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密级论文编号：：中国农业科学院学位论文中国和巴基斯坦部分地区牛梨形虫病病原分行病学、血清学研究ＭｏｌｅｃｕｌａｒａｎｄＳｅｒｏｌｏｉｃａｌＥｉｄｅｍｉｏｌｏｏｆＢｏｖｉｎｅＰｉｒｏｌａｓｉｍｇｐｇｙｐＳｅｃｉｅｓｆｒｏｍＳｅｔｅｓｏｆＣｈｉｎａａｎｄＰａｋｉｓｔａｎｐｌｅｃｄＡｒｅａｄｌｎｕｈａｍｍａＡｅＭｄｅ罗建勋导教师：请学位类别：博士专：预防兽医学研究生院业科学院兽医研究所 密级:论文编号:中国农业科学院学位论文中国和巴基斯坦部分地区牛梨形虫病病原分子流行病学、血清学研究MolecularandSerologicalEpidemiologyofBovinePiroplasmSpeciesfromSelectedAreasofChinaandPakistan博士研究生：HassanMuhammadAdeel指导教师：罗建勋申请学位类别：博士专业：预防兽医学研究方向：兽医寄生虫及其分子生物学培养单位：中国农业科学院研究生院、兰州兽医研究所提交日期2018年5月 Secrecy:No.ChineseAcademyofAgriculturalSciencesDissertationMolecularandSerologicalEpidemiologyofBovinePiroplasmSpeciesfromSelectedAreasofChinaandPakistanPh.D.Candidate：HassanMuhammadAdeelSupervisor：ProfessorLuoJianxunMajor：PreventiveVeterinaryScienceSpecialization：VeterinaryParasitologyandMolecularBiologyMay,2018 独创性声明本人声明所呈交的论文是我个人在导师指导下进行的研宄工作及取得的研宄成果。尽我所知，除了文中特别加以标注和致谢的地方外，论文中不包含其他人已经发表或撰写过的研宂成一果。，也不包含为获得中国农业科学院或其它教育机构的学位或证书而使用过的材料与我同工作的同志对本研究所做的任何贡献均已在论文中作了明确的说明并表示了谢意。ＤｅｃｌａｒａｔｉｏｎｏｆＯｒｉｇｉｎａｌｉｔｙＩｈｅｒｅｂｙｄｅｃｌａｒｅｔｈａｔｔｈｉｓｔｈｅｓｉｓｉｓｃｏｍｐｏｓｅｄａｎｄｏｒｉｇｉｎａｔｅｄｅｎｔｉｒｅｌｙｂｙｍｙｓｅｌｆｕｎｄｅｒｔｈｅｕｉｄａｎｃｅｏｆｍｙｓｕｐｅｒｖｉｓｏｒ．Ｔｏｔｈｅｂｅｓｔｏｆｍｙｋｎｏｗｌｅｄｅｉｎａｄｄｉｔｉｏｎｔｏｇｇ，ｔｄｔｅｉｓｓｏｔｔｈａｔｉｎｆｏｒｍａｉｏｎｅｒｉｖｅｄｆｒｏｍｈｐｕｂｌｉｓｈｅｄａｎｄｕｎｐｕｂｌｈｅｄｗｏｒｋｆｏｈｅｒｓｈａｓｂｅｅｎａｃｋｎｏｗｌｅｄｇｅｄｉｎｔｈｅｔｅｘｔａｎｄａｌｉｓｔｏｆｒｅｆｅｒｅｎｃｅｓｉｓｉｖｅｎｉｎｔｈｅｂｉｂｌｉｏｒａｈｙ．Ｔｈｅｇｇｐｔｍｈｅｓｉｓｄｏｅｓｎｏｔｃｏｎｔａｉｎａｎｙｏｔｈｅｒｐｕｂｌｉｓｈｅｄｏｒｕｎｐｕｂｌｉｓｈｅｄｒｅｓｅａｒｃｈｗｏｒｋｂｙｏｔｈｅｒｓ，ｏｒａｎｙａｔｅｒｉａｌｓｆｏｒａｎｏｔｈｅｒｄｅｇｒｅｅｏｒｄｉｐｌｏｍａｆｒｏｍｔｈｅＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＡｇｒｉｃｕｌｔｕｒａｌＳｃｉｅｎｃｅｓａｎｄｏｔｈｅｒｅｄｕｃａｔｉｏｎａｌｉｎｓｔｉｔｕｔｉｏｎｓ．Ｔｈｅｗｏｒｋｓｃｏｎｔｒｉｂｕｔｅｄｂｙｏｔｈｅｒｃｏｌｌｅａｇｕｅｓｈａｖｅｂｅｅｎｓｔａｔｅｄａｎｄａｃｋｎｏｗｌｅｄｇｅｄ．关于论文使用授权的声明本人完全了解中国农业科学院有关保留、使用学位论文的规定，即：中国农业科学院有权保留送交论文的复印件和磁盘，允许论文被查阅和借阅，苽以采用影印、缩印或扫描等复制手段保存、汇编学位论文。同意中国农业科学院可以用不同方式在不同媒体上发表、传播学位论文的全部或部分内容。ＡｕｔｈｏｒｉｚｅｄＵｓｅＡｇｒｅｅｍｅｎｔＩｆｕｌｌｙｕｎｄｅｒｓｔａｎｄｔｈｅｒｅｇｕｌａｔｉｏｎｓｃｏｎｃｅｒｎｉｎｇｒｅｓｅｒｖａｔｉｏｎａｎｄｕｓａｇｅｏｆｔｈｅｔｈｅｓｉｓｉｎｔｈｅＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＡｒｉｃｕｌｔｕｒａｌＳｃｉｅｎｃｅｓＣＡＡＳ）．ＣＡＡＳｒｅｔａｉｎｓｔｈｅｒｉｇｈｔｔｏｋｅｅｐｔｈｅｇ（ｃｏｐｉｅｓａｎｄｄｉｓｋｓｏｆｔｈｅｔｈｅｓｉｓ，ａｌｌｏｗｉｔｔｏｂｅａｃｃｅｓｓｅｄａｎｄｂｏｒｒｏｗｅｄ，ａｎｄｃｏｍｐｏｓｅｉｔｂｙｈｏｔｏｃｏｐｙａｎｄｓｃａｎ．ＣＡＡＳｃａｎａｌｓｏｄｉｓｓｅｍｉｎａｔｅａｎｄｕｂｌｉｓｈｔｈｅｆｕｌｌａｎｄｐａｒｔｏｆｔｈｅｔｈｅｓｉｓｐｐｉｎｄｉｆｆｅｒｅｎｔｗａｙｓａｎｄｏｎｄｉｆｆｅｒｅｎｔｍｅｄｉａ．’＾导师签名／Ｓｕｐｅｒｖｉｓｏｒｓｓ丨ｇｎａｔｕｒｅ：时间年／Ｙｅａｒ〈月／ＭｏｎｔｈｊＢ／Ｄａｙ２研’＊（＞究生签名／Ｓｔｕｄｅｎｔｓｓｉｇｎａｔｕｒｅ：时间／Ｄａｔｅ：Ｗ８年／Ｙｅａｒ月／Ｍｏｎｔｈ日Ｄａｙ 中国农业科学院博士学位论文评阅人、答辩委员会签名表ＭｏｌｅｃｕｌａｒａｎｄＳｅｒｏｌｏｇｉｃａｌＥｐｉｄｅｍｉｏｌｏｇｙｏｆＰｉｒｏｐｌａｓｍＳｐｅｃｉｅｓｆｒｏｍＳｅｌｅｃｔｅｄＡｒｅａｓ论文题ｇＣｈｏｆｉｎａａｎｄＰａｋｉｓａｎｔＨａｓｓａｎＰｒｅｖｅｎｔｉｖｅＶｅｔｅｒｉｎａｒｙＰａｒａｓｎｏｉｏ＾ａｎｄ论対＃Ｍｕｈａｍｍａ专业他＿町研究方尚，．，…ＭｏｌｅｃｕｌａｒＢｉｏｌｏｇｙｄ３“ｔＡｃｔｅｄＭｅｄｉｃｉｎｅＬａｎｚｈｏｕＶｅｔｏｍａｒｙ指导教师Ｐｒｏｆ，ＬｕｏＪｉａｎｘｕｎ培鮮位（研究所，＋心）ＲｅｓｅａｒｃｈＩｎｓｔｉｔｕｔｅ一￣￣￣￣￣姓名职称单位＾—硕导丽兽医学，赵俊龙教授ｓ华中农业大学ｗ评—壚發１７１Ｉ麵■学阅张練教授河南农业大学一———一导９巧英教授甘肃农业大学？＊＇１。；袭；％祝衆东教授兰州大学预防医学＾Ｌ博导，Ｊｎ嚴ｊｑ＾魏锁成教授西北民族大学预防＃医学答何玉琴教授甘肃农业大学生物丨中院Ｍ職员预防—＃严ｇ｜ｇ，｜！中委；研■肅＃医学ｇｇｇｇ—ｇ盍叫Ｍｒ４■教蕙疆燦職员￡Ｘ？颂博异导问Ｄ丨｜中国农业科学铜ｆ究院２膽３Ｋ１？＋－兽医学一部寺一嗎二＾＾、研—中院丨究员Ｓ男Ｉ；预防麵学考脅沒会议记谈（秘书）丨＋．．．Ｉ別？＿？‘ｉＨｚ 摘要泰勒虫病是由媒介蜱传播的泰勒虫寄生于宿主红细胞或网状内皮细胞所引起的血液原虫病，发病动物以发热、贫血、黄疸、体表淋巴结肿胀，甚至死亡，为典型临床症状。据报道，梨形虫病每年在全世界范围内造成数十亿美元的经济损失，包括动物死亡、生产性能下降及防治费用。因此，建立快速特异的诊断检测方法，并对牛泰勒虫病的流行情况进行调查，阐明中国和巴基斯坦两国部分地区的梨形虫种类与分布特征，可为牛梨形虫病的防控提供技术支撑和理论依据。依据环形泰勒虫烯醇化酶基因序列，设计引物，建立了可扩增大小281bp基因片段的RPA快速检测方法。对建立的RPA方法和常规PCR方法的检测效果进行了对比，通过对274份野外样本的检测，RPA方法检测出48份阳性样品（17.51%），常规PCR检测出21份环形泰勒虫阳性样品（7.66%），结果显示RPA检测方法的敏感性明显高于常规PCR。为证实RPA检测结果的正确性，对RPA检测呈阳性而常规PCR为阴性的样品进行了测序验证。在野外环境下，环形泰勒虫与东方泰勒虫经常以混合感染的情况出现。为能够同时并鉴别诊断两种泰勒虫，本研究中建立了能够同时检测环形泰勒虫和东方泰勒虫的多重RPA检测方法。根据两种泰勒虫的烯醇化酶基因序列，分别设计了特异性扩增引物，其中，环形泰勒虫的扩增片段长度为282bp，东方泰勒虫的扩增片段长度为229bp。特异性试验证实，该方法可特异性地扩增两种泰勒虫，而与其他梨形虫DNA无交叉反应，特异性良好。敏感性试验结果显示，该多重RPA检测方法最低能够检出100拷贝的烯醇化酶基因的质粒DNA，具有较好的检测敏感性。为验证多重RPA检测方法的应用价值，分别用建立的多重RPA方法和已报道的多重PCR检测方法，对188份样品中环形泰勒虫和东方泰勒虫的感染情况进行了检测。多重RPA检测出环形泰勒虫阳性样品45份（23.9%）和东方泰勒虫阳性样品5份（2.6%）。多重PCR方法检测出环形泰勒阳性32份（17%）和东方泰勒虫阳性3份（1.6%）。为了解巴基斯坦梨形虫病的流行情况，在Chakwal、Jhang和Faisalabad三个地区，选择有蜱叮咬但无临床症状的牛，用FTA卡共采集血液样品450份。应用已发表的针对梨形虫18SrRNAV4高变区的巢式PCR方法，对采集的样品进行检测。测序结果显示，在样品采集地区主要存在环形泰勒虫、东方泰勒虫和双芽巴贝斯虫。采集的样品中，梨形虫病病原的阳性率为28.44%（128/450）。其中环形泰勒虫阳性率为22.89%、东方泰勒虫阳性率为3.11%、双芽巴贝斯虫阳性率为2.44%。样品采集的三个地区中，Chakwal地区梨形虫病流行情况最为严重，梨形虫感染阳性率达58.51%。为进一步了解巴基斯坦和中国地区梨形虫病病原的基因相关性。从中国内蒙古地区的120份样品中筛选出梨形虫阳性DNA样品16份，与巴基斯坦地区筛选出的梨形虫基因序列进行基因相关性分析。核糖体18SrRNA全长序列和棒状体相关蛋白基因分别用于泰勒虫和双芽巴贝斯虫的基因相关性分析。采用以重组的主要梨形虫表面蛋白（rMPSP）为抗原建立的间接ELISA方法，对来自西藏、新疆和内蒙3个省份44个地区的840份牛血清样品进行了检测。结果显示，样品的平均阳性率为18.57%。新疆、内蒙和西藏的阳性率分别为27%、14%和10.94%。本项研究结果，可为巴基斯坦和我国牛泰勒虫病防控提供了理论依据。关键词：流行病学，PCR，ELISA，中国，巴基斯坦I AbstractPiroplasmosisinthelargeruminantsisconsideredasthesummernightmare.TheileriaandBabesiaarehaemoprotozoans(Apicomplexa:Piroplasmida),whicharetheetiologicalagentsoftheileriosisandbabesiosis,respectively.Ticks(Acari:Ixodidae)havebeenincriminatedasviciousbloodfeedersandefficientvectorsforthesementionedpiroplasms.ThesediseaseshavebeenreportedtocauseannualeconomiclossesofseveralbillionUSdollarsintermsofproductionlossesandcostofcontrolmeasuresworldwide.Thus,arapidandspecificdetectionmethodforpiroplasmsandthedataofprevalenceofpiroplasmsforsomeareasareneeded.Inthepresentstudy,arecombinasepolymeraseamplification(RPA)testmethodforT.annulatawasestablished.A281bpfragmentofenolasegeneofT.annulatawasamplifiedandthetestwasutilizedfor274fieldsampleswhilecomparingwiththeconventionalPCR.TheRPAresultswerepositivefor48of274(17.51%)samples.All274sampleswerescreenedusingconventionalPCRand21(7.66%)sampleswerepositiveforT.annulata.AllthesamplesthatwereRPApositivebutPCRnegativeweresequenced,whichconfirmedtheresultsofRPA.TheileriaannulataandT.orientalisareusuallyco-infectedinfieldsamples.AmultiplexRPAdetectionmethodforsimultaneousdetectionofT.annulataaswellasT.orientaliswasestablished.ThemultiplexRPAspecificallyamplified282bpand229bpfragmentsoftheenolasegenefromT.annulataandT.orientalisrespectivelyandhadnocross-reactionwithotherpiroplasmspecies.ThedetectionlimitofthemultiplexRPAwas100copiesofenolasegeneplasmidDNA.AimtoevaluatetheapplicationoftheestablishedmultiplexRPAmethod.188fieldsamplesweredetectedwithmultiplexRPAandapublishedmultiplexPCRmethod.Itwasfoundthat45(23.9%)and5(2.6%)outof188bloodsampleswerepositiveforT.annulataandT.orientalis,respectivelythroughRPA.ThemultiplexPCRdetectionshowedthat32(17.0%)and3(1.6%)bloodsampleswerepositiveforT.annulataandT.orientalis,respectively.InordertostudytheprevalenceofpiroplasmsinPakistan,450cattleseverelyinfestedwithticksbutasymptomatic,wereselectedforcollectionofbloodfromvariousregionsofPakistanincludingChakwal,JhangandFaisalabad.Apublishedsemi-nestedPCRwhichtargetsV4hypervariableregionof18SrRNAgene,wasusedfordetectiontheDNAofpiroplasms.Dependingupontheshortamplifiedsequence,T.annulata,T.orientalisandB.bigeminawerefoundtobeprevalentinthestudyareas.AndthepositiverateforpiroplasmsoftheselectedsamplesfromPakistanwas128/450(28.44%).AimtoknowthegeneticrelationshipofpiroplasmsbetweenPakistanandChina,120DNAsamplesfromInnerMongoliawereusedtoscreenthepiroplasmpositivesamplesshowing16/120(13.33%)aspositive.Largefragmentof18SrRNAgenewasamplifiedfortheconfirmationofTheileriaspecies;whilerhoptryassociatedprotein(RAP-1c)genewasamplifiedforconfirmationofB.bigemina.AimingtostudytheprevalenceofthebovinetheileriosisfromthreeprovincesinChina,anELISAmethodbasedonrecombinantmajorpiroplasmsurfaceprotein(rMPSP)wasusedfordetectionoftheselectedfieldserasamples.Total840fieldbovineserasampleswerecollectedfrom44II prefecturesof3provincesinChinaincludingTibet,XinjiangandInnerMongolia.Fromthedetectionresults,theoverallpositiveratewas18.57%.HighestprevalencewasfoundinXinjiangprovince105/389(27%),followedbyInnerMongolia7/50(14%)andTibet44/401(10.94%).ThedescribediELISAmightbeanappropriatetooltoidentifythebovinetheileriosis,andtheresultsalsojotdowntheimportantdataregardingthecurrentoccurrenceoftheileriosisinthestudyareasofChina.Keywords:Epidemiology,PCR,ELISA,China,PakistanIII ContentsCHAPTERIINTRODUCTION..........................................................................................11.1Taxonomyandbiologicalfeaturesofpiroplasms............................................................11.2Piroplasminfectingruminants.........................................................................................21.2.1Theileria....................................................................................................................21.2.2Babesia......................................................................................................................41.2.3LifeCycleofPiroplasms...........................................................................................51.3Distributionofpiroplasmspecies....................................................................................91.4Diagnosticmethodstodetectandidentifypiroplasmspecies.......................................101.4.1Conventionaltechniques.........................................................................................101.4.2Serologicalassays...................................................................................................111.4.3Molecularassays.....................................................................................................121.5Piroplasmdetectionstudies............................................................................................151.5.1PiroplasmdetectioninChina..................................................................................151.5.2PiroplasmdetectioninPakistan..............................................................................151.6PiroplasmaControl........................................................................................................161.6.1Vectorcontrol..........................................................................................................161.6.2BabesiaandTheileriacontrol.................................................................................17CHAPTERIIDEVELOPMENTOFRECOMBINASEPOLYMERASEAMPLIFICATION..............................................................................................................202.1Introduction....................................................................................................................202.2Materialsandmethods...................................................................................................212.2.1Parasites...................................................................................................................212.2.2Samplescollection...................................................................................................212.2.3DetectionofTheileriaannulatabyRPA................................................................222.2.4SensitivityofRPA...................................................................................................232.2.5SpecificityofRPA..................................................................................................232.2.6Fieldsamplesscreening..........................................................................................232.2.7Statisticalanalysis...................................................................................................232.3Results............................................................................................................................242.3.1SpecificityofRPA..................................................................................................24IV 2.3.2SensitivityofRPA...................................................................................................242.3.3Resultsoffieldsamples...........................................................................................252.4Discussion......................................................................................................................26CHAPTERIIIDEVELOPMENTOFMULTIPLEXRECOMBINASEPOLYMERASEAMPLIFICATION..............................................................................................................293.1Introduction....................................................................................................................293.2Materialsandmethods...................................................................................................303.2.1Parasites...................................................................................................................303.2.2Samplescollection...................................................................................................303.2.3SimultaneousdetectionofT.annulataandT.orientalis........................................313.2.4SensitivityofmultiplexRPA..................................................................................323.2.5SpecificityofmultiplexRPA..................................................................................323.2.6Fieldsamplesscreening..........................................................................................333.3Results............................................................................................................................333.3.1SpecificityofmultiplexRPA..................................................................................333.3.2SensitivityofmultiplexRPA..................................................................................343.3.3Resultsoffieldsamples...........................................................................................343.4Discussion......................................................................................................................35CHAPTERIVMOLECULAREPIDEMIOLOGYOFPIROPLASMSPECIES...............384.1Introduction....................................................................................................................384.2Materialsandmethods...................................................................................................394.2.1Samplescollection...................................................................................................394.2.2GenomicDNAextraction........................................................................................414.2.3Primerdesign...........................................................................................................414.2.4PCRamplification,cloningandsequencing...........................................................414.2.5Confirmationandsequenceanalysisofpositivesamples.......................................444.2.6Phylogeneticanalysis..............................................................................................444.2.7Speciesconfirmation...............................................................................................444.2.8Nucleotideaccessionnumbers................................................................................454.3Results............................................................................................................................454.3.1DetectionandidentificationofparasiteinsamplescollectedfromPakistan..........454.3.2DetectionandidentificationofparasiteinsamplescollectedfromChina..............464.4Discussion......................................................................................................................49V CHAPTERVSEROLOGICALDETECTIONOFTHEILERIOSIS.................................525.1Introduction....................................................................................................................525.2Materialsandmethods...................................................................................................535.2.1Parasites...................................................................................................................535.2.2SeraandgenomicDNAs.........................................................................................535.2.3CloningandexpressingMPSPgene.......................................................................535.2.4Westernblotting......................................................................................................545.2.5ELISAprocedure.....................................................................................................545.3Results............................................................................................................................555.3.1SerologicalprevalenceofTheileriosis....................................................................555.4Discussion......................................................................................................................56CONCLUSION...................................................................................................................58REFERENCES....................................................................................................................59ACKNOWLEDGEMENTS................................................................................................75CURRICULUMVITAE......................................................................................................76VI 