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分类号：密级：＿爭十嫩摩ＨＵＡＺＨＯＮＧＡＧＲＩＣＵＬＴＵＲＡＬＵＮＩＶＥＲＳＩＴＹ硕士学位论文ＭＡＳＴＥＲＳＤＥＧＲＥＥＤＩＳＳＥＲＴＡＴＩＯＮ湖北省儿童和乳牛中隐孢子虫和贾第虫的分子流行病学研究ＭＯＬＥＣＵＬＡＲＩＮＶＥＳＴＩＧＡＴＩＯＮＳＯＦＡＮＤＧＩＡＲＤＩＡＩＮＣＨＩＬＤＲＥＮＡＮＤＣＡＬＶＥＳＩＮＨＵＢＥＩＰＲＯＶＩＮＣＥＣＨＩＮＡ，££研究生：樊ＣＡＮＤＩＤＡＴＥ：ＦＡＮＹＩＮＧＹＩＮＧ学号：２０１５３０２１１０１２８ＳＴＵＤＥＮＴＮＯ．：专业：预防兽医学ＭＡＪＯＲ：ＰＲＥＶＥＮＴＩＶＥＶＥＴＥＲＩＮＡＲＹＭＥＤＩＣＩＮＥ导师：ＳＵＰＥＲＶＩＳＯＲ：ＰＲＯＦＥＳＳＯＲＲＯＢＩＮＢ．ＧＡＳＳＥＲ中国武汉ＷＵＨＡＮ，ＣＨＩＮＡ二〇一八年六月ＪＵＮＥ２０１８， HUAZHONGAGRICULTURALUNIVERSITYMASTER’SDEGREEDISSERTATIONMolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaCandidate：FanYingyingtudentID:XinYangStudentID：2015302110128Supervisor：ProfessorRobinB.GasserGuidinggroup:ProfessorHuMinStudentID:2015302010012DoctorWangTaoSupervisor:ProfessorRobinB.GasserGuidinggroupProfessorMinHuMAJOR:PREVENTIVEVETERINARYMEDICINEFIELD:MOLECAULARPARASITOLOGYDEGREENAME:DOCTOROFAGRONOMYDEGREEGRANTINGTIME:JUNE,2018COLLEGEOFVETERINARYMEDICINEHUAZHONGAGRICULTURALUNIVERSITYJUNE，2018 华中农业大学学位论文独创性声明及使用授权书一￣ＩＳＳＩ如需保密，解密时间年月曰Ｉ丨｜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究工作及取得的研究成果。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究成果，也不包含为获得华中农业大学或其他教育机构的学位或证书而使用过的材料师对此进行了审定一。与我同工作的同志对本研究所做的任，指导教何贡献均已在论文中做了明确的说明，并表示了谢意。《研究生签名（？：时间：２丨发年月５曰学位论文使用授权书本人完全了解华中农业大学关于保存、使用学位论文的规定，即学生必须按照学校要求提交学位论文的印刷本和电子版本，；学校有权保存提交论文的印刷版和电子版＇、并提供目录检索和阅览服务，可以采用影印、缩印或扫描等复制手段保存ｒ编学位论文。本人同意华中农业大学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，同，为存在馆际合作关系的兄弟高校用户提供文献传递和交换服务时本人保留在其他媒体发表论文的权力。注：保密学位论文（即涉及技术秘密、商业秘密或申请专利等潜在需要提交保密的论文）在解密后适用于本授权书。学位论文作者签名：Ｈ导师签名：ｊ签名日期：Ｘ）丨ｇ年《月／日签名日期：年月日／ ThissubjectwassupportedbyHuazhongAgriculturalUniversityScientific&TechnologicalSelf-innovationFoundation(ProgramNo.2015RC005) MolecularinvestigationsofCryptosporidiumandGiardiainhumansandcattleinHubeiProvince,ChinaTableofContentsAbstract...........................................................................................................................i中文摘要.....................................................................................................................iiiCHAPTER1-LITERATUREREVIEW......................................................................11.1.Introduction.............................................................................................................11.2.Historicalbackgroundandsignificance..................................................................21.2.1.Cryptosporidium....................................................................................21.2.2.Giardia...................................................................................................31.3.Cryptosporidiumtaxonomyandclassification.......................................................41.3.1.Nomenclaturehistory.............................................................................41.3.2.Cryptosporidiumspecies........................................................................51.3.2.1Cryptosporidiumspeciesofmammals.................................................51.3.2.2Cryptosporidiumspeciesofbirds......................................................151.3.2.3Cryptosporidiumspeciesofreptiles...................................................171.3.2.4Cryptosporidiumspeciesoffish........................................................191.3.2.4Cryptosporidiumspeciesofamphibians............................................201.3.3.Giardiaclassification...........................................................................201.4.Generallifecycles.................................................................................................221.4.1.Cryptosporidiumspecies......................................................................221.4.2.LifecycleofGiardia............................................................................231.5.Pathogenesis..........................................................................................................231.6.Clinicalmanifestations..........................................................................................241.7.Diagnosis...............................................................................................................251.7.1Conventionaldiagnosis.........................................................................251.7.1.1Directmicroscopy..............................................................................251.7.1.2Flotationmethods...............................................................................251.7.1.3Stainingtechniques............................................................................261.7.2.Immunologicaltools............................................................................261.7.3.Moleculartools....................................................................................271.7.3.1Nested-PCR........................................................................................281.7.3.2RFLPanalysis....................................................................................281.7.3.3Real-timePCR...................................................................................281.7.3.4Multiplexedtandem-PCR(MT-PCR)................................................291.7.3.5OtherPCRmethods...........................................................................291.8.Epidemiology........................................................................................................311.8.1.MolecularepidemiologyofCryptosporidiumspeciesandgenotypesinhumans...........................................................................................................311.8.2.Cryptosporidiumparvumsubtypesandzoonotictransmission...........321.8.2.1Cryptosporidiumparvumsubtypesinhumans,andzoonotictransmission...................................................................................................321.8.2.2.Cryptosporidiumparvumsubtypesinanimals.................................34I HuazhongAgriculturalUniversityMaster’sDegree20181.8.3EpidemiologyofGiardia......................................................................351.8.3.1ZoonoticpotentialofGiardia............................................................401.8.4.Epidemiologicalevidenceforzoonoticcryptosporidiosisandgiardiasis........................................................................................................................421.8.4.1Exposure............................................................................................421.8.4.2Routesoftransmission.......................................................................431.9.RecentinsightsintoCryptosporidiumofcattleinChina......................................451.9.1.CryptosporidiumspeciesandsubtypesindairycattleinChina..........561.9.2.CryptosporidiumspeciesandsubtypesinyaksinChina.....................571.9.3.Cryptosporidiumspecies/subtypesinbeefcattleandwaterbuffaloinChina..............................................................................................................571.9.4.AgedistributionsofCryptosporidiumspeciesincattle.......................631.10.RecentinsightsintoCryptosporidiumofhumansinChina................................631.11.Aspectsoftreatment,prevention,andcontrol.....................................................761.12.Conclusionsfromtheliteratureandfutureprospects.........................................78CHAPTER2-FIRSTSURVEYOFCRYPTOSPORIDIUM,GIARDIAANDENTEROCYTOZOONINDIARRHORICCHILDRENFROMWUHAN,CHINA..792.1.Introduction...........................................................................................................802.2.Materialsandmethods..........................................................................................812.3.Resultsanddiscussion..........................................................................................852.3.1.Cryptosporidium..................................................................................852.3.2.Giardia.................................................................................................872.3.3.Enterocytozoon.....................................................................................882.4.Concludingremarks..............................................................................................89CHAPTER3-MOLECULARINVESTIGATIONOFCRYPTOSPORIDIUMANDGIARDIAFROMPRE-ANDPOST-WEANEDCALVESINHUBEIPROVINCE..903.1.Introduction...........................................................................................................913.2.Materialsandmethods..........................................................................................923.3.Resultsanddiscussion..........................................................................................943.3.1.Cryptosporidium..................................................................................953.3.2.Giardia...............................................................................................1013.4.Conclusions...........................................................................................102CHAPTER4-GENERALDISCUSSION................................................................103References..................................................................................................................109ACKNOWLEDGEMENTS.......................................................................................148CURRICULUMVITAE............................................................................................150II MolecularinvestigationsofCryptosporidiumandGiardiainhumansandcattleinHubeiProvince,ChinaAbstractParasiticprotozoaoftheCryptosporidiumandGiardiaaretwosignificantzoonoticpathogens.Theseprotistsareusuallydirectlytransmittedtothehumanandotheranimalhostsviathefaecal-oralroute,followingtheingestionofinfectiveoocystsorcysts,andcancausecryptosporidiosisandgiardiasis-oftenassociatedwithdiarrhoea.Findingsofrecentepidemiologicalresearchindicatethatzoonotictransmissionplaysasignificantroleincryptosporidiosisandgiardiasis.Cattle,particularlyyoungcalves,infectedwiththeseprotistscanrepresentasignificantsourceofzoonoticinfectionsanddiseases.Nevertheless,epidemiologicalinformationonthespeciesandgenotypesthatoccurinbothhumansandanimalsarelackinginsomeareassuchasHubeiprovince,China.Toaddresstheaboveissues,weconductedthefollowingstudies:InChapter1,Iprovidethesalientbackgroundonthesetwoprotists,keyaspectsofthespecies,lifecycles,routesoftransmission,pathogenesis,diagnosisandthecontrolofcryptosporidiosisandgiardiasis,andsummariseknownspeciesandgenotypes,andprovideaperspectiveonfutureresearchtowardsthepreventionandcontrolofcryptosporidiosisandgiardiasis,guidedbymolecularbiologicalstudies.InChapter2,IconductedastudytodeterminetheprevalenceofCryptosporidium,GiardiaandEnterocytozooninfectionindiarrhoeicchildrenfromWuhan,Chinaforthefirsttime.Faecalsampleswerecollectedforanonymous(individual)childrenwithdiarrhoeaattheOutpatientDepartmentinWuhanPediatricHospitalandRenminHospitalofWuhanUniversity(June2016andAugust2016).ThesurveyshowedthatC.meleagridis,GiardiaassemblageAandEnterocytozoongenotypeDpredominatedinthisstudyofdiarrhoeicchildreninWuhan.Itisnoteworthythat,C.meleagridis(mainlyinfectingavianhosts)wasfoundtodominateinthechildrenstudiedinWuhan,thisfindingprovidethebasicsoftransmissionofthisentericpathogeninhumansinChina.InChapter3,calveswereinvestigatedforCryptosporidiumandGiardiainHubeiprovince,China.Sampleswerecollectedfrompre-andpost-weanedcalves(1to12weeksold)fromonebeeffarmandfivedairyfarmsinthenorthern,eastearnandwesternregionsofHubei(SeptembertoDecember2016).TheaimwastoexplorethespeciesandgenotypesofCryptosporidiumandassemblagesofGiardia.C.bovis,C.i HuazhongAgriculturalUniversityMaster’sDegree2018andersoni,C.bovisandGiardiaassemblageEwereidentifiedandquestionsregardingzoonotictransmissionwerediscussed.Chapter4providesageneraldiscussionoftheresultsachievedandprovidesaperspectiveonimplicationsandfutureresearch.Thisthesiscontributestoknowledgeinthefieldinfectious/parasiticdiseasesinHubei,China,andproposesthatfurtherworkbeconductedtoexploreCryptosporidiumandGiardiaonmuchlargerscaletobetterunderstandtheirepidemiologythroughoutChina.Keywords:Cryptosporidium,Giardia,Human,Calves,PCR-basedsequencing,NuclearribosomalRNAgenes,Hubeiii MolecularinvestigationsofCryptosporidiumandGiardiainhumansandcattleinHubeiProvince,China中文摘要隐孢子虫（Cryptosporidium）和贾第虫（Giardia）是两种重要的人畜共患肠道原虫，它们通常是经过粪-口途径直接进入人或动物的宿主体内，伴随着卵囊的摄取，最终引起人和动物的隐孢子虫病和贾第虫病，并伴随着宿主的腹泻；最近的流行病学研究表明，在隐孢子虫病和贾第虫病中，人畜共患之间的传播起着重要的作用；牛，尤其是乳牛，是作为隐孢子虫病和贾第虫病在人和动物之间传播的重要来源。然而，在一些地区，例如中国湖北省，关于人和犊牛的隐孢子虫和贾第虫的流行病学的数据（比如优势虫种和基因型）却仍是一片空白。为此，本文针对这些问题开展了如下的研究：在第1章中，对这两种原生寄生虫的背景知识、关键的物种、生活史、传播途径、病原体的发病机制、诊断及其防控进行详细的介绍；并总结了已有的物种和基因型。通过对分子流行病学调查的总结，提供了对隐孢子虫和贾第虫病防控的研究方向。在第2章中，对湖北省武汉市腹泻儿童的隐孢子虫、贾第虫和微孢子虫的分子流行病学调查进行了首次报道。在2016年6月至2016年8月，对武汉市儿童医院和武汉大学人民医院门诊部的腹泻儿童新鲜粪便样品共计500份进行了隐孢子虫、贾第虫和微孢子虫的分子流行病学调查，基于SSU基因，tpi基因和ITS基因位点采用Nested-PCR方法对所有样品进行扩增，序列比对结果经遗传进化分析，鉴定出隐孢子虫为C.meleagridis，贾第虫为assemblageA型，微孢子虫的基因型为D型。值得一提的是，在隐孢子虫中，发现了C.meleagridis是该地区的优势虫种，而该虫种主要的宿主是在鸟类中，该发现也为后续对该地区人和鸟之间病原的传播研究奠定了基础。在第3章中，进一步对湖北省犊牛进行了隐孢子虫和贾第虫的分子流行病学调查。在2016年9月至2016年12月，对湖北省北部、东部和西部地区的5个奶牛场和1个肉牛场的1-12周龄的断奶前和断奶后的犊牛进行了采样，旨在探索隐孢子虫和贾第虫的优势虫种和基因型。共鉴定出3种隐孢子虫，分别为C.bovis,C.andersoni和C.ryanae；在贾第虫中，确立了贾第虫的assemblageE型；在该研究中，并针对人畜共患之间的传播问题进行了讨论。在第4章中，对目前所取得的成果进行了综合性的讨论，并提供了对未来研iii HuazhongAgriculturalUniversityMaster’sDegree2018究的视角和方向；本论文对隐孢子虫和贾第虫在中国湖北省的分子流行病学的调查提供了数据参考；获得的数据提示我们，仍需开展更多的流行病学调查和采集更多的样品进行遗传多样性分析，以便更好的了解这些病原体在中国的传播。关键词：隐孢子虫，贾第虫，人，牛，PCR，核糖体RNA基因，湖北省iv MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaCHAPTER1-LITERATUREREVIEW1.1.IntroductionCryptosporidiumandGiardiaaretwoimportantgeneraofparasitesthatcaninfecthumansandalargescaleofvertebratesglobally(Fayer,2004;XiaoandFeng,2008).Generally,infectionwitheitherofthesetwoparasitesisoftenviathefaecal-oralroutetransmission(Korichetal.,1990;Fayeretal.,2000).Diarrhoeaisthemajorclinicalsignofcryptosporidiosisandgiardiasis,togetherwithdehydration,fever,inappetence,nausea,anorexia,malabsorption,weightlossand/orwasting(Fayer,2010;RyanandCaccio,2013).Althoughthetwodiseasesareoftenself-limitinginimmunocompetentindividuals(ChalmersandDavies,2010;FengandXiao,2011),subclinicalinfectionscanoccurininfants,elderlypeopleandimmunocompromisedindividualssuchasHIV/AIDSpatients.Inmanycases,clinicalsymptomscandisappearasimmunitydevelops(Riggs,2002).Furthermore,sincechemotherapyaloneisnoteffective(Zardietal.,2005;CacciòandPozio,2006),cryptosporidiosisandgiardiasiscouldbeseriousordeadly,especiallyinpatientssubjectedtolast-stageHIV/AIDS(Amadietal.,2001).Currently,therehavebeenhundredsofdocumentedoutbreaksofcryptosporidiosisinhumansworldwide,andanappraisaloftheliteratureshowsthatreportsofcryptosporidiosishavebeenreachedmorethan1000recordsandcoveratleast95countries(Fayeretal.,2000).Giardia,isthecommonestintestinalparasiteindevelopingcountriesincludingAsiaandAfrica,andapproximately280millioncasesofgiardiasisreportedperyear(YasonandRivera,2007).In2012,accordingtothejointFoodandAgricultureOrganization(FAO)andWorldHealthOrganization(WHO)expertcommittee(http://www.who.int/foodsafety/micro/jemra/meetings/sep12/en/),Cryptosporidiumwasrankedfifthamongthemost24significantfoodborneparasitesatagloballevel(Ryanetal.,2014).Therefore,cryptosporidiosisisawidespreadandhighlyprevalentdiseaseinhumans.Also,Giardiadistributedgloballycausingapproximately2.8×108casesofdiseaseeveryyear(LaneandLloyd,2002).Indevelopedcountries,cryptosporidiosisisoneofthemainreasonscausingtheoutbreaksofwaterborneandfoodbornehumandiseases(BaldurssonandKaranis,2011;Scallanetal.,2011).Forexample,forcryptosporidiosisintheUSA,in1999-2010,thetotalamountofcryptosporidiosiscasesreportincreasedfrom2,769for1999to19,7941 HuazhongAgriculturalUniversityMaster’sDegree2018for2007(Hlavsaetal.,2005;YoderandBeach,2007;Yoderetal.,2010;Yoderetal.,2012).Forgiardiasis,estimatesofprevalenceintheUSAindicateabout750,000casesannually;fromthesestatistics,wecanconcludethatcryptosporidiosisandgiardiasisareextremelywidespreadandhighlyprevalentdiseasesinhumans.Zoonotictransmissionisalsoveryimportant.Cattle,especiallypre-weanedcalves,canbeamajorcontributortohumaninfections(XiaoandFayer,2008).InChina,withtheexapansionofthelivestockindustry,concernsaregrowingaboutzoonotictransmissionofcryptosporidiosisandgiardiasisinrecentyears.However,therearelimitedmolecularepidemiologicaldataonthespecificand/orgenotypicidentityofCryptosporidiumandGiardiaofanimalsandhumansinsomecountries,includingChina.Theaimsofthisliteraturereviewwereto:(a)provideabriefintroductiononthesetwoprotistsbyaddressingtheirhistory,classification,lifecycle,pathogenesisaswellasdiagnosis;(b)reviewthecurrentepidemiologicalstudiesonthesetwoprotistsinhumansandlivestock,withanemphasisoncattle;(c)summarisethespeciesandgenotypesintheirgeographicdistributionsandprevalenceworldwide,particularlyinChina;(d)comparethedifferenceofCryptosporidiumspecies/genotypesandsubgenotypesdistributedinhumansandcalvesinvariousgeographicregionsacrossChinaand(e)provideaperspectiveonfutureresearchtowardsthepreventionandcontrolofthesetwodiseases,guidedbybiologicalandmoleculardataorparameters.1.2.Historicalbackgroundandsignificance1.2.1.CryptosporidiumCryptosporidiumwasfirstfoundbyTyzzerin1907;heisolatedtheparasiticprotistfromthegastricglandsofmice(Tyzzer,1907),andin1910,henamedCryptosporidiummurisasanewspecies(Tyzzer,1910);later,heprovidedamorecomprehensiveanddetaileddescriptionofthelifecycleofC.muris(Tyzzer,1910).Subsequently,in1912,hediscoveredanothernewspecies,alsofromlaboratorymice,whichwasgiventhenameCryptosporidiumparvum.ThistaxonwasdifferentmorphologicallyfromCryptosporidiummurisinoocystsize(Tyzzer,1912).AftertheinitialreportofCryptosporidiumbyTyzzer,itspathogenicrolewasnotrecognisedandoftenconfusedwithotherapicomplexangenera(e.g.,Sarcocystis)(Xiaoetal.,2004).In1955,Cryptosporidiumwasfirstfoundinadiarrhoeicturkey(Slavin,1955).In2 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China1971,forthefirsttime,Cryptosporidiumwasreportedinthediarrhoeicyoungcalves(Pancieraetal.,1971).Althoughtheparasitewasreportedinawidespectrumofanimals,thediscoveryinhumanswasneglecteduntilin1976,whentwohumancasesofcryptosporidiosisweredocumented(Meiseletal.,1976;Nimeetal.,1976).In1982,whentheCentersforDiseaseControl(CDC)reportedcryptosporidiosisin21AIDSpatientsintheUSA(MMWR,1982).Theroleofthispathogenasaseverepathogenwasestablished.Afurther11casesfrommorethan90countriesandonallcontinentsexceptAntarcticawerereportedoverthenext6years(Ungar,1990).Sincethen,Cryptosporidiuminfectionshavebeenattractedwideattentiononagloballevel(Abeywardenaetal.,2015).1.2.2.GiardiaIn1681,GiardiawasfirstfoundbyaDutchscientist,vamLeeuwenhoek(1632-1723)inhisowndiarrhoeicfaeces(Dobell,1920).In1859,Lambl(1824-1895)rediscoveredGiardiaanddefinedthemorphologicalcharacteristicsoftrophozoite(Lamel,1859).Then,Filice(1952)proposedataxonmicgroupingofGiardia.Hedescribedthemorphometricsofthetrophozoite(Fig.1-1),andnamedthreemajorgroups:G.agilis,G.muris,andG.duodenalis(Filice,1952).Filice’spapermarkedagreatadvanceforward,andwasthebasisforalargenumberofensuingstudies.Since,Giardiawasrecognisedtohavezoonoticpotentialbasedontheepidemiologicalinformationandcross-infectionstudies(WHO,1979).Fig.1-1:Giemsa-stainedtrophozoiteofGiardiaduodenalisshowingmultipleflagella,nucleiandmedianbodies(×400).