14]罗建勋,殷宏,关贵全,等.小亚璃眼蜱和亚洲璃眼蜱对环形泰勒虫传播能力的研究[J].中国兽医科技,2003,33(2):22-23.LUO Jian-xun,YIN Hong,GUAN Gui-quan,et al.Studies on ability to transmit Theileria annulata with Hyalomma anatolicum anatolicum and Hyalomma asiaticum asiaticum[J].Chinese Journal of Veterinary Science and Technology,2003,33(2):22-23.(in Chinese)
[15] HULLIGER L,BROWN C G,WILDE J K.Transition of developmental stages of Theileria parva in vitro at high temperature[J].Nature,1966,16(7):328-329.
[16]兰州兽医研究所、宁夏回族自治区农科所兽医系.牛泰勒焦虫病人工免疫的研究[J].畜牧兽医科技资料,1972,2(2):43-65.Lanzhou Veterinary Research Institute,Ningxia Veterinary Medicine of Agriculture Institute.Study on artificial immunity of theileriosis[J].Chinese Journal of Animal Husbandry and Veterinary,1972,2(2):43-65.(in Chinese)
[17] HULLIGER L.Cultivation of three species of Theileria in lymphoid cells in vitro[J].The Journal of Protozoology,1965,12(4):649-655.
[18]罗金,刘光远,田占成,等.基于18S rRNA基因序列的我国马梨形虫分类学地位分析[J].动物分类学报,2011,36(1):99-103.LUO Jin,LIU Guang-yuan,TIAN Zhan-cheng,et al.Taxonomic status of equine piroplasmid assays based on 18S rRNA gene sequencing in China[J].Acta Zootaxonomica Sinica,2011,36(1):99-103.(in Chinese)
[19] ALLSOPP M T,CAVALIER-SMITH T,DE WAAL D T,et al.Phylogeny and evolution of the piroplasms[J].Parasitology,1994,108(2):147-152.
[20]罗金,刘光远,田占成,等.马泰勒虫EMA1基因的克隆及生物学特性分析[J].中国兽医科学,2012,42(4):352-356.LUO Jin,LIU Guang-yuan,TIAN Zhan-cheng,et al.Cloning and phylogenetic analysis of EMA1 gene of Theileria equi[J].Chinese Veterinary Science,2012,42(4):352-356.(in Chinese)
[21] GLASCODINE J,TETLEY L,TAIT A,et al.Developmental expression of a Theileria annulata merozoite surface antigen[J].Molecular and Biochemical Parasitology,1990,40(1):105-112.
[22] TANAKA M,ONOE S,MATSUBA T,et al.Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA[J].Journal of Clinical Microbiology,1993,31(10):2565-2569.
[23] D’OLIVEIRA C,VAN DER WEIDE M,JACQUIET P,et al.Detection of Theileria annulata by the PCR in ticks(Acari:Ixodidae)collected from cattle in Mauritania[J].Experimental&Applied Acarology,1997,21(5):279-291.
[24] D’OLIVEIRA C,FEENSTRA A,VOS H,et al.Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems[J].Vaccine,1997,15(16):1796-1804.
[25] GUBBELS M J,D’OLIVEIRA C,JONGEJAN F.Development of an indirect Tams1 enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle[J].Clinical and Vaccine Immunology,2000,7(3):404-411.
[26]吕文祥,吕文顺,张其才,等.我国牛蜱传性血液原虫的虫种调查和分布特征的研究[J].中国兽医科技,1995,25(8):13-16.LUWen-xiang,LUWen-shun,ZHANG Qi-cai,et al.Species investigation and distribution characteristics of bovine tickborne protozoa in China[J].Chinese Journal of Veterinary Science and Technology,1995,25(8):13-16.(in Chinese)