英文缩略表ListofAbbreviations英文缩写英文全称中文名称bpBasepairs碱基对CAASChineseAcademyofAgriculturalSciences中国农业科学院DNADeoxyribonucleicAcid脱氧核糖核酸E.coliEscherichiacoli大肠杆菌ELISAEnzymeLinkedImmunosorbentAssay酶联免疫吸附试验Fig.Figure图LAMPLoop-MediatedIsothermalAmplification环介导等温扩增LVRILanzhouVeterinaryResearchInstitute兰州兽医研究所rap-1Rhoptry-AssociatedProtein-1棒状体相关蛋白-1RFLPRestrictionFragmentLengthPolymorphism限制性片段长度多态性RLBReverseLineBlotting反向线状印迹RPARecombinasePolymeraseAmplification重组酶聚合酶扩增rpmRoundPerMinute每分钟转数18SrRNA18SRibosomalRibonucleicAcid18S核糖体核糖核酸PCRPolymeraseChainReaction聚合酶链反应µLMicrolitre微升VII 中国农业科学院博士学位论文ChapterIChapterIIntroduction1.1TaxonomyandbiologicalfeaturesofpiroplasmsAlargegroupofeukaryoticorganismsareincludedinthephylumApicomplexa,knowntobetheparasitesofinvertebratesandvertebrates.Theseorganismsarecategorizedbypossessinganapicalcomplexwhichhasseveralsecretoryorganelleswhichareinvolvedinattachmentprocessinthevertebrateorinvertebratehost(Bishopetal.,2004).ThePhylumApicomplexaincludestheordersPiroplasmorida(piroplasmids),Haemospororida(haemosporidians),Gregarinasina(gregarines)andCoccidia(Adletal.,2012).Thenomenclature„piroplasmids‟describesthemorphologyofmultiplicationstageoftheparasiteinvertebratehostbloodi.e.pear-shaped.Piroplasmidsarenon-pigmentforminghemoparasitesincontrastwithpigment(hemozoin)forminggenera,suchasHaemoproteusandPlasmodium(Uilenberg,2006).ThePiroplasmoridagroupconsistsofmainlythegeneraTheileriaandBabesia.Thedifferentiationandidentificationofpiroplasmspeciesisbasedonphenotypiccharacteristicsbyusingmicroscopicexamination.Thesesignificantcharacteristicsincludethedaughtercellsnumberinmerozoitedivisionandpiroplasmidsize.Thus,bytheirmorphology,piroplasmscanbecategorizedintothreegroups:TheileriaandlargeBabesiaparasites(<2.5-5µm)andsmallBabesia(>1-2.5µm)(Homeretal.,2000).Butafewexceptionsoccur(likeB.divergensandB.gibsoniarealthoughsmallinsize,yetincludedinBabesiasensustricto).Therefore,onlymorphologycannotactastaxonomic.LargeBabesia(Babesias.s.)aswellasparasitesdivideintotwomerozoitedaughtercellswhileTheileriaandSmallBabesia(Babesiasensulato)budintofourmerozoitedaughtercells(Uilenberg,2006).Theclassificationofpiroplasmidbasedonthespecificityofvertebrateandinvertebratehost,couldonlybesuggestiveinepidemiologicalstudiesbutnotforspeciesidentificationbecausemanypiroplasmidspeciescaninfectonevertebratehostsimultaneously.Meanwhile,asingletickspeciesmaytransmitseveralpiroplasmidspeciesandviceversa(Guanetal.,2009;Zaeemietal.,2011).Piroplasms,thetick-bornehaemoprotozoa,actassummernightmarefordifferentdomesticandwildanimals.Afewspeciesoftheseparasitesalsoinfecthumanbeingsaswell.Pathogenesisofthetheileriosisisassociatedwithradicaldivisionofinfectedleucocytes.Theileriaannulatainduceslympho-destruction.AfewmembersofT.1 中国农业科学院博士学位论文ChapterIorienatlisgrouparenon-pathogenicormildpathogenicforlargeruminants(Uilenberg,1981a).Enlargementofthelymphnodes,conjunctivitis,increasedheartrate,anorexia,non-rumination,weakness,reducedproductivity,diarrhea,ocularandnasaldischargesarethesignsofacutetheileriosis(Mehlhornetal.,2008).Animaldeathmayoccurwithin3to4daysincaseofper-acuteform,whileicterus,anemia,undulatingfeverandemaciationmaybevisibleinchronicformforalongperiod(Levine,1985).Clinicalsignsofbabesiosisdependonthecausativespecies.Forinstance,B.bovisandB.bigeminaareverydangerousleadingtothesignslikehaemoglobinuria,fever,anemiaandicteruswithdeathraterangingfrom30-50%oftheinfectedanimals(Friedhoff,1997;Hashemi-Fesharki,1997).1.2Piroplasminfectingruminants1.2.1TheileriaTheileriaisthecausativeagentoftheileriosis,whichincludesmanyspeciesthataresignificantparasitesoflivestockanimals,transmittedbyhard(ixodid)ticks.Theyinfectdomesticandwildmammals,butmainlytheruminants.Membersofthisfamilyareirregular,ovoidorbacilliforminshapeandsmallerinsize.TheileriaaremostcloselyassociatedtoBabesia,butunlikeBabesia,theseinvolveleucocytesforcompletionoflifestagesbeforeinfectingtheredbloodcells.IncomparisonwithBabesiidaefamily,apicalcomplexismuchreducedanditincludesrhoptriesonly.Sub-pelliculartubules,micronemes,conoidandpolarringareabsent.MicroporeisalsopresentonlyintheRBCsstage(Uilenberg,1981a).ThetopographicaldistributionofTheileriaislargelycontrolledbytheclimatethatfavorstherespectivetickspecies.TransmissionofTheileriatoruminantsbyticksoccurtransstadially.Theileriacanbecategorizedintoschizont“non-transforming”and“transforming”species(Sivakumaretal.,2014).Thenon-transformingTheileriaarenon-malignant(benign),butstillcapableofcausingdiseaseasaresultofanemia(Sivakumaretal.,2014).Schizont“transforming”groupincludeshighlypathogenicspecies,likeT.lestoquardi,T.annulataandT.parvawhichareresponsibleforactivationandproliferationoftheinfectedWBCsand,bybracketingwithmitoticspindleduringcelldivision.Theileriaalsodividesimultaneously,assuringthatinfectionissustainedinthedaughtercells(McKeever,2009);Theileriasp.(bougasvlei),Theileriasp.(buffalo),T.taurotragi,whichareunabletocauseschizontrelatedpathology(Pienaaretal.,2014);2 中国农业科学院博士学位论文ChapterITheileriasp.(sable)causinglymphoidhyperplasiatypicallyrelatedwiththetransformingTheileria(Nijhofetal.,2005).HighlypathogenicTheileriaspeciescantransformhostcells,andthisabilityfacilitatesparasitepropagation,butitdoesnotresultindisease.ThischaracteristicisbestillustratedbyT.parva,whichinfectsandtransformstheleukocytesofAfricanbuffalo(S.caffer)andcattleinvitrowithsameefficacy(Baldwinetal.,1986),yetbuffalocellsinfectiondoesnotexhibitthedisease.Thisreflectsalongtimeofevolutionaryeditionofbuffalo,whichhelpthemtoresisttheinfection.BovinetheileriosisisbelievedtobecausedbyfiveimportantspeciesincludingT.sinensis,T.sergenti,T.mutans,T.parvaandT.annulata.Amongthese,T.annulataiswidespreadgeographicallycoveringbroadsubtropicalzoneintheNorthernHemisphereextendingfromnorthernAfricaandsouthernEuropethroughtheMiddleEasttoAsia.Thisparasitecausesanacutedisorder(Mediterraneanortropicaltheileriosis)whichinfectscamels,waterbuffalo,yaksandcattle.Europeanbreedsofcattleareparticularlypronetotheileriosisandmayresultinhighlevelsofmorbidityandmortality.Intropicaltheileriosis,mildtomoderateanemiacanbeobserved.ThevectorsofT.annulataincludeH.marginatum,H.dromedarii,H.excavatum,H.detritum,H.asiaticumandH.anatolicum(Sayinetal.,2003).TheileriaparvaistheetiologicalagentofEastCoastfever,transmittedbyRhipicephalusappendiculatus.Thisdiseasecauseshighmortalityinfullysusceptiblestock.Thediseaseiswidelyspreadincludingsouthern,central,andeasternpartsofAfrica,resultingthousandsofcattledeathseachyear(LawrenceJAetal.,1992).Theileriasergentiisthemajorvector-borneprotozoanparasiteofgrazingbuffaloandcattleinKoreaandJapan,includedinthegroupofT.orientalis/buffeli/sergenti.ThisparasitehasalsobeenreportedinChinaandisconsideredtobethemostprevalentbenignTheileriawhichistransmittedbyHaemophysalislongicornis.SeveralstudiesinJapanreportthatsomeT.sergenticanalsocausetransientanaemia,alongwithotherclinicalsignsandmortalityupto2.5%(Chaisietal.,2014).TheclinicalsignsofT.sergenti/buffeli/orientalisinfectionarehaemolyticanaemia,feverandalsocausingdeathinsomeseverecases.Theileriasergentiinfectionisalsoassociatedwithstillbirths,abortionandremarkablelossinmilkproductioninaffectedanimals(Morrison,2015).Theileriasinensisisauniquebenignspecies,discoveredinGansuprovinceofPeoplesRepublicofChina(Baietal.,2002b).ThisparasiteinfectsyaksandcattleandistransmittedbyHaemophysalisqinghaiensis(Liuetal.,2010).Theileriamutansrarelycausesdisease,ithasoccasionallybeenreportedinassociationwithclinicaldisease,3 中国农业科学院博士学位论文ChapterImanifestingasfever,severeanaemiaandicterus,withmortalityinafewanimals.OlderanimalsthatwerenotpreviouslyexposedtotheparasitearesusceptibletoT.mutansinfection.Theclinicalsignsareonlyobservedincaseofmixedparasiticinfections.Thisparasitehasbeenreportedinsouthern,central,easternandwesternAfrica.AmblyommavariegatumandfourotherAmblyommaspecieshavebeenreportedtobevectorofT.mutans(Bishopetal.,2004;Morrison,2015).AnimportantcharacteristicofalltheTheileriaspeciesistheirabilitytodeveloppersistentinfectionsasaresultofimmuneresponseofhostanimal.IncaseofT.annulataandT.parva,theseinfections(knownascarrierstate)areusuallynotdetectableinmicroscopicexamination,butcanbedetectedbypolymerasechainreaction(Skiltonetal.,2002).CarrierinfectionswithTheileriaspeciesthatdonotmultiplyduringthepiroplasmstagerelyonpersistenceofsmallnumbersofschizontinfectedcells.Thesiteofpersistenceofthesecellsandthemechanismthathowtheypersisttheimmuneresponseofthehostanimal,areyettobestudied.1.2.2BabesiaVictorBabeswasthefirstwhodiscoveredthemicrobesinsidebovineRBCsinRomaniancattlewhichpresentedhemoglobinuria.Later,hefoundsimilarorganisminovineblood(Babes,1888).Followinghim,SmithandKilbournereportedanintraerythrocyticmicroberesponsiblefortick-borneTexasCattleFeverintheSouthernUS(SmithandKilborne,1893).Thatwasthefirstexplanationofanarthropod-bornepathogeninvertebrates.ThemicroorganismsdiscoveredbyBabesandreportedbySmithandKilbournewerenamedasBabesiabovis,B.ovisandB.bigemina,respectively(Mihalcaetal.,2010).Lateron,otherdomesticatedanimalswerereportedtobeinfectedbyseveralBabesiaspeciessuchasdogsbyB.canis(PianaandGalli-Valerio,1895)andhorsesbyB.caballi(Koch,1904).Withthepassageoftimeandadvancementofthediagnostictechniques,over100Babesiaspecieshavebeendiscovered(Criado-Fornelioetal.,2004;Lacketal.,2012).Babesiainfectionsareseriousthreattowildanimals,domesticanimalsandhumans.Thisisthemostcommonhaemoparasiteinfectingmammalsaftertrypanosomeswhichcausetrypanosomiasis(TelfordSRetal.,1993).Theseverityofdiseasedependsontheimmunologicalstatus,age,geneticfactorsofhostandco-infectionswithotherpathogens(Schnittgeretal.,2012).Bovinebabesiosisisoneofthemostimportanttick-4 中国农业科学院博士学位论文ChapterIbornediseasesoflargeruminantsandiscausedbyB.bigemina,B.bovis,andB.major.Theseparasitesarewidelydistributedgeographicallyinsubtropicalandtropicalregionsoftheworld.BabesiabigeminaisdistributedinAsia,EuropeandAfrica(Uilenberg,2001).SeveralbovineBabesiaspecies(B.bigemina,B.bovis,B.major,B.ovata,B.divergens,BabesiaUsp.KashiandB.orientalis)havebeendescribedtoinfectyak,buffaloandcattle(Luoetal.,2005a).BabesiabigeminaandB.bovisaremainlyresponsibleforcausingbovinebabesiosis.BabesiabigeminahasbeenreportedinAustralia,Europe,AsiaandAfricawhileB.bovishasalsobeenreportedinthesecontinentsexceptAfrica(Bocketal.,2004).BabesiabigeminaandB.bovisaretransmittedbyR.geigyi,R.annulatusandR.microplus(Liuetal.,2014).BabesiaovataandB.majorhavecomparativelylowvirulence;B.ovatahasbeenreportedinAsia,whileB.majorinEurope,includingtheGermany,France,theNetherlandsandUK(Uilenberg,2006).HaemophysalispunctataandH.longicornisareconsideredtobethepotentialvectorsoftheseparasites.BabesiaorientalisishighlypathogenicforbuffaloandhasbeenreportedinAsia.TicksofRhipicephalusgenusareresponsiblefortransmission(Heetal.,2012).BabesiaUsp.KashiistransmittedbyHy.anatolicum,whichisreportedonlyinChina(Luoetal.,2005b).BabesiadivergenshasbeenreportedinEurope.ItistransmittedbyIxodesricinusandfoundinanaemicpatientsyetnotincattle(Moreauetal.,2015).1.2.3LifeCycleofPiroplasmsTheileriaspp.requirestwohostsforthecompletionoftheirlifecycle,whichareticks(invertebrates)andmammals(vertebrates).Sexualreproductiontakesplaceintickswhileasexualreproductionoccursinbothvertebrateandinvertebratehost.Lifecyclecanalsobecharacterizedintothreestageswithrespecttoreproduction,whicharegamogony,sporogonyandmerogony.Ingamogony(sexualreproduction)formationandfusionofgametstakesplaceinsidethetickgutwhileasexualreproductiontakesplaceinticksalivarygland,termedassporogony.Asexualreproductioninthevertebratehostiscalledasmerogony(KakomaandMehlhorn,1993).Babesiaspecieshavebeendefinedbytheabsenceofschizontsandtransovarialtransmission,whilethepresenceofschizontsandexclusivetransstadialtransmissionplacedaparasiteinthegenusTheileria.5 中国农业科学院博士学位论文ChapterIFig.Figure2.1:Lifecycleofpiroplasmosisinbovinesinvolvingtwohostsincludestickandbovines.Gametogonyand2:lifecycleofpiroplasmosisinbovinesinvolvingtwohostsincludestickandbovines.Gametogonyandsporogonyoccurintickswhilemerogonytakesplaceinsporogonyoccurintickswhilemerogonytakesplaceinbovinesbovines1.2.3.1LifecyclestagesinhostThetickvectorinfectsthecattlehostwhiletakingabloodmeal.Occasionally,infectionmaybeacquiredcongenitallybycalves;consequentlyallcasesoccurringduringthefirstweekoflifemaybeconsideredcongenital(Levine,1985).Theinfectivestage,sporozoite,isanovalshapedbodymeasuring1μminlength.Sporozoitesareformedinthesalivaryglandoftheadulttickfollowinginoculation,whilethetickistakingabloodmeal,invadeleucocytesanddevelopintotheuninucleatetrophozoite.DifferentbovineleucocytepopulationsareknowntobepreferentiallyinfectedbyT.annulataandT.parva.ForTheileriaspp.parasiteproliferationandstagedifferentiationtakesplaceinthehostcellcytoplasm,asopposedtoaparasitophorousvacuoleasoccurswithsomeotherapicomplexanssuchasPlasmodiumandToxoplasma(MehlhornandSchein,1985).Thetrophozoitedevelopswithinthehostcelltoformthemacroschizont,amultinucleatestageoftheparasitethatinduceshostcelldivisionandsynchronisesitsown6 中国农业科学院博士学位论文ChapterIproliferationtothatoftheinfectedleucocytebybindingtothemitoticspindle.Inculture,macroschizontscontain,onaverage,twelvenuclei,eachnucleusmeasuringabout1.5μmindiameter(MehlhornandSchein,1985).Theproliferatinginfectedleucocytesquicklyproducealargepopulationofinfectedcells.Proliferationinitiallyoccursinthelymphnodesbutsubsequentlyinfectedcellsmaybefoundinthebloodstreamorinvadearangeoftissuesandorgansgivingthediseasea„cancer-like‟quality,i.e.uncontrolledhostcellreproductionand„metastasis‟.Macroschizontinfectedcellsmaybedetectedinsmearsfromsuperficiallymphnodesandtheliver,7to28dayspost-infection.Invivo,aproportionofmacroschizontsundergoesaprocessknownasmerogonywheretheschizontbecomesenlargedfollowedbytheproductionofalargenumberofuninucleatemerozoites,whichliefreeinthehostcellcytoplasm.Anelectron-microscopicstudyinT.parvademonstratedtheprocessbywhichthesyncytialdifferentiatingmacroschizontpackagestheprescribedassortmentoforganellesintofreemerozoites(ShawandTilney,1992).Ingeneral,eukaryoticcellsareunabletosynthesizeorganellesdenovo,soitiscriticalthatthefullcomplementoforganellesispresentineachmerozoite.Toensurethisoccurs,theorganellesareboundtothenuclearenvelope,whichthenassociateswiththeschizontplasmamembrane.Followingthis,merozoitesdetachfromthemainsyncytialbodyinanorderlymannerknownas„budding‟(ShawandTilney,1992).AnanalogousprocessisbelievedtooccurinT.annulata.Themerozoitecontainsasinglenucleus,oneortwomitochondria,amicronemeandbetweenthreeandsixrhoptries(Levine,1985;MehlhornandSchein,1985).Followingthebreakdownofthehostcellplasmamembrane,maturemerozoitesareliberatedintothebloodstream,whichtheninvaderedcellsandformintra-erythrocyticmerozoites,knownaspiroplasms.Thesemaybedetectedonetothreedaysafterthefirstappearanceofmacroschizonts(8–10daysfollowinginfection),andmaypersistforyearsininfectedcattle(PipanoandShkap,2000).Piroplasmsaregenerallyspherical,ovalorcommashapedbodiesboundedbyasingle-layeredcellplasmamembrane(MehlhornandSchein,1985).Inculture,itwasdemonstratedthatpiroplasmsundergoadivisionprocesscharacteristicofmerogony,i.e.karyokinesisisfollowedbycytokinesis(Conradetal.,1987).Fourdaughterpiroplasmsmaybeproduced,withthesamefeaturesasmerozoitesgeneratedwithinleucocytes,howeverthisintra-erythrocyticdivisionisconsideredtobelimitedinT.parvaandT.annulata.Inthesespecies,redcellsarecontinuallyinvadedbymerozoitesproducedfrominfectedleucocytesresidinginimmunologicallyprivileged7 