[FigurecourtesyofNicoletteBinz.].3 HuazhongAgriculturalUniversityMaster’sDegree20181.3.Cryptosporidiumtaxonomyandclassification1.3.1.NomenclaturehistoryTraditionally,thenomenclatureforapicomplexanprotozoawasbasedonhostspecificityandparasiticstagesofendogenousstages.Therefore,Cryptosporidiumwasbasedonhostspecificitytoidentifybefore,forexample,C.agniinsheep(Barkeretal.,1974),C.garnhamiorC.enteriditisinhumans(QadripirandKlose,1985),C.anserinumingeese(ProctorandKemp,1974),C.bovisincalves(Barkeretal.,1974),C.cuniculusinrabbits(InmanandTakeuchi,1979),andC.rhesiinmonkeys(Levine,1980).However,duetoalackofsufficientinformation,thisnomenclaturewasinvalid.Simplybasedonmorphology,oocystscanbeidentifiedbybigorsmall.Therefore,theycannotbedistinguishedbasedonshape.Duringtheperiodfrom1970sto1990s,onlyC.muriswasrecognisedtoinfectgastricglands,whichwasdifferentfromC.parvumthatinfectedthesmallintestine;sointhisbriefperiod,limitedevidencefortransmissionwasavailableforthegenusCryptosporidium.C.parvum(orC.parvum-like)waswidelyfoundinallkindsofmammalsincludinghumanbeings(Tziporietal.,1980).Duringorbeforetheperiod,severalCryptosporidiumparasiteswerenamed,suchasC.felisincats(Ise,1979),C.meleagridisinturkeys(Slavin,1955),C.wrairiinguineapigs(Vetterlingetal.,1971a),whichweredifferentfromC.parvumandC.muris.Additionally,areviewofliteraturerevealedareportthatamong71casesinimmune-compromisedpatientshadcryptosporidiosis(Casemoreetal.,1985);allcaseswerecausedbyhuman-to-humantransmission(anthroponotictransmission).However,among159casesofCryptosporidiuminfectioninimmune-competentpatients,notonlywasanthroponotictransmissiondetected,butalsozoonotictransmissionwasconfirmedin26cases(Casemoreetal.,1985).Later,theexistenceofhuman-to-animaltransmissionforcryptosporidiosisinurbancitiessupportedtheevidencepresentedbyCasemoreandJackson(1984).Subsequently,peoplebasedonmorphologicaldata,nameda“humangenotype”(type1)viahuman-to-humantransmission,anda“bovinegenotype”(type2)viaanimal-to-humantransmission.Thereafter,thesetypeswereformallynamedasC.hominisandC.parvum,respectively.Morerecently,basedonbiologicalcharacterization,otherCryptosporidiumspeciesinbirdsandreptileswerealsonamed,suchasC.baileyi(Currentetal.,1986)andC.saurophilum(KoudelaandModry,1998).4 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaWiththedevelopmentofnewimmunologicalormolecularmethods,manyspecies/genotypesofCryptosporidiumarenowrecognisedandcharacterized.Currently,thereare31speciesofthisparasiteidentified(Table1-1)-18taxaarenotrecognisedasspecies(Ryanetal.,2014).Inaddition,morethan40genotypesofunknowntaxonomicstatusand,thus,havenotbeennamedasspeciesbasedoncriteriaoftheInternationalCodeforZoologicalNomenclature(ICZN).Withtheincreasingmolecularandbiologicalcharacterization,webelievemostofthemwillbefinallyassignedspeciesstatus(RyanandHijjawi,2015).1.3.2.Cryptosporidiumspecies1.3.2.1CryptosporidiumspeciesofmammalsBasedonhostspecificity,the19speciesinfectingmammalsincludeC.andersoni(cattle),C.canis(domesticdog),C.felis(domesticcat),C.hominis(monkeysandhuman),C.muris(rodents),C.parvum(mammalianandhumans),C.wrairi(guineapigs),C.suis(pigs),C.bovis(cattle),C.fayeri(marsupials),C.ryanae(cattle),andC.macropodum(marsupials),C.xiaoi(sheepandgoats),C.ubiquitum(cattle),C.cuniculus(rabbitsandhuman),C.tyzzeri(rodents),C.viatorum(human),C.scrofarum(pigs),C.erinacei(hedgehogsandhorses).Here,eachCryptosporidiumspecieswillbeintroducedseparately.1.3.2.1.1CryptosporidiummurisIn1907,thistaxonwasdiscoveredbyErnestEdwardTyzzer,anditwasdescribedinthestomachglandsofmiceinthelaboratory.Theendogenousphasehasextracellularproperties,withoocystsmeasuring5-7μm,andfaecal-oraltransmissionwasdemonstrated(Tyzzer,1907).Threeyearslatter,Tyzzerreportedthedetailsofthisspecies;heexpandedthegeographicdistributionsofCryptosporidiummurisfromNorthAmericatotheUK,andthelifecycleofallstageswasdescribedinmoredetails.However,intermsofpathologicalchangesininfectedmice,therewasonlythegastricglandsswellingwithoutacuteinflammation.5 HuazhongAgriculturalUniversityMaster’sDegree2018Table1-1:ValidspeciesofCryptosporidiumspp.(31species)SpeciesMajorhostTypehostReferenceC.murisRodents,bactriancamelsMusmusculus(housemouse)Tyzzer(1910)C.parvumRuminants,huamansMusmusculus(mouse)Tyzzer(1912)C.meleageidisBirdsandhumansMeleagrisgallopavo(turkey)Slavin(1955)C.canisCanidsCanisfamiliaris(domesticdog)Fayeretal.(2001)C.hominisHumansHomosapiens(human)Morgan-Ryanetal.(2002)C.bovisCattleBostaurus(domesticcattle)Fayeretal.(2005)C.baileyiBirdsGallusgallus(chicken)Currentetal.(1986)C.felisCatsFeliscatis(domesticcat)Iseki(1979)C.fragileAmphibiansDuttaphrynusmelanostictus(Toad)Jirkuetal.(2008)C.galliBirdsGallusgallus(chicken)Pavlasek(1999)aC.ryanaeCattleBostaurus(Cattle)Fayeretal.(2008)C.scrofarumPigsSusscrofa(Pig)Kváčetal.(2013)Elapheguttata(cornsnake)C.serpentisSnakesElaphesubocularis(ratsnake)Levine(1980)Sanziniamadagascarensus(Madagascarboa)C.xiaoiSheepandgoatsOvisaries(Sheep)FayerandSantín(2009)C.varaniiReptilesandLizardsVaranusprasinus(EmeraldMonitor)Pavláseketal.(1995)C.viatorumHumansHomosapiens(Human)Elwinetal.(2013)C.wrairiRodentsCaviaporcellus(Guineapig)Vetterlingetal.(1971)C.scophthalmicFishesSparusaurataL.andDicentrarchuslabraxL.Alvarez-Pelliteroetal.(2014)C.fayeriMarsupialsMacropusrufus(redkangaroo)Ryanetal.(2008)6 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaC.molnariFishesSparusaurata(giltheadseabream)Alvarez-PelliteroandSitja-Bobadilla(2002)C.andersoniCattleBostaurus(Cattle)Lindsayetal.(2000)C.suisPigsSusscrofa(Pig)Ryanetal.(2004)C.tyzzeriRodentsMusmusculus(Mouse)Renetal.(2012)bC.erinaceiHedgehogsandhorsesEuropeanhedgehog(Erinaceuseuropaeus)Kváčetal.(2014)C.ubiquitumRodents,primatesBostaurus(Cattle)Fayeretal.(2010)C.cuniculusRabbits,humanOryctolaguscuniculus(Europeanrabbit)InmanandTakeuchi(1979)C.viatorumHumansHomosapiens(Human)Elwinetal.(2012)C.rubeyiRodentsSpermophilus(Californiagroundsquirrel)Lietal.(2015)C.huwiFishesPoeciliareticulate(Guppy)Ryanetal.(2015)C.aviumBirdsNovaezealandiae(Red-crownedparakeet)Holubováetal.(2016)C.proliferansRodentsTachyoryctessplendens(Mice)Kváčetal.(2016)Note:aSeeRobinsonetal.(2010).bSeeRyanetal.(2003).cNomoleculardataavailableforthis‘species’.7 HuazhongAgriculturalUniversityMaster’sDegree2018Inthelate1980s,theresearchersusedtheoocystsofCryptosporidiumisolatedfromwildratscapturedinOsaka(Japan)toinfectlaboratorymice,andfoundthattheseoocystsdevelopedsolelyinthegastricglandsofmice.OocystsfromthisfindingwasrecognisedasC.muris,then,incrosstransmissionexperiments,notonlyinfectivetorats,butalsotoguineapigs,rabbitsandcats(Iseki,etal.,1989).Numerousreportsindicatedthatthisparasitesmaynaturallyinfectabroadspectrumofhosts,suchassquirrels,ratsinSiberia,Algerianrats,commonrats,woodmice,bankvoles,procavia,bactriancamel,wildwhitegoats,cynomolgusmonkey,bilby,pigsanddogs(Chalmersetal.,1997;Xiaoetal.,1999a;Morganetal.,2000d;Torresetal.,2000;Dubeyetal.,2002;Warrenetal.,2003;Zintletal.,2007;Lupoetal.,2008).Inaddition,sevenhumancasesreportedinFrance,Kenya,Thailand,andPerurelatedtoC.muris(Guyotetal.,2001;Gateietal.,2002;TiangtipandJongwutiwes,2002;Palmeretal.,2003;Muthusamyetal.,2006).ManycountrieshavefoundtheoocystsofC.murisfoundinthefaecesofcattle,includingtheUSA,Brazil,Japan,Iran,andScotland(Andersonetal.,1991;BukhariandSmith,1996;Penaetal.,1997;Kanetaetal.,1998;Nouri,2002).Duetolackingofgeneticandexperimentalverification,theseauthorsjustnamedtheseoocystsasC.muris-like.Recently,molecularresearchhaverevealedthatC.murisorC.muris-likeparasitesisolatedfrombovinearelikelyC.andersoni(Xiaoetal.,2004).1.3.2.1.2CryptosporidiumparvumIn1912,C.parvumwasfirstfoundinlaboratorymicebyTyzzer.Atthattime,althoughsimilarparasitescouldfoundinrabbitfaeces,itwasstilluncertainwhetherC.parvumcouldinfectotheranimals.C.parvumwasreportedtobedifferentfromC.murisintermsofthesizeofoocystandlocationofinfection(Tyzzer,1912).In2004,itwasreportedthatmorethan150speciesofmammalianhostsharbouredwasrecognisedasC.parvumorC.parvum-likeparasites,butmanyspecieswereconsideredasC.parvumwithoutmolecularcharacterisation.Withthedevelopmentofmolecularbiology,researchersbegantousemolecularmethodstodistinguishC.parvumfromC.hominis,withC.parvumformerlynamedthebovinegenotype(type2)andC.hominisnamedthehumangenotype(type1)(Xiaoetal.,2002;Fayer,2004).Today,C.parvumisknowntoinfectmanyanimals,including,forexample,theArabianantelope,eland,addax,cats,dogs,whitetaileddeer,mice,pigsandhumans(Morganetal.,1999a;Morganetal.,1999e;PerzandLiBlancq,2001;PerzandTrout,2001;Santinetal.,2008;XiaoandRyan,2008).C.parvum,thezoonoticspecies,hasbeenconfirmedtobethecommonestspeciesinpre-weanedcalves(Fayeretal.,2006).8 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaInterestingly,in2006,ŠlapetasuggestedthatC.parvumshouldberenamed“Cryptosporidiumpestis”;thiswasbecause“bovinegenotype”couldinfectcattle,miceandhumans,butthe“mousegenotype”failedtoinfectcattleandhumans.Recently,Cryptosporidiumoocystsfromthefaecesfrompanthers,blackneckedcranes,Barbarysheep,antelope,pandaandchickenswereshown,usingmoleculartools,tobegeneticallythesameas“Cryptosporidiumpestis”,whichindicatedthatthe“mousegenotype”hasawidehostrange(Karanisetal.,2007).WhatthisexampleshowsisthatrenamingC.parvummaycausecontroversyandapotentiallychaoticsituation.Accordingtotheprinciplesofmaintainingtaxonomicstability,popularityofnomenclatureandavoidingconfusion,C.parvumisstillusedasthespeciesnameofthe“bovinegenotype”(Šlapeta,2009).1.3.2.1.3CryptosporidiumandersoniOriginally,accordingtothesizeofoocysts,C.andersoniwasmistakenasC.muris.In2000,Lindsayandcolleaguesbasedontheendogenousstage(parasiticonabomasum),hostrangeandsequencinganalysisof3geneloci,delineateditasaseparatespecies,andthespecieswasnamedafterBruceAnderson(Lindsayetal.,2000).C.andersonicannotinfectoutbredorimmunocompetentorimmunocompromisedmice,chickensorgoats(Lindsayetal.,2000),norinfectioninBALB/Cmice,SCIDmice,commonvoles,Binhaivoles,fieldmice,Mongoliagerbils,desertmarmot,rabbits,orgoats(Koudelaetal.,1998).C.andersoniisdistributedincattleglobally(Enemarketal.,2002;Koyamaetal.,2005;Matsubayashietal.,2005;Fayeretal.,2006;Kváčetal.,2006;Robinsonetal.,2006).TheSSUrRNAandCOWPlociofC.andersoniisolatedfromthebactriancamelissimilartothosefromotherisolates,butthereweresomedifferencesintheactinlocus(homology99.2%).C.andersoniisolatedfromthiscamelspeciescouldnotinfectratsorbovinesbutinfectedMongoliangerbils(Wangetal.,2008).Interestingly,C.andersonihasbeenfoundinhumansinAustralia,China,France,MalawiandtheUK(Leonietal.,2000;Guyotetal.,2001;Morseetal.,2007;Waldronetal.,2011;Liuetal.,2014;Jiangetal.,2014).1.3.2.1.4CryptosporidiumcanisThroughpropagationstudiesandsequencinganalysisofSSUrRNAandhsp70genes,anewparasitehasbeennamedasaspecies,namelyCryptosporidiumcanis(Fayeretal.,2001),whichpreviouslynamedthe“doggenotype”(Casemoreetal.,1985;Morganetal.,2000c).Cryptosporidiumcanisinfectioncanoccurinhumansand9 HuazhongAgriculturalUniversityMaster’sDegree2018bovinesafteringestionofoocysts,butnotinmice(Fayeretal.,2001).SinceC.canisandC.parvumoocystscannotbedistinguishedbymicroscopy,manycanineinfectionswithC.canishadbeenreportedasC.parvum.Inaddition,itisreportedthatC.caniscannaturallyinfectfoxesandcoyotes(Zhouetal.,2004;Troutetal.,2006).Moreover,30sporadiccasesarefoundinhumansinEngland,Jamaica,Kenya,Peru,ThailandandtheUnitedStates(Zhouetal.,2004;Troutetal.,2006;Gateietal.,2008;Santinetal.,2008)hadC.canisinfecion.TheoocystsisolatedfromJapanesedogsweredifferentinsizeandshapefromC.canis,butthesequencingdatashowedatwo-basedifferencefromC.canis,indicatingvariationwithinC.canis(Satohetal.,2006).1.3.2.1.5CryptosporidiumhominisGenotypes1and2ofCryptosporidiumparasitewereshowntobedistinctspeciesusinggeneticmethods,andultimatelynamedC.hominisandC.parvum,respectively(Cacciò,2005).StudiesshowedthattheprepatentperiodofC.hominis(8.8days),whichislongerthanthatofC.parvum(5.4days)(Pereiraetal.,2002).Traditionally,C.hominiswasconsiderednoninfectiveforlambsandcalves,butthisspeciescanbemaintainedexperimentallywithrelativelyhighinfectivedosesofoocysts(Tziporietal.,1994;Mooreetal.,1995;Gilesetal.,2001;Tanriverdietal.,2003;Smithetal.,2005).Morerecently,studiesreportedthat,undernaturalconditions,C.hominisinfectsdugong,calvesandlambs(Morganetal.,2000a;Gilesetal.,2001;Ebeidetal.,2003;Parketal.,2006).C.hominishasbeenshowntobetransmissibletoneonatalpiglets(alsoathighinfectivedoses)(Tziporietal.,1994;Mooreetal.,1995;Pereiraetal.,2002).C.hominishasbeenidentifiedintherhesusmonkeyandclassifiedasCryptosporidiummonkeygenotype(Xiaoetal.,1999b;Xiaoetal.,2002).Clearly,C.hominisisofmajorpublichealthsignificance.1.3.2.1.6CryptosporidiumsuisOriginally,thisspecieswasnamed“CryptosporidiumpiggenotypeI”or“C.parvumporcinegenotype”(Morganetal.,1999a;Enemarketal.,2003)viaanalyzingSSU-rRNA,hsp70andactinloci,itwaseventuallynamedCryptosporidiumsuis(Ryanetal.,2004).Ithasbeendetectedinallagegroupsofpigs,withahigherprevalenceinpigsof<5weeksofage(Ryanetal.,2004),andhasapatentperiodof9-15days(Enemarketal.,2003).Thespeciesdoesnotappeartoinfectnudemice(Morganetal.,1999d),hasbeenfoundrarelyincattle,butthespeciescanbetransmittedexperimentallytocattlewithlownumbersofoocystsexcretedincattlefaeces10 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China(Enemarketal.,2003).Additionally,C.suiswasrecordedincowsandpeople(Xiaoetal.,2002;Geurdenetal.,2006b;Fayeretal.,2006;Leonietal.,2006;Camaetal.,2007).PigletsaremildlyaffectedbyC.suis,despitetheexcretionofhighnumbersofoocysts.However,itcancauseanexacerbationofclinicalsymptoms,andevencausedeath(Enemarketal.,2003).However,thisspecieshaslowzoonoticpotentialandisthusofminorpublichealthsignificance(Šlapeta,2013).1.3.2.1.7CryptosporidiumbovisThisspeciesisfoundincattle(Bostaurus),particularlyinpost-weanedcalves.DuetoC.parvumresemblingC.bovisinoocystsizeandmorphology,itwasoriginallynamedas“CryptosporidiumgenotypebovineB”(Santínetal.,2004).SequencingofthreegeneticlociprovidedevidenceforanewspeciesthatisdifferentfromC.parvum(seeFayeretal.,2005),andwasthennamedC.bovis.TheprepatentperiodofC.bovis(18days)islongerthanthatofC.parvum(4-12days)(Fayeretal.,2005).OocystsofthisspecieswerenotinfectivetoBALB/cmiceorlambsof2-3monthsofage.However,C.boviswasdetectedintwoweek-oldlambs(Fayeretal.,2005).Currently,thespeciesisrecognisedtobedominantincattleglobally.Forinstance,thespecieswascommonindairycattleinsomepartsofAustralia,China,India,Sweden,theUSAandSriLanka(Fengetal.,2007;Silverlaetal.,2010;Ngetal.,2011;Abeywardenaetal.,2014;Zhangetal.,2015).Recently,accordingtoSantínetal(2008),C.boviswasidentifiedtopredominantlyinfect3to14month-oldagedairycalves,withacumulativeprevalenceforC.bovisof80%,butnotbeingassociatedwithovertdisease(Santínetal.,2008).1.3.2.1.8CryptosporidiumryanaeC.ryanaewasformerlyrecognisedasthe“deer-likegenotype”(Santínetal.,2004).Usingthreegeneloci,thistaxonwasrecognisedasanewCryptosporidiumspecies,namedC.ryanae(Santínetal.,2008).Theprepatentaswellaspatentperiodsforthisparasiteare11and15-17days,respectively(Fayeretal.,2008).Actually,thespecieswasnotdetectedindeer,buttheSSU-rRNAsequenceofC.ryanaehadhighhomologytothatofthedeer-likegenotype(Xiaoetal.,2002).TheoocystsofC.ryanaearesmallforanyCryptosporidiumspeciesofmammaliananimals,andtheywereshownnottobeinfectiousto5day-oldBALB/cmiceorlambs(Fayeretal.,2008).IntheUSA,theprevalenceofC.ryanaewas3.8%incattle,beingpredominantincalvesof14weeksto6months(Santínetal.,2008).Inanothersurveyofheifers(1to2yearsofage)inthe11 HuazhongAgriculturalUniversityMaster’sDegree2018USA,1.8%(10/571)wereinfectedwithC.ryanae-beingrestrictedtocattleof12-17monthsofage(Fayeretal.,2006).Atpresent,C.ryanaehasnotbeenfoundanyotherhosts(includinghumans)otherthancattle,andcausesnon-symptomaticinfection(Silverlåsetal.,2010).1.3.2.1.9CryptosporidiumwrairiIn1966,acoccidiumfoundintheguineapig(Jervisetal.,1966)wasnamedCryptosporidiumwrairi(Vetterlingetal.,1971a).Infectionwasnotrelatedtodiarrhoeaorobvioussymptomsofcoccodiosis(Jervisetal.,1966;Vetterlingetal.,1971a).Vetterlingetal(1971b)describedtheasexualandsexualstages,andphotographedtheultrastructureofthesestagesfromfreshmucosalscrapings;thedevelopmentalstagesobservedwererecorded,butoocystswerenotseen(Vetterlingetal.,1971b).Exceptforthematuremicrogametes,fertilizedeggsandoocysts,theultrastructureofallstageswasdescribed(Vetterlingetal.,1971b).C.wrairiappearedtoinfectonlyguineapigs,anddevelopedinthebrushborderofthesmallintestine(distal)whengivento3week-oldrabbits,chickens,turkeysandguineapigsbygastricgavage.Thisspeciesisnotzoonotic;therefore,itisofnopublichealthsignificance.1.3.2.1.10CryptosporidiumfelisIn1979,Iseki(1979)firstdescribedtheoocystsofCryptosporidiumfromthefaecesfromcats.Basedonpreliminaryobservationsofendogenousdevelopmentalstages,thisauthornameditC.felis.Sincethen,C.felishasbeencommonlyfoundinasymptomaticandsymptomaticcatsworldwide(SantínandTrout2008).TheendogenousdevelopmentalstagesofC.felisinvadethesmallintestine,withnodevelopmentalstagesinthestomach,caecumorcolon.Theprepatentandpatentperiodsare5-6daysand7-10days,respectively(Iseki,1979).Oocystscouldnotinfectmice,ratsordogs,butcatswereinfectedsuccessfully(Iseki,1979;Asahietal.,1991).OnecasereportinPolandsuggestedthatacowwasinfectedbyC.felisbasedonthesheddingofoocystsoftheappropriatesize(Bornay-Llinaresetal.,1999).IntheUK,between2000and2008,duringroutinetypingof14,469humancasesofcryptosporidiosis,C.felisrankedthefourthcommonestspecies(Elwinetal.,2012).Accordingtoastudyin2007,58peoplewereinfectedwithC.felis(Raccurt,2007).Cacciòetal(2002)mentionedthattherewere14casesofC.felisinfectionintheAmericas,AfricaandEurope,inimmunocompetentaswellasimmunosuppressedpaitents(Cacciòetal.,2002),indicatingpublichealthimportanceofthisspecies.12 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China1.3.2.1.11CryptosporidiumfayeriInitially,C.fayeriwasrecognisedas“CryptosporidiummarsupialgenotypeI”(Poweretal.,2004),andRyanetal(2008)describedtheCryptosporidiumfrommarsupials,includingkangaroos,wallabiesandkoalasinAustraliaasanewspeciesandnameditC.fayeri.Forthespeciesisolatedfromthefaecesfromredkangaroo,oocystsarenotdistinguishablefromthoseofC.parvum(Ryanetal.,2008).Experimentaltransmissiontolaboratoryanimalswasunsuccessful;nodevelopmentalstageshavebeendescribedindetail,andthepatentperiodisunknown(Ryanetal.,2008).Genetically,C.fayeriappearstoberestrictedtomarsupials(Morganetal.,1999b;Morganetal.,1999c;Poweretal.,2004;Poweretal.,2005),exceptforindividualreportsinsheep(Ryanetal.,2005).AsystematicepidemiologicalsurveyoftheeasterngreykangaroointheAustralianbasindetectedoocystsofCryptosporidiumin6.7%ofkangaroos.Thesequencingdatashowedthat61%ofsamplescontainedC.fayeri(Poweretal.,2005).Inaddition,in2009,anAustraliawomanwasfoundtobetest-positiveforC.fayeri,indicatingthatthisspecieshaszoonoticpotential(Waldronetal.,2010).Thiswasthefirstreportofthisspeciesinhumans,indicatingthatthisspeciesislikelyzoonotic.1.3.2.1.12CryptosporidiummacropodumTheoocystsofC.macropodum(previouslyrecognisedas“CryptosporidiummarsupialgenotypeII”)werefoundinthefaecesofwildeasterngreykangaroosandrecordedasanewspecies(Poweretal.,2004;PowerandRyan,2008).Morphologically,itislikelythatthespeciesisdistinguishablefromotherspecies,suchasC.parvumandC.hominis(Poweretal.,2008),andtheprecisepatentperiodisnotknown.C.macropodumhasbeenfoundinthefaecesfromwesterngreykangaroo,swampwallaby,westerngreykangarooandtheredkangaroo(Poweretal.,2008).Todate,thisspecishasnotbeendetectedinhumans;therefore,thespecieshasnoapparentzoonoticpotentialorpublichealthimportance.1.3.2.1.13CryptosporidiumubiquitumC.ubiquitumwaspreviouslyrecognisedasthe“Cryptosporidiumcervinegenotype”(or“deer”or“genotype3”).Thisparasiteinwidespreadgeographically,andcaninfectabroadspectrumofmammalianhostsandhumans(Fayeretal.,2010).C.ubiquitumwasfirstrecognisedinfaecalsamplesfromsymptomaticpaediatricpatients(lessthan10yearsofage)inonestudyfromCanada(Ongetal.,2002).Itisseemsthat13 