中国农业科学院博士学位论文ChapterIsites(Ilhanetal.,1998).Itislikelythatwithintheerythrocyte,theprocessofdifferentiationtogametesisinitiated,pre-adaptingtheparasiteforthetickphaseofthelife-cycle.Itistheparasitizedredcell,whichisinfectivetothefeedingtickvector.1.2.3.2LifecyclestagesinvectorBetweenoneandfourdayspost-repletion,piroplasmswithiningestederythrocytesdevelopintoslender,spindle-shapedmicrogamontswithinthetickgut(Scheinetal.,1975).Uptofournucleiareformedwithinthesemicrogamontsalongwithseveralflagellum-likeappendages,whichbreakawaytocreatefiliformmicrogametes.Bythistime,sphericalstages,termedmacrogametes,havealsoappeared.Itispresumedthatthemicro-andmacro-gametesunitetoformazygote,althoughthisfusionhasnotbeenobserved(Levine,1985;Scheinetal.,1975).Zygoteshaveavacuole-likecentreandappearfromfivedayspost-repletioninthegutepithelium.Afterafurthergrowthperiodofaround12–15days,motileelongatedforms,termedkinetes,areliberatedintothehaemolymphandmigratetothesalivaryglandofthenymph(ScheinandFriedhoff,1978).Thezygoteandthekinetearepresumedtobetheonlydiploidforms,intheprimarilyhaploidlifecycleofthisparasite.WithintypeIIandIIIacini,kinetesinvadetickcellsandtransformintofissionbodies.Thesestructuresgrowinsizeastheirnucleusdividesuntiltheonsetofvectormoultingwhendevelopmentceases(ScheinandFriedhoff,1978).Theactoffeedingactivatessporogonyandallowsparasitedevelopmenttoprogress.AstudyofHyalomma(H.)anatolicum(a.)anatolicumdemonstratedthatunfedtickswereincapableofproducinginfectioninexperimentalcalves.Sporozoitesappearedinthesalivaryglandafteradulttickshadbeenfeedingfortwotothreedaysandwereatamaximallevelat72hours(Singhetal.,2001).Ithasbeenshownthatatemperatureof37°Candarelativehumidityof95%isinitselfsufficienttostimulatetheproductionofinfectivesporozoitesininfectedadultHyalomma(H.)excavatumwithouttheneedforabloodmeal(Samish,1977),suggestingsometicksmaybealreadycontaininginfectivesporozoitesbeforefeedingcommences.Oncesporogonyisactivated,aseriesoffissileeventsresultsintheproductionofsporozoites,whichareliberatedintotheticksalivaasthehostacinarcellsdegenerate.Ithasbeenestimatedthat40,000sporozoitesmaybeformedfromeachinfectedacinus(Youngetal.,1992).Themolecularprocessesthatgovernandcontroltheformationofdifferentstagesthroughoutthelife-cyclearenotfullyunderstood.However,ithasbeen8 中国农业科学院博士学位论文ChapterIproposedthattheprocessesofsporogony,merogony(intheleucocyte)andpiroplasmdifferentiation(intheerythrocyte)occurbyamorphologicallysimilarmechanismwhichmaybeunderthecontrolofasinglecassetteofgenes(ShawandTilney,1992).1.3DistributionofpiroplasmspeciesPiroplasmosisisoneofthewidelydistributedchallengestotheanimalhealth.TheilerioisisgeographicallydistributedinfivecontinentsoftheworldincludingAsia,Africa,Australia,EuropeandNorthAmerica(Alimetal.,2012;Criado-Fornelioetal.,2009;Jamesetal.,1984;ReidandBell,1984;ZiamandBenaouf,2004).Ontheotherhand,babesiosisisgeographicallydistributedinsixcontinentsoftheworldincludingAsia,Africa,Europe,Australia,NorthAmericaandSouthAmerica(Blaschitzetal.,2008;Gomesetal.,1991;Hilpertshauseretal.,2007;Molloyetal.,1998;Shenetal.,1997;Vargasetal.,1997).InChina,fourBabesiaspecies,Babesiabovis,Babesiabigemina,Babesiamajor,andBabesiaovataweredescribedasresponsibleforcattle,buffalo,andyakbabesiosis(Luoetal.,2005b;Qinetal.,2015).Recently,Liuetal.,(2017)detectedanewbovineBabesia,whichisquitesimilartoB.venatorumandHeetal.,(2017)reportedtheepidemiologicalstudyofB.orientalisinChina.AmongdifferentmembersofTheileria,T.annulata,T.orientalis,T.sinensishavebeenreportedtocausethediseaseinChina(Abdallahetal.,2017).Recently,T.luwenshunihasbeenreportedforthefirsttimeinyaks(Qinetal.,2016).InPakistan,twoBabesiaspecies,BabesiabovisandBabesiabigeminaweredescribedasresponsibleforcattle,buffalo(Jabbaretal.,2015).AmongdifferentmembersofTheileria,T.annulatahasonlybeenreportedtocausethediseaseinPakistan.Recently,amolecularstudywasconductedtojotdownthepresenceofT.orientalisinPakistan(Gebrekidanetal.,2016).However,thisstudyreportedthepresenceofT.orientalisonlyincattleexportedfromAustralia,whileallthenativeanimalswerenegative.Todate,thereisnoreportofT.sinensisfromPakistan.Hassanetal.,(2018a)reportedtheT.orientalisinnativecattleofPakistanforthefirsttimeusingrecombinasepolymeraseamplificationandpolymerasechainreaction.9 中国农业科学院博士学位论文ChapterI1.4DiagnosticmethodstodetectandidentifypiroplasmspeciesTherehasdifferenttypeofassaysusedforpiroplasmosisdiagnosis,includingclinicalsymptoms,microscopicdetection,serologicalandDNAdetection.Intheacutecases,piroplasmosiscouldbediagnosedbyusingclinicalsymptomsandbloodsmeardetection.Butforthechroniccasesorrecoveredanimals,serologicalorDNAdetectionmethodsshouldbybeemployedfordetectionofthecausedpathogen.1.4.1Conventionaltechniques1.4.1.1ClinicalexaminationIntheileriosis,theclinicalsignsincludeenlargementoflymphnodes,pyrexia,inappetenceandlossofconditionwithdeclineinmilkproduction.Ecchymoticandpetechialhemorrhagesmaybeseenonthemucousmembranes.Lacrimation,nasaldischarge,cornealopacityanddiarrheaarealsoobserved.Fatallyillanimalsoftendeveloppulmonaryedema,severedyspneaandafrothynasaldischarge.Animalsthatrecoverfromthisdiseaseoftenbecomecarrierswithreducedproductionandstuntedgrowth.Theileriaspp.demolishredbloodcells,leadingtojaundice,anemia,andinfewinstances,hemoglobinuria.Hemorrhagicdiarrhea,neurologicalsignsandabortionsmaybeobservedintheterminalstagesofdisease(RadostitsO.M.,2007).Bovinebabesiosisischaracterizedbyweightloss,anorexia,restlessnessandfever.Clinicalsignsmainlydependupontheetiology.Thus,thediseasecausedbyB.bovis,B.bigeminaandB.divergencemaybeacuteand/orchronic,B.ovatamaybenon-pathogenicorhaveaverylowpathogenicity.Acutestageofdiseaseshowsthesignsofhemoglobinuriawithmortalityrangingfrom30%to50%ininfectedhost(Friedhoff,1997;Hashemi-Fesharki,1997).1.4.1.2OpticalmicroscopyTraditionally,mostofthepiroplasmspecieswerefirstidentifiedbasedonlightmicroscopicexaminationofthinbloodsmears.ThismethodinvolvesidentificationofthepiroplasminGiemsa-stainedthinbloodsmearsorbiopsysamplesfromlymph-node.Thistechniquerequiresonlyamicroscope,soveryconvenientforsamplescreeningespeciallyinthefieldconditions.Themajorsnagsassociatedwiththismethodincludethelowsensitivityofthemethod,personnelwithhighexpertiseisrequired,falsenegativeresultsintheconditionswhenparasitemiaislowandthedifferentiationbetweenthespeciesof10 中国农业科学院博士学位论文ChapterItheparasites.Evennow,thediscoveryofnovelspeciesstillutilizesmorphologyaspartoftheirofficialdescription(Paparinietal.,2012),soopticalmicroscopyisstillafasttechniquetoprovideabasicdifferentialdiagnosisinclinicalcases(Izzoetal.,2010).However,usingthismethod,itisdifficulttodetectthecarrieranimals,sohindersitsuseinepidemiologicalinvestigations(Liuetal.,2014).Itisnotpracticabletodifferentiatethevariousspeciesofparasitesinthemixedinfections.Underlightmicroscopy,Giemsa-stainedbloodsmearsofTheileriaisseenassmalloval,roundordots-likeinsideredbloodcellsandasmacroschizontsinlargelymphocytes.Babesiaiscomparativelylargerandsingleorpairedpyriformforminganacuteangleinsidetheerythrocytes(ZanganaandNaqid,2011).MicroscopicinspectionofGiemsa-stainedbloodsmearsat100Xcoulddetectaparasitemiaupto0.01%to0.05%(Rjeibietal.,2014).1.4.1.3Bloodinoculation,ticktransmissionandisolationofpiroplasmsTickcollectionandtransmissionlinkedwithclinicaldiseasecanbeusedtoconfirmthedisease-causingspeciesandthetickvector.Thedirectinjectionofinfectedbloodmaygiveinformationaboutthespeciesthatcanproliferateduringparasitestage.Thisapproachisusefulonresearchandepidemiologicalstudyandmaybetheonlywaytoconfirmtickvectordesignationandclinicalpathologyofspecificparasitestrainorasafirststepinparasiteisolation.However,itisnotsuitableforhigh-throughputorroutineanalysisandstillinthedomainofspecializedresearchgroups(Steyletal.,2012).1.4.2SerologicalassaysIndirectserologicaltechniques,suchasenzyme-linkedimmunosorbentassay(ELISA),westernblotting,complementfixationtest(CFT)andImmunofluorescentantibodytest(IFAT),whichdetecttheantibodiesinthecarrierphaseoftheinfectionwhentheparasitemiadecreases,areappropriateandconvenienttoperform.TheIFATremainsthegoldstandardassayrecommendedbytheOIEformosteconomicallyimportantparasites.Thistestuseswhole-bodyantigensandneedsafewchemicalsandmoderateskills.Antigencanbepreparedfromparasiteswhichderivedfrominfectedanimalsorcellculture.However,Theileriainvitroculturemaybemoreusefulfordetectionincarrieranimals,todetecttransformingparasites,whichactasmeanstoproduceIFATantigen,anddifferentiatevariousspeciesandstudydifferentparasitesusingmonoclonalantibodies.However,IFATalsohassomeshortcomingssuchus,operatordependentexplanationofresults,difficultyinstandardization,low11 中国农业科学院博士学位论文ChapterIthroughputandcross-reactivitybetweencloselyrelatedspecies(Pienaaretal.,2014).Thismethodisnotappropriateoptiontodetectthecloselyrelatedspecies,forexamplethegenotypesfoundinT.velifera,T.mutansandT.buffeliclades.However,IFATmaystillbeusedinepidemiologicalstudies(Mbizenietal.,2013).Theenzymelinkedimmunosorbentassay(ELISA)hasbeenappliedtodetectparasitespecificantibodies.ThistechniquemaybeusedforthedetectionofantibodiesagainstTheileriaandBabesiaspecies.ELISAcandetectantibodiesforalongerperiod,andismoresensitiveandspecificthanIFAT.ELISAisasinglemoleculeassaythatisundertakingmorespecificantigensversatiletohighthroughputanalysis.Itischeapandfastmethodtoscreenanddiagnoselargenumbersofsamplesandremainstheworkhorseforlaboratorieswheremolecularinfrastructuredoesnotexist.Todate,manyproteinssuchasTasp,Tams-1,Spm2andPIMwereidentifiedandusedforthedevelopmentofELISAtodetectdifferentTheileriaspecies(Gubbelsetal.,2000;Mohamedetal.,2012;RajendranandRay,2014;Schnittgeretal.,2002;Seitzeretal.,2010).Yet,thebiggestdisadvantageofthistechniqueislowspecificity,particularlywhenthereisco-infectionofparasites(OIE,2014).Moreover,detectionbyELISAcouldnotgivetherightproposalfordrugtreatment,becauseevenintheabsenceofthepathogens,theantibodiesarestillpositiveagainstantigens(Mansetal.,2015).1.4.3MolecularassaysPolymeraseChainReaction(PCR)isaradicalinventionbyKaryMullisin1980s.PCRusestheabilityofDNApolymeraseenzymeforsynthesisofnewstrandsofDNAwhicharecomplementarytothetemplatestrand.AsDNApolymerasemayaddthenucleotideonlytoalreadyexisting3'-OHgroup,itrequiresaprimerontowhichitmayaddthefirstnucleotide.Thisprerequisitemakesitpossibletodescribeaspecificregionoftemplatesequencetobeamplifiedbytheresearcher.TowardstheterminationofthePCRreaction,billionsofcopiesofthespecificsequencewillbeaccumulatedwhichareknownasamplicons.Thepolymerasechainreaction(PCR)isabasicmoleculartechniqueusedforamplifyingtargetsequencesfromaDNAtemplateinanexponentialmanner.Thisisaccomplishedbyusingthermalcycling,aprocessinwhichasolutionthatincludesDNAisrepeatedlyheatedandcooledinorderto(1)melttheDNA,(2)annealshortDNAfragmentscalledprimers(typicallyartificiallydesignedoligonucleotides)tothecomplementaryDNAtarget,and(3)enzymaticallyreplicatetheprimer-boundsequencesusingtemperature-dependentDNApolymerasessuchasTaqpolymerase.PCRhasawide12 中国农业科学院博士学位论文ChapterIvarietyofapplicationsandisconsideredastapleofageneticist‟stoolkit.PCRisthemostfrequentlyusedmethodforthedetectionofhaemoparasitesindevelopedcountries.InPCR,variousregionsandproteingenesareamplifiedforthedetectionofpiroplasmspecies.ThemicroscopicexaminationisconsideredasconventionalmethodandisverylesssensitiveandspecificthanPCR.Thelatterisabletodetectthecarrieranimals,trophozoitestageofparasiteandparasitemiarateupto0.00001%(Aktaşetal.,2005).Polymerasechainreaction(PCR)hasemergedasaprecisetooltodetectvariousparasiticorganismsfrombloodoftheirhostsandvector(Bishopetal.,1992;d'Oliveiraetal.,1997;DeKoketal.,1993;Gubbelsetal.,1999;Ilhanetal.,1998;Shayanetal.,1998;Tanakaetal.,1993)andisthemostabundantlyusedmethodforthedetectionofbloodparasitesindevelopedcountries.Species-specificPCRandPCR-basedreverselineblot(RLB)hybridizationmethodshavebeenusedforthedetectionofTheileriaandBabesiaspecies(Aktaşetal.,2005;Altayetal.,2008;Garcia‐Sanmartinetal.,2008;Georgesetal.,2001;Gubbelsetal.,1999;M‟ghirbietal.,2008).Severalreal-timePCRapplicationswerepreviouslydescribedfordetectingTheileriaandothercloselyrelatedBabesiaparasites,usinggenomictargetssuchasthe18SrRNAandcytochromebgenes(Criado-Fornelioetal.,2009;Jeongetal.,2003;Paplietal.,2011).VariousPCRandhybridizationassaysarecurrentlyusedforthedetectionofpiroplasmosis(Gubbelsetal.,1999).However,quantificationoftheparasitecanbedonebynoneofthese.Real-timequantitativePCRhasagreatpotentialforanalyticalapplicationsinquantitativeDNAanalysis,butprovidingquantitativedatarequiresamorecomplicateddataanalysisthanqualitative.Real-timePCRtechniqueshavebeendesignedforarapiddetectionoftheinfectionwithreducedcontaminationriskandgenerallywithhighersensitivity(Criado-Fornelioetal.,2009).However,simplePCRisunabletodetectmixedinfections.Toovercomethisproblem,areverselineblot(RLB)assaywasestablishedtodetectvariousspeciesofthepiroplasms(Gubbelsetal.,2000).Prevalenceofpiroplasmswasfoundbyusingreverselineblothybridizationasadiagnostictechnique(36.08%)inTurkey.VariousscientistshaveusedRLBtechniqueforconcurrentdetectionofvariouspiroplasmspecies(Altayet13 中国农业科学院博士学位论文ChapterIal.,2008;Altayetal.,2007;Garcia‐Sanmartinetal.,2008;Georgesetal.,2001;Kouametal.,2010;Nagoreetal.,2004;Nijhofetal.,2005;Ouraetal.,2004).Reverselineblot(RLB)hybridizationhasbeencommonlyusedforthesimultaneousdetectionofseveralpiroplasmspecies.Inthisassayofconservedregion,the18SrRNAgeneisamplifiedbyconventionalPCRandthehybridizationofthesePCRproductsisaccomplishedwithmultiplespeciesandgenusspecificprobesimmobilizedonasinglemembrane,whichenablestheconcurrentdetectionofvariousspeciesofpiroplasm(Gubbelsetal.,2000)(Gubbelsetal.,2000).RLBhybridizationtechniqueofdiagnosisisquickandmorenumberofspeciescanbeidentifiedatatime.So,itiscomparativelyabettermethodfordiagnosisforresearchpurposes.Previouslyreportindicatedthatthesensitivityofreverselineblothybridizationismuchhigher(48.6%)thanmicroscopicdetection(37.4%)afterprevalencestudyofpiroplasmsinNorthernTunisia(M‟ghirbietal.,2008).However,applicationofthePCRandRLBrequirespecialequipmentandthecostofreagentsishigher.Inaddition,welltrainedpersonnelarealsorequired.Thereforetheyarenotcommonlyusedinresourcepoorlaboratories(Liuetal.,2012).Loopmediatedisothermalamplification(LAMP)amplifiesDNArapidlywithhighsensitivityunderisothermalconditions(Notomietal.,2000).DifferenttypesoftemplatescanbeamplifiedbyitsuchasincludingpurifiedgenomicDNA,blooddriedonfilterpaperandheat-treatedblood(Kubokietal.,2003).TheLAMPreagentsarefairlystableatroomtemperaturei.e.25to36°C,whichmaketheirusepossibleinresourcepoorlaboratoriesandfieldconditions(Thekisoeetal.,2010).(Salihetal.,2008)reportedaspecificLAMPassayforthedetectionoftropicaltheileriosis.(Liuetal.,2012)developedtwoLAMPassaysforthespecificdetectionofT.annulatainChinatargetingthe18SrRNAgene.ThesensitivityofLAMPcanbewelljustifiedwiththepointthatitcandetect0.1pgwhereasPCRcandetect10pgDNAinthesample(Liuetal.,2012).Recombinasepolymeraseamplification(RPA)isanewlydevelopedassay.Thistechniqueusesspecificoligonucleotideprimers,whichareabletobindtothetemplateDNAwiththeassistanceofarecombinaseincombinationwithstrand-displacementDNAsynthesis.AttemperaturesjustaboveroomtemperatureanamplificationofcomplexDNAtargetscanbeachievedinlessthan30minutes(Euleretal.,2012).14 中国农业科学院博士学位论文ChapterI1.5PiroplasmdetectionstudiesSeveralstudieshavebeenconductedinthesetwocountriesforepidemiologyofpiroplasmspecies.Afewofthemarebeingmentionedasunder.1.5.1PiroplasmdetectioninChinaVariousstudieshavebeenconductedinPakistanfordetectionofbovinepiroplasmosis.ThesummaryofthesestudieshavebeendescribedinTable1.HostTechniqueReferenceBuffaloELISAYaoetal.,2002BuffaloLAMPHeetal.,2009BuffaloReal-timePCRHeetal.,2011CattleLAMPLiuetal.,2012CattlePCRLiuetal.,2014Cattle,buffaloPCR,ELISALietal.,2014BovinesPCRLiuetal.,2015YaksELISAQinetal.,2015BovinesPCR-RFLPTianetal.,2015BovinesELISAYangetal.,2015Yaks,cattle,dairycattlePCRNiuetal.,2015BovinesELISAZhaoetal.,2017YaksPCRLiuetal.,20171.5.2PiroplasmdetectioninPakistanVariousstudieshavebeenconductedinPakistanfordetectionofbovinepiroplasmosis.ThesummaryofthesestudieshavebeendescribedinTable2.HostTechniqueReferenceCattleMicroscopyAshfaqueetal.,1983BuffaloMicroscopyBuriroetal.,1994CattleMicroscopyMuhammadetal.,1999Cattle,buffaloMicroscopyKhanetal.,2004CattleMicroscopyAfridietal.,2005CattleMicroscopyZahidetal.,2005BuffaloMicroscopyDurranietal.,2006CattleMicroscopy,PCRDurranietal.,200815 中国农业科学院博士学位论文ChapterICattleMicroscopyQayyumetal.,2010BuffaloMicroscopy,PCRShahnawazetal.,2011BuffaloMicroscopyBhuttoetal.,2012CattleMicroscopyAtifetal.,2012CattleMicroscopy,PCRKhattacketal.,2012Cattle,buffaloPCRKhanetal.,2013CattlePCRGebrekidanetal.,2016CattleRecombinasepolymeraseamplificationHassanetal.,2018a1.6PiroplasmaControlAnabsolutelyefficaciouscontrolmethodagainstpiroplasmosishasnotyetbeenachievedbecauseofseveralissuessuchas,thedeficiencyofknowledgeconcerningthebiologyandthecompleximmunemechanismofpiroplasma(Gohiletal.,2013),aglobaldistributionofpiroplasmaspeciesandtheirvectors(Zintletal.,2003),acaricidesresistancebytickpopulationscombinedwithresidueproblemsofdrugsandlow-efficiencyoflivevaccines(Mosquedaetal.,2012).Uptodate,thecontrolofpiroplasmosisiscarriedoutbyeradicationofthevectortickfromtheshedandanimalvicinity,chemotherapeuticdrugsandvaccinestargetingticksand/orpiroplasmaspecies(Mosquedaetal.,2012).1.6.1VectorcontrolThebestwaytocontrolthepiroplasmosisistheeradicationofthevector(tick)fromtheshedandanimalvicinity.Inthisregard,differentscientistsaroundtheworldhavedesignedthestrategiestocontrolthevector.Followingaresomeimportantpointswhichmayhelptocontrolthevectorpopulationassuggestedby(Muhammadetal.,2008):(i)Maketheshedsoftheanimalstickproof.Forthispurpose,properlycaulkthewallsofshedifthesearenotcementedbecausethecrevicesarefavoriteplacesforarthropodsliketicks.