HuazhongAgriculturalUniversityMaster’sDegree2018C.ubiquitumisfrequentlydetectedbothindrinkingandwastewatersamples(McCarthyetal.,2008;Chalmersetal.,2010).1.3.2.1.14CryptosporidiumxiaoiOriginally,C.xiaoiwasidentifiedasC.bovis,recordedinsheepinSpain,Tunisia,EnglandandtheUSA(Santínetal.,2007;Navarro-i-Martinezetal.,2007;ElwinandChalmers,2008;Mueller-Dobliesetal.,2008).Onthebasisofdataandparametersonmorphologyandbiology,thiswidelydistributedspecieswasdetectedinsheep,andwasnamedC.xiaoi.TheprepatentandpatentperiodforC.xiaoiinlambsis7-8daysand13-15days,respectively(FayerandSantín.2009).1.3.2.1.15CryptosporidiumscrofarumCryptosporidiumscrofarumhasoriginallyrecognisedasthe‘‘CryptosporidiumpiggenotypeII’’(Ryanetal.,2003).Onthebasisofsequencingdataof3genesloci(SSU-rRNA,actin,andHSP70),itisgeneticallyquitedifferentfromallrecognisedCryptosporidiumspecies,andwasnamedC.scrofarum(Kváčetal.,2013).ThisspeciesinfectsdomesticpigsandEurasianwildboars.PigsinfectedwithC.scrofarumareasymptomatic,andnopathologicaleffectshavebeendetected(Kváčetal.,2013).Attemptstoinfectexperimentalanimalswerealsonotsuccessful(Kváčetal.,2013).Inonereport,a29-yearoldpersonsufferingfromdiarrhoeawasshowntohaveaC.scrofaruminfection(Kváčetal.,2009).ThisreportofC.scrofaruminahumanindicatesthatthespecieshasminorzoonoticpotential.1.3.2.1.16CryptosporidiumcuniculusThespecieswasfirstreportedfromapparentlyhealthylaboratoryrabbitsintheUSA(InmanandTakeuchi,1979).In2010,Robinsonetal(2010)redescribedthespeciesindetails.Experimentalrabbitsshowednoclinicalsigns(Robinsonetal.,2010).Thepatentperiodis7days(Robinsonetal.,2010).During2007-2008,itwasreportedthattheseasonaldistributionofC.cuniculusinfectionpeakedinsummertoautumn(Chalmersetal.,2011).Inaretrospectivestudy,C.cuniculuswaslinkedtoawaterborneoutbreakinthesummerof2008inEngland(Chalmersetal.,2009);thus,thisspecieshaspublichealthsignificance.14 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China1.3.2.1.17CryptosporidiumviatorumRecently,anovelCryptosporidiumgenotypewasidentifiedamongtravelersreturningtoEnglandfromtheIndiansubcontinent,andwasthennamedCryptosporidiumviatorum(Elwinetal.,2012).Theprepatentperiodisunknown,andthepatentperiodis13-30days(median21days)(Elwinetal.,2012).InonecaseidentifiedC.viatorum,clinicalsymptomsincludeddiarrhoea,stomachpain,nauseaandvomiting.ThesignsofthesecasesweredistinctlydifferentfromthosewithC.parvumandC.hominis,forwhichvomitingisreportedless,andgastro-intestinalsignspersistedlonger.Additionally,C.viatorumwasalsorecordedintwopersonswhotravelledabroad(Insulanderetal.,2013).ComparedwithC.hominisorC.pestis,C.viatorumislikelytoinfectpeopleintheagegroupof20-29yearsofage(Hunteretal.,2004;Elwinetal.,2012b).Duetotheanthroponotictransmissionisnotindefinitelyrecordedandlimitedinformationofthisemergingspecies,thus,itisrecognisedofmediumpublichealthimportance.1.3.2.2CryptosporidiumspeciesofbirdsCryptosporidiumhasbeenrecordedinmorethan30speciesofbirdsworldwide.However,C.meleagridis,C.baileyiandC.galliarethespeciesrecordedtodate.Theycanaffecttherespiratorysystem,bursaofFabriciusorthealimentarytract(cf.Lindsayetal.,1990;O'Donoghue,1995;StreterandVarga,2000).BothCryptosporidiumbaileyiandCryptosporidiumgallicaninfectmanykindsofbirds,buttheirhostrangeandparasiticsitearedistinct.Cryptosporidiummeleagridisisfoundinturkeys,andCryptosporidiumbaileyiinchickens.C.tyzzerifromthechickensandC.anserinumfromgeesearerecognisedasinvalid(Fayeretal.,1997).C.tyzzeriappearstobea“juniorsynoym”ofC.meleagridis(Šlapeta,2012).1.3.2.2.1CryptosporidiummeleagridisThefirstavianspeciesofCryptosporidiumwasdescribedbyTyzzerin1929(Tyzzer,1929).However,26yearslaterthatin1955,Slavin(1955)isolatedCryptosporidiumfromwildturkey(Meleagrisgallopavo)poults,andnameditCryptosporidiummeleagridis,whichwaslaterfoundinnumerousspeciesofbirdandmammal(Morganetal.,2000b;Pagès-Mantéetal.,2007;RyanandXiao,2008).TheoocystsofC.meleagridisare4.5-4.0μminsizeandindistinguishablefromthoseofC.parvum(Lindsay,1989).Thespeciesinfectsthesmallintestineandcausesenteritis15 HuazhongAgriculturalUniversityMaster’sDegree2018(Slavin,1955;Pagès-Mantéetal.,2007).C.meleagridisinfectionisusuallyasymptomaticinchickensbutsymptomaticingnotobioticpiglets,leadingtodiarrhoeaandoccasionalmortalities(Akiyoshietal.,2003).ThelifecycleofC.meleagridisispoorlyunderstood,butasexualandsexualphasesweredescribedbySlavin(1955).BothimmunocompetentandimmunosuppressedpeoplecanbesusceptibletoC.meleagridis(Chappelletal.,2011),anditsvirulenceinmammalianhostsseemstobesimilartothatofC.parvumandC.hominis(Akiyoshietal.,2003),whichsuggeststhatmammalsmighthavebeentheoriginalhostsofC.meleagridis(seeXiaoetal.,2002,2004b).AC.meleagridisinfectioncasewasrecordedinadogintheCzechRepublic(Hajduseketal.,2004),butwhethercanidsrepresentnaturalhostsforC.meleagridisremainstobeverified.Cryptosporidiummeleagridisisthethirdcommonestspecies,afterC.parvumandC.hominis,tocausehumancryptosporidiosis(seeLeonietal.,2006;Akiyoshietal.,2012;Xiao,2010).Currently,onthebasisofsequencinganalysesofSSU-rRNAandhsp70loci,thereisevidencethatC.meleagridisistransmissiblefromchickentohumanonafarminSweden(Silverlåsetal.2012).Duetoitszoonotictransmissibility,thisparasitespeciesisofpublichealthsignificance.1.3.2.2.2CryptosporidiumbaileyiAnotherspeciesofCryptosporidium,initiallyisolatedfromabroilerchicken(Gallusgallus)intheUSA,wasnamedC.baileyionthebasisofmorphology(Currentetal.,1986).Itwasidentifiedasavalidspecies,becauseofitsoocystfeatures,inaddition,itdevelopedprimarilyintherespiratorytract(Currentetal.,1986);severeinfectionsdevelopedonlyinthebursaofFabriciusin1-day-oldducks(Currentetal.,1986).Moreover,thelifecyclewasre-describedinmoredetail(Currentetal.,1986;Cheadleetal.,1999).Theprepatentandpatentperiodsare3daysand4-24days,respectively(Currentetal.,1986).Inblack-headedgulls,C.baileyiwasfoundmainlyinthebursaofFabricius,cloaca,colonandrespiratorysites(Pavlásek,1993),butbirdsweremorelikelytobeinfectedinthisbursaandcloaca(Lindsayetal.,1987).Cross-transmissionexperimentsofC.baileyiweresuccessfulinvariousbirdsbutnotthebobwhitequail(Currentetal.,1986;Lindsayetal.,1987,Lindsayetal.,1989).CryptosporidiumbaileyiappearstobeageneralistavianCryptosporidiumspp.inbirds.Theprevalenceisvariable,dependingonhostspeciesandlocality,andassociatedmorewithrespiratorycryptosporidiosis(Goodwin,1989).Birdswithrespiratory16 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,Chinacryptosporidiosisbecomedepressed,anorexicandemaciated,andlossesonfarmsarerelatedtoimpairedgrowthrateanddeaths(LindsayandBlagburn,1990).Thisspeciesdoesnotinfecthumansand,therefore,isofnopublichealthsignificance.1.3.2.2.3CryptosporidiumgalliThethirdavianCryptosporidiumspecieswasinitiallyidentifiedinhens(Pavlásek,1999;Pavlásek,2001).Recently,thespecieswasredescribedfromavarietyofbirdsincludingthedomesticchicken(Ryanetal.,2003b).LittleisunderstoodaboutthelifecycleofC.galli.Differentfromotheravianspecies,C.gallidevelopsintheepithelialcellsofproventriculus(Pavlásek,1999;Pavlásek,2001).Oocystsmeasureapproximately8x6µm,whichisconsiderdlargeforavianspeciesofCryptosporidium(Pavlásek,2001).Cross-transmissionexperimentsrevealedthatoocystsofC.gallicaninfectnine-day-oldchickens(Pavlásek,2001).Moresurveysareneededtoconfirmthehostrangeofthisspecies.C.gallidoesnotappeartobeassociatedwithclinicalsigns,althoughithasbeensuggestedtocausechronicgastritisincanaries,lesserseed-finchesandcockatiel,leadingtomortalities(Antunesetal.,2008).AspeciesofCryptosporidiumreportedinanAustraliandiamondfiretailfinchcausingdiseaseintheproventriculusassociatedwithacutediarrhoeaanddeath,wascalledCryptosporidiumblagburni(cf.Blagburnetal.,1990;Morganetal.,2000a).However,basedonmolecularanalysisusingthethreeloci,Pavlásek(1999,2001)thistaxonwasconsistentwithC.galli.OthermolecularworkhasshownthatC.blagburniandC.galliarethesamespecies(Ryanetal.,2003b).SinceC.galliwaspublishedearlierthanC.blagburni,C.galliissuperiortoC.blagburni.1.3.2.3CryptosporidiumspeciesofreptilesAtpresent,morethan80specieshavebeendiscoveredinsnakes,lizardsandturtlesinfectedwithCryptosporidium.Reptilesfrequentlydevelopchronicinfections.AlthoughhighinfectionratesofCryptosporidiumhavebeenseeninreptiles,mostsurveyshavefailedtoidentifythestructuresofreptilianCryptosporidium.Untilnow,onlytwospeciesofCryptosporidiumarenamed:C.serpentis(thestomachCryptosporidium)andC.saurophilum(theintestinalCryptosporidium).SomegenotypeshavebeencharacterizedsuchastortoisegenotypeIandsnakegenotypesIandII(cf.RyanandXiao,2014).17 HuazhongAgriculturalUniversityMaster’sDegree20181.3.2.3.1CryptosporidiumserpentisCryptosporidiumserpentiswasidentifiedbyLevine(1980)onthebasisofaresearchreportedbyBrownsteinandcolleagues(Brownsteinetal.,1977).AccordingtotheICZNnamingrules,C.serpentisremainedaninvalidnameuntilin1990,whenitwasvalidatedbyTillyetal(1990)whoprovidedthemorphologicalandbiologicaldatatosupportitsstatus.Anumberofreptilianspecies,includingsnakes,lizardsandtortoises,canbeinfectedwithC.serpentis.Differentfromavianandmammaliancryptosporidiosis,infectionwithC.serpentisoftenappearsinmaturesnakes,buttheclinicalsignsareoftensustained,thatistosay,onceinfected,mostsnakesmaintaininfection(Brownsteinetal.,1977).Inlizards,infectionisgenerallyasymptomatic.Infectedhostsareknowntoshedoocystsforyears(CranfieldandGraczyk,1994).During1986-1988,528speciesofreptilesweretestedforCryptosporidium,and14specimensbelongingtoeightgenerafoundtobeinfected(Uptonetal.,1989).MostCryptosporidiumisolatesfromsnakeswereassociatedwithgastriccryptosporidiosis.Forexample,basedontheSSU-rRNAsequencingdatafrom48snakes,24lizardsandthreeturtleeggs,theresultsrevealedninespeciesofCryptosporidium:C.serpentis,C.saurophilum,C.muris,C.parvum,mousegenotype,C.serpentis,respectively,andthreenewgenotypes(Uptonetal.,1989).Morerecently,C.serpentiswasfoundincalvesinChinaandsubsequentlyshowntobeinfectivetomice(ChenandQiu,2012).ThisistheonlydescriptionofthecharacteristicsofC.serpentisisolatefromamammal,althoughpseudoparasitismwouldneedtoberuledoutinthiscase.Nevertheless,futurestudiesshouldbeconductedtoverifythisfinding.1.3.2.3.2Cryptosporidiumvaranii(syn.Cryptosporidiumsaurophilum)C.varaniiwasisolatedfromanemeraldmonitorintheCzechRepublicbutoriginallyfoundinNewGuinea(Pavlaseketal.,1995).Accordingtothemorphologyofoocysts,thehistologicalfeaturesofendogenousstageswereinthesmallintestine.Pavlaseketal(1995)describedthespeciesindetail,withnoevidencethatitinfectedmice(Pavlaseketal.,1995).Developmentstageswereconfinedtotheintestine,particularlythecaudalsection(Pavlaseketal.,1995).Clinicalsigns,ifpresent,havebeendescribedforlizardsandincludemainlyemaciationandanorexia(Terrelletal.,2003).Subsequently,thespecieswasalsodetectedinotherlizardsandsnakes(Pavlaseketal.,2008).In1998,KoudelaandModry(1998)foundanewspeciesfromastudyof220wildandcaptivelizardhostsof67speciesworldwide,andnameditCryptosporidiumsaurophilum(KoudelaandModry,1998).18 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaInterestingly,C.varaniiandC.saurophilumwerecomparedmolecularly(Xiaoetal.,1999a;Xiaoetal.,2004b;Pavlaseketal.,2008),andtheywerefoundtobegeneticallythesame.SinceC.varaniiwaspublishedearlier,C.varanniisusedmorecommonlythanC.saurophilum.1.3.2.4CryptosporidiumspeciesoffishCurrently,duetolackingofmolecularandbiologicaldataaboutCryptosporidiumfromfishandavailablemuseumspecimens,theclassificationofCryptosporidiumspeciesinfishisunclear.Atpresent,onlytwospeciesarerecognisedinfishes,namelyCryptosporidiummolnari(Alvarez-PelliteroandSitjà-Bobadilla,2002)andCryptosporidiumscophthalmi(Alvarez-Pelliteroetal.,2004).Otherspecies/genotypes(n=13)havebenfoundinfishusingmolecularmethods,includingC.parvum,C.hominis,C.xiaoiandC.scrofarum,piscinegenotypes1-8,ratgenotypeIII(cf.Koinarietal.,2013;RyanandXiao,2014).1.3.2.4.1CryptosporidiummolnariC.molnariwasfirstfoundingilt-headseabreamintheMediterraneanandCantabricandAtlanticSpanishwaters(Alvarez-PelliteroandSitjà-Bobadilla,2002).C.molnarifromgilt-headseabreamhasbeentransmittedexperimentallytotheEuropeanperch(Sitjà-BobadillaandAlvarez-Pellitero,2003).Theparasitewasshowntobelocatedintheparasitophorousvacuolesassociatedwithhostcelladjacenttoadoublehostmicrovillarmembrane(Alvarez-PelliteroandSitjà-Bobadilla,2002).C.molnarihasanimportantrelationshipbetweenfishweightandseason,demonstratedinalong-termepidemiologicalstudy.Thehighestincidenceoccurredinthespringtime,followedbysummer,anddecreasedwithincreasingweightoffish(peakinfishof30gto100ginweight)(Sitjà-Bobadillaetal.,2005).C.molnariisdefinedbyitsuniqueSSU-rRNAsequenceandlocationinthestomachoffish(Alvarez-PelliteroandSitjà-Bobadilla,2002;Palenzuelaetal.,2010).Asthespecieshasnozoonoticpotential,itisofnopublichealthsignificance.1.3.2.4.2CryptosporidiumscophthalmiAnotherpiscinespecies,C.scophthalmi,wasfoundin2004inculturedturbotsfromfishfarmsatdifferentsitesinSpain(AtlanticOcean)(Alvarez-Pelliteroetal.,2004).ContrastingC.molnari,thisspecieswasdiscoveredprimarilyintheintestinal19 HuazhongAgriculturalUniversityMaster’sDegree2018epitheliumratheringastricglands.Thespecieswasdescribedbasedonitsintestinallocationandpresumedtohaveastrictspecificitytoturbot(Alvarez-Pelliteroetal.,2004).However,itisconsideredanomennudumuntilitsstatusbecomesclear(cf.Ryanetal.,2014).Inanepidemiologicalstudy,thepoorconditionofyoungfishduringspringorsummerwassignificantlyassociatedwiththepresenceofC.scophthalmi(seeAlvarez-Pelliteroetal.,2009).Thereisnoevidencetosuggestthatthisspeciesistransmissibletohumans.Therefore,ithasnopublichealthimportance.1.3.2.5Cryptosporidiumspeciesofamphibians1.3.2.5.1CryptosporidiumfragileIn2008,Jirkuetal(2008)describedanaturalCryptosporidiuminfectioninthestomachoftheblack-spinedtoad,andnameditC.fragile.ThisisthesolespeciesofCryptosporidiumknowntoinfectamphibians(cf.Valigurováetal.,2008).C.parvumandC.serpentisarenottransmissibletoamphibians(Graczyketal.,1996;Graczyketal.,1998).C.fragilecaninfecttheSouthAfricanfrog;infectedfrogsshowclinicalsignsofweightloss,weaknessand/orhyperplasticgastritis(Greenetal.,2003).Thepatentperiodis22to46days(Jirkuetal.,2008).Todate,therearenosequencedataavailable.Asthespecieshasnoknownzoonoticpotential,itisofnopublichealthimportance.1.3.3.GiardiaclassificationSpeciesofGiardiaareestablishedonthebasisofmorphologicaldataandhostorigin.SpeciesincludeG.agilisinamphibians,G.ardeaeandG.psittaciinbirds,G.microtiandG.murisinrodents,G.duodenalisinmammals(Table1-2)(Thompson,2011).Interestingly,auniquespecies,G.varani,wasreportedinawatermonitor(UptonandZien,1997);however,themajorityofspeciesspecifictoreptilesareofnopublichealthimportance(Magninoetal.,2009).Thus,theseGiardiaspeciescansometimesbedistinguishedonthebasisoftheirmorphologicalfeatures(Adam,2000).G.duodenaliscancausezoonoticinfectionsandisthereforeofgreatestpublichealthsignificance.G.duodenalis,G.intestinalis,aswellasG.lambliaaresynonymousandexchangeableinuseinthepresentliterature(XiaoandFayer,2008).G.duodenalisisusedinthischapter.20 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaUsingmolecularapproaches,G.duodenalisrepresentsmultipleassemblages(A-H)(Table1-3).AssemblagesAandBinfecthumansandhaverelativelybroadhostspecificity.AssemblageAcontainstwosubgroups,AIandAII(Monisetal.,1999;Thompson,2000;Monisetal.,2003;Sulaimanetal.,2003;Readetal.,2004;Cacciòetal.,2005;WielingaandThompson,2007),whileassemblageBcomprisestwosubgroups,BIIIandBIV(Monisetal.,1996).However,assemblages(C-G)areknowntobehost-specific;forexample,assemblagesCandDarecommonlyfoundincanines(Hopkinsetal.,1997;Leonhardetal.,2007),whereasassemblageEisdetectedincattleandotherungulates(Eyetal.,1997),assemblageFhasbeenidentifiedincats,andGisfoundinrodents(Monisetal.,1999).Table1-2:CurrentlyrecognisedspeciesofGiardia.SpeciesHostrangeTrophozoiteDimensionsReferenceLengthWidthGiardiaagilisAmphibians20~30mm4~5mmHegner(1922)GiardiaBirds~10mm~6.5mmNoller(1920)ardeaeGiardiaMuskratsand12~15mm6~8mmBenson(1908)microtivolesGiardiamurisRodents9~12mm5~7mmBenson(1908)GiardiaBirds~14mm~6mmErlandsenandpsittaciBemrick(1987)GiardiaMammalsand12~15mm6~8mmDavaine(1875)duodenalishumansNote:FromRef.Abeywardenaetal.(2015).21 HuazhongAgriculturalUniversityMaster’sDegree2018Table1-3:Geneticgroupings(assemblages)withinGiardiaduodenalisG.duodenalis/assemblagesHostAssemblageAHumans,primates,dogs,cats,cattle,rodents,wildmammalsAssemblageBHumans,primates,dogs,cattle,somespeciesofwildmammalsAssemblageCDogsAssemblageDDogsAssemblageECattleandotherhoofedlivestockAssemblageFCatsAssemblageGRodentsAssemblageHMarinevertebratesNote:FromRef.Abeywardenaetal.