(ii)Avoidthegarbageheapsneartheanimalhousesastheseheapsmayhelptheticksinreproduction.(iii)Theburningofwastesneartheanimalhousemayhelpintheeradicationofticks.16 中国农业科学院博士学位论文ChapterI(iv)Keepthelocalandexoticanimalsindifferentsheds.Certainstudieshaveshownthatexoticandcrossbredanimalsaremoresusceptibletotickbornediseasesthanthenativeanimals.So,bytheabovementionedpracticethegoatsmayberearedsafely.(v)Thenewlypurchasedanimalsshouldnotbemixedwiththealreadypresentanimalssuddenly.Ifthenewanimalsareinfestedbyticks,treatthemproperlyandthenaddtotheshed.(vi)Rotationalgrazingmayhelpintheeradicationofticks.(vii)Thefarmworkerorfarmmanagershouldregularlyobservetheanimalscloselyandiftheticksarepresentontheanimals,thenremovethemmechanicallyandtreatthembeforetheoccuranceofclinicaldisease.(viii)RemovethevegetationaroundanimalhousemayhelptoeradicatetheticksasthebladesofgrassesarefavoriteplacesforcertaintickslikeBoophilusspecies.(ix)Regularuseofacaricidaldrugsisahelpfultechnique.Anyhow,certainprecautionarymeasuresmustbekeptinmindfortheuseofthesedrugs.(x)Periodicdippingofanimalsintheacaricidaltankisbeneficialincontrollingtheectoparasites.(xi)Thedippingshouldnotbepracticedoncold,rainyandcloudydays.(xii)Theorganicmatterlikedungshouldnotbeputinornearthedippingtankasthiscandecreasestheconcentrationofdrug.(xiii)Theanimalsshouldbedrainedproperlybeforegoingtothefieldtoavoidcontaminationoffeedwithdrug.(xiv)Useofvaccinemayalsobeeffectiveincontrollingtheticks.(xv)OneinjectionofvaccineshouldbegiventoallthesusceptibleanimalsannuallyinAprilorMayi.e.onetotwomonthsbeforetheonsetofdiseaseseason.(xvi)Organophosphorousorarseniccompoundsshouldbeusedtocontroltheticksinsummerandautumn.(xvii)Cementand/ormudshouldbeusedforfillingofcrevicesattheendofseasonofdisease.1.6.2BabesiaandTheileriacontrol1.6.2.1ChemotherapyChemotherapyisanimportanttooltocontrolpiroplasminfectioninthevertebratehostsincenoeffectivevaccineagainstbabesiosisiscurrentlyavailable(Mosquedaetal.,2012).17 中国农业科学院博士学位论文ChapterIForthetreatmentofbabesiaosis,severalchemicaldrugssuchas,suinuroniumsulfate,amicarbalide,trypanblue,diminazeneaceturateandimidocarbdiproprionate,havebeenused.However,suinuroniumsulfateandamicarbalidehavebeenretiredbecauseofsafetymatter.Imidocarbdipropionateisusedandremainthedrugofchoice(2.4mg/kgbodyweight).Diminazenediaceturatecanbeusedassecondoption(3.5mg/kg),becauseitsefficacyislessthantheImidocarbdipropionate(Rashidetal.,2010).Disadvantagesofimidocarbisitshightoxicityforanimalaswellasitsresidues(Mosquedaetal.,2012).Variousnewdrugs(e.g.Triclosan;Nerolidol;Artesunate;Epoxomicin;GossypolandAtovaquone)weremorerecentlydevelopedtocontrolandtreatbabesiosiswithefficiency,byinhibitingthegrowthofBabesiaparasites.Forthetreatmentandprophylaxisofallformsoftheileriosis,buparvaquone(marketedasBuparvex)isthedrugofchoice(2.5mg/kgbodyweight),butitsuseislimitedbycostandtheneedtotreatanimalsduringtheearlystagesofdiseasetobeeffective.Moreover,therearerecentreportsoftheemergenceofdrug-resistantstrainsofT.annulata(Mhadhbietal.,2010).Oxytetracyclinecanalsobeusedassupportivetherapy(20mg/kg).Thecombinationofbuparvaquoneandoxytetracyclinerevealtobemoreeffective(Shahzadetal.,2013).However,thedrawbacksofdrugtreatmentmotivatesearchesformoreeffectivewaysagainstpiroplasmosis.1.6.2.2VaccinesBecauseoftheshortcomingsofchemotherapeuticdrugs,vaccinationisthemostsustainableoptionforcontrolofthepiroplasmosis.ItwasreportedthattheanimalscouldrecoverfromnaturallyacquiredinfectionwithBabesia.Manyteststoproducevaccineweredoneusingbloodcollectedfromrecoveredanimals(DeWaalandCombrink,2006).Forinstance,thecurrentAustralianchilledtickfevervaccineandthreeliveattenuatedvaccinesusingB.bovisstrainswereproducedandofferedprotectionforB.bovisandB.bigemina(Gohiletal.,2013).However,somedisadvantagessuchastheriskoftheseliveattenuatedparasitestobecomevirulent,shortshelf-life,coldstorageconditions,potentialcontaminationwithotherbloodparasites,virusesorbacteria,limittheirbroadapplication(Hopeetal.,2005).Thedevelopmentofrecombinantorsubunitvaccinesagainstanumberofpiroplasmspecieshasbecometheconcernofresearchers.Thesevaccineshavetheintentofblockingeithertheparasitetransmissionbetweenticksandvertebratehostsorthe18 中国农业科学院博士学位论文ChapterIparasitemultiplicationwithinthevertebratehost.ThesecondapproachwasfavorableforBabesiavaccinesdevelopment,sincethetransmissionofBabesiatoticksisstillpoorlystudied.Severalvaccinecandidatesactingonparasiteasexualmultiplicationinthevertebratehosthavebeendescribed.ThisisthecaseforexampleofRhoptryAssociatedProtein-1(RAP-1),thathasbeendescribedinbovineBabesia(Zhouetal.,2007).Atlast,thereisnopropersinglemethodtocontrolthecomplextick-bornediseaseintheworld.Combiningtheapplicationofanti-tickchemicalacaricides,tickappropriatevaccinewithanti-Babesiaoranti-Theileriadrugsandvaccinesdevelopmentbasedonparasiteasexualgrowthcycleofbloodstagewerethoughtpracticalandideal(Gohiletal.,2013).19 中国农业科学院博士学位论文ChapterIICHAPTERIIDevelopmentofRecombinasePolymeraseAmplification2.1IntroductionPiroplasmosisiscausedby2generaofhaemoprotozoa,Theileria,andBabesia,andisamongtheimportantimpedimentstothelivestockindustryintropicalandsubtropicalareasoftheworld(Sivakumaretal.,2014).Withinthegenus,TheileriaannulataandTheileriaparvaareconsideredtobethemostpathogenicspecies,causingsignificantdiseaseincattleandbuffalopopulationsoftropicalandsubtropicalareaswhereasTheileriaorientalisandTheileriamutansinfectionsaregenerallyasymptomatic(Uilenberg,1981a;Uilenberg,1981b;Uilenbergetal.,1977).ThehardticksofgenusHyalomma(Acari:Ixodidae)actasvectorsoftheseparasitesintheendemicareasofAsiaandAfrica(Abdallahetal.,2017;Jabbaretal.,2015).Clinicalmanifestationsofthetropicaltheileriosisincludeamarkedriseinbodytemperatureto40-41.5C,followedbydepression,waterysecretionfromeyes,andnasaldischarge(Gubbelsetal.,2000).Pathologicalconsequencesincludeinflammationofthelymphnodes,hemoglobinuria,andanemia(Gubbelsetal.,2000).DiagnosisofpiroplasmaspeciesisconventionallydoneusingGiemsastainedbloodsmears.SpecificityandsensitivityofparasitedetectionbasedonopticalmicroscopyofGiemsastainedbloodsmearsaremainlydependentontheproficiencyandexperienceoftheexaminer.Carrieranimalsareasymptomaticbutinfectedandmaybeasourceofinfectionforalongtime(Oliveira-Sequeiraetal.,2005),butbloodsmearexaminationoftenisnotsuccessfulinidentifyingcarrieranimalsbecauseoftheirlowlevelsofparasitemia(ShayanandRahbari,2005).Serologicalassayshavealsobeendevelopedforthedetectionofpiroplasmosis.Indirectfluorescentantibodytests(IFAT)andcomplementfixationtests(CFT)arelesssensitivethanenzyme-linkedimmunosorbentassays(ELISA)whichareeasytostandardizeandthuspreferredforepidemiologicalstudies(Zhaoetal.,2017).However,lowspecificityofserologicaltestssuchasELISAreducestheirefficacyindetectingcarrieranimals(Junlongetal.,2015).Adequatedetectionofcarrieranimalsiscrucialinepidemiologicalsurveys(Fahrimaletal.,1992).Moleculartechniqueslikespecies-specificPCRareacomparativelyreliablewaytodetectmultipleparasiticspeciesinpopulation-basedhoststudies.Thedevelopmentofreverselineblot(RLB)hybridizationhasenabledthesimultaneousdetectionofmultiplepiroplasmspecies(Iqbaletal.,2013;M‟ghirbietal.,20 中国农业科学院博士学位论文ChapterII2008),butrequiresspecializedequipmentandextendedtestingtimes.Recombinasepolymeraseamplification(RPA)isanisothermaltestwhichcanbeconductedat37–42C(Yinetal.,2017).TheRPAmethoduses2target-specificoligonucleotideprimersthatbindtothetemplateDNAwiththeassistanceofarecombinaseincombinationwithstrand-displacementDNAsynthesis.AmplificationofcomplexDNAtargetscanbeachievedinlessthan30minutesattemperaturesjustaboveroomtemperature(Kerstingetal.,2014).Inthepresentstudy,anRPAmethodwasoptimizedfordetectionofT.annulatafromcattlebloodsamplesanditsefficacywascomparedtodiagnosisusingconventionalPCR.Highsensitivity,specificity,therequirementoflesslaboratoryequipmentandconvenienceinoperationmakethistechniqueusefulforepidemiologicalstudiesunderfieldconditions.2.2Materialsandmethods2.2.1ParasitesPositiveDNAsamplesofbovinesinfectedwithT.annulata,T.orientalis,T.sinensis,B.bovis,B.bigemina,B.major,T.ovisandAnaplasmaoviswereprovidedbyVectorsandVectorBorneDiseasesLaboratoryofLanzhouVeterinaryResearchInstitute.2.2.2SamplescollectionDuringJulyandAugust2016,274bloodsampleswererandomlycollectedfromapparentlyhealthybuttick-infestedcattleofseveralcountiesofChakwal(Talagang,KalarKahar,ChoaSaidanShah),Faisalabad(Jaranwala,ChakJhumra,Sammundari)andJhang(AtharaHazari,Shorkot,GarhMaharaja)districtsofPunjabProvinceofPakistanasshowninFig.2.Bloodwascollectedfromjugularveinusing10mLdisposable®syringe.Approximately2to3mLwasdividedamong4circlesofWhatmanFTAcard(GEHealthcareLimited,Buckinghamshire,U.K.)andallowedtoairdryafterlabeling,whiletheremainingbloodwasusedforserumseparationtobeusedinfurtherinvestigations.DriedbloodsampleswereshippedtoLanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciencesLanzhou,Chinaforprocessing.DNAwasextractedfromFTAcardsusingaQiagenGenomicDNApurificationKit®(Qiagen,Hilden,Germany)accordingtothemanufacturer‟sinstructions.Briefly,3circlesof3mmdiameterwerecutanddigestedby180µLofBufferATLat85°Cfor10min,followedbytheadditionof20µLofproteinaseKandincubatedat56Cfor1h.Followingthis,200µLofBufferALwasaddedandincubatedat70°Cfor10min.20021 中国农业科学院博士学位论文ChapterIIµLofabsoluteethanolwasaddedforpelletingofDNA.Thefinalsolutionwascarefullytransferredtothespincolumnandwashingwasdoneusing500µLofBufferAW1andBufferAW2eachrespectivelywithcentrifugationatfullspeedfor1min.Finally,elutionwasdonebyadditionof70µLBufferAEandcentrifugationat13000Xgfor1min.TheDNAconcentrationwasdeterminedwithaNanoDrop2000spectrophotometer®(NanodropTechnologies,Wilmington,Delaware).DNAwasstoredat-20°C.2.2.3DetectionofTheileriaannulatabyRPAInordertoamplifytheEnolase(Eno)geneofT.annulata,differentprimersetsweredesignedfromthetargetnucleotidesequence(AccessionNo.HQ646253).Onthebasisofinternaltrials,primersetT.ann-1F(5‟-GAAATTCTTGGTAAGTAGATCCTTTTGTGTTA-3')andT.ann-1R(5‟-®AAGGTATCTAATTCCTTCTGATTCAACGTATC-3')wasmostsuitable.TwistAmpBasickit(TwistDx,Cambridge,England)wasusedforRPA.Pre-preparedreactionmixture(47.5µL),consistingof29.5µLofRPAbuffer,2.4µLofeachprimer(24µmol),2µLofDNAand11.2µLnuclease-freewaterwastransferredtotubeswithanenzymepellet,mixed,and2.5µLof280mMMgOAcwasaddedtostartthereaction.Sampleswereincubatedat39°Cfor25minutes.TheRPAproductwaspurifiedusinganTM®EZgeneGel/PCRExtractionKit(BiomigaInc.).Thefinalelutedproductwasloadedin2%agarosegelwithloadingbufferandpositiveproductswereexcisedandpurifiedTM®usinganEZgeneGel/PCRExtractionKit(Biomiga,SanDiego,California).TheDNA®fragmentwasclonedintopGEM-TEasyvectors(Promega,Madison,Wisconsin).The®EscherichiacoliTrans5α(TransGenBiotech,Beijing,China)wastransformedandplasmidDNAfromtheselectedcloneswasidentifiedusingPCRwiththesetofvectorprimers;T7(5‟-TAATACGACTCACTATAGGG-3‟)andSP6(5‟-ATTTAGGTGACACTATAG-3‟)toverifythepresenceofcorrectinsertsintotheselectedclonesbeforesequencing.ThePCRwasoptimizedfor1stepofinitialdenaturationat95°Cfor3min,followedby35cyclesofdenaturation(94°Cfor1min),primerannealing(54°Cfor30sec),extension(72°Cfor1min),andthefinalextension®at72°Cfor5min.CloneswereshippedtoGenescript(Shanghai,China)forsequencing(Liuetal.,2016).22 中国农业科学院博士学位论文ChapterII2.2.4SensitivityofRPAForanalyticalsensitivity,apairofprimerswasdesignedtoamplifythelongregionofthesamegene,i.e.,HSN-1F(5‟-AGCCCGGGAAATTCTTGGTAA-3')andHSN-1R(5‟-ATTGGGTGCAAAGCCTCCTT-3')toamplifya682bpfragmentofEnousingthefollowingcyclingparameters:initialdenaturationat95°Cfor3min,followedby35cyclesofdenaturation(94°Cfor45sec),primerannealing(53°Cfor45sec),extension(72°Cfor1min),andfinalextensionat72°Cfor5min.ThePCRproductwasrecoveredfromthegelandclonedusingthemethodsdescribedabove.AfterconfirmationTMwithsequenceresults,plasmidDNAwasextractedfromculturedclonesusingAxyPrep®PlasmidMiniprepKit(Axygen,UnionCity,California).Quantificationoftheplasmid®DNAwasdoneusingNanoDrop2000spectrophotometer(NanodropTechnologies).ThenumberofcopiesofDNAwascalculatedusingtheformula:239No.ofcopies=(AmountX6.022x10)/(FragmentLengthX1x10X650)SerialdilutionsoftheplasmidDNAwerepreparedbyadditionofnucleasefreewateruntilasinglecopyofDNAperµLwasobtained.TheRPAtestwasusedtodetectthetargetfragmentfromdifferentserialdilutions.2.2.5SpecificityofRPAInordertoexaminespecificity,thesameprimerswereusedtoamplifythetargetsequencefromtheDNAofT.sergenti,T.sinensis,Babesiabovis,BabesiamajorandBabesiabigemina.2.2.6FieldsamplesscreeningInordertovalidatethetestunderfieldconditions,274samplescollectedfromapparentlyhealthybuttick-infestedanimalsofChakwal,FaisalabadandJhangdistrictsofPunjabprovinceofPakistanwereused.TocompareourmethodwiththeresultsofconventionalPCR,thesamesampleswerescreenedusingapublishedprimerset,AnCb(forward:5′-CGGTTGGTTTGTTCGTCTTT-3′;reverse:5′-GCCAATGGATTTGAACTTCC-3′),toamplifya393bpsequenceusingthecyclingparametersofLiuetal.(2015).AllthesampleswhichwerepositivebyRPAdetectionbutnegativebyPCRamplificationwereclonedandsequencedtoconfirmtheRPAresults.2.2.7StatisticalanalysisThedataregarding95%confidenceintervalwascalculatedusingWilsonscoreintervals.TheloweranduppervaluesareshowninTable3.23 中国农业科学院博士学位论文ChapterII2.3Results2.3.1SpecificityofRPAInordertoexaminespecificity,thesameprimerswereusedtoamplifythetargetsequencefromtheDNAofT.sergenti,T.sinensis,Babesiabovis,BabesiamajorandBabesiabigemina.Nocross-reactivitywasobserved,confirmingthespecificityofthetest.Specificitytrialsdemonstratednocross-reactionbetweentheDNAsamplesofcloselyrelatedpiroplasmsincludingT.sergenti,T.sinensis,B.bovis,B.majorandB.bigemina(Fig.2).Fig.2:SpecifictestoftheprimersetT.ann-1.M,DL500TMMolecularmarker(TaKaRa);lane1T.annulata,2T.sergenti,3T.sinensis,4B.bovis,5B.major,6B.bigemina,7water.Thebandshowsthe281basepairsequencesgeneratedfromT.ann-1primerset2.3.2SensitivityofRPAAnalyticsensitivitycanbedescribedintermsofminimumnumberofcopiesofDNAdetectedbyatest.SerialdilutionsoftheplasmidDNAwerepreparedbyadditionofnucleasefreewateruntilasinglecopyofDNAperµLwasobtained.TheRPAtestwasusedtodetectthetargetfragmentfromdifferentserialdilutions.OurmethodcoulddetectasinglecopyofDNAinareaction,demonstratingabsolutesensitivity(Fig.3).Fig.3:SensitivitytestoftheprimersT.ann-1.M,DL500TMMolecular87654321marker(TaKaRa);lane110,210,310,410,510,610,710,810,94,101,110numberofcopies.Thebandshows281basepairsequence24 中国农业科学院博士学位论文ChapterII2.3.3ResultsoffieldsamplesOf274samplesscreenedforT.annulata,RPAresultsshowedthat48(17.51%)werepositive.ConventionalPCRofthesamesamplesdetectedfewerpositivesamples(21of274or7.66%)(Fig.4).ThehighestprevalenceofT.annulatawasfoundinChakwaldistrict(25.83%and9.16%byRPAandPCRrespectively),followedbyFaisalabad(18.30%and11.26%)andJhang(4.81%and2.40%)asshowninTable3.Theamplifiedsequenceresultswere99%identicaltotheT.annulataElaziqstrain(AccessionNo.HQ646253).Fig.4:TwopercentagarosegelimageofTheileriaannulatabyconventionalPCR,wells3-9amongthepositivelyidentifiedfieldsamples25 中国农业科学院博士学位论文ChapterIITable3:PrevalenceofT.annulatainselectedcattlepopulationofstudyareasduringJulyandAugust,2016usingrecombinasepolymeraseamplificationandPCRNo.ofDistrictAreaPCR(+ve)95%CIRPA(+ve)95%CISamplesTalagang342(5.88%)0.02,0.196(17.65%)0.08,0.34KalarKahar476(12.76%)0.06,0.2516(34.04%)0.22,0.48ChakwalChoaSaidan393(7.69%)0.03,0.209(23.07%)0.13,0.38ShahSubTotal12011(9.16%)0.05,0.1631(25.83%)0.19,0.34ChakJhumra232(8.69%)0.02,0.274(17.39%)0.07,0.37Jaranwala212(9.52%)0.03,0.293(14.28%)0.05,0.35FaisalabadSammundari274(14.81%)0.06,0.326(22.22%)0.11,0.41SubTotal718(11.26%)0.06,0.2113(18.30%)0.11,0.29GarhMaharaja321(3.12%)0.01,0.162(6.25%)0.02,0.20Shorkot261(3.84%)0.01,0.191(3.84%)0.01,0.19JhangAtharaHazari250(0.00%)0.00,0.131(4.00%)0.01,0.20SubTotal832(2.40%)0.01,0.084(4.81%)0.02,0.12Total27421(7.66%)0.05,0.1148(17.51%)0.13,0.222.4DiscussionInPakistan,smallbovineherdsarerearedforbothhouseholdrequirementsandsmall-scalecommerce.Recentyears,haveseenatransitiontomoderncommerciallivestockfarmingwithapreferenceforimportedratherthanlocalcattlebreeds.However,theimportedanimalsaremorepronetotickinfestationandtick-bornediseasethanarelocalbreeds.Studieshaveshowncasefatalityoftick-bornediseaseupto80%inimportedbreeds,while20%fatalityiscommoninlocalbreedsinPakistan.Inthecountry,theileriosisismainlycausedbyT.annulata(Jabbaretal.,2015).Todate,moststudiesoftheileriosishavebeenconductedusingconventionalmicroscopy,whichunderestimatesprevalenceduetothelowsensitivityandspecificityofopticalmicroscopy.PCRmethodshavealsobeenused,butareimpracticalasafieldtechniqueinaresource-poorcountrylikePakistan.HereinwedemonstratedthefeasibilityoffieldapplicationforanewlydevelopedRPAthatismoresensitiveandspecificthanconventionalPCR.TheEnogenewastargetedasadiagnostictoolinthepresentstudy.Apicomplexanparasiteslackaerobicmetabolicpathways,thustheironlypathwayfor26 中国农业科学院博士学位论文ChapterIIenergyproductionisglycolysis.Inglycolysis,theEnoenzymecatalyzestheconversionof2-phosphoglycerate(2-PGA)tophosphoenol-pyruvate(PEP).Therefore,Enohasavitalimportanceinthelifeofparasitebecauseofitsroleinglycolysis(Akatetal.,2014).ThefirststudytoidentifytheileriosisincattleinPakistanwasconductedinMayandJuneof1982andreported100%prevalenceoftheileriosisdiagnosedbymicroscopicexaminationofbloodsmears(Ashfaqueetal.,1983).ThefirstconventionalPCRbasedsurveyoftheileriosisinPakistanwaspublishedin2008reportedaprevalenceof36%(Durranietal.,2008).ArecentstudyreportedtheprevalenceofT.orientalisfromimportedandnativebovinepopulationsinPakistanat24.5%(Gebrekidanetal.,2017).ThepresentstudydiagnosedT.annulatain25.83%ofcattlesampledusingtheRPAmethodbutonlyin9.16%usingconventionalPCRdiagnosis.SequenceanalysisofproductsconfirmedthatRPAisamoresensitivediagnostictoolthanthePCRmethod.TheRPAtechniquehasbeenusedbymanyscientistsfordiagnosisofparasitessuchasPlasmodiumfalciparum(Kerstingetal.,2014),Giardialamblia,EntamoebahistolyticaandCryptosporidiumspp.(Crannelletal.,2015).ThestudiesrevealedthatthesensitivityofRPAismuchhigherthanLAMPwhichisalsoanisothermalamplificationtechnique.TheoptimumreactiontemperatureforLAMPassaysrangesfrom60to65Cwithalongruntimeof30–60min(Notomietal.,2000).TheReal-timeqPCRassayisasimplewaytodetectT.annulatawithhighsensitivityandspecificity,butitisdifficulttoperforminresource-poorandremoteareas(Ros-Garciaetal.,2012).Thespecificitytrialsdescribedhereinfoundnocross-reactionbetweentheDNAsamplesofcloselyrelatedpiroplasmspeciesincludingT.sergenti,T.sinensis,Babesia(B.)bovis,B.majorandB.bigemina(Fig.3).Theanalyticalsensitivityofthemethodextendstosinglecopydetection(Fig.4).Incontrasttoothertechniques,RPAassayhasmanyadvantages.Itislesstimeconsumingandcanbeperformedinawaterbathorevenusingbodyheatinfieldconditionslackinglaboratoryequipment(Kerstingetal.,2014).Amongthe3studydistricts,thehighestrateofT.annulatawasfoundinChakwal(25.83%)followedbyFaisalabad(18.30%)andJhang(17.51%).ThehigherrateinChakwalmaybeduetothefeedingmanagementoftheanimalswhicharefieldgrazedwithahigherriskoftickinfestationwhilestallfeedingispreferredinFaisalabadandJhangdistricts.ThehighestinfectionrateamongdifferentcountieswasfoundinKalarKaharofChakwaldistrict(34.04%),whilelowestinShorkotofJhangdistrict(3.84%).Inthegeneamplificationassays,themostimportantfactorsincludetheefficiencyofprimersandlengthofthefragmentbeingamplified.Thefragmentlengthtargetedin27 中国农业科学院博士学位论文ChapterIIRPAisshorterthanthePCR,sothesemaybethereasonsofhighersensitivityofRPA.Meanwhile,RPAformulationhasamacromolecule,polyethyleneglycol(Piepenburgetal.,2015).Thiscomponentmodulatestheefficiencyofbiochemicalprocessesincludinganenhancementofenzymecatalyticactivity(Ellis,2001).IfRPAreactionmixtureisshakenduringincubation,itincreasesthesensitivityofreaction(Lillisetal.,2016).Duringthisstudy,theRPAreactionmixtureswerealsomixedvigorouslyandspunaftereverytwoandhalfminutesduringincubation.ThesefactorsmighthaveplayedtheirroletoincreasethesensitivityofRPA.SeveralscientistshavecomparedthesensitivityofRPAwithPCR.ManyofthemreportedthatthesensitivityofRPAisequaltoPCR(Wangetal.,2017;Yinetal.,2017)while,manyotherscientistshavereportedthatRPAismoresensitivethanPCR(Liuetal.,2017;Wambuaetal.,2017).Ourresultsarealsoinaccordancewiththelater.Onthebasisofpresentstudyresults,itcanbeconcludedthatRPAmaybeasuitablediagnosticmethodforfieldstudiesandlargeareaepidemiologicalstudies.Itmaybethemostconvenientmethodforfieldstudiesespeciallyinresource-poorcountrieslikePakistan.28 中国农业科学院博士学位论文ChapterIIICHAPTERIIIDevelopmentofMultiplexRecombinasePolymeraseAmplification3.1IntroductionLivestockindustryisoneofthemostimportantprofessionsinPakistan,whichcontributedfor11.8%ofthetotalGDPduringtheeconomicyear2014-15.Accordingtothestatisticalanalysisoftheeconomicsurvey,thereare76.8millionheadsofcattleandrdbuffalosinPakistan(Anonymous.,2015-16).Althoughitisestimatedthat2/3ofthepopulationofPakistanisattachedtothelivestock,directlyorindirectly,yetthepreventivemanagementpracticesandcontrolarenotuptothemark.Theileriosisisoneofthemostimportantdiseasesthateconomicimpactonthelivestockindustryintropicalandsubtropicalareasoftheworld(Sivakumaretal.,2014).Theileria(T.)annulataandT.parva,thepathogensoftropicaltheileriosisandEastCoastfever,areconsideredtobethemostpathogenicparasitespeciesincattleandbuffalo;whereas,T.orientalisandT.mutansareresponsibleforasymptomaticdisease(Uilenberg,1981a).Theileriosisiseconomicallyimportantdiseaseduetoitsexistenceinclinicalandsub-clinicalform.ThereportedlossduetoacutetheileriosisincattleisUS$179percattlefor305daysinAustralia(Pereraetal.,2014).Whileinanotherstudy,itslossesare22.84%,0.24%and3.15%peryearduetomortality,medicationandcontrolrespectivelyinCappadociaregionofTurkey(Incietal.,2007).Pakistanisdividedintotenagro-ecologicalzones,influencingtemporalandspatialpatternsofdiseases.Beinglocatedinsubtropicalzone(30°N,70°E)inSouthAsia,mostpartsofferfavorableconditionsforticks,whichtransmitseveraldiseasestohuman,livestockandcompanionanimals.DairyinvestorsinPakistanareimportinghighproducingcattlefromoverseastoimprovetheirmilkproductionandindigenousbreeds.Theexoticcattlebreedsaresusceptibletoticksandtick-bornediseases(Jabbaretal.,2015).AnumberofmethodsarebeingusedworldwideforthecontrolofTTBDsincludingchemotherapyandvaccinationtoreducetheireconomiclosses,buttherelianceonchemicalsisdecreasingduetoitscontinuoususeleadingtoemergenceofacaricideresistantticks.InPakistan,thereisnoscheduleofchemoprophylaxisforTTBDs.Hereothersmethodsfortickscontrolincludegrooming,burningofcattledungsandspraywithcypermethrineonanimalsbodyandsurroundingareasduringhighriskmonths(MaytoSeptember)arebeingpracticed(Jabbaretal.,2015).Severeformoftropicaltheileriosis29 中国农业科学院博士学位论文ChapterIIIcausesmortalityanditvariesincattlefrom5%inindigenousbreedsto90%inexoticbreeds(Durranietal.,2008).Thesignsoftheileriosisincattleconsistofhighriseofbodytemperature(40-o41.5C),nasaldischarge,lacrimation,depressionandswellingofsuperficiallymphnodes.Generaldiagnosisofhemoparasitesisbasedonthinbloodsmear(Iqbaletal.,2013).Thespecificityandsensitivityofeachmethoddependsupontheproficiencyandexperienceoftheexaminer.Smearexaminationisnotsuccessfulinidentificationofthecarrieranimalsbecauseoflowerlevelofparasitemia(ShayanandRahbari,2005).ThemolecularmethodslikePCR,RLB(Abdallahetal.,2017)andimmunologicalassayslikeIFAT,ELISAandCFTaremuchsensitiveandhelpfulfordifferentiationoftheinfectionsatspeciesandsubspecieslevels(M‟ghirbietal.,2008;Zhaoetal.,2017).However,thesemethodsneedadvancelaboratoryequipmentwhichisnotaccessibleforthelivestockfarmersbelongingtoremoteareas.ArecentlydevelopedisothermaltestknownasRecombinasepolymerase°amplification(RPA),whichcanbecompletedat37-42C(Yinetal.,2017)inlessthan30minutes(Kerstingetal.,2014).Thistestishighlysensitiveandspecificascomparedtootherdiagnosticmethods.Forthis,twotargetspecificoligonucleotideprimersareused,whichareabletobindtothetemplateDNAwiththeassistanceofarecombinaseincombinationwithstrand-displacementDNAsynthesis.ThereisdireneedtodevelopamethodthatcanidentifymoreTheileriaspeciesinasingletest.Inthepresentstudy,amultiplexRPAmethodhasbeenestablishedforthedetectionofT.annulataandT.orientalisfrombloodsamples,andcomparedwiththealreadystudiedconventionalPCR.3.2Materialsandmethods3.2.1ParasitesPositiveDNAsamplesofbovinesinfectedwithT.annulata,T.orientalis,T.sinensis,B.bovis,B.bigemina,B.major,T.ovisandAnaplasmaoviswereprovidedbyVectorsandVectorBorneDiseasesLaboratoryofLanzhouVeterinaryResearchInstitute.3.2.2SamplescollectionDuringJulyandAugust2016,188bloodsampleswererandomlycollectedfromapparentlyhealthybuttick-infestedcattleofseveralcountiesofChakwal(Talagang,KalarKahar,ChoaSaidanShah)districtofPunjabProvinceofPakistanasshowninFig.2.Bloodwascollectedfromjugularveinusing10mLdisposablesyringe.Approximately30 中国农业科学院博士学位论文ChapterIII®2to3mLwasdividedamong4circlesofWhatmanFTAcard(GEHealthcareLimited,Buckinghamshire,U.K.)andallowedtoairdryafterlabeling,whiletheremainingbloodwasusedforserumseparationtobeusedinfurtherinvestigations.DriedbloodsampleswereshippedtoLanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciencesLanzhou,Chinaforprocessing.DNAwasextractedfromFTAcardsusinga®QiagenGenomicDNApurificationKit(Qiagen,Hilden,Germany)accordingtothemanufacturer‟sinstructions.Briefly,3circlesof3mmdiameterwerecutanddigestedby180µLofBufferATLat85°Cfor10min,followedbytheadditionof20µLofproteinaseKandincubatedat56Cfor1h.Followingthis,200µLofBufferALwasaddedandincubatedat70°Cfor10min.200µLofabsoluteethanolwasaddedforpelletingofDNA.Thefinalsolutionwascarefullytransferredtothespincolumnandwashingwasdoneusing500µLofBufferAW1andBufferAW2eachrespectivelywithcentrifugationatfullspeedfor1min.Finally,elutionwasdonebyadditionof70µLBufferAEandcentrifugationat13000Xgfor1min.TheDNAconcentrationwas®determinedwithaNanoDrop2000spectrophotometer(NanodropTechnologies,Wilmington,Delaware).DNAwasstoredat-20°C.3.2.3SimultaneousdetectionofT.annulataandT.orientalisTargetingontheEnolase(Eno)genesequencesofT.annulata(AccessionNo.HQ646253)andT.orientalis(AccessionNo.XM009693846),twopairsofRPAprimesweredesigned,Tann-2F/2R(T.annulata)andTornt-1F/1R(T.orientalis)asshownin®Table4.TwistAmpBasickit(TwistDx,England)wasusedtoperformRPAusingreferenceDNAsprovidedbyVectorsandVectorBorneDiseasesLaboratoryofLanzhouVeterinaryResearchInstitute,China.Thepre-preparedreactionmixtureof47.5µL,consistingof29.5µLofRPAbuffer,2.4µLofeachprimer(24µmol),3µLofDNAand5.4µLnucleasefreewaterwastransferredtothetubeswithenzymespelletandthenmixedproperly.Followingthat,2.5µLof280mMMgOAcwasaddedtostartthereaction.ImmediatelyafteradditionofMgOAc,thesampleswereplacedinThermalCyclerforincubationat39ºCfor25minutes.TheRPAproductswerepurifiedusingBiomigaTMEZgeneGel/PCRExtractionKit(SanDiego,CA,USA)accordingtomanufacturer‟sinstructions.Finally,elutedproductswereloadedin2%agarosegelafteradditionoftheloadingbuffer.ThepositiveproductswereexcisedfromthegelandpurifiedusingaTMBiomigaEZgeneGel/PCRExtractionKit(SanDiego,CA,USA).TheDNAfragment®wasclonedintothepGEM-TEasyvectors(Promega,USA).TheEscherichiacoliTrans®5α(TaKaRa,China)wastransformedandplasmidDNAfromtheselectedcloneswas31 中国农业科学院博士学位论文ChapterIIIidentifiedusingPCRwiththesetofvectorprimers;T7(5‟-TAATACGACTCACTATAGGG-3‟)andSP6(5‟-ATTTAGGTGACACTATAG-3‟)toverifythepresenceofcorrectinsertsintotheselectedclonesbeforeproceedingforthesequencing.ThePCRwasoptimizedforonestepofinitialdenaturationat95°Cfor3min,followedby35cyclesofdenaturation(94°Cfor1min),primerannealing(54°Cfor30s),extension(72°Cfor1min),andthefinalextensionat72°Cfor5min.Thecorrect®insertedcloneswereshippedtoGenescript(Shanghai,China)forsequencing.TheobtainedsequenceswerealignedandanalyzedusingtheMegAligncomponentoftheDNAStarsoftwareprogram(Version4.0DNAStar,Madison,USA)andBLASTninNCBI.3.2.4SensitivityofmultiplexRPAForanalyticsensitivity,2pairsofprimers,HSN-1F/1RandADL-2F/2R(Table4),weredesignedtoamplify682bpand900bpfragmentsofEnogeneforT.annulataandT.orientalis,respectively.ThereactioncyclinginthePCRwasoptimizedforonestepofinitialdenaturationat95°Cfor3min,followedby35cyclesofdenaturation(94°Cfor45s),primerannealing(53°Cfor45s),extension(72°Cfor1min),andthefinalextensionat72°Cfor5min.ThePCRproductwasrecoveredfromthegelandcloningwasdonebythesamemethodasmentionedabove.Afterconfirmationofcloneshavinggeneofinterestbythesequenceresults,plasmidDNAwasextractedfromculturedclonesTMusingAXYGENAxyPrepPlasmidMiniprepKit(UnionCity,CA,USA).QuantificationoftheplasmidDNAwasdoneusingNanoDrop2000spectrophotometer®(NanodropTechnologies,Wilmington,DE,USA).Numberofcopieswascalculatedusingthefollowingformula:239No.ofcopies=(ConcentrationX6.022x10)/(FragmentLengthX1x10X650)SerialdilutionsoftheplasmidDNAwerepreparedbyadditionofnucleasefreewateruntilsinglecopyoftheDNAµLwasobtained.TheRPAtestwasusedtodetectthetargetfragmentfromdifferentserialdilutions.3.2.5SpecificityofmultiplexRPAInordertoexaminethespecificity,sameprimerswereusedtoamplifythetargetsequencefromtheDNAsofT.sinensis,Babesia(B.)bovis,B.majorandB.bigemina.ThereferenceDNAswereprovidedbyVectorsandVectorBorneDiseasesLaboratoryofLanzhouVeterinaryResearchInstitute.Table4:DetailsoftheprimersusedinthemultiplexRPA32 中国农业科学院博士学位论文ChapterIIIPrimerProductParasiteSequence(5’-3’)ReferenceNameSize(bp)T.ann-2FGAAATTCTTGGTAAGTAGATCCTTTTGTGTTAT.annulata282ThisStudyT.ann-2RGGTATCTAATTCCTTCTGATTCAACGTATCT.ornt-1FGTCGGAGTTCTTCGTGAAGGAGAAGGGAGCGT.orientalis229ThisStudyT.ornt-1RCACCAGCAGGTCGTCTCCCACTATTTGCACCHSN-1FAGCCCGGGAAATTCTTGGTAAT.annulata682ThisStudyHSN-1RATTGGGTGCAAAGCCTCCTTADL-2FAGGACCTGGACAACCTGATGT.orientalis900ThisStudyADL-2RCCTCCGTTTCTCCTGACCTGAnCb-FCGGTTGGTTTGTTCGTCTTTT.annulata393Liuetal.2015AnCb-RGCCAATGGATTTGAACTTCCSerITS-FCAACCCAGCTGCTTTTGAT.orientalis818Liuetal.2015SerITS-RCAACAGAATCGCAAAGCGGT3.2.6FieldsamplesscreeningInordertovalidatethetestunderfieldconditions,188samplescollectedfromapparentlyhealthybuttick-infestedanimalsofChakwaldistrictofPunjabprovinceofPakistanwereused.TocompareourmethodwiththeresultsofconventionalPCR,thesamesampleswerescreenedusingapublishedprimersets;AnCb-F,AnCb-Ramplifying393bpfragmentofT.annulataandSerITS-F,SerITS-Ramplifying818bpfragmentofT.orientalisasshowninTable4(Liuetal.2015).AllthesampleswhichwerepositivebyRPAdetectionbutnegativebyPCRamplificationwereclonedandsequencedtoconfirmtheRPAresults.3.3Results3.3.1SpecificityofmultiplexRPAThespecificitytrialsdescribedthatthereisnocrossreactionamongtheDNAsamplesofcloselyrelatedpiroplasmsincluding:T.sinensis,B.bovis,B.majorandB.bigemina(Fig.5).33 中国农业科学院博士学位论文ChapterIIIFig.5:SpecifictestoftheprimersetsT.ann-2(a)andT.ornt-1(b).M,DL500TMMolecularmarker(TaKaRa);lane1T.annulata&T.orientalis,2T.annulata,3T.orientalis,4T.sinensis,5B.bovis,6B.major,7B.bigemina.Thebandsfromaandbshowsthe282and229basepairsequencesgeneratedfromT.ann-2andT.ornt-1primersets,respectively3.3.2SensitivityofmultiplexRPATheanalyticsensitivitycanbedescribedintermsofminimumnumberofcopiesofDNAdetectedbyatestanditwasobservedthatourmethodcoulddetectthe100copiesofDNAinareactionthatshowsthetestissensitive(Fig.6).Fig.6:SensitivitytestoftheprimersT.ann-2(a)andT.ornt-1(b).M,DL500TMMolecularmarker(TaKaRa);lane1-7No.ofcopiesofpDNA.3.3.3ResultsoffieldsamplesOf188samplesscreenedbyRPA,45(23.93%)andfive(2.65%)werepositiveforT.annulataandT.orientalis,respectively.However,conventionalmultiplexPCRofthesamesamplesrevealedlowernumberofpositivesamples,32(17.02%)andthree(1.59%)showingtheproductsizeof393bpand818bpforT.annulataandT.orientalis,respectively(Fig.7).RPAwasfoundhighlysensitiveandsensitivediagnosticmethodascomparedtoconventionalPCR.34 