(2015).1.4.Generallifecycles1.4.1.CryptosporidiumspeciesThelifecycleofCryptosporidiumisdirect.Oocystsareingested,sporozoitesarereleasedinthesmallintestineandinvadeepithelialcells.Oncetheyareintracellular,theystartthefirstphaseofasexualreproduction.First,theydevelopintotrophozoites,andthentrophozoitesundergothreenuclearfissionsanddeveloptotype-Imeronts(schizonts),whichundergoasexualreproduction.EachofthetypeImerontscontains16merozoites.TypeImerontscontinueschizogonyandrepeatthecycle,ordevelopintotypeIImeronts,andinitiatesexualreproduction.Thisstagecontainsfourmerozoites,eachofwhichproducemicrogamonts(male)ormacrogamonts(female).Oncemicrogametesmature,theyfertilisethemacrogamonts,andthefertilisedmacrogametedevelopsintoazygote,whichsubsequentlydevelopsintoanoocystcontaining,fournakedsporozoites(O’Donoghue,1995;FayerandXiao,2008).22 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaTherearetwotypesofoocysts-thick-walledoocysts(80%),whichareresistantcyststhataremainlyexcretedwiththefaecesandspread,andthethin-walledoocysts(20%),whichremaininthehostandinitiateautoinfection.Onceexcreted,oocystsareinfective,thusenablingimmediatefaecal-oralrotetransmission(deGraafetal.,1999;Koseketal.,2001).1.4.2.LifecycleofGiardiaForGiardia,lifecycleissimpleanddirect.Followingingestionbyasusceptiblehost,cystsenterthesmallintestine,especiallytheduodenum,wheretheyexcystbyfollowigtheactionofgastricacidsandpancreaticenzymes(Thompsonetal.,2008).Excystationreleasestrophozoites.Inthesmallintestine,thetrophozoitesreproduceasexuallyandeitherswimfreelyorattachedtothemucosaoftheintestine.Bothcystsandtrophozoitesarethenpassedthroughintestinaltractandspreadintotheenvironment.Theycanbeinfectiousimmediatelyorshortlythereafter.SomestudieshavesuggestedthatGiardiacanreproducesexually(Birky,2005;Thompson,2011).Atleast10oocystscancauseinfection(Adam,1991).Theseoocystsareoftentakenupviatheconsumptionofcontaminatedfoodorwater.Theycanstayaliveinawet,coolenvironmentforseveralmonths,andcyststhatareresistanttochlorination,ultravioletexposureandfreezing(Birky,2005;Thompson,2011).1.5.PathogenesisCryptosporidiumcaninfectthegastrointestinaltractsofavarietyofmammals,birdsandreptiles,causingmalabsorptivediarrhoea.Inimmunocompetenthosts,Cryptosporidiuminvadesintheepithelialcellsoftheterminaljejunumandileum(Koseketal.,2001),whereasinimmunocompromisedindividuals,thewholegastrointestinaltract,includingbiliaryandpancreaticducts,canbeinfected(HunterandNichols,2002).Duetoparasiteinvasionandinflammatoryreactions,themostpronouncedmorphologicalalterationsinthegastrointestinaltractarevillousatrophy,whichcancausemalabsorptionanddiarrhoea(Koseketal.,2001;Thompsonetal.,2003).EarlysurveysshowedthatC.parvumisfoundtocauseapoptosisinbileandintestinalepitheliacells(Chenetal.,1999;McColeetal.,2000).Bothinnateandacquiredimmunitiesdevelopagainstcryptosporidiosis(Pantenburgetal.,2008;Onetal.,2011).Mucosalepithelialcellsprovideinitial23 HuazhongAgriculturalUniversityMaster’sDegree2018defenceagainstCryptosporidiuminfectionbyproducingdifferentinflammatorymediators(Onetal.,2011).AdaptiveimmunityisactivatedbyrecognitionofspecificantigensbyT-cellsandB-cells.Onceexposed,a/b,bandCD8+,andg/dT-cellscanstimulatetheintestinalvilli,whileinchallengingimmuneanimals,onlyCD8+T-cellswasdetectedinanincreasingnumber(Abrahamsenetal.,1997).Mucosalantibodiesarealsoinvolvedinthecontrolofinfection.Therefore,humoralaswellascellularcomponentsofimmunityplayacrucialroleincontrollingcryptosporidiosis(cf.Abeywardenaetal.,2015).Inaddition,Cryptosporidiumisknowntocauserespiratoryorrenalinfectionsinbirds(O’Donoghue,1995;Ryan,2010).Giardiasisisoftenmultifactorial(O’HandleyandOlson,2006),anddependsonhostandparasitefactors(Cottonetal.,2011).Theparasiteusuallycolonisestheduodenumandjejunumofthesmallintestine.HostdefencesagainstGiardiainvolveanumberofimmunologicalandnon-immunologicaleventsinthegut(Roxstrom-Lindquistetal.,2006),andstudiesinmicehavedemonstratedthatIL-6isnecessaryforearlycontrolofacuteG.duodenalisinfections(Zhouetal.,2003a).1.6.ClinicalmanifestationsTheclinicalsymptomsofcryptosporidiosisandgiardiasismainlyrelatetothehostfactors(includingimmunestatus)andpathogenspecies,variant,virulence,originand/orinfectivedose(Goodgameetal.,1993;Okhuysenetal.,1999).Humanpatientswhoareimmunocompromised(e.g.,HIV-infectedpatients,malnutritionand/ordefectsintheCD40-CD154system)canhaveaself-limitinginfection;itmayleadtochronicenteritis/diarrhoeaandsometimescanleadtodeath(HunterandNichols,2002).Inaddition,theCD4+counthasadirectclinicaleffectoncryptosporidiosisinHIV-infectedpatients.Flaniganetal(1992)notedthatHIV-infectedcaseswithCD4+counts>180μLhaveself-limitingdiseases.Incontrast,inpatientswithCD4+counts<180μL,whodevelopcryptosporidiosis,87%developpersistentdisease(Flaniganetal.,1992).AnotherstudyalsofoundthatCD4+countsoflessthan50μLwereatriskofdevelopingbiliarycryptosporidiosis(Vakiletal.,1996).Therefore,theCD4+countsareconnectedwiththeclinicalcategoryintheseverelyimmunocompromisedgroup.However,cryptosporidiosisinimmunocompetentindividualsisusuallyself-limiting,andalatencyperiodlastsfor5-10days(Pantenburgetal.,2008).Inaddition,wasting,abdominalcramps,fatigue,anorexia,feverandvomitingarecommonsigns(MacKenzieetal.,1994;Koseketal.,2001).24 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaCryptosporidiosisincattleistypicallysymptomaticinneonatesandpre-weanedcalves,butcattleatolderagesareusuallyasymptomatic(Ramirezetal.,2004).Cryptosporidiosisinneonatalcalvesismanifestedbyseverediarrhoea,depression,anorexiaandabdominalpain.Prepatentandpatentperiodsare3-6and4-13days,respectively(Fayeretal.,1998),whichimpliesthatcalvestendtobeinfectedduringorshortlyafterbirth.Clinicalsymptomscanlastfor4-14days(Thompsonetal.,2008).Morbiditiesareoften100(Thompsonetal.,2008).Thedurationofgiardiasiscanrangefromafewdaystoseveralmonths,andintermittentexcretionofGiardiacystshasbeenobservedinbothhumansandanimals(Thompson,2011).Indogsandcats,thedominantclinicalsymptomsofgiardiasisisdiarrhoea,whichtendstobeacuteorchronic,discontinuousorpersistentintheenvioronment.1.7.DiagnosisDiagnosisiscentraltostudyingtheepidemiologyofcryptosporidiosisandgiardiasisandtothepreventionandcontrolofthesediseases.Inthefollowing,conventionalandadvancedmethodsarereviewed.1.7.1Conventionaldiagnosis1.7.1.1DirectmicroscopyInthefirstearlyyearsafterthediscoveryoftheseparasitesinhuman,microscopybecamethemainmethodtodiagnosecryptosporidiosisandgiardiasis.MicroscopicmethodsarepracticalforthediagnosisofCryptosporidiumandGiardiainaclinicalsetting(e.g.,faecaltissue,environmentaland/orwatersamples).1.7.1.2FlotationmethodsTheflotationmethodisusedtoisolateoocystsfromfaecalmatter.Thecommonlyusedsolutionsarezincsulphate,magnesiumsulphate,sodiumchlorideandsugar.Usingdifferentsolutions,thepurity,activityandrecoveryrateofoocystsobtainedvary.Thereareparticularkindsofoocystconcentrationapproaches,includingSheather’ssugarflotationandzincsulfateflotation(Arrowood,1997).25 HuazhongAgriculturalUniversityMaster’sDegree2018Althoughtheflotationmethodusingsaturatedsodiumchlorideissimple,andtheseparationspeedisfast,thepurificationefficiencyandtherecoveryefficiencyarerelativelylow.TheconcentrationbyusingSheather’ssugarflotationandtheviscosityareveryhigh,thusaffectingtheseparationofoocystsfromfaecalmatter.1.7.1.3StainingtechniquesDuetothesmallsizeofoocyst,itisdifficulttodistinguishbydirectmicroscopy.Stainingtechniquescanbeemployedtoaidthedetection.SeveralstainingmethodsareestablishedforshowingtheoocystsofCryptosporidiumandGiardiainstoolspecimens:modifiedacid-faststainingandnegativestaining.Alsomodifiedacid-faststainingsuchasmodifiedKynouinandmodifiedZiehl-Neelsenacidfaststainingareused(seeCacciòandPozio,2014).Acidfaststainingtechniquesarecommonlyusedandeffective;oocystwallsarestainedred,whereastheyeastcellsshowbackgroundstaining(Fig.1-2A)(seeCacciòandPozio,2014),whichhasadvantagesoverotherapproaches.Fig.1-2:(A)Oocystsstainedbythemodifiedacid-fastmethod.Againstablue–greenbackground,theoocystsstandoutinabrightredstain.(B)Oocystsseenwiththedifferentialinterferencecontrastmicroscope.(C)Oocystslabeledwithimmunofluorescentantibodies.(Cacciò&Pozio,2014)1.7.2.ImmunologicaltoolsTodate,theusageoffluorescencemicroscopyanddirectfluorescenceantibody(DFA)testarecommon;wholeorganismsaredetectedusingmonoclonalantibodies(Fig.1-2C).Forexample,themethodsrecogniseepitopesoncysts,andhavebeenreportedtoachieverelativelyhighspecificity(99.8-100%)andsensitivity(93-100%)26 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,Chinaforthetestofcystsinfaecalsmearsandenvironmentalspecimen(Riggsetal.,1983;ZimmermanandNeedham,1995;GarciaandShimizu,1997;Johnstonetal.,2003;Lemosetal.,2005;Mekaruetal.,2007;Rimhanen-Finneetal.,2007).ThemajorrestrictionofDFAassaysisthattheyrequireanexpensivefluorescencemicroscope(seeCacciòandPozio,2014).Thedetectionofantigensoftheseparasitesinstoolspecimen(i.e.,copro-antigens)isasecondmethod.Avarietyofenzyme-linkedimmune-assays(includingELISA)havebeenused(Ungaretal.,1984;Stibbsetal.,1988;ZimmermanandNeedham,1995;Schunketal.,2001;Weitzeletal.,2006;Schuurmanetal.,2007).ELISAisoneofthebroadlyappliedassay,whichisbasedonthedetectionofsolubleantigensofCryptosporidiumandGiardiainfaecalsamplesbyELISA.Theassayisreadilyusedtoscreenlargesamplesizes.However,crossreactionscanoccurwithotherparasites.Theindirectimmune-fluorescentantibody(IFA)detectionmethodismainlyusedforthedetectionandcountingofCryptosporidiumandGiardia,andcanhavehighsensitivity,specificityandrepeatability(Garciaetal.,1987;Garciaetal.,1988;Weberetal.,1991);nonetheless,specificitycanbereducedbycrossreactionswithotherantigensofparasitesandnon-parasiticcomponents,whichproducefalsepositivereactions.Therefore,in2007,Graczyketal(2007)combinedFISHwithIFAmethods,andthusimprovedthespecificityandsensitivityofdetection(oneactiveoocystscanbedetected)(Graczyketal.,2007).Inaddition,IFAcanbeappliedtoserafrompatients(GomezMoralesetal.,1992).Immunochromatographicallateral-flowimmuno-assaysareusedforthediagnosisofCryptosporidiumandGiardiainfections.Thismethodhasbecomeapopulardiagnostictool,becausetheynolongerrequiremicroscopesorexpensiveequipments.Thismethodcanbeperformedin10min,insteadof1-2hbyusingDFAandELISA(seeCacciòandPozio,2014).1.7.3.MoleculartoolsMoleculartools,particularlythosereliantontheamplificationofnucleicacidsinvitro(e.g.,PCRandrelatedmethods),havebeenused.PCRtechniquesofferhighlysensitiveandspecifictests,andcanbeextendedtogeneticallycharacteriseCryptosporidiumandGiardiaspecies,genotypesorassemblages(CacciòandRyan,2008).Anumberofmolecularassssaysareavailable(e.g.,Gasser,2006),andselectedmethodsappliedtoCryptosporidiumandGiardiaaredescribedinthefollowing.27 HuazhongAgriculturalUniversityMaster’sDegree20181.7.3.1Nested-PCRIn1996,Mayeretal(1996)appliednestedPCRmethodtotestthesewageofCryptosporidiumusingCOWPastargetsequencetodetectCryptosporidium,andthesensitivitywas100oocysts/Lsewage(Mayeretal.,1996).In1999,GibbonsandcoworkersusednestedPCRmethodbasedontheDHFRgenetoestablishamethodwithhighersensitivityandspecificityforthetestofCryptosporidium(Gibbonsetal.,1999).Currently,nestedPCRiscombinedwithrestrictionfragmentlengthpolymorphism(RFLP)analysistodistinguishamongCryptosporidiumspeciesandgenotypes.In2004,Coupeetal(2004)appliedNestedPCR-RFLPmethodtodetectandgenotypeCryptosporidiumwith18S-rRNAasthetarget,andthesensitivityreached5x10-5ngDNA,equivalenttotheamountofDNAofanoocyst(Coupeetal.,2004).1.7.3.2RFLPanalysisThismethodisusedbyresearchgroupstoresolveindividualspeciesandgenotypesorassemblages:ForGiardia,thegenetargetincludingSSUrRNA,gdh,bgandtriosephosphateisomerase(tpi)gene;viathesegeneticlociforgenotypingofG.duodenalisaswellasthesequencingperformancearereviewedrecently(Wielingaetal.,2007).Frequently,asidefromtheSSUgene,additionalmarkerscanachieveassemblageorsub-assemblageidentification(reviewedbyWielingaandThompson,2007;FengandXiao,2012;RyanandCacciò,2013).SomecommonlyusedprimersinrecentstudiesfortheidentificationareshowninTable1-4.PCR-RFLPgenotypinghasbeenappliedtoGiardiagenotyping(e.g.,Monisetal.,1996;Homanetal.,1998;Amaretal.,2002).Comparedwiththemicroscopymethod,thisdiagnostictoolprovidesrelativelyhighsensitivityandspecificitythatcandistinguishassemblagesofGiardia(McGladeetal.,2003).Meanwhile,PCR-RFLPhastheabilitytodetectandrecognizetheexistenceofmixedgenotypes.1.7.3.3Real-timePCRReal-timePCR,whichissuperiortoconventionalPCR,includesthefollowingsteps:(1)genomicDNAisisolatedfromfaecalsamples;(2)andregionswithinthesecondprimersspecifillyamplifiedbyreal-timePCR;(3)aftercompletionofPCRand28 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,Chinamelting-curveanalysis,(4)anestimateofthecontributionofindividualCryptosporidiumspeciesandGiardiaassemblagesismade.SeveralsurveyshaveemployedthemethodforGiardia(seeBertrandetal.,2004;Haqueetal.,2007).1.7.3.4Multiplexedtandem-PCR(MT-PCR)Thetechniquecontainstwostages:(i)aprincipal‘targetenrichment’stage,guidedbyemployingdifferentprimerpairs;(ii)anamplificationstage,containingofthetargetedamplification,intandeminsteadofmultiplex,ofallgeneticlociemployingspecific,nestedprimers(Gateietal.,2007).Thistechnologycouldprovidearobustdetectionapproachformultiplexassayswithoutstandingaccuracyandspeed,whichshouldallowforthedesignandimplementationofeffective,integratedmonitoringandsurveillanceprogrammesfortheongoingsafeguardingofwaterandtheenvironment.1.7.3.5OtherPCRmethodsMultilocussequencetypingisatypingtoolthatcanassessthegeneticdiversityofspecies(Gateietal.,2007).Onthebasisofhousekeepinggenesandthevariousmarkersforgenotyping,aMLSTtoolforC.murisandC.andersonihasbeenreportedbyFengetal(2011).Therefore,itwillbeapplicabletosubtypingusingvariousgeneticmarkersincludingmicro-satellitesandotherDNArepeats(RyanandCacciò,2013).MLSTisahelpfulmethodforstudyinggeneticstructures(Cacciòetal.,2008;Lebbadetal.,2010).Inconclusion,majorprogresshasbeenmadetobetterunderstandtheidentificationandgeneticcharacterisationoftheseprotists,andthenbetterdiagnosisofthem.Inparticular,identificationandclassificationofCryptosporidiumhasimprovedthroughtheuseofgenemarkers,suchasSSU,LSUaswellasgp60(Xiaoetal.,1999;Stensvoldetal.,2014;Koehleretal.,2017).ForGiardia,genemarkers,suchasbg,gdh,tpi,incombinationwithvariablelociinSSUgenehavebeenemployed(Table1-4).Inthefuture,awiderangeofmarkersfromgenomescouldassistclinicaldiagnosisincryptosporidiosisandgiardiasis.DiagnosticmethodsshouldofferpowerfultoolstosupportinvestigationsoftheepidemiologyofCryptosporidiumandGiardia.29 HuazhongAgriculturalUniversityMaster’sDegree2018Table1-4:GeneticmarkerscommonlyusedforG.duodenalisgenotypingtools.GenemarkersSequenceofprimers(5'-3')SizeRelativedegreeofLevelofidentificationReference(bp)sequencevariabilitytpiAL3543(AAATIATGCCTGCTCGTCG)605HighSpecies,assemblageandSulaimanetal.(2003)AL3546(CAAACCTTITCCGCAAACC)sub-assemblageAL3544(CCCTTCATCGGIGGTAACTT)532AL3545(GTGGCCACCACICCCGTGCC)gdhGhd1(TTCCGTRTYCAGTACAACTC)754ModerateSpecies,assemblageandCacciòetal.(2008)Gdh2(ACCTCGTTCTGRGTGGCGCA)sub-assemblageGdh3(ATGACYGAGCTYCAGAGGCACGT)530Gdh4(GTGGCGCARGGCATGATGCA)bgG7(AAGCCCGACGACCTCACCCGCAGTGC)753ModerateSpecies,assemblageandCacciòetal.(2002)G759(GAGGCCGCCCTGGATCTTCGAGACsub-assemblageGAC)GiarF(GAACGAACGAGATCGAGGTCCG)511GiarR(CTCGACGAGCTTCGTGTT)SSUrRNARH11(CATCCGGTCGATCCTGCC)292LowSpeciesandassemblageHopkinsetal.(1997)(18sgene)RH4(AGTCGAACCCTGATTCTCCGCCCAGG)GiarF(GACGCTCTCCCCAAGGAC)130GiarR(CTGCGTCACGCTGCTCG)30 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China1.8.Epidemiology1.8.1.MolecularepidemiologyofCryptosporidiumspeciesandgenotypesinhumansUntilnow,morethan20Cryptosporidiumspeciesandgenotypesarefoundinhumans.However,fiveCryptosporidiumspeciesareresponsibleforhumancryptosporidiosis,includingC.hominis,C.parvum,C.meleagridis,C.felis,andC.canis(XiaoandFeng,2008).Amongthem,thefirsttwospeciesaretwodominantspeciesthatinfecthumans,especiallyinindustrializednations(XiaoandRyan,2004).SometimesC.meleagridisisalsoresponsibleforsomecasesinhumans;forexample,itwasdetectedinonestudywiththeinfectionrateashighasC.parvum(Camaetal.,2007),whereastheremainingspeciesincludingC.muris,C.suis,C.andersoniaswellasC.canisareinfrequentlyfoundinhumaninfections(XiaoandFeng,2008;XiaoandRyan,2008).C.hominisandC.parvumareconsideredtobethemainspeciescausinghumancryptosporidiosis,butinfectionandprevalenceratesarevariableindifferentgeographicallocationsglobally.InEuropeancountries,includingCzechRepublic,England,NewZealand,France,Portugal,andNorthernIreland,C.parvumismorelikelytocausemoreinfectioninhumans(57%,onaverage)(McLauchlinetal.,2000;Loweryetal.,2001;Alvesetal.,2003;Hajduseketal.,2004;Learmonthetal.,2004;Ngouanesavanhetal.,2006;Leonietal.,2007);nevertheless,C.hominisisdominantspeciesinhumansincountriessuchasAustralia,Canada,andtheUSA(Pengetal.,1997;Morganetal.,1998;Sulaimanetal.,1998;Ongetal.,1999,2002;Xiaoetal.,2004a).Moreover,C.hominisispredominantinbothpartsofindustrialcountriesanddevelopingcountries(Xiao,2010;Nazemalhosseini-Mojaradetal.2012).Also,thedistributionsofC.parvumandC.hominisinurbanandruralareasarediffer.Inurbanareas,C.hominisinfectionismorecommon,whereasC.parvumisdominantinruralregions(McLauchlinetal.,1999,2000;Learmonthetal.,2004;Feltusetal.,2006).ThehighinfectionrateofC.parvuminhumansinruralareasprobablybecauseofthepoolmanagementandintensivehusbandrypracticesforanimalsandrelatedovercrowedlivingconditions(XiaoandFeng,2008),ormaybeduetothedifferencebetweeninfectionoriginsandtransmissionpatterns(Xiao,2010).31 HuazhongAgriculturalUniversityMaster’sDegree2018Additionally,reportedstudieshaveshownthatindevelopingcountriessuchasinAsia(i.e.,India,ThailandaswellasVietnam),SouthAmericacountries(e.g.,Brazil,Peru)andAfricancountries(e.g.,Kenya,Malawi),70-90%ofhumaninfectionareresultfromC.hominis(Pengetal.,2001;Xiaoetal.,2001;Leavetal.,2002;Camaetal.,2003;Gateietal.,2003;Pengetal.,2003a;Tumwineetal.,2003;Dasetal.,2006;Muthusamyetal.,2006;Bushenetal.,2007).However,thediseaseburdenactonC.parvumislowerthanC.hominis.BecauseoftheinfectionratesofC.parvuminhumansarelowerindevelopingcountries,thisfindingclearlyindicatesthatzoonoticinfectionismoreprevalentinindustrialnationsthandevelopingcountries.Also,dependingongeographicalareas,theprevalenceofhumaninfectionsdiffers.Forexample,theprevalenceofcryptosporidiosisinasymptomaticpeople(e.g.,immunocompetentorhealthyindividuals)hasbeenestimatedtorangefrom0to6.4%indevelopedcountries,andfrom2.3%to7.5%indevelopingcountries(reviewedbyJexetal.,2011).Interestingly,therearealsoseasonalandage-associateddistributionsbetweenC.parvumandC.hominis(seeChalmersetal.,2009;2011).IntheUKandNewZealand,C.parvumwascommoninspringtime(McLauchlinetal.,2000;Learmonthetal.,2004),whereasthelatersummer-earlyautumn,C.hominiswaspredominantinIrelandandtheNetherlands(Wielingaetal.2008;Zintletal.2009),indicatingthatseason-associateddifferenceisofsignificanceinspecifictransmissionroutesmightexist.Also,intheNetherlands,C.hominiswasdomianantinchildren,whereasC.parvuminadults(Wielingaetal.,2008),suggestingthatage-associateddifferencemightalsoexist.1.8.2.CryptosporidiumparvumsubtypesandzoonotictransmissionPresently,morethan14C.parvumsubtypesarerecognised(IIa-IIo).Amongthem,IIaandIIdaretwomaindominantsubtypeinzoonotictransmission,whereasIIcandIIeareanthroponoticsubtypes.Inaddition,therestofsubtypesareinfrequentlyfoundinzoonoticinfections(Xiao,2010;Wangetal.,2011).1.8.2.1Cryptosporidiumparvumsubtypesinhumans,andzoonotictransmissionOfthesesubgenotypefamilies,IIaisadominantsubtypelinkedtozoonotictransmissionglobally.Forexample,itisprevalentinhumaninfectionsinruralregionsintheUSAandEuropeancountries(Glabermanetal.,2002;Alvesetal.,2003;32 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaStantic-Pavlinicetal.,2003;Chalmersetal.,2005;Alvesetal.,2006;Feltusetal.,2006;Ngetal.,2008;Waldronetal.,2009;Zintletal.,2009),IIaA15G2R1,isthemainC.parvumsubgenotypeinhumaninfectioninthesereports(Alvesetal.,2006;SobaandLogar,2008;Wielingaetal.,2008).Inaddition,someoftheIIasubtypesdetectedinhumansalsodetectedincattleinthesameregion.Forinstance,IIaA15G2R1,oneC.parvumsubtypefoundinhumaninfection,alsofoundincattleandruminantsinPortugal(Alvesetal.,2003,2006).Also,inNorthernIreland,IIasubtypesfoundincalvesalsofoundinhumanoutbreaks,withIIaA18G3R1asthepredominantsubtype(Glabermanetal.,2002;Thompsonetal.,2007;Zintletal.,2009).Anothertwosubgenotypes,IIaA16G2R1andIIaA19G3R1,accordingtoareviewbyJexandGasser(2010),havebeenrecordedinbothhumansandanimals,withtheIIaA19G3Rasthepredominantsubtypesinhumansandsecondlymostcommonincalvesworldwide,also,ithasbeenreportedinhumansincountriesincludingAustralia(Jexetal.,2008;Ngetal.,2008;O'Brienetal.,2008;Waldronetal.,2009;Ngetal.,2010a,b),theUK(Bouzidetal.,2010)andNorthernIreland(Glabermanetal.,2002).ThissubtypesbothfoundinhumansandcattleinAustralia(Nolanetal.,2009;Pangasaetal.,2010;Ngetal.,2011),Canada(Trotz-Williamsetal.,2006),theNetherlands(Wielingaetal.,2008)andNorthernIreland(Thompsonetal.,2007).IIaA16G2R1arereportedinhumansincountriessuchasKuwait,theUK,andtheNetherlands(Sulaimanetal.,2005;JexandGasser,2008;Wielingaetal.,2008),andinotheranimalssuchasPortugal,Brazil,NorthernIreland,theUSA,Germany,Spain,theNetherlandsandAustralia(Alvesetal.,2003;Alvesetal.,2006;Meirelesetal.,2007;Thompsonetal.,2007;Xiaoetal.,2007;Brogliaetal.,2008;Quílezetal.,2008b;Wielingaetal.,2008;Nolanetal.,2009),suggestingapossibleconnectiontozoonotictransmission.ApartfromfamilyIIasubtypes,C.parvumsubtypefamilyIIcarealsodetectedfrequentlyandreportedincountriessuchasAustralia,Guatemala,Japan,Kuwait,Malawi,Peru,Portugal,SouthAfrica,Spain,theNetherlandsandUganda(Pengetal.,2001;Leavetal.,2002;Pengetal.,2003;Sulaimanetal.,2005;Abeetal.,2006;Akiyoshietal.,2006;Alvesetal.,2006;Camaetal.,2008;JexandGasser,2008;O'Brienetal.,2008).Uptodate,thesubtypefamilyonlyhasonesubtype:IIcA5G3R2,andonlyhasbeenrecordedinhumansaccordingtothenumberofTCAmicrosatellites,indicatingthatIIcisspecifictohumaninfection(JexandGasser,2010).SubtypefamilyIIdofC.parvum,isalsoberesponsibleforzoonoticinfectionsreportedinAsia,Australia,EuropeancountriesandMiddleEasterncountries(Xiaoand33 HuazhongAgriculturalUniversityMaster’sDegree2018Ryan,2008;Xiao,2010;Wangetal.,2011;Ameretal.,2012;Iqbaletal.,2012;Nazemalhosseini-Mojaradetal.,2012;Imreetal.,2013;Insulanderetal.,2013).Nevertheless,IIdsubtypes,areabsentincattleaswellashumansinCanada,NorthernIreland,andtheUSA(Glabermanetal.,2002;Pengetal.,2003b;Trotz-Williamsetal.,2006;Thompsonetal.,2007;Xiaoetal.,2007).Interestingly,inPortugal,fourIIdsubtypesweredetectedinHIV+patients(Alvesetal.,2003,2006).Therefore,furthersurveysshouldbeconductedinmorecountriestoestablishwhetherIIdplaysaroleinzoonotictransmissionofCryptosporidium.Ofthesesubgenotypefamilies,IIcwasabsentincalves(Alvesetal.,2006);anotherstudyinSloveniaalsorevealedthesamesituation,whichgeneticdiversityofC.parvumismuchhigherinhumans,andofthefivesubtypefamilies,three(Ia,Ib,IIc)wereabsentfromcalves(SobaandLogar,2008).1.8.2.2.CryptosporidiumparvumsubtypesinanimalsToidentifyzoonotictransmissionofC.parvum,amultitudeofstudiesinvestigatedthesubgenotypesofC.parvuminfarmanimals,especiallyincattle.Subgenotypeanalysisonthebasisofsequencingdataforthegp60genehasshownthatIIaisadominantsubtypeincattleworldwide.IIaA15G2R1isparticularlycommoninpre-weanedcalvesinmostareas(Xiao,2010)andisfoundinhumans.IIaA16G1R1isanothermajorsubgenotypeinBelgrade,Serbia,andMontenegro,HungaryaswellasSlovenia.IIaA18G3R1isalsopredominantinNorthernIrelandandAustralia(summarizedinTable1-5).ApartfromfamilyIIasubtypes,IIdisalsoreportedincalvesinsomecountries.Generally,IIdislesscommonlyrecordedthanthezoonoticsubtypeIIa(Xiao,2010).Sofar,ithasonlybeenfoundindairycalvesinBelgium,Belgrade,Egypt,Germany,Hungary,Montenegro,Portugal,Serbia,SwedenandSpainhavebeenfoundindairycalves(Misicetal.,2007;Broglia,etal.,2008;Quilezetal.,2008a;Ameretal.,2010;Silverla˚setal.,2010;Xiaoetal.,2010).Additionally,IIlsubtypesareoccasionallyfoundincountriesincludingtheNetherlands(IIlA24),Belgrade,MontenegroandSerbia(IIlA17);Slovenia(IIlA18R2)(Misicetal.,2007;SobaandLogar,2008;Wielingaetal.,2008)(Table1-5).Forotherfarmanimals,therearefewerreportsofC.parvumsubtypefamilies,butIIaandIIdsubtypefamilieshavebeendetectedinsheepfromBelgiumandPortugal;amongthem,IIdismoreprevalentthantheIIasubtypefamily(Quilezetal.,2008b).34 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaInChina,subtypingofC.parvumfromcattleandhumanshasbeenconducted(Lvetal.,2009;Wangetal.,2011;Zhangetal.,2013;Huangetal.,2014;Wangetal.,2014;Qietal.,2015;Zhangetal.,2015;Lietal.,2016),gp60sequencingdatahaverevealedthatalloftheC.parvumisolatedfromdairycattleidentifiedbelongingtoIId,suchasIIdA15G1inBeijingandNingxia(Huangetal.,2014;Lietal.,2016),IIdA19G1indairycattleinBeijing,HeilongjiangandHenan(Wangetal.,2011a;Zhangetal.,2013;Lietal.,2016).IIdA17G1indairycattleinBeijing(Lietal.,2016),andinterestingly,thisisthefirstsuchsubtypereportedincattleworldwide.Inaddition,fiveIIasubtypeswereidentifiedinyaksinQinghai(Mietal.2013),revealingthespecialcharacterofIIdsubtypeofC.parvuminChina.1.8.3EpidemiologyofGiardiaOftheestablishedGiardiaduodenalis,onlytwogenotypes,namelyassemblageAandBareinfectedtohumans,also,bothgenotypesarefoundinawidespectrumofmammals(Mayrhoferetal.,1995).Therefore,itisnoteworthythatzoonotictransmissionaresupposedtobeimportanceinmolecularepidemiologicalsurveysofhumangiardiasis.However,assemblagesC-Gseemtobehost-specificlimitedtolivestocks(summarizedinTable1-3).ArecentreviewbyRyanandCaccio(2013)onzoonotictransmissionofGiardiahasreportedthattheprevalenceofassemblageAinEngland,France,GermanyandItalyrangedfrom28%to61%.Inaddition,anotherreviewbyXiaoandFayer(2008)havefocusedonassemblagesAandB,particularlyforassemblageA,whichhasbeenfoundinstoolsfromnumerousmammaliananimalsincludingcattle,sheep,dogsandcats.However,incontrast,inthereviewbyRyanandCacciò(2013)indicatedthatassemblageB(~58%)wasmorecommonthanassemblageA(~37%)globally.AssemblageAhasbeendividedtofoursub-genotypes(AI-IV)(Monisetal.,2003).ThemostcommontwosubtypesareAIandAII,whichdifferinhostdistribution.Forexample,humansarelikelytoinfectAII,thoughAIisalsooccasionallydetectedinseveralregionssuchasAustralia,Bangladesh,BrazilandMexico(ReadetAl.,2004;Haqueetal.,2005;Lalleetal.,2005;Volotaletal.,2007).Incontrast,animalsisolatesbelongingtoAI,similarlytheAIIsubtypeisfoundoccasionallyinIndia,PortugalandtheUnitesStates(Traubetal.,2004;Mendoncaetal.,2007;Fengetal.,2008).Similarly,sub-assemblageBI-BIVweredescribedinassemblageB,humanisolatesgenerateBIIIandBIV,whereasanimalisolateswereBIandBII(Monisetal.,2003).35 HuazhongAgriculturalUniversityMaster’sDegree2018Table1-5:DistributionofCryptosporidiumspeciesandC.parvumgenotypesincattleindifferentcountries.LocationIdentifiedspeciesNo.subtypedC.parvumsubtypesMajorsubtypeReferenceUnitedStatesC.parvum(175)175IIaA15G2R1(135),IIaA15G2R2(11),IIaA11G2R1IIaA15G2R1Xiaoetal.(2007)(11),IIaA17G2R1(10),IIaA18G2R1(7),IIaA19G2R1(4)UnitedStatesC.parvum(6),C.bovis(9),C.ryanae(5),6IIaA15G2R1(6)IIaA15G2R1Fengetal.(2007)C.bovis-C.ryanae(3)UnitedStatesC.parvum(110),C.bovis(45),C.ryanae91IIaA15G2R1(91)IIaA15G2R1Santinetal.(2008)(33),C.andersoni(2)CanadaC.parvum(44)36IIaA15G2R1(10),IIaA16G2R1(9),IIaA16G3R1IIaA15G2R1Trotz-Williamsetal.(8),IIaA16G1R1(4),IIaA13G2R1(2),IIaA17G2R1(2006)(2),IIaA18G3R1(1)Belgrade,Serbia,C.parvum(62)18IIaA16G1R1(6),IIlA16(4),IIaA18G1R1(2),IIaA16G1R1MisicandAbe(2007)andMontenegroIIaA20G1R1(2),IIdA18G1b(2),IIlA17(2)AustraliaC.parvum(7)7IIaA15G2R1(2),IIaA18G3R1(5)IIaA18G3R1O’Brienetal.(2008)AustraliaC.parvum(124)122IIaA19G3R1a(80),IIaA20G2R1(9),IIaA20G4R1(9),IIaA23G3R1(9),IIaA16G3R1(7),IIaA18G2R1a(2),IIaA18G2R1b(1),IIaA18G4R1(1),IIaA19G3R1aNolanetal.(2009)IIaA19G3R1b(1),IIaA20G3R1(1),IIaA21G3R1(1),IIaA17G2R1(1)36 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaAustraliaC.parvum(26),C.bovis(28),C.24IIaA17G2R1(17),IIaA18G3R1(1),IIaA20G3R1(6)IIaA17G2R1Ngetal.(2011)ryanae(15),piggenotypeII(1),C.ryanae/C.parvum(1)AustraliaC.parvum(14),6IIaA16G3R1(3),IIaA17G2R1(1),IIaA18G3R1(5),IIaA18G3R1Ngetal.(2008)C.bovis-C.parvum(1)IIaA19G4R1(2),IIaA20G3R1(1),IIaA21G3R1(1),IIaA21G3R1(1)N.IrelandC.parvum(215),C.bovis(8),215IIaA18G3R1(120),IIaA15G2R1(28),IIaA17G2R1IIaA18G3R1Thompsonetal.(2007)C.ryanae(3)(19),IIaA19G4R1(15),IIaA20G3R1(6),IIaA19G3R1(5),IIaA17G3R1(5),IIaA20G5R1(3),IIaA18G2R1(2),IIaA20G2R1(2),IIaA16G3R1(1),IIaA17G1R1(1),IIaA18R1(1),IIaA19G2R(1),IIaA20G4R1(1),IIaA21G2R1(1),IIa-unknown(5)HungaryC.parvum(21),C.ryanae(1)21IIaA16G1R1(15),IIaA17G1R1(3),IIaA18G1R1IIaA16G1R1PlutzerandKaranis(2007)(1),IIdA19G1(1),IIdA22G1(1)BelgiumC.parvum(90),C.bovis(9),90IIaA15G2R1(84),IIaA16G2R1(3),IIaA14G2R1IIaA15G2R1Geurdenetal.(2007)C.suis(1)(1),IIaA13G2R1(1)IIdA22G1(1)PortugalC.parvum(72)72IIaA15G2R1(61),IIaA16G2R1(7),IIdA17G1(4)IIaA15G2R1Alvesetal.(2006)SpainC.parvum(147),C.bovis(2)140IIaA15G2R1(106),IIaA16G3R1(14),IIaA18G3R1IIaA15G2R1Quilezetal.(2008)(8),IIaA16G2R1(4),IIaA19G3R1(2),IIdA23G1(2)SpainC.parvum(27)27IIaA15G2R1(26),IIaA13G1R1(1)IIaA15G2R1Díazetal.(2010)37 HuazhongAgriculturalUniversityMaster’sDegree2018GermanyC.parvum(53)53IIaA15G2R1(43),IIaA14G2R1(2),IIaA17G2R1IIaA15G2R1Brogliaetal.(2008)(2),IIaA18G2R1(2),IIaA21R1(1),IIaA22G1R1(1),IIaA16G1R1(1),IIdA22G1(1)EgyptC.parvum(24),C.andersoni(2)26IIdA20G1(23),IIaA15G2R1(1)IIdA20G1Ameretal.(2010)ItalyC.parvum(62)62IIaA15G2R1(34),IIA18G2R1(10),IIaA17G2R1(9),IIaA15G2R1Durantietal.(2008)IIaA14(5),IIaA13(4)EnglandC.parvum(50),C.bovis(3),51IIaA15G2R1(35),IIaA17G1R1(7),IIaA16G3R1IIaA15G2R1Brooketal.(2009)C.ryanane(1)(4),IIaA19G1R1(2),IIaA18G1R1(2),IIaA14G2R1(1)EnglandandC.parvum(29),C.bovis(2)13IIaA17G1R1(10),IIaA15G2R1(3)IIaA17G1R1Smithetal.(2010)WalesEgyptC.parvum(24),C.andersoni(2)26IIdA20G1(23),IIaA15G2R1(1)IIdA20G1Ameretal.(2010)SwedenC.parvum(15),C.bovis(83),73IIaA18G1R1(2),IIaA21G1R1(2),IIdA20G1e(2),IIaA18G1R1Silverla˚setal.(2010)C.ryanae(10),C.andersoni(2)IIdA23G1(2),IIdA16G1(1),IIaA16G1R1(1),IIdA22G1c(1),IIaA15G1R1(2)NetherlandsC.parvum(160)129IIaA15G2R1(89),IIaA17G1R1(14),IIaA16G3R1IIaA15G2R1Wielingaetal.(2008)(6),IIaA13G2R1(2),IIaA14G2R1(2),IIaA17G2R1(2),IIaA18G4R1(2),IIaA18R1(2),IIaA19G2R1(2),IIaA11G2R1(1),IIaA12G2R1(1),IIaA16G1R1(1),IIaA16G2R1(1),IIaA18G3R1(1),IIaA19G1R1(1),IIaA21G3R1(1),IIlA24(1)38 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaSloveniaC.parvum(45),C.bovis(3),45IIaA15G2R1(27),IIaA16G1R1(6),IIaA13R1(5),IIaA15G2R1SobaandLogarC.ryanae(3)IIaA16R1(3),IIIA16R2(2),IIlA18R2(2)(2008)JapanC.parvum(3)3IIaA15G2R1(3)IIaA15G2R1Abeetal.(2006)IndiaC.parvum(1),C.bovis(11)8IIaA15G2R1(5),IIaA13G2R2(1),IIaA14G2R1a(1),IIaA15G2R1Fengetal.(2007)IIaA14G2R1b(1)39 HuazhongAgriculturalUniversityMaster’sDegree20181.8.3.1ZoonoticpotentialofGiardiaZoonotictransmissionbetweencattleandhumansarefoundinseveralsurveys(e.g.,Uehlingeretal.,2006;Khanetal.,2011).Forexample,inonesurveyinWestBengal,genotypeA1wasrecognisedincattleandworkersatthesamedairyfarm,suggestingthatthereisapossibletransmissionofassemblageAbetweencattleandhumans(Khanetal.,2011).Besidesdirectlycontactedwithanimals,transmissionofzoonoticassemblagesfromcattleisalsolikelytooccurthroughthecontaminationofdrinkingwater(FengandXiao,2011;Budu-Amoakoetal.,2012).1.8.3.1.1GiardiainfarmanimalsAmajorityofcattle,sheepaswellaspigsweremainlyinfectedwithassemblageE(Barigyeetal.,2008;Thompsonetal.,2008;XiaoandFayer,2008;FengandXiao,2011;Fayeretal.,2012).Ofthe562samplesofbovinestudiesinEurope,422werepositivefortheassemblageE(Sprongetal.,2009).AlongitudinalsurveyofcattleinanAustralianfarmshowedthat100%calvesinfectedwithGiardiawithin12weeksandallofthemwereassemblageE(Becheretal.,2004).ThoughassemblageEmainlypredominatesincattle,assemblageAisincreasingbeingfoundinBelgium,Europe,FranceGermany,Italy,UKandtheUSA(Geurdenetal.,2008;Sprongetal.,2009;Santínetal.,2009;Geurdenetal.,2012).Forexample,Geurdenetal.(2008,2012)conductedmolecularepidemiologicalsurveysincattleinBelgium,France,Germany,Italy,andtheprevalenceofassemblageArangedfrom61%to59%.Furthermore,31%and32%prevalencehadamixedgenotype(assemblagesAandE)usingtheassemblage-specificPCRin2009and2012,respectively,suggestingthatcalvesmightbeunderestimatedasapotentialhostforhumangiardiasis.AssemblageBwasnotoftendetectedinanimals(FengandXiao,2011);exceptinNewZealand,besidesassemblageA,assemblageBwasalsofoundincattle,whereasassemblageEwasnotidentifiedatall(Huntetal.,2000;Winkworthetal.,2008).However,recently,asurveyisolatesfromNewZealandfoundthatbesidesassemblageA,assemblageEwasalsoidentified,whichisthefirstreportofthisassemblageinNewZealand,suggestingdairycattleinthisregionhave‘zoonotic’genotypes,thusitmaybeofsignificancetopublichealth(Abeywardenaetal.,2012).Recently,anothersurveyconductedincattleinHeilongjiangprovinceidentifiedanoverallinfectionrateof5.2%(42/814)forGiardia,ofwhich42.9%(18/42)wereassemblageB(Liuetal.,2012).Inadditiontothisregion,inNingxiaAutonomousRegion,bothassemblageEand40 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaassemblageBfoundinpre-weanedcalves(Huangetal.,2014).Forsheepandgoats,theyareresponsibleforassemblageE(Ruizetal.,2008;FengandXiao,2011),also,assemblageAaredetectedinnumerouscountriessuchasAustralia,Italy,theUSA,JapanandChina(Abeetal.,2005;Giangasperoetal.,2005;Aloisioetal.,2006;Abeetal.,2010;Nolanetal.,2010;Zhangetal.,2012).AlthoughassemblageBisnotdominantinthesetwohosts,recently,ithasbeenfoundinsheepinseveralnationssuchasChina,Norway,andItaly(Aloisioetal.,2006;Robertsonetal.,2010;Zhangetal.,2012).Forpigs,bothassemblagesAandEaredetectedinthehost,withassemblageEbeingfoundthecommonest(FengandXiao,2011).ForassemblageB,itwasrarelyreported(Sprongetal.,2009).Nevertheless,recently,asurveywasconductedinswinefarmsinCanada.ForGiardia,92.1%isolatedwereassemblageB,whereas7.9%wereassemblageE(Farzanetal.,2011).ThisfindingindicatesthatassemblageBislikelytohavezoonotictransmissionbetweenpigsandhumans.1.8.3.1.2GiardiaincompanionanimalsRecentgenotypingsurveysofGiardiaincompanionanimalssuchasdogsandcatshaveidentifiedthereservoirsofseveralzoonoticassemblagesexistintheseanimals,suchasassemblageA,B,CandD(FengandXiao,2011).Withanincreasingamountofpetshopsorclinicsareexpandedwordwide,however,someofwhichlackofpropermanagementaswellasgoodhygienemeasures,andthusconsciousnessandpreventionofzoonoticdiseasesisofincreasingsignificance.Fordogs,earlystudiesshowedthatdogsmainlyinfectedwithGiardiaofassemblagesCandD(Itagakietal.,2005;Palmeretal.,2008;Overgaauwetal.,2009).However,recentstudiesshowedthatassemblageBwasrarelyfoundindogs.SomeofthedogsmaybeinfectedwithassemblageB,thereasonsproballyisthatdogsingestingtheirowners’faecesanddisseminatingtheoocystsofGiardia(Traubetal.,2003).Presently,aswithotheranimals,dogsaremainlyinfectedwiththesub-assemblageAI,Forexample,inonestudyinEurope,itwasreportedthatsub-assemblageAIin73%of120isolatesand27%wasAII(Sprongetal.,2009).SeveralstudiesconductedinArgentina,India,Italy,Thai,andThailandhaveshownthatpotentialzoonotictransmissionbetweendogsandhumans(Traubetal.,2004;Inpankaewetal.,2007;Minvielleetal.,2008;Traubetal.,2009;Marangietal.,2010).Forexample,asurveywasconductedfromhumansanddogsinBangkokandThailand.ThelargestproportionofGiardiaisolatedfromdogspopulationwasassemblageA,whilehumanisolateswere41 HuazhongAgriculturalUniversityMaster’sDegree2018assemblagesAandB,indicatingthatdogsharboredazoonoticpotentialtohumaninfection(Inpankaewetal.,2007).InasecondreportinsouthernItaly,whichcollectedsamplesfromaRomcommunityinacamp,identifiedGiardiaassemblageA199.5%homologyfromdogs(n=8)andhumansamplestyped(n=6)(Marangietal.,2010).Theseresultsrevealedthepresenceofpotentiallypublichealthriskofgiardiasisindogsseemdtobesignificance,butitneedsmoretesting.Inaddition,duetothedifferentlivingconditions,thedistributionofassemblagesofGiardiaindogsaredifferent,suggestingthattwotransmissionrotesmayexist(ThompsonandMonis,2004).Forcats,mainlyinfectedwithassemblagesAandF,withassemblageFbeingthecommonest(FengandXiao,2011).ForassemblageA,itwasrecognisedsub-assemblagesAIIandAIIIincatsaccordingtoMLSTtechnique(Caccioetal.,2008;Sprongetal.,2009;Lebbadetal.,2010;Suzukietal.,2011).Currently,somesurveysalsodetectedassemblageBincats(Readetal.,2004;Palmeretal.,2008;Sprongetal.,2009;Jarosetal.,2011;McDowalletal.,2011),orassemblageDincats(Readetal.,2004;Jarosetal.,2011).1.8.3.1.3GiardiainwildmammalsBeaversarepossiblyrecognisedastheanimalsourceofhumangiardiasis,thusleadtheWorldHealthOrganization(WHO)tolistgiardiasisaszoonosis(Thompson,2004).Twostudiesscreenedfaecalsamplesfromcompanionanimals,aquaticwildlife(e.g.,beaversandmuskrats)andonerat,identifiedassemblageBatthetpilocusinallofbeavers(n=11)(Sulaimanetal.,2003;Fayeretal.,2006).AnotherstudyconfirmedtherecognitionofassemblageAinbeavers(Appelbeeetal.,2002).1.8.4.Epidemiologicalevidenceforzoonoticcryptosporidiosisandgiardiasis1.8.4.1ExposureGenerally,thetransmissionofthesetwoparasitestohumansareusuallyoccurredbywhichtheoocystsareexcretedinfaecesfrominfectedhosts,andhavetheabilitytoinfectsusceptiblehostsbyswallowing.42 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China1.8.4.2RoutesoftransmissionCryptosporidiumandGiardiaoocystsremarkablystableforlongperiodsandcanremaininfectiveformonthsuptooneyear,particularlyincoldandmoistenvironments(Smithetal.,2006).Oocystsimmediatelypossesstheinfectionwhenexcretedinfaeces.Inaddition,thedosesofinfectiontotheirhostsareverylow(<10oocysts)(Rendtorff,1954;Okhuysenetal.,1999).Humanscanobtaininfectiondirectlyfromcontactingwithinfectedhumans(human-humantransmission)oranimals(human-animaltransmission),orindirectlyfrompollutedfoodborneorwaterbornesources(Smithetal.,2007).1.8.4.2.1Person-to-person(anthroponotic)Casesofhuman-humantransmissionarerecordedinfamilymembers,sexualpeers,hospitalemployeewiththeirpatients,childreninnurserycentersorpeopletotravelplaceswherediseasesareendemic.Amongthem,childrenindaycarecentersisthemostcommon(PickeringandEngelkirk,1990;Artiedaetal.,2012;Fengetal.,2012),mainlybecauseofthelowerstandardsofpersonalhygienichabits.Forexample,asurveyconductedbyNascimentoetal(2009),inapublicdaycarecenterinRecife,amongthe182samplesanalyzed,59(32.4%)werepositiveregardingthepresenceofoocystsofCryptosporidium,thedirectcontactbetweenchildrenorbetweenchildrenandstaffarethemainriskfactorsofthehighfrequencyofsamplespositiveinthedaycare-centers.ThemostimportantinfectionrouteforCryptosporidiumisperson-to-persontransmission,whichiswellillustratedindaycarecenters.Additionally,travelerstoareaswherediseasesisendemicalsoeasytoinfectcryptosporidiosisandgiardiasis.Forexample,intheUnitedKingdom,Cryptosporidiuminfectionwasdetectedmorefrequentlyinpeoplewithahistoryoftravelingabroad(McLauchlinetal.,2000;Gohetal.,2004;Hunteretal.,2004,2007).Indeed,duringafootandmouthdiseaseoutbreakintheUK,duringthisperiodinSpring2001,strictrestrictionontheaccesstovisitingfarmsandcullingfarmherds,sharplyreducedCryptosporidiuminfectioninhumansafter2001(Hunteretal.,2003;Smerdonetal.,2003).1.8.4.2.2.Animal-to-personandviceversa(zoonotic)Cryptosporidiuminfection,whichiscausedbydirectcontactbetweenhumansandanimals,occursmainlyinfarmfarmers,petbreeders,vets,andfarmvisitorsand43 HuazhongAgriculturalUniversityMaster’sDegree2018correspondinganimals.Izadietal(2014)investigatedtheprevalenceinIrandairyfarmsaswellasindividualsworkinginthesefarmsandtheirhouseholdmembers,foundthattheprevalencefarmerswiththeirhouseholdmembers,andthecalves,was8.5%,14.2%,respectively.Furthermore,C.parvumwasthepredominantspecies,indicatingpossiblezoonotictransmissioninthesefarmsexisted.ContactingwithcalveswasthemostimportantriskfactorofC.parvuminfection(Izadietal.,2014).Additionally,zoonoticoutbreaksofC.parvuminfectioninveterinarystudentsarewelldocumented(Pohjolaetal.,1986;Reifetal.,1989;Preiseretal.,2003;Kiangetal.,2006;Gaitetal.,2008;Grinbergetal.,2011;Rimšelienėetal.,2011;Langeetal.,2014;Galuppietal.,2016).Forexample,in2009andin2012,NorwegianschoolchildrenwereinfectedintwooutbreaksofCryptosporidium,andtheresultsshowedthatcontactwiththefarmanimalsmaybethemaintransmissionofCryptosporidiuminfection(Rimšelienėetal.,2011;Langeetal.,2014).1.8.4.2.3.FoodbornetransmissionFoodisanothermajorvectorofCryptosporidiumandGiardiatransmissiononanumberofoccasions,Foodmaycausecontaminationwithinfectedstageinsomefruitsandvegetablecropsgrowingstages;application,irrigation,andevenfarming,cancauseinfectedbythesetwoprotists.Also,duringharvesting,packing,transportation,sellingand/orpreparationunderunhygienicconditionsmayleadtocontaminatedfoodofinfectedstages(Escobedoetal.,2010;RobertsonandChalmers,2013).Inaddition,duetoeatingfruitsnotcompletelycleanorvegetablesnotcookedandshellfishseafood(Ethelbergetal.,2009;Pönkaetal.,2009;Gherasimetal.,2012;Halletal.,2012),drinkingunpasteurizedmilkandfruitwineandeatingrawmeatcanalsoresultintheoutbreakofCryptosporidiumandGiardia(Gelletlieetal.,1997;Yoshidaetal.,2007).Forexample,inJapan,anoutbreakbycryptosporidiosisandgiardiasiswascausedbyeatenrawmeatdishandrawliver(Yoshidaetal.,2007).1.8.4.2.4WaterbornetransmissionWaterbornetransmissionisparticallyimportantmeansassociatedwithcryptosporidiosis,becausetheoocystsofCryptosporidiumareresistanttousedwaterpurification(e.g.,chloride)(Peetersetal.,1989;Carpenteretal.,1999).Amongthem,drinkingandrecreationalwaterarebothtwomainmediaforwatertransmission.AccordingtoareviewbyD'Antonioetal.