中国农业科学院博士学位论文ChapterIIIFig.7:Agarosegel(1.5%)electrophoresisofproductsamplifiedbyPCR:1=NTC;2=PositiveControl(Both);3=Theileriaannulata(393bp);4=Theileriaannulata(393bp)&Theileriaorientalis(818bp)(Both)3.4DiscussionInPakistan,smallholdercattlefarmingsystemisfulfillingthehouseholdrequirementsorcommercialpurposesoftheresource-poorruralcommunities.Intherecentyears,peopleareadoptingcommercialfarmingusingmoderntechniquesandimportedbreedsofcattle(Bostaurus)duetotheirhigherpotentialofmilkyield.However,theimportedanimalsaremorepronetotheticksandtick-bornediseasesascomparedtothelocalbreeds(Bosindicus);thecasefatalityintheformerisupto80%ascomparedtolater,whichislessthan20%.Amongmanytick-bornediseases,sub-clinicaltheileriosisisconsideredamongthemosteconomicallynotoriousdiseases,predominantlycausedbyT.annulata(Jabbaretal.,2015).Todate,mostofthestudieshavebeenconductedthroughconventionalmicroscopywhich,duetolowsensitivityandspecificity,isnottrulyrepresentingtheactualdistribution.Asfarascouldhaveascertained,thereisnopublishedreportoftheserologicaldistributionoftheileriosisinPakistan.PCR,ifusedinfewstudies,hasnotbeenfoundconvenientatgroundlevelinaresource-poorcountrylikePakistan.Keepinginviewthesefactors,establishmentofahighlyspecific,sensitive,economicalandfield-friendlydiagnostictestfordeterminingtheprevalenceofvariousspeciesofpiroplasmsishence,justified.TheEnogenewastargetedasadiagnostictoolinthepresentstudy,whichisresponsibleforencodingofenolaseenzyme.Apicomplexanslackaerobicsystemand35 中国农业科学院博士学位论文ChapterIIIanaerobicpathwayi.e.glycolysisistheonlysourceoftheirenergyproduction.Inglycolysis,Enoenzymecatalyzestheconversionof2-phosphoglycerate(2-PGA)tophosphoenol-pyruvate(PEP).Therefore,Enohasavitalimportanceinthelifeofapicomplexans(Akatetal.,2014).InPakistan,firstpublishedstudytoidentifytheileriosisincattlewasconductedinMay-June,1982whichreported100%prevalenceoftheileriosisthroughopticalmicroscopyofthebloodsmears(Ashfaqueetal.,1983).FirstconventionalPCRbasedreportoftheileriosiswaspublishedin2008,showing36%prevalence(Durranietal.,2008).Tillnow,thereareonlyafewmolecularstudiesforidentificationofTheileriaspeciesinPakistan.However,nopublishedreportusingahighlysensitivetechniquelikeRPAisavailable.Recently,astudypresentedtheprevalenceofT.orientalisfromimportedandnativebovinepopulationofPakistandescribing24.5%positiveresults.ThatstudyjotsdownabriefdescriptionofTheileriaspeciesinPakistanincludingT.annulataandT.orientalis(Gebrekidanetal.,2017).Thepresentstudyrevealedthat45(23.93%)and5(2.65%)werepositiveforT.annulataandT.orientalisrespectively.However,conventionalmultiplexPCRofthesamesamplesrevealedlowernumberofpositivesamplesi.e.32(17.02%)and3(1.59%)showingtheproductsizeof393bpand818bpforT.annulataandT.orientalisrespectively.AllthesampleswhichwerepositiveinRPAdetection,whilenegativebyPCRamplification,wereusedforcloningandsequencingforconfirmationoftheRPAresults.SequenceanalysissuccessfullyverifiedtheRPAresults.TheRPAtechniquehasbeenusedbymanyscientistsfordiagnosisofparasitessuchasPlasmodiumfalciparum(Kerstingetal.,2014),Giardialamblia,EntamoebahistolyticaandCryptosporidiumsp.(Crannelletal.,2015).ThestudiesrevealedthatthesensitivityofRPAismuchhigherthanLAMP,whichisalsoanisothermalamplification◦technique.Therequiredoptimumreactiontemperaturerangesfrom60to65Candalongruntimeof30–60minutes(Notomietal.,2000).Althoughreal-timeqPCRassay,asimplewaytodetectT.annulata,hasalsobeendevelopedwithhighsensitivityandspecificity;however,itisdifficulttoperformintheresourcepoorandremoteareas(Ros-Garciaetal.,2012).Incontrasttoothertechniques,RPAassayhavemanyadvantageslikelesstimeconsuming,easytoperforminfieldconditions,doesnotneedanylaboratoryequipmentexceptwaterbath(Kerstingetal.,2014).SimultaneousamplificationofdifferentDNAtargetsinthesamereactionisadvantageoustohavemoreinformationfromeachsampleandthusreducescostsandtimeforeverydiagnosis.To36 中国农业科学院博士学位论文ChapterIIIdate,mostoftheisothermalnucleicacidamplificationmethodsareusuallyabletoamplifyonlyonetargetsequence(Kerstingetal.,2014).ThespecificitytrialsdescribedthatthereisnocrossreactionbetweentheDNAsamplesofcloselyrelatedpiroplasmspeciesincludingT.sinensis,Babesia(B.)bovis,B.majorandB.bigemina(Fig.5)TheanalyticalsensitivityofamethodcanbecalculatedbyminimumnumberofcopiesofplasmidDNAdetected,whichincaseofRPAisupto100DNAcopiesdetection(Fig.6).Inthegeneamplificationassays,themostimportantfactorsincludetheefficiencyofprimersandlengthofthefragmentbeingamplified.ThefragmentlengthtargetedinRPAisshorterthanthePCR,sothesemaybethereasonsofhighersensitivityofRPA.Meanwhile,RPAformulationhasamacromolecule,polyethyleneglycol(Piepenburgetal.,2015).Thiscomponentmodulatestheefficiencyofbiochemicalprocessesincludinganenhancementofenzymecatalyticactivity(Ellis,2001).IfRPAreactionmixtureisshakenduringincubation,itincreasesthesensitivityofreaction(Lillisetal.,2016).Duringthisstudy,theRPAreactionmixtureswerealsomixedvigorouslyandspunaftereverytwoandhalfminutesduringincubation.ThesefactorsmighthaveplayedtheirroletoincreasethesensitivityofRPA(Hassanetal.,2018a).SeveralscientistshavecomparedthesensitivityofRPAwithPCR.ManyofthemreportedthatthesensitivityofRPAisequaltoPCR(Wangetal.,2017;Yinetal.,2017)while,manyotherscientistshavereportedthatRPAismoresensitivethanPCR(Liuetal.,2017;Wambuaetal.,2017).Ourresultsarealsoinaccordancewiththelater.37 中国农业科学院博士学位论文ChapterIVChapterIVMolecularEpidemiologyofPiroplasmSpecies4.1IntroductionTick-borneprotozoandiseases(e.g.,theileriosisandbabesiosis)poseimportantproblemsforthehealthandmanagementofdomesticcattleinthetropicsandsubtropics(JongejanandUilenberg,1994).ThemostwidespreadandmalignantTheileriaspeciesisTheileriaannulata,causingtropicaltheileriosis,whichoccursaroundtheMediterraneanbasin,intheMiddleEast,andinSouthernAsia.TheothermalignantTheileriaspeciesisT.parva,whichoccursinEastandSouthernAfricaandcausesEastCoastfever.BovinebabesiosisiscausedbyBabesiabovisandB.bigemina,bothofwhichoccurworldwideintropicalandsubtropicalregions.B.divergensoccursincattleinEuropeandextendsintoNorthAfrica(BouattourandDarghouth,1996).Inaddition,benignformsoftheileriosisarecausedbyT.mutans,T.velifera,andT.taurotragi,whicharemainlylocatedinAfrica,whereasparasitesoftheT.sergenti-T.buffeli-T.orientalisgroup,referredtobelowastheT.orientalisgroup,occurworldwide(Uilenberg,1981).Severaltechniquesfordetectionofthesehemoparasiteshavebeendevelopedseparatelyforeachspecies.FortheT.orientalisgroup,severalsensitivePCRassaysbasedonthegeneencodingthemajorpiroplasmsurfaceproteinhavebeendeveloped(Kawazuetal.,1992).AsimilarapproachusingtheTams1genewasfollowedforT.annulata(d‟oliveiraetal.,1995).PCRdetectionofT.parvausingrepetitiveDNAsequencesorgene-specificprimershasalsobeendeveloped.SensitivePCRmethodsareavailableforB.bovis(Calderetal.,1996).AssaystodifferentiateTheileriaspeciesonthebasisoftheirrRNAgeneshavebeendescribedbyAllsoppetal.(1993),buttheseassaysdidnotincludeallTheileriaspecies.Moreover,theseassaysweredevelopedmerelyforthedifferentiationofspeciesratherthanfordetectionincarrieranimals.Todate,screeningforthepresenceofbenignTheileriaspecies,suchasT.mutans,T.velifera,andT.taurotragi,occurringincattledependsontheimmunofluorescentantibodytestandmaygiverisetocross-reactions(Papadopoulosetal.,1996).Moreover,sinceserodiagnosisdoesnotdetecttheparasiteitself,theanimalmayhavealreadyclearedthepathogenbutremainedseropositive.ThefirstintegratedapproachtosensitivedetectionanddifferentiationofB.bigemina,B.bovis,andAnaplasmamarginaleina16S–18SrRNAgenemultiplexPCRwasreportedbyFigueroaetal.(1993).38 中国农业科学院博士学位论文ChapterIVDespitetheusefulnessoftheseassays,itwouldbeverypracticaltohaveauniversaltesttosimultaneouslydetectanddifferentiateallprotozoanparasitesthatcouldpossiblybepresentinthebloodofcarriercattle.Semi-nestedPCRassaywasusedtodetectthepiroplasmspeciesinthesamplescollectedfromvariousregionsofPakistanandChina.Semi-nestedPCRisamodifiedformofPCR,inwhichtwosetsofprimersareusedtocompletetheamplification.ThismodifiedformofPCRismuchsensitivethanconventionalPCR,soitcandetectthelowlevelofparasitemia(Gubbelsetal.,1999).4.2Materialsandmethods4.2.1SamplescollectionInordertocollectthesamplesfromPakistan,anofficialtourwasmadeinsummer®seasonof2016.TheWhatmanFTAcard(GEHealthcareLimited,Buckinghamshire,U.K.)wereprovidedbyVectorsandVectorBorneDiseasesLaboratoryofLanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciences,China.ThreegeographicalregionsofPunjabprovinceofPakistanincludingChakwal,JhangandFaisalabadwereselectedforrandomizedpurposivesampling.ChakwalliesinthearidzoneofPunjab;FaisalabadistheindustrialcityandcalledasManchesterofPakistan,whileJhangisthelandofrivershavingtwobigriversChenabandJhelum.Tick-infestedbutapparentlyhealthycattlewereselectedforsampling.Intotal450samples(Chakwal188;Faisalabad93andJhang169)werecollectedfromPakistan,while120gDNAsamplesofcattlecollectedfromInnerMongoliawereprovidedbyVectorsandVectorBorneDiseasesLaboratoryofLanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciences,China.Bloodwascollectedfromjugularveinusing10mL®disposablesyringe.Approximately2to3mLwasdividedamong4circlesofWhatmanFTAcard(GEHealthcareLimited,Buckinghamshire,U.K.)andallowedtoairdryafterlabeling,whiletheremainingbloodwasusedforserumseparationtobeusedinfurtherinvestigations.DriedbloodsampleswereshippedtoLanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciencesLanzhou,Chinaforprocessing.Theremainingbloodwasspuntoextractserumsamples.39 中国农业科学院博士学位论文ChapterIV40 中国农业科学院博士学位论文ChapterIV4.2.2GenomicDNAextractionDNAwasextractedfromFTAcardsusingaQiagenGenomicDNApurification®Kit(Qiagen,Hilden,Germany)accordingtothemanufacturer‟sinstructions.Briefly,3circlesof3mmdiameterwerecutanddigestedby180µLofBufferATLat85℃for10min,followedbytheadditionof20µLofproteinaseKandincubatedat56℃for1h.Followingthis,200µLofBufferALwasaddedandincubatedat70℃for10min.200µLofabsoluteethanolwasaddedforpelletingofDNA.Thefinalsolutionwascarefullytransferredtothespincolumnandwashingwasdoneusing500µLofBufferAW1andBufferAW2eachrespectivelywithcentrifugationatfullspeedfor1min.Finally,elutionwasdonebyadditionof70µLBufferAEandcentrifugationat13000gfor1min.TheDNAconcentrationwasdeterminedwithaNanoDrop2000spectrophotometer®(NanodropTechnologies,Wilmington,Delaware).DNAwasstoredat-20℃.4.2.3PrimerdesignTheprimersalreadydescribedby(Gubbelsetal.,1999)wereusedinthepresentstudytotargettheV4hypervariableregionof18SrRNAgene,inordertodetectthepiroplasmspeciesfromgDNA.Inthefirstcycleofsemi-nestedPCR,RLB-F2(5‟-GACACAGGGAGGTAGTGACAAG-3‟)andRLB-R2(5‟-CTAAGAATTTCACCTCTGACAGT-3‟),whileinthesecondPCRcycle,RLB-R2andRLB-Fint(5‟-GACAAGAAATAACAATACRGGGC-3‟)wereused.4.2.4PCRamplification,cloningandsequencingInthepresentstudy,V4hypervariableregionof18SrRNAgenewastargetedtodetectTheileriaandBabesiaspecies.InthefirstcycleapairofprimersRLB-FandRLB-Rwereusedinatotalreactionmixtureof25µLcontaining12.5µLofPCRmastermixincludingdye,1µLofeachprimer(10mmol),3µLofDNAtemplateand7.5µLofnuclease-freewater.Thecyclicconditionswerefollowedasdescribedby(Gubbelsetal.,1999).Initialdenaturationat95°Cfor3min,followedby35cyclesincludingdenaturationat95°Cfor30sec,annealingat52°Cfor30secandextensionat72°Cfor1min.Finally,theextensionwasdoneat72°Cfor5min.FollowingthefirstroundofPCR,thesecondPCRwassetupwithalltheconditionsasmentionedabove,excepttheuseofRLB-FintprimerinsteadofRLB-F,whilethecyclicproductofthefirstroundwasusedastemplateinthesecondround.50µLPCRmixturecontaining25µLofmaster41 中国农业科学院博士学位论文ChapterIVmixincludingdye,1µLofeachprimers,3µLofPCRproductoffirstround(astemplate)and20µLofnucleasefreewater.Theamplifiedproductswereanalyzedon1.5%agarosegels.Forthepreparation®of1.5%agarosegel,1.5gofthemoleculargradeagarosepowder(Biowest,GeneCompany,HongKong)wasweighedandthenmixedwith100mlof1xTrisacetateEDTA(TAE)bufferinaflask.Gelwaspouredonthegeltrayavoidingbubblesandsplash.Afterthegelwassolidified,thecombwastakenoutofthecastingtrayandthetraywithgelwastransferredtotheelectrophoresistank.TheelectrophoresistankwasconnectedwiththePowerPacand120voltswereprovidedtothegel.After30minutesthegelwasshiftedtothegeldocumentationsystem.Anexpectedlengthofabout393bpwasobtainedasshowninFig.9.TherightpositionbandsofthegelwerecutdownandTMtheproductwaspurifiedforfurtherprocess.Forthispurpose,EZgeneGel/PCR®ExtractionKit(Biomiga,SanDiego,California,USA)wasusedasprescribedbythemanufacturer.Briefly,thepartofgelplacedina1.5mLtubewasadded600µLBufferGCandwarmedat65°Ctillthegelwasliquefiedcompletely.ThenthemixtureofgelandbufferGCwastransferredtospincolumnandcentrifugedat13000xgfor1min.Theflowthroughwasdiscardedand700µLwashingbuffer(ethanoladded)wasaddedandcentrifugedat14000xgfor30sec.Thesamestepwasrepeatedtoensurethecompletewashing.Thenthespincolumnwasplacedina2mLcollectiontubewithopenlidandcentrifugedatmaximumspeedfor2minfordryingofthecolumnmembrane.Finally,theproductwaselutedin30µLBufferAEbycentrifugationat13000xgfor1min.®TheyieldedproductwasligatedintopGEM-TEasyvectors(Promega,Madison,Wisconsin,USA).5µLof2Xbufferwasmixedwith3µLofDNAand1µLofthevectorwasadded.Atlast,1µLofT4DNAligaseincludedinthekitwasaddedtocatalyzetheligationprocess.Intotal,10µLmixturewaspreparedinPCRtubeandincubatedat16°Covernight.Nextday,30µLofcompetentcellsEscherichiacoliTrans®5α(TransGenBiotech,Beijing,China)wasaddedtotheligatedproductandincubatedinicefor30minutes,followedbyincubationat42°Cfor90secandagainincubationinicefor2min.500µLofLBmediumwasaddedandincubatedat37°Cwithshakingat200rpmfor1hour.400mLLBmediumwaspreparedbymixing4goftrypton,2gofyeastextractand4gofsodiumchlorideindistilledwaterandautoclavedat121°Cfor20min.42 中国农业科学院博士学位论文ChapterIVFollowingtheincubationat37°Cwithshaking,thesamplewasshortspunandthesupernatantwasdiscardedwhile,4µLofIPTG(1M)and16µLofX-galwereadded.Thefinalproductwaspouredon1.5%LBagarplates(Ampicillinadded)andspreadedusingglassbeads.Theplateswereincubatedat37°Cfor14-16hoursandwhite(correctlyinserted)cloneswerepickedupandincubatedin1000µLofLBmedium(Ampicillinadded)andincubatedat37°Cwithshakingfor4to5hours.TheculturedcloneswereconfirmedbyPCRusingvectorprimers;T7(5‟-TAATACGACTCACTATAGGG-3‟)andSP6(5‟-ATTTAGGTGACACTATAG-3‟).ThePCRwasoptimizedfor1stepofinitialdenaturationat95°Cfor3min,followedby35cyclesofdenaturation(94°Cfor1min),primerannealing(54°Cfor30sec),extension(72°Cfor1min),andthefinalextensionat72°Cfor5min.Cloneswere®shippedtoGenescript(Shanghai,China)forsequencing.Fig.9:1.5%agarosegelimageofpiroplasmspeciesbysemi-nestedPCR,wells3-9amongthepositivelyidentifiedfieldsamplesFig.10:1.5%agarosegelshowingthePCRproductsusingvectorprimers43 中国农业科学院博士学位论文ChapterIV4.2.5ConfirmationandsequenceanalysisofpositivesamplesThesequencingresultswereanalyzedusingEditSeqversion7.1.0(DNASTAR,®Lasergene,Madison,WisconsinUSA).Afterreceivingthesequencingresultsfromthecompany,thevectorpartwasdeletedtogettheexactsequenceamplified.ThecorrectedsequenceswereconfirmedbyBLASTatNCBI.Thesequenceswereconsideredaccordingtothereferencesequenceswithmostidentityofmorethan99%.Thefoundsequenceswerethensubmittedinthegenbankandaccessionnumbersweregot.4.2.6PhylogeneticanalysisTheevolutionaryhistorywasinferredusingtheNeighbor-Joiningmethod(SaitouandNei,1987).Thepercentageofreplicatetreesinwhichtheassociatedtaxaclusteredtogetherinthebootstraptest(1000replicates)areshownnexttothebranches(Felsenstein,1985).TheevolutionarydistanceswerecomputedusingtheKimura2-parametermethod(Kimura,1980)andareintheunitsofthenumberoftransitionalsubstitutionspersite.Theanalysisinvolved28nucleotidesequences.Allambiguouspositionswereremovedforeachsequencepair.Therewereatotalof2030positionsinthefinaldataset.EvolutionaryanalyseswereconductedinMEGA7(Kumaretal.,2016).4.2.7SpeciesconfirmationInthepresentstudy,samplingfromtick-infestedcattlewasdonefrom3differentregionsfromPunjabincludingChakwal,FaisalabadandJhang.MeanwhilethegDNAsamplesofthecattlecollectedfromInnerMongoliawereprovidedbyVectorsandVector-bornediseasesgroupofLanzhouVeterinaryResearchInstitute,China.Thepositivelyidentifiedsamplesfromtheresultsofsemi-nestedPCRasdiscussedearlierwereselectedrandomlyforamplificationofmorelengthofthegenesinordertoconfirmthedetectedspecies.Forthispurpose,almostfulllengthof18SrRNAgenewasamplifiedfortheTheileriapositivesamples,whileRAP-1c(rhoptryassociatedprotein)genewasamplifiedfromthesamplespositiveforBabesia.NBab-1Fand18SRev-BTwereusedforamplificationoflongfragmentof18SrRNAandB.birap-1cseq-FandB.birap-1cRcwereusedfortheRAP-1cgeneofB.bigemina.TheprimersusedinthisstudyhavebeenshownintheTable5.FollowingtheamplificationbyPCR,cloningwasdoneasdescribedasearlierandsequencingwasdonebythecompany.44 