(1985),anoutbreakofgastroenteritisoccurredinasuburbregioninTexasin1984,thecryptosporidiosisoutbreakdatedback44 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,Chinatothecommunitywatersupplypollution(D'Antonioetal.1985).Subsequently,inmanycountriesaroundtheworld,outbreaksofcryptosporidiosisrealatedtodrinkingwaterareemergingglobally(Hayesetal.,1989;Richardsonetal.,1991;Willocksetal.,1998;Widerströmetal.,2014).Amongthem,themostnotablemassiveoutbreakhappenedinWeissinWisconsin,Milwaukeecity(USA)in1993,whichwascausedbyCryptosporidiumoocyststhatflewthroughthefiltrationsystemofurbansewagetreatmentplant,finallycausing403,000personshadwaterydiarrheasuspectecdinthisincident,andcausedmorethan100peoplefatalitiesinthisoutbreak.Otherthandrinkingwater,recreationalwateralsoasanimportantmediumtothetransmissionofcryptosporidiosisandgiardiasis.Suchasswimmingpools,divingpools,waterparksandfountains.AccordingtoareviewbyBaldurssonandKaranis(2011),CryptosporidiumandGiardiahavebeenresponsiblefor120(60.3%)and70(35.2%)of199waterborneoutbreaksfrom2004to2010.Inaddition,between1992and2001,therewereonly29outbreaksassociatedwithswimmingpoolwater,butduring2002-2011,thenumberofoutbreaksassociatedwithswimmingpoolwaterhadrisento56(BaldurssonandKaranis2011),suggestingthattheoutbreakscausedbyrecreationalwaterwasslightlyincreasingglobally.1.9.RecentinsightsintoCryptosporidiumofcattleinChinaInChina,bovinecryptosporidiosisgainmoreincreasingconcernduetocattle,particularlycalves,aresignificantsourceofthesepathogens,whichisbecomingapotentialdangertopublichealthinrecentyears.AlthoughnumeroussurveyshavereportedCryptosporidiuminfectionsoncattleandhumans.Uptodate,therearenoefficienttherapeuticscheduleandvaccinesaccessibleagainstcryptosporidiosisinChina.Thus,byreviewingallavailablepublishedrecordsanddatainEnglishandChineselanguage,theaimsofthissectionarefocusonidentifytheprevalence,genotypes,andsubgenotypesofCryptosporidiumincattleandhumansinChina,assessageandbreed-relateddiscrepancyintheprevalenceincattle,andevaluatedifferenceintheregionaldistributionsofCryptosporidiumincattleandhumansinChina,andtobettergraspthezoonotictransmissionandelucidatepublichealthimportance.Meanwhile,weexploredtherelationshipbetweensubtypesandbreedsofcattle,thedatawillbehelpfultoavoidorreducetheoccurrenceofcattleandhumancryptosporidiosisinChinabymakingefficientcontrolstrategiesbasedonmolecular45 HuazhongAgriculturalUniversityMaster’sDegree2018resources.TheywillalsobevaluabletoassesstheriskthatcattleinfectedwithCryptosporidiumposeathreattohumans.Additionally,publichealthyauthorities,governmentalprofessionals,andcommonpeoplecanmakesomerecommendationsforfutureresearchtowardspreventingandcontrollingthisimportantprotist.In1986,Cryptosporidiumwasfirstisolatedfromcattleincasesofpre-weaneddiarrheiccalvesinLanzhou,GansuProvince(Chenetal.,1986).Sincethen,numerousreportsofcryptosporidiosisindifferentregionsofChinahavebeenpublished.Inrecentlyyears,withthedevelopmentofcattleindustryinChina,therearesomeconcernsthatcryptosporidiosisincattlearethreateningthesafetyofcattleandhumanbeings.Thusfar,accordingtotheavailablemolecularepidemiologicalstudiesofpublishedEnglishandChineseliteratures,whichsearchedPubMedandtheChinaNationalKnowledgeInfrastructure(CNKI).Here,thedatalistedinthispartwereoriginatedfrompeer-reviewedstudypublishedinChinese.Inordertoevalutetherichessbetweentheinfectionandspecies/genotypesofCryptosporidium,allpublishedrecordsdescribingtheinformationwerecompiled(Table1-6).Cryptosporidiumspp.aredistributedwithin21provinces,3municipality(Beijing,Shanghai,Tianjin)andTaiwaninChina,mainlyinthenortheast,northwestandcentralregionsofChina(Fig.1-3),includingnorthernChina(InnerMongoliaandTianjin)(Yangetal.,2004;Mengetal.2007);northeasternChina(e.g.,Heilongjiang,Jilin)(Liuetal.,2009;Zhangetal.,2013;Zhaoetal.,2014);northwesternChina(e.g.,Gansu,Ningxia,Qinghai,Shaanxi,Xinjiang)(Chenetal.,1989;Chenetal.,1991;Baoetal.,1997;Zhangetal.,2005;Huangetal.,2014;Zhangetal.,2015;Zhangetal.,2006);southwesternChina(e.g.,SichuanandTibet)(Qietal.,2015;Haoetal.,2016);EasternChina(Beijing,Fujian,Guangdong,Hebei,Tianjin,Jiangsu,Shandong,ShanghaiandTaiwan)(Suetal.,1991;Guoetal.,1993;Qinetal.,1994;GuoandLian,1999;LiangandHuang,2000;Xiangetal.,2004;Liuetal.,2005;Watanabeetal.,2005;Mengetal.,2007;Xuetal.,2007;Chenetal.,2011;ChenandHuang,2012;Maetal.,2015);centralChina(e.g.,Anhui,Henan,Hubei,HunanandJiangxi)(Lietal.,1998;Lietal.,1999;Heetal.,2001;Jianetal.,2005;Xuetal.,2007;Luetal.,2008;Wuetal.,2008;Zhouetal.,2008;Liangetal.,2009;Wangetal.,2011a;Wangetal.,2011b;ChenandHuang,2012;Chenetal.,2015;Maetal.,2015;Qietal.,2015)(Table1-6).46 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaTable1-6:StudiesofCryptosporidiumincattleinChina(AliteraturesearchedinPubMedandtheChinaNationalKnowledgeInfrastructure).AnimalLocationHostageDiagnostictechniquesInfectionrateCryptosporidiumspeciesReferencesDairyAnhui<6;6~24,Modifiedacid-faststainingtechnique15.0%(52/350)NotassessedChenandHuang(2012)>24months(MAFS)AnhuiPre-andpost-weanedSheather’ssugarflotation5.2%(26/502)C.parvum,C.murisXuetal.(2007)calve;yearlings;adultsAnhuiPre-andpost-weanedSheather’ssugarflotation6.4%(52/814)C.parvum,C.murisLietal.(1999)calve;yearlings;adultsAnhuiCalves,youngcows,Sheather’ssugarflotation16.8%(19/113)C.murisLietal.(1998)adultscowsAnhuiCalves,youngcows,Sheather’ssugarflotation13.6%(52/381)C.murisWuetal.(2008)(Hefei)adultscowsAnhuiCalves,youngcows,Sheather’ssugarflotation,MAFS31.7%(103/325)C.murisZhouetal.(2008)(Hefei)adultscowsBeijingCalf,heifer,adultMolecularanalysis;2.6%(21/822)C.parvum(12),Lietal.(2016)(SSU-rRNAandgp60)C.andersoni(9)FujianAdultscalvesMAFS6.8%(2/32)C.murisSuetal.(1991)GansuPre-weaned;3~11Molecularanalysis4.6%(58/1257)C.parvum(1),C.bovisZhangetal.(2015)months;1~2year;>2(SSU-rRNAandgp60)(29),C.andersoni(17),C.yearsryanae(11)47 HuazhongAgriculturalUniversityMaster’sDegree2018Guangxi1~6months,7~12Sheather’ssugarflotation,MAFS,11.7%(189/1612)C.andersoni(7)Taoetal.(2012)months,>1yearMolecularanalysis(PCR-RFLP)Guangxi1~6months,>1yearModifiedZiehle-Neelsen(MZN)8.2%(35/429)C.parvumJiangetal.(2008)Guangxi<2monthsSheather’ssugarflotation,MAFS9.2%(57/617)C.andersoniWeietal.(2014)Guangxi<2years,adultsSaturatedsodiumchlorideflotation35.4%(17/48)NotassessedLietal.(1992)Guangdong1~3months,4~6Sheather’ssugarflotation,MZN8.5%(92/1087)C.muris(92)Xiangetal.(2004)months,7~12month,>1yearGuangdong<1year,>1yearsMAFS26.7%(28/105)C.parvum(22),C.murisGuoandLian(1999)(9),bothinfection(3)Heilongjiang<2months,3-11Molecularanalysis5.3%(27/507)C.andersoni(26),Liuetal.(2009)months,12-24(18S-rRNA,HSP70andactingene)C.ryanae(1)months,>24monthsHeilongjiangPre-weanedcalvesMolecularanalysis47.7%(72/151)C.parvum(2)Zhangetal.(2013)(SSU-rRNAandgp60)C.bovis(34),C.andersoni(26),C.ryanae(5),C.meleagradis(5)48 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaHeilongjiang12~14monthsMolecularanalysis20.7%(87/420)C.andersoni(27)Zhaoetal.(2014)(PCR-RFLP,MLST)Henan1~8weeksMolecularanalysis21.5%(172/802)C.parvum(54),C.bovisWangetal.(2011a)(SSU-rRNAandgp60gene)(65),C.andersoni(12),C.ryanae(19),C.parvum-C.bovis(6)C.parvum-C.ryanae(4)C.parvum-C.andersoni(3)Henan3~11month,12~24Microscopicanalysisand7.9%(104/1315)C.bovis(20)Wangetal.(2011b)month,>24monthPCR-RFLPanalysisC.andersoni(84)Henan1~6months,yearling,Sheather’ssugarflotation,MZN11%(64/582)C.parvumJianetal.(2005)adultsC.andersoniHenan1~8weeks,>8weeksMZN,Microscopicanalysisand2.2%(14/624)C.bovis(1)Liangetal.(2009)PCR-RFLPanalysisC.andersoni(13)Henan<1month,2~6MZN,Microscopicanalysis6.7%(34/432)C.bovis(6)Chenetal.(2015)months,7~12(18S-rRNA)C.parvum(1)months,>12monthsC.andersoni(27)49 HuazhongAgriculturalUniversityMaster’sDegree2018Henan<30d,31~60d,61Sheather’ssugarflotation,MAFS4.8%(109/2268)C.andersoniQietal.(2015)~90d,91~180d,181~360d,>360dHenan<3months,3~12Microscopicanalysis(SSU-rRNA)36.2%(108/298)C.bovis(25)Maetal.(2015)months,13~24C.ryanae(5)months,>24monthsC.andersoni(68),C.ryanae-C.bovis(1),C.suis-like(2)Henan<2months,MAFS26.1%(152/582)C.parvum(7)Luetal.(2008)3~12monthsC.andersoni(25)Henan0~6months,7~16MAFS,Zincsulfateflotation20.6%(21/102)C.murisHeetal.(2001)months,>17months,adultsHenan<1year,1~8years,>8Molecularanalysis2.24%(14/624)C.andersoni(2)Liangetal.(2009)years(PCR-RFLP)C.bovis(1)Hunan1~12months,>1yearFlotationtechnique21.3%(63/296)NotassessedLiuetal.(1994)Innercalves,adultsSheather’ssugarflotation,MZN22.5%(16/71)NotassessedYangetal.(2004)MongoliaJiangsu<6;6~24,Modifiedacid-faststainingtechnique20.7%(251/1215)NotassessedChenandHuang.(2012)>24months(MAFS)Jiangsucalves,adultsSheather’ssugarflotation21.4%(27/126)NotassessedLiuetal.(2005)50 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaJiangsu1~3months,4~6Sheather’ssugarflotation29.1%(16/55)NotassessedLiangandHuang(2000)monthsJilinNotassessedMAFS14.3%(43/300)C.murisTianetal.(2000)Jilincalves,adultsFlotationtechnique64.3%(9/14)NotassessedXuetal.(2002)Ningxia3~11months,1~2Molecularanalysis1.7%(23/1366)C.parvum(15)Huangetal.(2014)year,>2years(SSU-rRNAandgp60gene)C.bovis(4)C.andersoni(4)NingxiaPre-weaned,3~11Molecularanalysis5.5%(92/1688)C.parvum(19)Zhangetal.(2015)months,1~2year,>2(SSU-rRNAandgp60gene)C.bovis(41),yearsC.andersoni(19),C.ryanae(13)Qinghai1~3months,4~5,Saturatedsodiumchlorideflotation36.0%(18/50)C.parvumChenetal.(1991)6~7,7~8,>9monthsQinghai0~3months,3~8,Sheather’ssugarflotation32.7%(36/110)NotassessedZhangetal.(2006)8~12months,youngcows,adultscowsQinghai1~3months,4~5Sheather’ssugarflotation12.7%(7/55)C.parvumChenetal.(1989)months,6~7monthsShandong<3months,3~12Microscopicanalysis(SSU-rRNA)24.3%(36/148)C.bovis(13)Maetal.(2015)months,13~24C.ryanae(10)months,>24monthsC.andersoni(11)51 HuazhongAgriculturalUniversityMaster’sDegree2018Shandong1~30,30~60daysMAFS25.3%(39/154)NotassessedGuoetal.(1993)(Jinan)calves,deliverycowShandongcalves,youngcows,Microscopicanalysis(RT-PCR)10.4%(59/565)NotassessedChenetal.(2011)(Yantai)adultscowsShaanxi<3months,3~11Microscopicanalysis(SSU-rRNAand2.6%(32/1224）C.andersoni(32)Zhaoetal.(2013)months,1~2year,>2COWPgenes),MLSTyearsShaanxicalves,youngcows,Flotationtechnique61.2%(442/722)NotassessedZhangetal.(1991)adultscowsShaanxi1~3monthsMAFS4.1%(3/73)NotassessedLietal.(1991)Shanghai<3months,3~12Microscopicanalysis(SSU-rRNA)12.5%(55/440)NotassessedChenandHuang(2012)months,13~24months,>24monthsShanghai<1months,1~12Sheather’ssugarflotation,MAFS36.5%(214/586)NotassessedXuetal.(2007)months,>12monthsTianjincalves,adultsSheather’ssugarflotation,MAFS8.1%(11/136)C.parvum(6),C.murisMengetal.(2007)(36)TaiwanCattleMZNS,IFA37.6%(173/460)C.parvum(173)Watanabeetal.(2015)52 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaXinjiangPre-andpost-weanedNest-PCR,RFLP16.0%(82/514)C.parvum(22),C.bovisQietal.(2015)calves(20),C.andersoni(25),C.ryanae(9),C.bovis-C.parvum(20),C.bovis-C.ryanae(4)Xinjiang<60days,61~180Multilocussequencetyping(MLST)4.3%(60/1391)C.andersoni(60)Qietal.(2016)days,181~450days,>450daysBeefHenan<3months,3~12Microscopicanalysis(SSU-rRNA)26.5%(44/166)C.bovis(16),Maetal.(2015)months,13~24C.andersoni(15)months,>24monthsC.ryanae(6)C.ryanae-C.bovis(1)Heilongjiang12~14monthsMolecularanalysis17.5%(71/405)C.andersoni(23)Zhaoetal.(2014)(PCR-RFLP;MLST)Shaanxi<3months,3~11Microscopicanalysis(SSU-rRNAand4.5%(38/847)C.andersoni(38)Zhaoetal.(2013)(Qinchuan)months,1~2year,>2COWPgenes),MLSTyearsAnhui<1year,1~2year,>2Sheather’ssugarflotation15.3%(42/275)C.parvum,C.murisLietal.(2003)yearsXinjiang<60days,61~180Multilocussequencetyping(MLST)2.3%(10/436)C.andersoni(10)Qietal.(2016)(Hecattle)days,181~450days,>450days53 HuazhongAgriculturalUniversityMaster’sDegree2018YaksQinghai<1year,1~2year,>2Molecularanalysis24.2%(162/586)C.bovis(31),C.andersoniMietal.(2013)years(SSU-rRNAandgp60gene)(15),C.ryanae(5),C.parvum(15),C.parvum-C.bovis(2),C.ryanae-C.bovis(1)QinghaiNotassessedSerologicaltest33.7%(368/1094)C.parvumMaetal.(2011)Qinghai1~2months,3~4Flotationtechniques,MAFS3.3%(13/396)C.bovisZhouetal.(2009)months,4~5monthsQinghai1~10,11~12monthsFlotationtechnique32.5%(26/80)NotassessedBaietal.(2001)Qinghai1~10,>11monthsFlotationtechnique,MAFS16.9%(32/190)NotassessedWangandLiu(2007)Qinghai1~2,3~44~5,5~6,Flotationtechnique,MAFS30.6%(26/85)NotassessedDou(2017)7~8,>9Qinghai<1year,>1yearMolecularanalysis4.0%(10/248)C.parvum(5),C.bovis(3),Qietal.(2015)(gp60gene)C.ryanae(1)QinghaiWeanedcalves,Molecularanalysis30.0%(98/327)C.bovis(56),C.ryanaeMaetal.(2014)yearlings,adults(gp60gene)(33),C.andersoni(2),C.ubiquitum(1),C.xiaoi(1),novelgenotype(2),C.bovis-C.ryanae(3)54 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaShanghaiadultsFlotationtechniques,MAFS0%(0/6)NotassesedZhouetal.(2009)GansuNotassessedMicroscopicanalysis(SSU-rRNA)5.3%(4/76)C.andersoni(1)Qinetal.(2014)C.bovis(3)Gansu<1year,>1yearMolecularanalysis(gp60gene)6.0%(7/117)C.bovis(2),C.ryanae(2),Qietal.(2015)C.parvum(2),C.ubiquitum(1)Sichuan<1year,>1yearMolecularanalysis(gp60gene)1.2%(1/84)C.parvum(1)Qietal.(2015)SichuanNotassessedSheather’ssugarflotation,MAFS14.8%(32/216)C.bovis(8),C.andersoniHaoetal.(2016)(13),C.ryanae(11)Tibet<1year,>1year.Molecularanalysis(gp60gene)4.2%(4/96)C.parvum(4)Qietal.(2015)BuffaloGuangxi<1year,>1year.Sheather’ssugarflotation,MAFS0.1%(1/1009)C.andersoni(1)Xieetal.(2013)Hunan<3months,3~12,Microscopicanalysis(SSU-rRNA)29.1%(231/793)C.bovis(3),C.ryanae(7)Maetal.(2015)13~24,>24monthsJiangxi1~7monthsdiarrhoeaZincsulfateflotation35.9%(65/181)C.parvum,C.murisXieetal.(2002)calvesHunan<1year,>1yearFlotationtechnique14.7%(26/177)NotassessedLiuetal.(1994)55 HuazhongAgriculturalUniversityMaster’sDegree2018Fig.1-3:DistributionsofCryptosporidiumindifferentregionsincattleinChinaAdditionally,inChina,therearefourdifferenttypesofcattlebreeds,dairycattle,beefcattle,yaksandbuffalo,respectively.FromTable1-6,wecanconcludethattheinfectionrateofCryptosporidiumindairycattlerangedfrom2.2%to61.2%,withtheaveragerateof12.6%(3277/25968).Inbeefcattle,theprevalenceofCryptosporidiumvariedfrom2.3%to26.5%,withtheaveragerateof9.6%(205/2129).Inyaks,theinfectionrateofCryptosporidiumvariedfrom1.2%to33.7%,withtheaveragerateof17.9%(783/4384).Inwaterbuffalo,theinfectionrateofCryptosporidiumrangedfrom0.1%to35.9%,withtheaverageinfectionrateof13.0%(323/2483).TheChi-sequaretestdidnotshowanymarkeddifferenceintheCryptosporidiuminfectionamongstcattlegroups(P>0.05).1.9.1.CryptosporidiumspeciesandsubtypesindairycattleinChinaSeveralspeciesofCryptosporidiumhavebeenfoundindairycattle,includingC.bovis,C.parvum,C.andersoni,C.ryanae,C.muris,C.melegradis,C.suis,andmixedinfection,havebeenfoundinChina.FromthestatisticdatainTable1-7,wecanconcludethatC.andersoni(n=460),C.bovis(n=258),C.parvum(n=312)andC.muris(n=128)werethecommonspeciesamongthespeciesdetected,withC.andersonibeingthecommonestspeciesindairycattle.56 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaAdiversityofCryptosporidiumsubtypeshavebeenfoundindairycattle,gp60sequencingdatarevealedthatalloftheC.parvumisolatedfromdairycattleinentifiedtodatebelongingtoIIdsubtypes,suchasIIdA15G1inBeijingandNingxia(Huangetal.,2014;Lietal.,2016),IIdA19G1inBeijing,HenanandHeilongjiang(Wangetal.,2011a;Zhangetal.,2013;Lietal.,2016).IIdA17G1isolatedfromC.parvumindairycattleinBeijing,andinterestingly,thiswasthefirstsubtypereportedincattleglobally(Lietal.,2016),thesefindingsconfirmtheuniquecharactersofC.parvumIIdsubtypesinChina(Table1-7).A4A4A4A1(n=19),A2A4A4A1(n=2),A2A4A2A1(n=5)andA4A4A2A1(n=2)inHeilongjiang(Zhaoetal.,2014).Inaddition,theidentifiedsubtypeofC.meleagridiswasIIIeA22G2R1inpre-weanedcalvesfromHeilongjiang,alsothisisthefirstsubtypeisolatedfromC.meleagridisinChina(Zhangetal.,2013).(Table1-7).Inaddition,IIdA15G1wasthedominantsubtypeofC.parvum,andA1A4A4A1wasmostcommonsubtypeofC.andersoni.1.9.2.CryptosporidiumspeciesandsubtypesinyaksinChinaInyaks,C.parvum,C.bovis,C.andersini,C.ryanae,C.ubiquitum,C.xiaoi,andnovelgenotypesarethemajorspeciesidentifiedinChina,amongthem,C.bovis(n=103)isthecommonestspecies,followedbyC.ryanaeandC.parvum.IIaA15G2R1isthecommonestsubtype,additionally,fiveIIasubtypes(i.e.,IIaA14G1R1,IIaA14G2R19,IIaA16G2R1,IIaA16G3R1)wereidentifiedinyaksinQinghaiprovince(Mietal.,2013).TheidentifiedsubtypeofC.ubiquitumwasXIIa(n=1)inGansuprovince,andIIdA15G1(n=3),IIdA18G1(n=1)werefoundinQinghaiprovince,andIIdA19G1(n=1)wasdetectedinTibetAutonomousRegion(Qietal.,2015)(cf.Table1-8).1.9.3.Cryptosporidiumspecies/subtypesinbeefcattleandwaterbuffaloinChinaForbeefcattle,numerousCryptosporidiumspecieswerecharacterized,suchasC.parvum,C.bovis,C.andersoni,C.ryanae,C.murisandC.bovis-C.ryanaemixedinfection.Amongthem,C.andersoniisthepredominantspecies.FourC.andersonisubtypeswereidentifiedfromQinchuancattleinShaanxiprovince,includingA4A4A4A1(n=25),A2A4A4A1(n=3),A2A4A2A1(n=1),A4A4A2A1(n=1)57 HuazhongAgriculturalUniversityMaster’sDegree2018(Zhaoetal.,2013).AlsoinXinjiang,threeC.andersonisubtypeswereidentifiedfromHecattle,includingA4A4A4A1(n=6),A2A4A2A1(n=1)andA4A4A2A1(n=3)(Qietal.,2016).Moreover,A4A4A4A1(n=14),A1A4A4A1(n=2)andA5A4A4A1(n=5)inHeilongjiangprovince(Zhaoetal.,2014),andA1A4A4A1wasdominantsubtypeofC.andersoni,whichisconsitentwithdairycattleinChina(Table1-9).Inbuffalo,fivespecies(e.g.,C.bovis,C.ryanae,C.andersoni,C.parvum,C.muris)ofCryptosporidiumhavebeenreportedinChina(Table1-9),withC.ryanaeconsideredasthepredominantspecies.Nevertheless,thereisnosubtypesreportedinChina,alsothezoonoticimportanceofwhichisunknown.Therefore,large-scaletemporalandspatialstudiesshouldbeconductedinthefuturetoevalutethemolecularinvestigationandzoonotictransmissionofCryptosporidium.Inconclusion,C.andersoniwasdominantspeciesindairyandbeefcattle.C.boviswascharacterizedasthegloballydistributedspeciesresponsibleforyaks,whileinbuffalo,C.ryanaewasshowntobethemostcommonlyspecies.Inaddition,C.parvumdisplaysagreaterinfectionsindairycalvesaswellasyaksinChina.58 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaTable1-7:DistributionofCryptosporidiumspeciesandsubtypesindairycattleinChina.LocationIdentifiedspeciesSubtypesReferencesHeilongjiangC.parvum(2)IIdA19G1(1)Zhangetal.(2013)C.bovis(34),C.andersoni(26),IIIeA22G2R1(3)C.ryanae(5),C.meleagradis(5)HeilongjiangC.andersoni(26),N/ALiuetal.(2009)C.ryanae(1)HeilongjiangC.andersoni(27)A4A4A4A1(19),A2A4A4A1(2),Zhaoetal.(2014)A2A4A2A1(1)A4A4A2A1(2)NingxiaC.parvum(15)IIdA15G1(15)Huangetal.(2014)C.bovis(4),C.andersoni(4)NingxiaC.parvum(19)IIdA15G1(19)Zhangetal.(2015)C.bovis(41),C.andersoni(19),C.ryanae(13)GansuC.parvum(1)IIdA15G1(1)Zhangetal.(2015)C.bovis(29),C.andersoni(17),C.ryanae(11)XinjiangC.parvum(22)IIdA15G1(11)Qietal.(2015)C.bovis(20),C.andersoni(25)IIdA14G1(4)C.ryanae(9),C.bovis-C.parvum(20)C.bovis-C.ryanae(4)XinjiangC.andersoni(60)A1A4A2A1(2);A2A4A2A1Qietal.(2016)(17);A2A4A4A1(1);A4A4A2A1(1);A4A4A4A1(13);A4A5A2A1(13);A4A5A4A1(1);A5A4A4A1(2);59 HuazhongAgriculturalUniversityMaster’sDegree2018HenanC.parvum(54)IIdA19G1(67)Wangetal.(2011a)C.bovis(65),C.andersoni(12)C.ryanae(19)C.parvum-C.bovis(6)C.parvum-C.ryanae(4)C.parvum-C.andersoni(3)HenanC.bovis(20)N/AWangetal.(2011b)C.andersoni(84)ShaanxiC.andersoni(32)A1A4A4A1(26),A4A4A4A1(1)Zhaoetal.(2013)ShaanxiC.bovis(26),C.andersoni(14)CRTypeI(9),CRTypeIIQietal.(2015)C.ryanae(12)(1),CRTypeIII(1),CRTypeIV(1)BeijingC.parvum(12)IIdA19G1(1),IId17G1(1),Lietal.(2016)C.andersoni(9)IIdA15G1(1)Anhui,Jiangsu,C.parvummousegenotypes(185)N/AChenetal.(2012)ShanghaiC.bovis(62),C.andersoni(112)C.honinis(24)C.serpentis(4)Shandong,HenanC.andersoni(79)N/AMaetal.(2015)C.bovis(38)C.ryanae(15)C.ryanae-C.bovis(1)C.suis-like(2)N/A,notavailable60 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaTable1-8:DistributionofCryptosporidiumspeciesandsubtypesinyaksinChina.LocationIdentifiedspeciesSubtypesReferenceGansuC.andersoni(1),N/AQinetal.(2014)C.bovis(3)GansuC.bovis(2),C.ryanae(2),C.parvumIIdA15G1(2),Qietal.(2015)(2),C.ubiquitum(1)XII(1)QinghaiC.bovis(56),C.ryanae(33),N/AMaetal.(2014)C.andersoni(2),C.ubiquitum(1),C.xiaoi(1),novelgenotype(2),C.bovis-C.ryanae(3)QinghaiC.bovis(31),C.andersoni(15),IIaA15G2R1(8),IIaA16G2R1(2),Mietal.(2013)C.ryanae(5),C.parvum(15),IIaA14G1R1(1),IIaA14G2R1(1),C.parvum-C.bovis(2),IIaA16G3R1(1)C.ryanae-C.bovis(1)QinghaiC.parvum(5),C.ryanae(1),C.bovisIIdA15G1(1),Qietal.(2015)(3),IIdA18G1(1)SichuanC.parvum(1)N/AQietal.(2015)TibetC.parvum(4)IIdA19G1(1)Qietal.(2015)TotalsC.parvum(27),C.bovis(103),IIaA15G2R1(8),IIaA16G2R1(2),C.andersoni(31),C.ryanae(52),IIaA14G1R1(1),IIaA14G2R1(1),C.ubiquitum(2),C.xiaoi(1),IIaA16G3R1(1),IIdA15G1(1),C.bovis-C.ryanae(4),C.IIdA18G1(1),IIdA19G1(1),parvum-C.bovis(2)XII(1)N/A,notavailableTable1-9:DistributionofCryptosporidiumspeciesandsubtypesinbeefcattleinChina.LocationIdentifiedspeciesSubtypesReferenceShaanxiC.andersoni(38)A4A4A4A1(25),A2A4A4A1(3),Zhaoetal.A2A4A2A1(1),A4A4A2A1(1)(2013)HeilongjiangC.andersoni(23)A4A4A4A1(14),A5A4A4A1(5),Zhaoetal.A1A4A4A1(1)(2014)XinjiangC.andersoni(10)A4A4A4A1(6),A2A4A2A1(1),Qietal.(2015)A4A4A2A1(3)61 HuazhongAgriculturalUniversityMaster’sDegree2018Table1-10:DistributionsofdifferentagegroupsofCryptosporidiumspecies/subtypesincattleinChina.AgeNo.ofNo.ofInfectionCryptosporidiumspeciesSubtypesReferencessamplespositiverate(%)1~3months6790128719.0%C.andersoni(77),C.ryanae(74)IIdA19G1(68)Zhangetal.(2015);Lietal.(2016);(pre-weaned)C.bovis(227),C.parvum(121),C.IIIeA22G2R1(3),IIdA15G1(92),Wangetal.(2011a);Zhaoetal.meleagradis(5),C.bovis-C.parvum(8),IIdA14G1(4),A4A4A4A1(3),(2013);Maetal.(2015);Qietal.C.bovis-C.ryanae(12),C.parvum-C.CRTypeI(9),CRTypeII(1),(2015);Maetal.(2014);Qinetal.andersoni(3)CRTypeIII(1),CRTypeIV(1)(2014);Liuetal.(2009);Huangetal.(2014);Zhangetal.(2015);3~11months518092217.8%C.andersoni(140),C.ryanae(10)IIdA15G1(2),A4A4A4A1(11),Zhangetal.(2015);Lietal.(2016);(post-weaned)C.bovis(38),C.parvum(2),C.bovis-C.A2A4A4A1(1),A2A4A2A1(1)Wangetal.(2011b);Zhaoetal.ryanae(3)A1A4A1A1(10)(2013);Mietal.(2013);Maetal.(2014);Qietal.(2016)1~2years560777113.8%C.andersoni(98),C.parvum(5),C.bovisA4A4A4A1(19),A2A4A4A1(2),Zhangetal.(2015);Lietal.(2016);(yearling)(33),C.ryanae(10),C.bovis-C.ryanaeA2A4A2A1(1)A4A4A2A1(2)Wangetal.(2011b);Zhaoetal.(2),C.xiaoi(1),(2013);Maetal.(2015);>2years758179410.5%C.andersoni(108),C.bovis(25),C.A4A4A4A1(6),A2A4A2A1(1),Zhangetal.(2015);Lietal.(2016);(adults)ryanae(17),C.ubiquitum(1),newA4A4A2A1(3)Wangetal.(2011b);Zhaoetal.genotype(2),C.parvum(7)(2013);Maetal.(2015)62 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China1.9.4.AgedistributionsofCryptosporidiumspeciesincattleTheagedistributionofCryptosporidiuminfectionincattlearesummarizedinTable1-10.Ingeneral,catllecanbeclassifiedintofourgroups,pre-andpost-weanedcalves,yearlingsandadults,theaverageinfectionratesrangingfrom10.5%to19.0%.Additionally,theCryptosporidiumspecieshaveage-associateddistributions.Inpre-weanedcalves,C.bovisandC.parvumshowntobethepredominantCryptosporidiumspecies,withC.bovisfoundmorecommonthanC.parvum,also,subtypesofIIdA14G1,IIdA15G1,IIdA19G1andIIIeA22G2R1arecommonlydistributed,withIIdA15G1beingthemostdominantsubtypes.Incontrast,C.andersoniandC.bovis,ofteninfectpost-weanedcalvesaswellasyearlings,withC.bovisfoundmorecommonthanC.bovis.Presently,inChina,C.andersoni,C.bovis,C.ryanae,andC.parvumarecommonestspeciesofCryptosporidiumfoundincattle.Similarwithothercountries,C.andersoniwascommonlydecectedinpost-weaned,yearlings,andadultcattle.Interestingly,C.bovisispredominantinginpre-weanedcalves(Table1-7,8,9).1.10.RecentinsightsintoCryptosporidiumofhumansinChinaInordertoassesstherichessbetweentheinfectionrateandspecies/genotypesofCryptosporidiuminhumansacrossChina(Fig.1-4),allpublishedrecordsdescribingtheinformationintheChinaNationalKnowledgeInfrastructure(CNKI)werecompiled(Table1-11,12).Obviously,fromTable1-8,wecanbrieflydrawsomeconclusions:(i)Age:Infectionrateishigheramongchildrenandelderlypatientsascomparedtotheotheragegroups.Thispredispositionmaybeduetolowimmunity,anyconcurrentdiseaseandenvironmentalconditions;(ii)Sanitation:Infectionrateishigherinpeoplefromruralareasascomparedtopeopleresidinginurbanareas,whichisassociatedwithmanyfactorslikeeating/drinkinghabitsandsanitaryconditions.(iii)Professionalconcern:Theinfectionrateishigherinthepersonnelsinvolvedinhandlingandporkprocessingasasourceofincomethanotherprofessionalgroups.(iv)Season:Ingeneral,theinfectionrateisfoundnotequallydistributedinseasonalvariation,assomestudiesindicatesitshigherprevalenceinsummerandautumnthaninwinterandspring.63 HuazhongAgriculturalUniversityMaster’sDegree2018Table1-11:StudiesofCryptosporidiuminhumansinChina.(AliteraturesearchedinPubMedandtheChinaNationalKnowledgeInfrastructure)LocationTotalHostgroup(agegroup)DiagnostictechniquesInfectionrateSusceptiblehost(area/season)ClinicalsymptomsReferences(Provinces)numberAnhui668Infant,students,teachers,merchant,Modifiedacid-faststainingMAFS:2.36%(15/635)i)Infant,students,farmers;DiarrheaWangetal.(2015)farmerstechnique(MAFS),ELISAELISA(IgG):21.81%ii)<14yearsoldpeople;iii)with(140/642)diarrheanearlyonemonthMAFS+ELISA:1.91%(12/628)Anhui5421Infant,Pupil,MiddleschoolAuramine-phenolstaining1.33%(74/5421)i)Infant,ii)Adultwithdiarrhea,Anorexia,vomiting,feverLietal.(2004)student,Collegestudent,Adultwithmethod,MAFS,ELISA,BSAwithimmunodeficency,iii)ruraldiarrhea,withimmunodeficiencyareas>urbanareas,Anhui827Diarrheapatients,includingAuramine-phenolstaining5.6%(46/827)i)infants;ii)chronicdiarrheapatients;Diarrhea,fever,vomiting,Caietal.(2003)children,acutediarrheaandchronicmethod,MAFSiii)rural>urban;iv)patientswithabdominalpain,diarrheapatientsintestinalparasiticnematodeinfectiondyspepsia,wateryfaeces,anorexiaAnhui4048Infants;pupils;middleschoolAuramine-phenolstaining1.3%(54/4048)i)Infants(3.15%)>othergroups;ii)Lightabdominalpain,Xuetal.(2005)students;collegestudentsmethod,MAFSRuralstudents(1.8%)>urbanlightdiarrhea,loosestoolstudents(0.8%).Beijing67i)HIV/AIDSpatientsMAFS6.0%(4/67)i)Ruralarea>urbanarea;ii)HIVProctitis,colitis,slightWangetal.(2011)(withchronicdiarrhea);advancedperiod>asymptomatic,iii)abdominalpain.ii)Urbanareas,RuralareaslowlevelofCD4cell64 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaBeijing1752Thekindergartenchildren,MAFS2.3%(41/1752)FamilydiarrheaDiarrhea,fever,Yanetal.(2014)(ruralarea)Primarystudentsabdominalpain,facesvariesfrommucustowaterydiarrhoeChongqing1238Slaughterhouse;HospitalMAFS3.4%(42/1238)i)Slaughterhousestaff,ii)Children,PersistentdiarrheaZhangetal.(2012)Outpatient;Ruarlareas;Outpatients;iii)Immunocompromised<14,>14yearsoldoldmanFujian161DiarrheapatientsandfamilyIodinestaining,MAFS,10.0%(16/161)AllpositivesampleswereinfantsDiarrhea(faecesvariesSuetal.(1989)(Fuzhou)members(infantsandadults)methylenebluestaiing(13.3%)frompasty,mucustowaterydiarrhea),inaddition,numerousredbloodcellandasmallwhitebloodcellinstools,fever,abdominalpain,poorappetite,thirstily.Fujian385DiarrheachildrenAldehydeethercentrifugal8.1%(31/385)i)ruralchildren;ii)higherincidenceNotmentionedZhengandXiao(Nanping)sedimentationmethod,MAFSwereinautumn,summer(1993)Fujian257DiarrheapatientsinhospitalMAFS3.1%(8/257)i)<1year;ii)patientsfromruralFaeceswithbloodandSuetal.(1991)(Zhangzhou)areas>urbanareas.mucusbloody,fever,vomiting,abdominalpain,nausea,lossofappetite.65 HuazhongAgriculturalUniversityMaster’sDegree2018Gansu600Patientswithhypertension,diabetesMAFS4.3%(25/600)0~10years;chronicpatients>NotmentionedWangetal.(2013)(Lanzhou)andmalignancy(n=160);Nonnon-chronicpatientschronicpatients(n=340)Guangdong348Diarrheachildren(<14years)NestedPCR3.5%(12/348)i)<3yearsold;ii)therearetwopeaksFaeceswithwatery,mucusYaoetal.(2014)inthedetectionseason:3~4,11~12bloody,fever,vomiting,season,respectively.InNov.istheabdominalpain,nausea,highest.WBC<15.Guangdong126AIDSpatientsMAFS,Immunofluorescent4.0%(5/126)i)50~59yr.group(4.0%)>30~39yr.NotmentionedYaoetal.(2008)staininggroup(1.8%);ii)antiviraltherapygroup(1.1%)>noantiviraltherapygroup(21.1%);iii)terminalAIDSpatients,especiallyCD4cell<100/lGuangdong556i)ChronichepatitisBpatientsAuramine-phenolstaining(i)5.8%(16/276);(ii)Infectionrate:i>ii>iii;RuralCHBDiarrheaLietal.(2007)(276);ii)Diarrheachildren(140);method,MAFS,PCR-RFLP1.4%(2/140);(iii)0.7%patients(10.3%)>urbanpatientsiii)Healthycases(140)(1/140)(3.7%);Patientscontactwithdogsandcats(43.8%)>Nocontactthem(17.3%);Positivecaseshavethediarrheahistory(63%)>Negativecaseshaveit(25%)Guizhou1739RuralresidentsMAFS2.3%(40/1739)i)<12yearsoldchildren,especiallyDiarrhea,faeceswithWangetal.(2006)(Qiannan)prechildrenchildren;ii)Thesurveywatery.was7~8months66 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaHeilongjiang931PediatricdiarrheaModifiedZiehl-Neelsen1.4%(13/931)i)Ruralchildren(2.68%)>urbani)The13caseswereZhaoetal.(1997)(Harbin)children,includingurbanchildren(MZN),Sheather’ssugarchildren(0.39%);ii)ArtificialfeedingoccurredinMayto(n=510),ruralchildren(n=421);flotation(8.33%)>Breast-feeding(0.24%)October,mainlyinrainlyBreast-feeding(n=409)andseasonofJulytoAugust;artificialfeeding(n=108)ii)diarrhea,abdominalpainandnausea,feverHeilongjiang330DiarrheapatientsRapidmodifiedacidfast3.3%(11/330)i)PreschoolchildrenandoldmanDiarrhea,fever,vomiting,Niuetal.(2006)(Qiqihar)stainingabdominalpain,anorexiaHubei1128Ruralchildren,includingMAFS3.3%(36/1128)i)familyhadthehistoryofparasitesDiarrhea,fever,vomiting,Heetal.(2013)(Chibi)kindergartenandprimarychildren.infectionnearly1yr.ii)familyhadabdominalpaindiarrheaordysenteryinrecent3months.Hubei1035Ruralchildren,includingMAFS5.6%(58/1035)i)ChildrenwhodonotoftenwashDiarrheaMinetal.(2014)(Xianning)kindergartenandprimarychildren.theirhands;ii)ChildrenwithpoornutritionalandsanitaryconditionsHubei2549Urbanresident,ruralresidentsi)Auramine-phenolstaining;4.7%(119/2549)i)Ruralarea>urbanarea;ii)Intermittentdiarrhea,mildZhuetal.(2009)(Shiyan)ii)MAFS;iii)methylenebluechildren>adultsabdominalpainandstaiing;iv)1uramine-phenolnausea,fever,faeceswithstainining-MAFSwatery,mucus.67 HuazhongAgriculturalUniversityMaster’sDegree2018Hubei941i)Elementaryschoolstudent,highMAFS6.6%(62/941)i)Elementaryschoolstudent;ii)ruralDiarrhea,abdominalpainZhuandSong(2007)(Shiyan)schoolstudent;ii)urbanstudents,students.andnausea,fever,faecesruralstudents.withwateryandmucus.Henan149HIV/AIDSpatientsMAFS16.1%(24/149)Ruralarea>urbanarea;HIVProctitis,colitis,slightWangetal.(2011)(withchronicdiarrhea)advancedperiod>asymptomatic,lowabdominalpain.levelofCD4cellHenan483DiarrheachildreninhospitalofMAFS,Safranin-methylene2.5%(12/483)<1,1~2,2~3,3~4havenosignificantDiarrhea,fever,vomiting,Suetal.(1991)(Kaifeng)outpatientandinpatientdepartmentbluemethoddifferenceabdominalpain,anorexia,thirsty,lossofappetite.Hunan37390~10,10~20,30~40,>40MAFS1.9%(69/3739)i)0~10aresusceptible;ii)higherDiarrhea,fever,vomiting,Luetal.(1992)male,femaleincidencewereinautumn,summer;abdominalpain,thirst,iii)driningunboiledwater,iv)faecesvariesfrompalepoultriestobebredinthefamilyyellowwithmucustoprofusewaterydiarrhoeaHunan2134urbanandruralresidentMAFS,1.9%(41/2134)i)Contactwithanimals,Diarrhea,fever,vomiting,Luetal.(2009)(Changsha)Iodinestainingii)Malnutritionabdominalpain,thirstHunan90320~30;30~40;>40yearsoldmaleRapidmodifiedacidfasttwo19.1%(172/903)i)Drugaddictingtimeover11years;DiarrheaHuangetal.(2003)drugabusersstepmethodsii)Self-employedlaborers;iii)<40yearsolddrugster68 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaHunan102OutpatientdiarrheachildrenFastimprovedanti-acid4.9%(5/102)i)Allagegroup(0~5,5~10,10~14)Diarrhea,wateryyellowHuangetal.(1998)(Changsha)two-stepmethodwerenosignificantdifference;ii)Allstool,durationof2~30positivecasesweredetectedindays,accompaniedbyMarch~May,whicharewarmerandabdominalpain,wettermonthsoftheyear.abdominaldistension,discomfort,nausea,vomiting,lossofappetite,feverandthirst.Jiangsu22680~6,7~12,>12outpatientchildrenAuramine-phenolstaining1.0%(22/2268)i)>12monthsishigherthan0~6,NotmentionedDuetal.(2009)(Nanjing)includingdiarrhea,UPIandothersmethod,MAFS7~12monthschildrenJiangsu2018Thepupilsofkindergarten(2~5)MZN0.8%(16/2018)i)2~3pupils(2.43%);ii)ruralDiarrhea,abdominalShenandGe(1991)(Nanjing)pupils>urbanpupils;iii)mostpainpositivecsaesliketoplaywithdogsandcats.Jiangsu4582OutpatientdiarrheapatientsAuramine-phenolstaining1.3%(59/4582)Insummerandautumn.Diarrhea(wateryHanetal.(1989)(Nanjing)method,MAFS,methylenediarrhea),dehydration,bluestaiinglossofappetite,abdominalpain,bowel,nauseaandvomiting,thirst,andfever69 HuazhongAgriculturalUniversityMaster’sDegree2018Jiangsu394OutpatientpatientsMAFS3.6%(14/394)Allpositivecaseswerechildren:1~2Acutegastroenteritis,ChenandYao(1990)(Xuzhou)yr.(n=4);4~9yr.(n=4);11yr.(n=2).wateryfaeces,whichappearsyellow,orlightgreenmucus,andredbloodcells,abdominalpain,andfeverJiangsu7393Urbanandruralcensus(6221),MAFS1.9%(136/7393)i)Incensus:0~3ishighest;inWateryandmucusChenetal.(1993)(Xuzhou)outpatients(1172);childrenandoutpatients:infants(65.4%);iii)Sep.diarrhea,fever,vomiting,adults~Nov.ishigherthanotherseasons.abdominalpain,inappetence,cough.Liaoning353Pediatricdiarrheapatients(n=283);MAFS,Iodinestaining,4.3%(15/353)0~18diarrheapatients.DifferentdegreesofLietal.(2006)(Shenyang)Drugaddicts(n=70)Pyrazoleblackstainingdiarrhea,mainlywatery,fever,vomiting,abdominalpain,inappetence.Inner1002Thechildrenof8kindergartenRIDA,Auramine-phenol4.2%(42/1002)i)ThechildrenusedtodrinkunboiledNotmentionedSun(2015)Mongoliastainingmethodwater(78.7%)>drinkboiledwater.(OngniudBanner)Shaanxi462PediatricdiarrheachildrenMAFS7.7%(36/462)i)0~3infants;ii)autumnandwinterWateryandstubbornHouetal.(2011)(Xian)seasonishigherthanhot,dryseasondiarrhea70 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaShanghai500i)Diarrheapatients,CommunityMAFS,IFA4.8%(24/500)i)highestincidencewasinNotmentionedZhouetal.(2005)residents,familyfeedwithdog;ii)September;ii)eatingfoodnot0~20,20~40,40~60,>60yearscompletelycleanornotcooked;iii)familyfeedwithdogsorcatsShandong108MalignanttumorpatientsAuramine-phenolstaining66.7%(72/108)i)Treatmentgroup:chemotherapyDiarrhoeasymptomisnotXinetal.(2007)method,MAFSgroup>radiotherapy+chemotherapy>apparent,alvineperistalsisradiotherapygroup,ii)Categories:isslow,appearDigestiveapparatus>lactealglandconstipationphenomenonandrespiratoryapparatusinstead.Shandong1835i)Pediatricdiarrheachildrenii)MAFS0.8%(15/1835)<5yearsoldchildrenIntermittentdiarrhoea,Chenetal.(1991)ruralresidents,pre-schoolstudents,fever,thefaecesareinfantsyellowandmushy.ShandongShanghai2817Diarhoeapatients,includingIFA,Dipstickof1.3%(37/2817)i)Migrantworkers;ii)Summerandi)Diarrhea,fever,Zhangetal.(2016)Migrantworkers,Students,immuno-chromatography6~9monthsarethepeakseasonandvomiting,abdominalpain;unemployedandretiredpeople,months;ii)eosinophilsandcookersandothersneutrophilsincrease;iii)faecescontainingwhitebloodcells,redbloodcells71 HuazhongAgriculturalUniversityMaster’sDegree2018Shanghai493i)Acutediarrheapatients,ii)liverMAFSi)5.3%(12/225)ImmunocompromisedpatientssuchasNotmentionedQianetal.(2011)cirrhosisaccompaniedwithdiarrheaii)10.1%(17/168)AIDSwithdiarrheapatientshasthepatients,iii)AIDSwithoutdiarrhea,iii)10.5%(6/57)highestinfectioniv)andwithdiarrheapatientsiv)16.3%(7/43)Sichuan406Diarrheachildren(<10years)MAFS3.0%(12/406)1~4yearsoldisthehighest.NotmentionedZhangetal.(1991)Xinjiang37HIV/AIDSpatientsMAFS10.8%(4/37)Ruralarea>urbanarea;HIVProctitis,colitis,slightWangetal.(2011)(withchronicdiarrhea)advancedperiod>asymptomatic,lowabdominalpain.levelofCD4cellYunnan30AIDS/HIVdiarrhoeicandMAFS60.0%(18/30)NotmentionedFever,diarrhea,WangandPu(2011)(kunming)non-diarrhoeicpatientsprogressiveweightloss,lymphnodeenlargementsymptomsYunnan378DiarrheapatientsMAFS,Iodinestaining,5.3%(20/378)i)Preschoolstudents(8.5%);ii)TheDiarrhea,wateryfaecesZhangetal.(2002)Bainationality(12.5%).Yunnan86AIDSpatientsMAFS,Immunofluorescent4.7%(4/86)i)50~59yr.group(4.0%)>30~39yr.NotmentionedYaoetal.(2008)staininggroup(1.8%);ii)antiviraltherapygroup(1.1%)>noantiviraltherapygroup(21.1%);iii)terminalAIDSpatients,especiallyCD4cell<100/lYunnan73PediatricdiarrheapatientsZincsulfateflotation12.3%(9/73)6~10(66.7%)ishighestNotmentionedZuoetal.(1990)72 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaZhejiang300DiarrheachildrenMAFS,IFA8.7%(26/300)NotassessedAcutegastroenteritis,Chengetal.(2000)(Hangzhou)fever,waterdiarrhea,faecescontainingwhitebloodcells,redbloodcellsandasmallamountoffatdropletZhejiang548PediatricdiarrheachildrenMAFS,Immunofluorescent10.4%(57/548)i)1~10yearsoldchildren(17.5%)>Acutegastroenteritis,Luetal.(2000)(Hangzhou)staining0~1infants(5.7%)fervescence,pyocyte,redandwhitebloodcell,andfatdropletwerefoundinthefaeces.Zhejiang1060PatientswithdiarrheainoutpatientAuramine-phenol-MAFS5.7%(60/1060)i)0~15(6.1%)>over15year(1.0%);Acutediarrhea(n=34),Xingetal.(1999)(Wenzhou)orhospitalizedpatientsmethod,ii)ruralarea(9.6%)>urbanareachronicdiarrhea(n=26),(2.0%).iii)thepatientsinfectedenteritis,dyspepsia,intestinalparasiticnematodenausea(n=9),vomiting(9.42%)>withoutnematode(4.3%);(n=5),abdominalpainiv)summerandautuman(n=12),poorappetite(n=11),fever(n=8),cough(n=3)73 HuazhongAgriculturalUniversityMaster’sDegree2018Table1-12:DistributionofCryptosporidiumspeciesandgenotypesinhumansinChinaLocationNumberHostgroupInfectionrateSpeciesGenotypesReferencesJiangsu232Diarrhealoutpatients1.3%C.andersoni(21),A+-370,A370Jiangetal.(2014)C.hominis(2)Shanghai252Diarrhealoutpatients13.5%C.andersoni(34)N/ALiuetal.(2014)Shanghai6,284Pediatricpatients51.4%C.meleagridis(4),IaA14R4(34),IdA19Fengetal.(2012)C.hominis(86)(37),IbA19G2(1),IdA14(1)Hubei500Diarrhoeicchildren2.0%C.meleagridis(10)IIIbA21G1R1(1),Wangetal.