中国农业科学院博士学位论文ChapterIVTable5:DetailsoftheprimersusedforconfirmationofpiroplasmspeciesProductNameSequenceReference(bp)NBab-1FAAGCCATGCATGTCTAAGTAGAAGCTTTT(Liuetal.,18SRev-BT~1600GAATAATTCACCGGATCACTCG2016)B.birap-TTACGCTGCTTACTACAGCTTCA(Niuetal.,1cseq-F10542015)B.birap-1cRcTTACGACGATCGTTTGAAGTACTTC4.2.8NucleotideaccessionnumbersThereferencesequencesofthepresentstudyweresubmittedtoNationalCenterforBiotechnologyInformation(NCBI),U.S.NationalLibraryofMedicinegenbankunderaccessionnumbers:T.annulata:KY626168,MF427698,MG585358-73,MG599086-87,MG599090-95,MG645810-13;T.orientalis:KY765557-58,MG585374-83,MG599088-89,MG599096-99;B.bigemina:KY646456,KY765561-62,MG874651-56.4.3Results4.3.1DetectionandidentificationofparasiteinsamplescollectedfromPakistanIntotal,450sampleswerecollectedfromPakistan,including188fromChakwal,169fromJhangand93fromFaisalabadregions.Amongthesesamples,128sampleswerepositivelydetectedbysemi-nestedPCRforpiroplasmspecies.Theileriaannulatawasfoundtobemostabundantspeciesinfecting103animalsi.e.80.47%oftheinfectedand22.89%ofthetotalsamplesscreened.Amongareawiseprevalence,T.annulatawasfoundtobemostprevalentinChakwal(88/188)i.e.46.80%ofthesamplesfromthisregion,followedbyFaisalabad(10/93)andJhang(5/169),accountingfor10.75%and2.96%ofthetotalscreenedanimalsineachregionrespectively.ThesecondmostabundantspecieswasT.orientalisinfecting14animalsi.e.10.93%oftheinfectedand3.11%ofthetotalsamplesscreened.Amongareawiseprevalence,T.orientaliswasfoundtobemostprevalentinChakwal(12/188)i.e.6.38%ofthesamplesfromthisregion,followedbyFaisalabad(2/93),accountingfor2.15%ofthetotalscreenedanimalsintheregion.NoneofthesamplesfromJhangregionwasfoundtobepositiveforT.45 中国农业科学院博士学位论文ChapterIVorientalis.TheleastabundantspecieswasB.bigeminainfecting11animalsi.e.8.60%oftheinfectedand2.45%ofthetotalsamplesscreened.Amongareawiseprevalence,B.bigeminawasfoundtobemostprevalentinChakwal(10/188)i.e.5.32%ofthesamplesfromthisregion,followedbyFaisalabad(1/93),accountingfor1.07%ofthetotalscreenedanimalsintheregion.NoneofthesamplesfromJhangregionwasfoundtobepositiveforB.bigemina.Ofthetotal450samples,322werenegativeforthepiroplasmspeciesusingsemi-nestedPCR.Overall,Chakwalregionsamplesweremostaffectedwithpiroplasmspecies,followedbyFaisalabadandJhangrespectively.4.3.2DetectionandidentificationofparasiteinsamplescollectedfromChinaIntotal,120sampleswerecollectedfromInnerMongoliaprovinceofChina.Amongthesesamples,16sampleswerepositivelydetectedbysemi-nestedPCRforpiroplasmspecies.Theileriaannulatawasfoundtobemostabundantspeciesinfecting12animalsi.e.75%oftheinfectedand10%ofthetotalsamplesscreened.TheotherspecieswereT.orientalisandB.bigeminaequallydistributed,infecting2animalsi.e.12.5%oftheinfectedand1.67%ofthetotalsamplesscreened.Table4:MolecularPrevalenceofPiroplasmSpeciesfromSelectedAreasofChinaandPakistanusingSemi-NestedPCRAreaT.annulataT.orientalisB.bigeminaNegativeTotalChakwal88(46.80%)12(6.38%)10(5.31%)78(41.48%)188Jhang05(2.96%)00(0.00%)00(0.00%)164(97.04%)169Faisalabad10(10.75%)02(2.15%)01(1.07%)80(86.02%)93InnerMongolia12(10.00%)02(1.67%)02(1.67%)104(86.67%)120Total115(20.17%)16(2.80%)13(2.28%)426(74.73%)57046 中国农业科学院博士学位论文ChapterIVFig.11:PhylogenetictreeofTheileriaandBabesiaspp.basedontheV4regionof18SrRNAgenesequences.Theparasiteidentifiedinthepresentstudyismarkedinbold.SamplesfromPakistan;SamplesfromChina47 中国农业科学院博士学位论文ChapterIVFig.12:PhylogenetictreeofTheileriaspp.constructedbasedonthe18SrRNAgenesequences.TheparasiteidentifiedinthepresentstudyismarkedinboldSamplesfromPakistanSamplesfromChina48 中国农业科学院博士学位论文ChapterIVFig.13:PhylogenetictreeofBabesiabigeminaconstructedbasedontherhoptryassociatedprotein(RAP-1c)genesequences.ThesequencesidentifiedinthepresentstudyismarkedinboldSamplesfromPakistanSamplesfromChina49 中国农业科学院博士学位论文ChapterIV4.4DiscussionInPakistan,smallholdercattlefarmingsystemisfulfillingthehouseholdrequirementsorcommercialpurposesoftheresource-poorruralcommunities.Intherecentyears,peopleareadoptingcommercialfarmingusingmoderntechniquesandimportedbreedsofcattle(Bostaurus)duetotheirhigherpotentialofmilkyield.However,theimportedanimalsaremorepronetotheticksandtick-bornediseasesascomparedtothelocalbreeds(Bosindicus);thecasefatalityintheformerisupto80%ascomparedtolater,whichislessthan20%.Amongmanytick-bornediseases,tropicaltheileriosisisconsideredamongthemosteconomicallynotoriousdiseases,predominantlycausedbyT.annulata(Jabbaretal.,2015).Todate,mostofthestudieshavebeenconductedthroughconventionalmicroscopywhich,duetolowsensitivityandspecificity,isnottrulyrepresentingtheactualdistribution.Recently,astudypresentedtheprevalenceofT.orientalisfromimportedandnativebovinepopulationofPakistandescribing24.5%positiveresultsonlyinimportedanimals,whileabsenceoftheinfectioninnativeanimals.ThatstudyjotsdownabriefdescriptionofTheileriaspeciesinPakistanincludingT.annulataandT.orientalis(Gebrekidanetal.,2017).Inthepresentstudy,acomparisonofthepiroplasmsspeciesinfectionsfromselectedareasofPakistanandChinahasbeendescribed.ThreestudyareasfromPakistan,whileonefromChinawasincludedinthepresentstudy.Overall,highestinfectionratewasfoundinChakwalregionofPakistan(58.51%),followedbyFaisalabad(13.98%)andInnerMongolia,China(13.34%),whilelowestinfectionratewasfoundinJhangregionofPakistan(2.96%).ThehigherrateinChakwalregionofPakistanmaybedueitsclimaticconditionswhicharesuitableforticksprevalence(Hassanetal.,2018).ThephylogeneticanalysisbasedontheshortamplifiedsequencesofV4hypervariableregionof18SrRNAgene,threepiroplasmsspeciesincludingT.annulata,T.orientalisandB.bigeminawerefoundtobeprevalentinthestudyareasofChinaandPakistan.Inordertoconfirmthesespecies,thelongerregionofthegeneswereamplified.Inthisregard,thelongersequenceof18SrRNAgene(about1600bp)wasamplifiedforTheileriaspecies,whilerhoptryassociatedprotein(RAP-1c)wasusedforconfirmationofB.bigemina.MostoftheT.annulatasequenceswerefoundtobecloselyrelatedtoalreadyreportedsequencesfromPakistan(Khanetal.,2013)andIran(AccessionNo.50 中国农业科学院博士学位论文ChapterIVKF429799).Ontheotherhand,MG599091,MG599092fromPakistanandMG599087fromChinadidnotshowtheclosedrelationshipwithothersequencesreportedinthisstudy.SomeofT.orientalissequencesreportedfromPakistanarecloselyrelatedtoalreadyreportedsequencesfromChina(AccessionNo.KU363043),whileafewsequencesarenotcloselyrelatedtothealreadyreportedsequencesofthegenbankandappearasdistinctfromotherduringphylogeneticanalysis(AccessionNos.MG599098,MG599099).TheB.bigeminasequenceswereanalyzedbyusingsomeotherreferencesequencesofthesamegene(Niuetal.,2015).TheseshowrelationshipwiththereportedsequencesfromChinaandArgentina.ThepresentstudyreportsthesimultaneousdetectionofmultiplepiroplasmspeciesfromPakistanandtheircomparisonwiththesamespeciesdetectedfromChina.Meanwhile,thisisthefirstreportofT.orientalisfromnativebovinepopulationofPakistan.51 中国农业科学院博士学位论文ChapterVCHAPTERVSerologicalDetectionofTheileriosis5.1IntroductionBovinetheileriosisisatick-bornehaemoprotozoandiseasecausedbytheparasitesofthegenusTheileria.Itinfectsbovineleukocytesanderythrocytes(Liuetal.,2009).ThediseasehaswidegeographicrangeofdistributionfromSouthEurope,NorthAfrica,India,MiddleEastandAsia(Bilgicetal.,2010).Itcauseslossesinlivestockindustrybydecreaseproduction,morbidityandmortalityinexoticbreed,whilelessinindigenousandcrossbred(Bakheitetal.,2004).Thediseaseischaracterizedbyfever,lymphnodeswelling,anemiaandjaundice(Xuetal.,1997).TheimportantTheileriaspeciesforbovineinfectionincludesTheileria(T.)annulata,T.parva,T.sergenti,T.sinensis,T.mutans,T.veliferaandT.taurotragi(Onumaetal.,1998;Sugimotoetal.,1991).TropicaltheileriosisiscausedbyT.annulata,whichistransmittedbyticksofgenusHyalomma(Dolan,1989).Thisinfectsespeciallycrossbredcattlewithclinicalsignoffever,lymphadenopathy,splenomegaly,anemia,lossofbodyweightandfinallydeath(Omeretal.,2002;El-DeebandYounis,2009).TheileriasergentiiswidelydistributedinAustralia,Japan,KoreanPeninsulaandNew-Zealandwithclinicalsignoffever,lymphadenectasisandanemia(Wangetal.,2010a;Liuetal.,2010;Kamauetal.,2011a;Eamensetal.,2013;Pulfordetal.,2016b;Jinetal.,2007).TheileriasinensiswhichtransmitedbyHaemaphysalisqinghaiensisandisthoughttooriginatefromGansuProvinceofChina.Itisnotharmfulforanimalsduetoitslimiteddistribution.Earlyphaseofhaemoprotozoancanbeeasilydiagnosedbymicroscopyofthinbloodsmear.APCRassayisalsobeingusedforthedetectionofmanygenesoftheileria.Thismethodcandifferentiatethecasesoftruepositiveandfalsepositiveresultsduetooldinfectionandcrossreaction(Martin-Sanchezetal.,1999;Aktasetal.,2002).Othermethodslike,RLBandIFATarealsobeingusedasadiagnosticmethod.Alltheabovemethodsarenotsuitabletoperforminfieldconditionbecausetheyareeconomical,needexpensiveinstrumentsandaretimestaking.Serologicalassaysaresuitableforthediagnosisoftheseparasitesantibodytiterininfectedanimals.Enzyme-linkedimmunosorbentassay(ELISA)isasuitableandeconomicalmethodfordetectionofTheileriaspeciesinlatterphaseofdiseaseandinrecoveredcarrieranimals.Thisassayismoresensitiveandquickthanmicroscopy,PCR,RLB,andIFAT(Bakheitetal.,2004).TheileriagenesusedforPCRidentificationare18SrRNA,ITS,majorpiroplasmasurfaceprotein(MPSP)andT.annulatamerozoitesurfaceprotein1(Tams1)(Tanakaetal.,1993;52 中国农业科学院博士学位论文ChapterVd‟Oliveiraetal.,1995;Hanetal.,2009;Kamauetal.,2011b).TheMPSPisglycoproteininnature,containingaconservedregiononthesurfaceoftheileriawiththesizerangingfrom30to34kDa.Theyhaveexcellentimmunogenicityandstimulateastrongimmuneresponseininfectedanimals.Itissuggestedthattheseproteinsarebiologicallysignificantinparasitedevelopment(Shielsetal.,1995;Onumaetal.,1997).Theseproteinsmaybecandidateantigensforsero-diagnosticandvaccinedevelopment.Inthepresentstudy,anELISAdetectionmethodwhichbasedonrecombinantMPSPprotein,wasappliedfordiagnosisofbovinetheileriosis.ThismethodcouldidentifyantibodiesagainstT.annulata,T.sergentiandT.sinensisintheserumsamples.Atotalof840serasamplesthatcollectedfromthreeprovincesofChinaincludingTibet,XinjiangandInnerMongolia,weredetectedbyELISA5.2Materialsandmethods5.2.1ParasitesBovinebloodsamplesinfectedwithT.sinensis(DingxistrainfromGansuprovince)wascryopreservedinliquidnitrogenattheLanzhouVeterinaryResearchInstitute(LVRI),China.5.2.2SeraandgenomicDNAsReferencepositiveseraandgenomicDNAofbovinepiroplasms(T.sinensis,T.orientalis,T.annulata,Babesia(B.)major,B.bigeminaandB.bovis),ovinepiroplasms(T.uilenbergi,T.luwenshuniandB.motasi)andAnaplasmaoviswereprovidedbytheVectorsandVector-borneDiseases(VVBD)LaboratoryofLVRI.Animalexperimentationwasapproved(permitSYXK2010-0001)bytheScienceandTechnologyDepartmentofGansuProvince,China.Field-collectedbovineserawerecollectedfrom840cattleandyaksin44prefecturesof3provincesfrom2012to2014.TheprovincessampledincludedInnerMongolia(Hulunbeier,Baotou,Tongliao,Xing‟anmengandXilinguolemeng),Tibet(Lhasa,QuShuiXian,RiKeZi,QieWeiXian,ShanNanandDaZi)andXinjiang(Yili,SaiKeXin,ALeTai,WenSuXian,AKeSu,TaCheng,XinjingTianPi,KaShi,HeTianandBuErJin).5.2.3CloningandexpressingMPSPgeneThespeciesspecificprimerswereusedtoamplifyMPSPCDSbasedonthesequencesofT.sinensis(accessionnos.GQ180193.1)inGenBankasdescribedbyZhaoetal.,(2017).Thesetofprimerwas(Forward:5′-CCGGAATTCATGTTGTCCAAGAGATATCTTAACG-3′andReverse:5′-53 中国农业科学院博士学位论文ChapterVCCGCTCGAGCTAAAGATAGTAGAAAACGGCG-3)′toamplify873bpfragment.Subsequently,thePCRproductswerepurified,digestedwithrestrictionenzymesEcoRIandXhoIandfinallyinsertedintoapET-30avector.TherecombinantpET-30a-MPSPplasmidwasconfirmedbydoublerestrictionenzymedigestionandsequencing.TherecombinantvectorwasthentransformedintocompetentE.coliBL21(DE3)toexpresstherMPSPasHis-fusionproteins.300µLofrecombinantE.coliwasinoculatedinto30mlfreshLBmediumwith50μg/mlofkanamycinandcultivatedat37°Cwithshakingat200rpm.WhentheOD600ofthebacteriumculturereached0.6,IPTGwasaddedatafinalconcentrationof1.0mmol/Ltoinduceproteinexpressionat37°Cfor8h.Finally,therMPSPwasidentifiedandanalysedbySDS-PAGE.TherMPSPwaspurifiedaccordingtotheinstructionsoftheNi-NTAPurificationSystem(CatalogueNo.K950-01,Invitrogen,CarlsbadCity,CA,USA).Briefly,theE.coliwerepelletedandresuspendedinnativebindingbuffer(denaturingbufferforproteinsupernatant)andthenlysedbyfreezethawingandsonication.Followingthis,thesupernatantwascollected.Afterdenaturationbyguanidiniumlysisbuffer,thesupernatantissoluble.ThesolubleproteinfromsupernatantwascombinedwiththeNi-NTAresinfor1–2hundergentlerotation.Followingthreewasheswithnativeordenaturingwashbuffer,theeluatewascollectedintoseparatetubesforidentificationbySDS-PAGE.5.2.4WesternblottingThecross-reactivitybetweentherMPSPandpositivereferenceseraagainstT.sinensis,T.orientalis,T.annulata,T.uilenbergi,T.luwenshuni,A.ovis,B.motasi,B.major,B.bigeminaandB.boviswereexaminedbywesternblotting.TherMPSPshowedstrongreactionswiththepositiveseraagainstT.sinensis,T.orientalisandT.annulatabutnocross-reactivitywithotherpiroplasmspeciesandA.ovissera.5.2.5ELISAprocedureAnELISAproceduredescribedpreviouslybyChauvinetal.(1995)andGuanetal.(2010)wasusedinthepresentstudy.Briefly,96-wellmicrotiterplates(Nunc,Roskilde,Denmark)werecoatedwith50μlrecombinantantigenataconcentrationof2.8μg/mlinacoatingbuffer(0.1Mcarbonate–bicarbonatebuffer,pH9.6).TheplateswerewashedthreetimeswithPBScontaining0.1%Tween20(PBST)andincubatedwith100μl/wellofablockingsolution(1%BSAinPBST)for1hat37°C.Afterdryingtheplate,thesamples,blanks(PBST)andstandardpositiveandnegativecontrols(dilutionof1:200)weredistributedinduplicateandtheplateswereincubatedat37°Cfor1h.Afterwashingasdescribedabove,aperoxidaseconjugateofrabbitanti-bovineIgG(A-5295,54 中国农业科学院博士学位论文ChapterVSigma)dilutedat1:40,000withPBSTwasaddedtoeachwellandtheplateswereagainincubatedat37°Cfor1h.Theplateswerewashedthreetimesasdescribedabove,andsubsequently,50μlofTMB(T0440-1L,Sigma,USA)wasaddedtoeachwellandincubatedatroomtemperaturefor20min.Thereactionwasstoppedbytheadditionof50μlof0.2MH2SO4andtheopticaldensity(OD)wasmeasuredwithanELISAreader(microplatereaderModel680,Bio-Rad,USA)atawavelengthof450nm.Theresultsareexpressedasthepercentageofthespecificmeanantibodyrate(AbR%),determinedusingtheformula,AbR%=(SamplemeanOD−NegativecontrolmeanOD)/(PositivecontrolmeanOD−NegativecontrolmeanOD)×100%Cutoffpoints,specificityandsensitivityoftheELISAwereevaluatedbyreceiveroperatingcharacteristic(ROC)analysisusingMedCalcstatisticalsoftwarewith47negativeseraand67positiveseraagainstT.annulata,T.orientalisandT.sinensisfromcattle.5.3Results5.3.1SerologicalprevalenceofTheileriosisUsingtheabovementionedsetofprimers,an893bpfragmentwasamplifiedbyPCRforcdsregionofMPSPofT.sinensis.Followingthedoublerestrictionenzymedigestion,therecombinantMPSPwasexpressedusingE.coliBL21(De3).ThewesternblotresultsforspecificityofproteinareexpressedinFig.14.Aftertheproteinpurification,itwasobservedthattheexcessproteinispresentinthesupernatant.Later,theproteinwasusedtooptimizetheindirectELISA.Keepinginconsiderationthesensitivityis95.5%(0.875∼0.991under95%CI)andspecificityoftheassayis95.7%(0.855∼0.995under95%CI)whenthethresholdvalueis28.8%,840samplesfrom3provincesshowedtheoverallpositiverateof18.57%(156/840).HighestprevalencewasfoundinXinjiangprovince27%(105/389),followedbyInnerMongolia14%(7/50)andTibet10.97%(44/401).MT.annulataT.orientalisT.sinensisB.bigeminaB.bovisB.majorFig.14:WesternblotimageofrMPSPspecificityusingT.annulata,T.orientalis,T.sinensis,B.bigemina,B.bovisandB.majorpositivesera.55 中国农业科学院博士学位论文ChapterV5.4DiscussionTropicaltheileriosisisatickbornehaemoprotzoandiseasewhichiswidespreadinthesubtropicalregionoftheworld.Thediseaseisfatalleukoproliferativeinthebovinepopulation,mainlytargetingthecattle.Theileriaannulataisthemaincausativeagentoftropicaltheileriosisresultinginhugeeconomicloss,whileT.sergentiandT.sinensisplayvitalroleinthedevelopmentofsubclinicaldisease(Schnittgeretal.,2002).Traditionally,opticalmicroscopyofthethinbloodsmearswasusedtoidentifytheileriainfection.However,duetolackofsensitivityandspecificityofthismethod,manymolecularandserologicalassayshavebeendevelopedfordiagnosis.(Salihetal.,2007)developedtheRLBassayforsimultaneousdetectionofT.annulata,T.buffeli,T.taurotragi,T.velifera,T.mutansandT.parva.ManyscientistsfromdifferentregionsusedPCRassaysforidentificationoftheileriosis.MultiplexPCRmethodisalsodevelopedforsimultaneousdetectionofT.annulataandT.orientalis(Junlongetal.,2015),whileLAMPassayisalsodevelopedfordetectionofT.orientalisandT.sinensis(Liuetal.,2013).Duringterminalstageofthedisease,itisdifficulttodiagnosebymicroscopicexaminationandPCR,astheparasitemiaisverylow.SerologicalmethodcanbethebestchoiceatthisstagebecausetheantibodytiterismuchhigheranditcanbedetectedeasilybyELISA.Alltheseadvancedtechniquesaremuchsensitiveandspecificincomparisonwiththeconventionaltechniques.However,thesetechniquesaremuchtimeconsumingandmaynotbeconvenientforlargescaleepidemiologicalstudies.WeexpressedMPSPproteinofT.sinensiswhichishighlysensitiveandisusedfordetectionofT.annulata,T.sergentiandT.sinensisinfectionwithoutdifferentiationasdescribedpreviouslybyZhaoetal.,(2017).ThispurifiedproteinshowedastrongreactivitywiththepositiveseraoftheabovementionedthreetheileriaspeciesandtherewasnoreactionwiththereferenceserasamplesoftheotherpiroplasmspeciesandA.ovis.Thehigherconcentrationofproteinwasfoundinthesupernatantthanpellet.ThecalculatedspecificityandsensitivityoftheELISAofthisproteinwere95.7and95.5%respectively,withathresholdvalueof28.8%.TherMPSPhadastrongreactionwithanti-polyhistidineantibodydemonstratedbywesternblot,whichindicatestheexactexpressionoftherMPSPintheprokaryoticexpressionsystem.Thisresultshowsthatthe56 中国农业科学院博士学位论文ChapterVrMPSPcanbeusedtodevelopauniversalserodiagnosticmethodforevaluatinginfectionwiththethreebovineTheileriaspecies.However,thismethoddoesnotdifferentiatebetweenthethreespecies.Evaluatingthecross-reactivity,specificity,sensitivityandthresholdvalueofELISAGiventheconcentrationofthepurifiedrMPSP,therMPSPofT.sinensiswasusedasanantigentodevelopauniversalELISAfordetectingtheinfectionofthethreebovineTheileriaspecies.ThemeanAbRsandstandarddiversity,calculatedwithExcel2007forELISAresults,showedtherMPSPofT.sinensishadstrongreactionswiththepositiveseraagainstT.annulata,T.orientalisandT.sinensisbutnocross-reactionswithotherpiroplasmandA.ovispositivesera,whichwasconsistentwiththeresultsfromthewesternblot.Followingtheaboveprocedure,840fieldserasampleswereprocessedbyindirectELISA,keepinginconsiderationthresholdvalueis28.8%.ThespecificityandsensitivityvaluesofourstudycorrelatewithalreadyreportedvaluesofTaSP(Bakheitetal.2004).Thus,thepurifiedproteinwasusedinindirectELISAonthefieldserasamplesfrom46localitiesof3provincesofChinaincludingTibet,XinjiangandInnerMongolia.Theoverallsero-prevalencewas18.57%,withhighestrateinXinjiang(21.29%),followedbyTibet(17.26%)andInnerMongolia(10.44%).Formaximumconfidenceregardingseraresult.Eachsamplewasprocessestwiceandwasconsideredwhenit‟sbothreadingsfellbeloworabovethreshold.ThereportedseroprevalenceofT.annulatabasedonTams1was11%whichiscomparativelylessthanthepresentstudy(Jianetal.,2011).Inotherstudies,sero-prevalenceofT.annulatawas87.5and75%bytargetingTaSPandTams1inSudanandEgyptrespectively(Bakheitetal.,2004;Muhammadetal.,2012).Thedifferenceofseroprevalencebetweenstudiedandreportedmightbespecificityandsensitivityoftargetgene.ThepresentstudyprovidesacomprehensivedataontheseroprevalenceofthethreetheileriaspeciesinthreelargeprovincesofChina.Thisstudyattractstheattentionofpolicymakertodevelopcontrolstrategiesfortheileriosistoavoidtheeconomiclossestodairyfarmers.ItisconcludedthattheileriosisissignificantlypresentinmajorthreeprovincesofChina.OurstudystrengthenstheevidenceofMPSPofT.sinensisspecificityandsensitivity,whichcanbeusedforlargeepidemiologicalstudies.57 中国农业科学院博士学位论文ConclusionConclusionRecombinasepolymeraseamplificationmethodwasoptimizedfordetectionofT.annulataandT.orientalis.Thisisaconvenient,rapid,highlyspecificandsensitivemethodforidentificationoftheseparasitesespeciallyinthefielddiagnosis.Onthebasisofpresentstudyresults,itcanbeconcludedthatRPAmaybeasuitablediagnosticmethodforfieldstudiesandlargeareaepidemiologicalstudies.Itmaybethemostconvenientmethodforfieldstudiesespeciallyinresource-poorcountrieslikePakistan.However,furthersimilarmethodsshouldbeoptimizedtohavethecosteffectivediagnosticmethod.ThepresentstudyreportsthesimultaneousdetectionofmultiplepiroplasmspeciesfromPakistanandChina.Meanwhile,thisisthefirstreportofT.orientalisfromnativebovinepopulationofPakistan.ThespeciesreportedinthepresentstudyincludeT.annulata,T.orientalisandB.bigemina.However,alargescalesamplingshouldbedoneespeciallyfromPakistantohavetheclearideaofpiroplasmspecies.TheindirectenzymelinkedimmunosorbentassaymethodbasedonrecombinantMPSPwasutilizedforserologicalepidemiologyofimportantTheileriaspecies.TherMPSPofT.sinesisishighlysensitiveandcanbeusedtodetecttheinfectioncausedbyT.annulata,T.orientalisandT.sinensis.TheoverallpositiverateofTheileriaspeciesusingindirectELISAwasfoundtobe18.57%.However,species-specificdiagnosticmethodwouldbebetterforproperunderstandingoftheetiology.58 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中国农业科学院博士学位论文AcknowledgementsACKNOWLEDGEMENTSIamthankfultothemostGracious,Merciful,andAlmighty“ALLAH”whogavemethehealth,thoughtsandopportunitytocompletethework.IbowmycompassionateendowmentstotheHolyProphet“MUHAMMAD”(Peacebeuponhimandhisdescendants)andAALEMUHAMMAD(AlaihumUssalam)whoareever,torchofguidanceandknowledgeforhumanityasawhole.Theworkpresentedinthismanuscriptwasaccomplishedunderthesympatheticattitude,fatherlybehavior,animatedirections,observantpursuit,scholarlycriticismandenlightenedsupervisionofProf.Dr.JianxunLuo,DeputyDirector,LanzhouVeterinaryResearchInstitute,Lanzhou.Ideemitutmostpleasuretoavailtheopportunitytoexpresstheheartiestgratitudeanddeepsenseofdevotion.Withhumble,profoundanddeepsenseofdevotion,IwishtorecordmysincereappreciationtoProf.Dr.GuiquanGuan,Dr.JunlongLiu,Mr.JifeiYangfortheirreliablecomments,dynamicsupervision,sincerehelpandinspiringguidancethroughoutthecourseofthisresearchwork.EarnestanddevoutappreciationtoDr.MuhammadSohailSajid,AssociateProfessor,DepartmentofParasitology,UniversityofAgricultureFaisalabad,Pakistan,forhistimetotimeconstructivecriticismandvaluablesuggestions.IwillbefailingmydutiesifIdonotappreciateProf.Dr.HongYin,DirectorGeneralofLVRI,Lanzhouforprovisionoffavorableenvironmentforproductiveresearch.IamthankfultoallmylabfellowsespeciallyShuaiyangZhao,SitongLi,PengfeiGuo,Hanyuan,YinyinChen,MuhammadRashid,NaveedIqbalandJinmingWangastheypotentiallytoleratedagonyandallmiseriesandprovidedmethecharmingcompanyandpositivehelpinthisstudy.HereIwillnotforgettoacknowledgemynephewsMuhammadAliAwan,MuhammadAliAjwadandniecesEshaalZahra,HurremFatima,ZahraAli,AaimaAli,FatimaAliandSabeenZahraforspecialprayersformysuccess.Icannotforgettheaffectionandloveofsomeonespecial,whoencouragedmeateverystepthroughoutmyPh.D.IamspecialthankfultomyfatherMalikGhulamHassanAwan(Mayhissoulrestinpeace!)forhisstruggletofulfilallneedsofmylife.ImustacknowledgemymotherMrs.GhulamHassanAwan(Mayshelivelong!)forspecialprayers,withoutthoseIwouldneverbesuccessful.Overall,thepresentdoctoralstudywasofferedbyChineseScholarshipCouncilandhostedbyStateKeyLaboratoryofVeterinaryEtiologicalBiology,KeyLaboratoryofVeterinaryParasitologyofGansuProvince,LanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciences,China.TheexperimentswerefinanciallysupportedbytheNationalKeyR&DProgramofChina(2017YFD0501200);973Program(2015CB150300);NBCIS(CARS-37);ASTIP（CAAS-ASTIP-2016-LVRI）;JiangsuCo-innovationCenterprogrammeforPreventionandControlofImportantAnimalInfectiousDiseaseandZoonose.MuhammadAdeelHassan75 中国农业科学院博士学位论文CVMuhammadAdeelHassanChineseAcademyofAgriculturalSciences,ChinaAreaofResearch:Epidemiologyofticksandtick-borneparasiticdiseasesAcademicRecord:Ph.D.VeterinaryParasitologyandChineseAcademyofAgriculturalSciences,ChinaMolecularBiology2015~2018Ph.D.ResearchTopic:MolecularandSerologicalEpidemiologyofPiroplasmSpeciesfromSelectedAreasofChinaandPakistan.M.Phil.ParasitologyUniversityofAgriculture,Faisalabad,Pakistan2013~2015M.Phil.ResearchTopic:MolecularEpidemiologyofPiroplasmosisinSmallRuminantsPopulationofDistrictRahimYarKhan,Punjab,Pakistan.DVM(DoctorofVeterinaryMedicine)UniversityofAgriculture,Faisalabad,Pakistan2008~2013ResearchExperience:WorkedasPostgraduateStudentFellowinaUSAIDfundedresearchprojectundertitle“ArthropodFunctionalGenomics:BuildingCommunityResourcesforAnimalHealth”from18-11-2013to16-08-2014,performingmydutiesincludingfieldsampling,DNAextractionfrombloodandtissues,tickdissectionandPCR.ResearchExpertise:Fieldsamplecollection,microscopy,nucleicacidextraction,PCR,recombinasepolymeraseamplification,cloning,SDS-PAGE,westernblotting,proteinexpressionandpurification,ELISA,tickdissection,managementandpreservationofsamples,epidemiology,geneticdiversityPublications:Hassan,M.A.,J.Liu,M.S.Sajid,M.Rashid,A.Mahmood,Q.Abbas,G.Guan,H.YinandJ.Luo.2018.SimultaneousdetectionofTheileriaannulataandTheileriaorientalisinfectionsusingrecombinasepolymeraseamplification.TicksandTick-BorneDiseases.DOI:10.1016/j.ttbdis.2018.03.028(IF:3.230)Tian,Z.,X.Du,J.Du,S.Gao,R.Yu,M.A.Hassan,G.Liu,J.LuoandH.Yin.2018.DevelopmentofanindirectELISAbasedontherecombinantspm2proteinfordetectionoftropicaltheileriosis.ActaTropica.182:232-236.DOI:10.1016/j.actatropica.2018.03.003(IF:2.218)Hassan,M.A.,J.Liu,M.S.Sajid,A.Mahmood,Q.Abbas,G.Guan,H.YinandJ.Luo.2018.MoleculardetectionofTheileriaannulataincattlefromdifferentregionsofPunjab,PakistanusingrecombinasepolymeraseamplificationandPCR.JournalofParasitology.DOI:10.1645/17-173(IF:1.326)76 中国农业科学院博士学位论文CVHan,R.,J.Yang,Z.Liu,S.Gao,Q.Niu,M.A.Hassan,J.LuoandH.Yin.2017.CharacterizationofAnaplasmaovisstrainsusingthemajorsurfaceprotein1arepeatsequences.ParasitesandVectors10:447.DOI:10.1186/s13071-017-2363-6(IF:3.080)Yang,J.,Z.Liu,Q.Niu,J.Liu,R.Han,G.Guan,G.Liu,M.A.Hassan,J.LuoandH.Yin.2017.AnovelzoonoticAnaplasmaspeciesisprevalentinsmallruminants-potentialpublichealthimplications.ParasitesandVectors10:264.DOI:10.1186/s13071-017-2182-9(IF:3.080)Abdallah,M.O.,Q.Niu,J.Yang,M.A.Hassan,P.Yu,G.Guan,Z.Chen,G.Liu,J.LuoandH.Yin.2017.Identificationof12piroplasmsinfectingtentickspeciesinChinausingreverselineblothybridization.JournalofParasitology.103(3):221-227.DOI:10.1645/16-161(IF:1.326)Zhao,S.,J.Liu,H.Zhao,Y.Li,J.Xie,A.Liu,M.A.Hassan,H.Yin,G.GuanandJ.Luo.2017.EvaluatinganindirectrMPSPenzyme-linkedimmunosorbentassayforthedetectionofbovinetheileriainfectioninChina.ParasitologyResearch,116(2):667-676.DOI:10.1007/s00436-016-5332-7(IF:2.329)Abbas,M.T.,M.Arif,M.Saeed,M.Reyad-ul-Ferdous,M.A.Hassan,M.A.ArainandA.Rehman.2016.Emulsifiereffectonfatutilizationinbroilerchicken.AsianJournalofAnimalandVeterinaryAdvances,11(3):158-167.DOI:10.3923/ajava.2016.158.167Raza,S.H.A.,K.M.Arain,M.Saeed,S.Yalçin,M.E.A.ElHack,M.A.Arain,R.N.Soomro,H.M.Zakria,M.A.Hassan,I.H.R.Abbasi,S.S.Faraz,S.A.Fazlani,G.H.Jaffar,M.H.Baloch.2015.StudyonproductionandmarketingofbeefinQuettadistrictPakistan.InternationalJournalofInnovativeandAppliedResearch,3(12):8-21.Ali,N.,I.Ulla,S.M.Suhail,S.H.A.Raza,M.Saeed,S.U.Rehman,F.Ahmad,M.A.Arian,R.N.Soomro,M.Arif,H.M.Zakria,M.A.Hassan,Md.Reyad-ul-Ferdous.2015.Effectofcoolingonproductionandphysiologicalperformanceofdairycows(Holstein-Friesian&Jersey)insubtropicalenvironmentofPeshawar,Pakistan.InternationalJournalofInnovativeandAppliedResearch,3(11):54-62.ConferencePapers:Hassan,M.A.,J.Liu,M.S.Sajid,M.Rashid,C.Yin,S.Zhao,Q.Abbas,G.Guan,H.YinandJ.Luo.2018.SeroprevalenceofbovinetheileriosisinnorthwesternChinausingindirectELISA.Proceedingsoftwodays“InternationalConferenceonDairyAnimalHealthChallenges(DAHC-2018)”onJanuary17-18,2018organizedbyFacultyofVeterinaryScience,UniversityofAgriculture,Faisalabad,Pakistan.P.33.Hassan,M.A.,M.S.Sajid,Z.Iqbal,A.Kausar,W.Shahzad,M.Saqib,Q.AbbasandM.Maqbool.2017.MolecularepidemiologyofsmallruminanttheileriosisinRahimYarKhandistrict,Punjab,Pakistan.IranianJournalofParasitology,12(1):51(IF:0.758)Maqbool,M.,M.S.Sajid,Z.Iqbal,M.SaqibandM.A.Hassan.2017.Roleofwildbirdsindistributionoftoxoplasmosis.IranianJournalofParasitology,12(1):25(IF:0.758)Sajid,M.S.,H.M.Rizwan,N.Iqbal,S.Tahira,A.Kausar,M.A.Hassan,A.Qudoos,R.NadeemandM.N.Khan.2014.PrevalenceofintestinalparasiticinfectionsinrangelandsheepofdistrictChakwal.5thInt.Conferenceon77 中国农业科学院博士学位论文CV“Agriculture,FoodSafetyandClimateChange”heldonSep.09-11,2014,UniversityofPoonch,RawalakotandPAS-Forum.P.238.Shamim,A.,M.S.Sajid,R.M.Siddique,W.Ahmad,M.Jawad-ul-Hassan,M.A.HassanandM.N.Khan.2014.Impactofglobalclimatechangeonepidemiologyofticksandtick-bornediseases.5thInt.Conferenceon“Agriculture,FoodSafetyandClimateChange”heldonSep.09-11,2014,UniversityofPoonch,RawalakotandPAS-Forum.P.238.Extensionarticle:Hassan,M.A.,M.S.SajidandZ.Iqbal.2014.Babesiosisinsmallruminants:Asummernightmare.TheWeeklyVeterinaryNews&Views,Vol.9,No.14.P.07.Participation:1)Participationintwodays“InternationalConferenceonDairyAnimalHealthChallenges(DAHC-2018)”onJanuary17-18,2018organizedbyFacultyofVeterinaryScience,UniversityofAgriculture,Faisalabad,Pakistan.2)Participationinonedayseminaron“5DiseasesAilingResearch-andHowtoCureThem”onNovember15,2017organizedbyElsevierPublishingCampus,online.3)Participationin“InternationalParasitologyConference:RecentAdvances&EmergingIssuesinParasitology”onOctober25-26,2017organizedbyPakistanSocietyofParasitologyatUniversityofVeterinaryandAnimalAdvances,Lahore,Pakistan.4)Participationin15days“InternationalTrainingWorkshoponMajorTransboundaryAnimalDiseases(TAD)DiagnosisTechnology”fromSeptember10-24,2017organizedbyDepartmentofInternationalCooperation,MinistryofScienceandTechnology,ChinaatChineseAcademyofAgriculturalSciences,LanzhouVeterinaryResearchInstitute,Lanzhou,China.5)Participationin20days“InternationalTrainingWorkshoponChineseTraditionalVeterinaryMedicineandTechniques”fromJuly10-29,2017organizedbyDepartmentofInternationalCooperation,MinistryofScienceandTechnology,ChinaatChineseAcademyofAgriculturalSciences,LanzhouInstituteofHusbandryandPharmaceuticalSciences,Lanzhou,China.6)Participationintwodays“WorkshopforAppliedPhysiologyandPharmacologicalTechniquesforYoungScientists”onFebruary12-13,2016organizedbyInstituteofPharmacy,PhysiologyandPharmacology,UniversityofAgriculture,Faisalabad,Pakistan.7)Participationinoneday“3rdAnnualWorkshoponScientificWriting&Plagiarism”onNovember20,2014organizedbyOfficeofTheSeniorTutor,UniversityofAgriculture,Faisalabad,Pakistan.8)Participationin3dayshands-ontrainingon“AdvancesinTickandTick-BorneDiseasesPrevention”onAugust5-7,2014OrganizedbyDepartmentofParasitology,UniversityofAgriculture,Faisalabad,Pakistan.78 中国农业科学院博士学位论文CV9)Participationinonedayseminaron“MalariaThreats:PresentScenarioandFutureProspects”onJune15,2014organizedbyDepartmentofParasitology,UniversityofAgriculture,Faisalabad,Pakistan.10)Participationinonedaysymposiumon“TheTicksandTick-BorneDiseases”onMarch15,2014organizedbyMolecularParasitologyLaboratory,UniversityofAgriculture,Faisalabad,Pakistan.Honors:1)InvitedSpeakerattwodays“InternationalConferenceonDairyAnimalHealthChallenges(DAHC-2018)”onJanuary17-18,2018organizedbyFacultyofVeterinaryScience,UniversityofAgriculture,Faisalabad,Pakistan.2)LifetimeMembershipof“PakistanSocietyofParasitology”.3)CertificateofAppreciationbyDirectorGeneral,LanzhouVeterinaryResearchInstitute,ChineseAcademyofAgriculturalSciencesforParticipationinaSixWeeks“BasicEnglishLanguageTrainingforChineseStudents”asResourcePersonfromMay10toJune14,2017.4)Memberof“TheAmericanSocietyofParasitologists”2017-18.5)CertificateofHonorforActivityWorkingGroupforScientificandProfessionalProgressawardedbytheDean,FacultyofVeterinaryScience,UniversityofAgriculture,Faisalabad,Pakistan,2015.6)AwardoffullyfundedChineseGovernmentScholarshipforPh.D.2015.7)AwardoflaptopfromPrimeMinisterofPakistanforEducationalAchievements,2014.8)RegistrationwithPakistanVeterinaryMedicalCouncilasAuthorizedVeterinaryPractitioner,2013.9)QualifiedGraduateAssessmentTest(GAT)-GeneralbyNTSPakistanheldon18thAugust,2013obtaining69/100markswith97.71percentilethroughoutthecountry.10)Editorofcollegemagazine“TheMuse”ofChenabCollegeJhangforthesession2006-2007.79
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