(2017)IIIbA22G1R1(8),IIIbA26G1R1(1)Henan10N/A10(posotive)C.hominis(9),IbA16G2(1),Wangetal.(2011)C.felis(1)IbA19G2(2),IbA20G2(3),IaA9R3(1),IdA21(2)Guangdong348Diarrhoeicchildren3.5%C.hominis(8),C.Ia(6),Ib(2),Yaoetal.(2014)parvum(4)IIa(3),IId(1)Shanghai90Rawurbanwastewater70.0%C.hominis(47),C.IbA19G2,Fengetal.(2009)meleagridis(5),C.IbA20G2,parvum(1),C.IbA21G2hominis-C.baileyi(3),C.hominis-C.meleagridis(2),C.hominis-C.muris(1),C.hominis-C.suis(1),C.hominis-ratgenotype(1),ratgenotype-aviangenotypeIII(1),C.hominis-newgenotype(1)Tianjin5Patients100%C.hominis(5)Ib,Id,IIbPengetal.(2001)Note:N/A,notavailable74 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaFig.1-4:DistributionsofCryptosporidiumindifferentregionsinhumansinChinaHowever,oftheserecordsinChina,thereislittleinformationofthespecies/genotypesofCryptosporidium,bysearchingthePubMeddatabase,allthedescribingspecies/genotypeswerecompiled.Thereareonlyafewreportsavailable,andtheinformationofthesestudiesareconcludedinTable1-12[exceptYaoetal.(2014)fromCNKI].ThetableshowedtheexistenceofthefollowingfourspeciesofCryptosporidiuminhumansinChina:C.hominis(n=157),C.meleagridis(n=19),C.andersoni(n=55),C.felis(n=1),andseveralmixedCryptosporidiuminfection,withC.hominisasthecommonestdetectedspecies/genotypeoverall.TheresultsofsubtypesanalysisindicatethatsubtypefamilyIa,Ib,IdfoundinShanghai,IbinHenan,IIIbinHubei,Ib,Id,IIbinTianjin(Table1-12),Ia,Ib,IdfoundinthisreviewisconsistentwithareviewbyJexandGasser(2010),Ia-Igareusuallyseeninhumansglobally,withIbasthemostfrequentlyrecordedaccordingtotheavailabledatabase.Incontrast,IIIdfoundinChinaareconsideredlessfrequentlyrecordedcomparedwiththeformersubtypes,whichusuallyfoundinhumansinAsia,suchasChina,India,IndonesiaandThailand(Stensvoldetal.,2014),illustratingthatthisgeographicsegregationcouldbeduetodifferencesinthedistributionofthespeices.Additionally,thefindingIIbbyPengetal(2001)inhumans,whichusuallyfoundinC.parvum,illustratingthatC.hominismaytobeinfectivetoanimals,butitisonlysuppositionalandremainstobeproved.Therefore,furthersurveysshouldbeconductedinChinatoestablishwhetherIIbplaysaroleinzoonotictransmissionofCryptosporidium.75 HuazhongAgriculturalUniversityMaster’sDegree2018Forthefirsttime,wehavedetailedlysummarizedtheprevalenceandspecies/genotypesandsubgenotypesinvariouscattlebreedsaswellashumansindifferentregionsofChina,whichrepresentthefirstcomprehensiveandspecificanalysisofCryptosporidiumindifferentcattlebreedsandhumansinChina,mayimproveustohaveanemphasisonthecharacteristicandzoonotictransmissionofCryptosporidiuminChina,Nevertheless,thelackofcompleteepidemiologicinformation,particularlyinhumans,preventedusfromlookingintotherelationshipbetweengenotype/subgenotype,epidemiologicanddemographicfactors,whichwouldgreatlypromoteunderstandingofCryptosporidiumepidemiology.Therefore,expandedresearchshouldbeconductedinChinatograspmoreinformationofCryptosporidiumepidemiologyinmuchmoredetails.1.11.Aspectsoftreatment,prevention,andcontrolCryptosporidiumandGiardiaarecharacterizedasthemainprotistscausingenteritisanddiarrhoeainhumansandanimals,particularlyinchildrenandHIVpatients.Therefore,treatmentcanhelpcontrolcryptosporidiosisandgiardiasis,buttreatmentisdifficult,numerousdrugsaretestingforthetreatmentofthesetwodiseases,includingparomomycin,azithromycinandnitazoxanide(Leder,2009).However,theUSFoodandDrugAdministration(FDA)hasapprovedtheonlyuseofnitazoxanidefornon-immunodeficientchildrenandadults,whichhavebecomethefirstdrugapprovedfortreatingthedisease(SmithandCorcoran,2004;Rossignol,2010;Striepen,2013).Todate,Halofuginone,asasyntheticquinazolinone,isknowntohaveatherapeuticeffectontheclinicalsymptomsofcryptosporidiosis,especiallyinruminants(Jarvieetal.,2005),whichhasbeeninvestigatedforprophylacticeffectagainstcryptosporidiosisinsucklingcalves,(Lefayetal.,2001).Thatstudyshowedevidencethathalofuginonelactate(Halocur)significantlyreducedoocystshedding,delayedonsetandreducedseverityofdiarrheaincalvesexposedtoC.parvuminfection(Lefayetal.,2001).Althoughhalofuginonehasrevealedhighefficacy,aneffectivedrugforthepreventionandtreatmentofcryptosporidiosisremainsabsent(Sréteretal.,2000;ShahiduzzamanandDaugschies,2012).ForGiardia,currently,nolicenseddrugareavailableforthetreatmentofGiardiainruminants,thoughitiscontroversialforthetreatmentinruminants(O’HandleyandOlson,2006).Manydrugs,includingnitrimidazolederivatives(suchasmetronidazoleortinidazole),nitrofuran,andbenzimidazolehavebeenshowntobeeffectiveagainstgiardiasisinhumansandanimals(Farthing,2006).AlbendazoleaswellasnitazoxanidehasshownefficacyagainstGiardia76 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,Chinabutneedforhighdosesanddailyadministration.NitazoxanidehasbecomethefirstdrugtestedforthetreatmentofGiardiainthelasttwodecades(Rossignol,2010).Inaddition,severalnewpotentialdrugtargetsinthesetwoparasiteshavebeenattempted,butitseemstobelittleactivitytowardsclinicalsignsfortreatingthesediseases(Rossignol,2010).Therefore,thedevelopmentofnewdrugsshouldbeencouraged.Clearly,acommercialvaccine(GiardiaVax™,OverlandPark,Kansas,USA)producedfromtrophozoitesisolatedfromsheepisaccessiblefordogsandcatsinNorthAmerica(Olsonetal.,2000).SubunitvaccinescouldreduceGiardiainfectionandcouldsignificantlyreduceoreliminateintestinaltrophozoitesorfaecalcystexcretion;therefore,vaccinationhasthefunctionofprotectinganimalsfrominfectionandreducingclinicalsymptoms(Olsonetal.,1996,1997).Atpresent,thereisnoavailableofGiardiavaccineforuseinruminants.Forcryptosporidiosis,severalantigenshavebeenexploredforuseasnewpotentialvaccinecandidates.Forexample,gp60(alsocalledgp40/15)isapolyproteincleavedbyaparasiteserineproteinaseintotwosurfaceproteins-GP15andGP40(Checkleyetal.,2015).VaccinesbasedonGP15aloneorincombinationwithotherantigensareindevelopment(Burtonetal.,2011).Also,threeoftheseantigensincludingGP15,profilinandapyrasemightbepursuedasnewvaccinesagainstcryptosporidiosis(Manqueetal.,2011).Vaccinesareshowntoreduceclinicalsymptoms,but,inmostcases,donotreduceoocystshedding(cf.Thompsonetal.,2008).Althoughdifferenttreatmentoptionsareavailable,preventionisthemosteffectivewaytoprotecthumansandanimalsfromCryptosporidiumandGiardiainfections.Inhumans,controlmeasuresshouldbeincludegoodhygienepracticesandavoidtheingestionofcontaminatedwaterand/orfood,werecommendfrequenthand-washingusingpropertechniqueanddissuasionfromeatingindirtyplacetominimizepossibleexposuretocontaminatedsurfaces.Also,inlivestocksuchascalves,cryptosporidiosisandgiardiasisarealsocontrolledthroughpropermanagementandhusbandrypractices.Inaddition,duetothehigherinfectionrateinpre-weanedcalves,additionalmeasuresshouldbetakenintoaccountineliminatingdiarrhoea.Suchastimelycolostrumfeeding,keepingthecalvesinindividualpens,withstrawcovered,andhigh-pressurecleaningatsetintervals;inordertodecreasethetransmissionriskandtherapidspreadofdiseasetohumans,onecanusean‘all-in-all-out’managementstrategy(Silverlåsetal.,2009).Therefore,properanimalmanagementisakeyfacorsforthecontrolofcryptosporidiosisandgiardiasis.77 HuazhongAgriculturalUniversityMaster’sDegree20181.12.ConclusionsfromtheliteratureandfutureprospectsRecentresearchhasmarkedlyenhancedourknowledgeofthegeneticdiversityandpublichealthimportanceofCryptosporidiumandGiardiainhumansanddomesticcattle.WenowhaveamorecomprehensiveunderstandingofthecomplexityofCryptosporidiumatthespecificandsubspecificlevels.Wearealsoabletouseadvancedmolecularmethodsfordetailedgeneticstudiesofthesetwoparasitesinhumansanddomesticanimals,butinvestigationsshouldfocusonvariousanimalstoassesstheirimportanceasreservoirs/sourcesforzoonotictransmission.Additionally,thereisalsoaneedtoexpandfutureresearchinsomecountries,wherecryptosporidiosisandgiardiasisareparticularlyprevalentinhumanpopulations,andwherethereislimitedepidemiologicalinformation.InChina,despitethemajorprogressinCryptosporidiumandGiardiaresearch,therestillexistmanychallenges.Forexample,thereislimitedinformationonthezoonotictransmissionoftheseprotists;inaddition,limitedmolecularepidemiologicalinformationispresentlyavailableforsomepartsofChina.Therefore,morecomprehensiveandsystematicstudiesshouldbeconductedtoincreasetherichessofdata,focusedonobtainingabetterunderstandingontheoriginsandsourcesoftransmissionofthesetwopathogensinChina.Inconclusion,thepresentliteraturereviewhasshownclearlythatverylittleisknownaboutCryptosporidiumandGiardiaforsomepartsofChina.Inordertoaddressaknowledgegap,thepresentthesisexploredthegeneticmakeupofthesetwoprotistsinhumans(Chapter2)andcattle(Chapter3)inpartsofHubeiprovinceinChina,anddiscussesthefindingsinageneralcontext(Chapter4).78 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaCHAPTER2-FIRSTSURVEYOFCRYPTOSPORIDIUM,GIARDIAANDENTEROCYTOZOONINDIARRHORICCHILDRENFROMWUHAN,CHINAAbstractIntestinalprotozoanpathogenscausesignificantdiarrhoealdiseasesinchildren.However,todate,therehasbeenlimitedgeneticstudyoftheintestinalpathogensCryptosporidium,GiardiaandEnterocytozooninhumansinChina,withtheexceptionofresearchinasmallnumberofcities/provinces.Inthepresentstudy,PCR-basedtoolswereusedtodetectandcharacterisetheseprotistanparasitesfrom500childrenwithahistoryofdiarrhoeainWuhanandenvirons,Hubeiprovince,China.GenomicDNAsfromfaecalsampleswerescreenedfortheparticularprotistsbyPCRutilisingregionsinthesmallsubunit(SSU)ofthenuclearribosomalRNA,the60kDaglycoprotein(gp60),theinternaltranscribedspacerofnuclearribosomalDNA(ITS)and/orthetriosephosphateisomerase(tpi)genesasmarkers.CryptosporidiummeleagridissubtypeIIIb(10/500,2.0%),GiardiaduodenalisassemblageA(7/500,1.4%)andEnterocytozoonbieneusigenotypeD(1/500,0.2%)wereidentifiedinsmallpercentagesofthe500samples.Nosignificantgender-orage-associateddifferencesintheprevalenceofCryptosporidiumandGiardiainfectionswerefound.Futurestudiesmightfocusontheoccurrenceoftheseprotistsinchildrenaswellasanimals,withanemphasisonCryptosporidiummeleagridisinpetsandagriculturallyimportantbirds,indifferentpartsofHubeiprovince.Keywords:Cryptosporidium,Giardia,Enterocytozoon,Human,China,PCR-basedanalyses,Sequencing79 HuazhongAgriculturalUniversityMaster’sDegree20182.1.IntroductionDiarrhoealdiseasesareconsideredtobethesecondleadingcauseofmorbidityandmortalityinyoungchildren(<5years)indevelopingcountries(Kotloffetal.,2013;deLucioetal.,2016).TheprotistsCryptosporidiumspp.,GiardiaduodenalisandEnterocytozoonbieneusiarerecognisedasbeingparticularlyimportantpathogensthatcausesignificantchildhooddiarrhoealdiseases(FengandXiao,2011;Kotloffetal.,2013;Checkleyetal.,2015).Alloftheseparasiteshaveawiderangeofhosts,includinghumans,domesticandwildanimals,andaretransmittedanthroponoticallyand/orzoonoticallyviathefaecal-oralroute(Mathisetal.,2005;Xiao,2010;FengandXiao,2011;RyanandCacciò,2013).Theaccuratedetectionandcharacterizationofdifferentspecies,genotypesorassemblagesoftheseparasitesarecentraltodeterminingtheirpotentialinfectionsourcesandtransmissionroutes(Koehleretal.,2014).Usingmoleculartools,suchaspolymerasechainreaction(PCR)-basedtechniques,Cryptosporidiumhominis,C.parvum,C.meleagridis,C.felis,C.canis,GiardiaduodenalisassemblagesAandBaswellasEnterocytozoonbieneusigenotypeDappeartoberesponsibleformosthumaninfections(SantínandFayer,2009;Xiao,2010;RyanandCaccio,2013).However,theoccurrencesofthespecies,genotypesorassemblagesoftheseparasitesareknowntovaryaccordingtohostage(Xiao,2010;FengandXiao,2011)andgeographicallocation(Fengetal.,2007;Ngetal.,2011).Despitetheirwidespreadoccurrence(in>20Chinesecities/provinces),limitedmolecularepidemiologicaldataofthesepathogensinhumansareavailableinChina,withtheexceptionofsomecitiesorprovinces,includingShanghai(Fengetal.,2012;Wangetal.,2013a;Liuetal.,2014),Tianjin(Pengetal.,2001),Henan(Wangetal.,2011;Zhuetal.,2012;Wangetal.,2013b),Jiangsu(Jiangetal.,2014),Yunnan(Zhangetal.,2016a),Jilin(Zhangetal.,2011),Heilongjiang(Yangetal.,2014;Lietal.,2015).Lietal(2012)conductedamolecular-basedsurveyofthecitysewersystemfrom2006to2009,andidentifiedseveralhuman-pathogenicspecies/genotypes/subgenotypes,includingC.hominis,G.duodenalissub-assemblageA-IIandE.bieneusigenotypeD,asbeingdominantinwastewaterfromWuhan,Hubeiprovince(Lietal.,2012).However,todate,thereisnopublishedmolecularinformationontheseparasitesfromhumansinHubeiprovince.Here,weconductedthefirstmolecularsurveyofCryptosporidium,GiardiaandEntercytozoonindiarrhoeicchildreninWuhan,Hubeiprovince,China.80 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China2.2.MaterialsandmethodsAtotalof500freshfaecalsamplesweretargetedforcollectionduringJune2016andAugust2016fromanonymous(individual)childrenwithdiarrheaweredonatedbytheOutpatientDepartmentinWuhanPediatricHospitalandRenminHospitalofWuhanUniversity,China.GenomicDNAwasextractedfrom0.2gofeachfaecalsampleusingthePowerSoilDNAIsolationKit(MoBio,Carlsbad,USA),accordingtothemanufacturer’sinstructions,andthenfrozenat-20ºCuntiluse.Thiskitwasused,asitishighlyeffectiveatremovingcomponentsthatareinhibitorytoPCR(Pontirolietal.,2011;Koschetal.,2013;Nolanetal.,2013).Aliquots(2μl)ofindividualgenomicDNAsamplesweresubjectedtonestedPCR-basedamplificationandsequencing,employing(individually)fourdistinctlociofnuclearDNAinseparateassays.ForCryptosporidium,aportionofthesmallsubunitofthenuclearribosomalRNAgene(designatedpSSU;~240bp)wasused(Xiaoetal.,1999;Nolanetal.,2010),andgenotypic/subgenotypicclassificationwasachievedemployingaregionofthe60kDaglycoproteingene(designatedpgp60;~900bp)(Stensvoldetal.,2014).ForGiardia,aportionofthetriosephosphateisomerasegene(designatedptpi;~530bp)wasemployedandgeneticassignmentwastothelevelofassemblage(Sulaimanetal.,2003).ForEnterocytozoon,theinternaltranscribedspacerofnuclearribosomalRNAgene(designatedITS;~390bp,including125bpofflankingnuclearribosomalDNA)wasused(Buckholtetal.,2002).GenotypesofE.bieneusiwerenamedaccordingtoanestablishednomenclature(SantínandFayer,2009).AllprimersusedinthestudyarelistedinTable2-1.PCRwascarriedoutinavolumeof50μlcontaining10mMTris-HCl(pH=8.4),50mMKCl(Promega,Madison,USA),2.0-3.0mMofMgCl2(dependingonthelocus),200μMofeachdeoxynucleotidetriphosphate,50pmolofeachprimerand1UofeitherGoTaq(Promega,USA)(forpSSU,ptpiandITS)orMangoTaq™(Bioline,USA)(forpgp60)DNApolymerase.Knowntest-positive,test-negativeandno-templatecontrolswereincludedineachstepofeachsetofPCRs.ForCryptosporidium,forprimaryamplification,ofSSUgeneutilizedthecyclingprotocol,94°Cfor5min(initialdenaturation),followedby30cyclesof94°Cfor45s(denaturation),45°Cfor2min(annealing)and72°Cfor1.5min(extension),withafinalextensionof72°Cfor10minwasemployed.Secondaryamplificationwasachievedemploying94°Cfor5min,followedby35cyclesof94°Cfor30s,55°Cfor30s,and72°Cfor30s,withafinalextensionof72°Cfor10min(Xiaoetal.,1999;Nolanetal.,2010).81 HuazhongAgriculturalUniversityMaster’sDegree2018Forsomesamples,Cryptosporidiumwasfurthercharacterizedusingalongerregion(~900bp)ofthegp60gene.Reactionconditionsincludedaninitialdenaturationof95°Cfor4minfollowedby35cyclesof95°Cfor30s,60°Cfor30s(primaryPCR)or58°Cfor30s(secondaryPCR),and72°Cfor1minfollowedbyafinalextensionstepat72°Cfor7min(Stensvoldetal.,2014).ForGiardia,theprimaryamplification,thecyclingprotocolwas94°Cfor5min(initialdenaturation),followedby35cyclesof94°Cfor45s(denaturation),50°Cfor45s(annealing),and72°Cfor1min(extension)andafinalextensionof72°Cfor10min.Secondaryamplificationoftpiwasachievedemploying94°Cfor5min,followedby35cyclesof94°Cfor45s,55°Cfor30s,and72°Cfor1min,withafinalextensionat72°Cfor10min(Sulaimanetal.,2003).ForEnterocytozoon,cyclingparametersforthefirstPCRwere35cyclesof94°Cfor30s,57°Cfor30s,and72°Cfor40s.Onemicroliterofthefirstreactionmixturewasusedasthetemplateforthenestedreactionwithcyclingparametersof30cyclesof94°Cfor30s,55°Cfor30s,and72°Cfor40s.AllnestedPCRproductsweredetectedbyelectrophoresisin1.5%agarosegels,stainedwithethidiumbromidebeforesequencing.Forsequencing,aliquots(5μl)ofindividualamplicons(undigested)weretreatedwiththeenzymesExoIandathermosensitivealkalinephosphatase(FastAP,Thermofisher,USA),accordingtothemanufacturer'sinstructions,andthensubjectedtodirect,automatedsequencing(BigDyeTerminatorv.3.1chemistry,AppliedBiosystems,USA)inbothdirectionsusingthesameprimers(separately)asemployedinPCR.Phylogeneticanalysisofpgp60sequencedataforCryptosporidium(includingarangeofreferencesequences;Table2-2)wasconductedbyBayesianinference(BI)usingMonteCarloMarkovChain(MCMC)analysisinMrBayesv.3.2.3.ThelikelihoodparameterssetforBIanalysisofpgp60datawerebasedontheAkaikeInformationCriteriatest(Darribaetal.,2012)injModeltestv.2.1.7.Thenumberofsubstitutions(Nst)wassetat6,withaproportionofinvariablesites.Posteriorprobability(pp)valueswerecalculatedbyrunning2,000,000generationswithfoursimultaneoustree-buildingchains.Treesweresavedevery100thgeneration.Attheendofeachrun,thestandarddeviationofsplitfrequencieswas<0.01,andthepotentialscalereductionfactorapproachedone.A50%majorityruleconsensustreeforeachanalysiswasconstructedbasedonthefinal75%oftreesgeneratedbyBI.Analyseswererunthreetimestoensureconvergenceandinsensitivitytopriors.TheoutgroupusedinphylogeneticanalysiswasC.meleagridissubtypeIIId(GenBankaccessionno.DQ0675701).Thechi-squaretestwasperformedusingSPSSStatistics24software(IBM,NewYork,USA).82 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaTable2-1:Primersusedforprotozoangeneamplificationinthisstudy.GenusGeneSequenceofprimers(5'to3')Ampliconsize(bp)ReferenceorsourceCryptosporidiumpSSUXF2:GGAAGGGTTGTATTTATTAGATAAAG~240Xiaoetal.(1999)XR2:AAGGAGTAAGGAACAACCTCCApSSUf:AAAGCTCGTAGTTGGATTTCTGTTNolanetal.(2010)pSSUr:ACCTCTGACTGTTAAATACRAATGCpgp60CRSout115F:GATGAGATTGTCGCTCGTTATC~900Stensvoldetal.(2014)CRSout1328R:AACCTGCGGAACCTGTGATGFmod:GAGATTGTCGCTCGTTATCGGATR2:GATTGCAAAAACGGAAGGGiardiaptpiAL3543:AAATTATGCCTGCTCGTCG~530Sulaimanetal.(2003)AL3546:CAAACCTTTTCCGCAAACCAL3544:CCCTTCATCGGTGGTAACTTAL3545:GTGGCCACCACTCCCGTGCCEnterocytozoonITSEBITS3:GGTCATAGGGATGAAGAG~390Buckholtetal.(2002)EBITS4:TTCGAGTTCTTTCGCGCTCEBITS1:GCTCTGAATATCTATGGCTEBITS2.4:ATCGCCGACGGATCCAAGTG83 HuazhongAgriculturalUniversityMaster’sDegree2018Table2-2:SummaryinformationoftheCryptosporidiummeleagridissubtypesusedinthephylogeneticanalysis.SpeciesSubtypeHostLocalityIsolateGenBankNo.ReferenceCryptosporidiummeleagridisIIIaA24G3R1aTurkeyUSA295AF401499Glabermanetal.(2001)CryptosporidiummeleagridisIIIaA24G3R1bHumanKenya1920AF401500Glabermanetal.(2001)CryptosporidiummeleagridisIIIbA21G1R1HumanChina145KY575458PresentstudyCryptosporidiummeleagridisIIIbA21G1R1aHumanThailand/VietnamSwec030KJ210610Stensvoldetal.(2014)CryptosporidiummeleagridisIIIbA22G1R1HumanChina149KY575457PresentstudyCryptosporidiummeleagridisIIIbA22G1R1cHumanIndiaSwec060KJ210614Stensvoldetal.(2014)CryptosporidiummeleagridisIIIbA23G1R1bHumanThailandSwec022KJ210609Stensvoldetal.(2014)CryptosporidiummeleagridisIIIbA23G2R1HumanIndonesia(Bali)Swec052KJ210612Stensvoldetal.(2014)CryptosporidiummeleagridisIIIbA24G1R1HumanChinaSwec737KU852729J.Beser,unpublisheddataCryptosporidiummeleagridisIIIbA26G1R1HumanChina244KY575459PresentstudyCryptosporidiummeleagridisIIIbA26G1R1bHumanAfricaSwec063KJ210615Stensvoldetal.(2014)CryptosporidiummeleagridisIIIdA6R1HumanIndiavlr200418DQ067570Muthusamyetal.(2006)CryptosporidiummeleagridisIIIeA17G2R1HumanIndia/NepalSwec011KJ210608Stensvoldetal.(2014)CryptosporidiummeleagridisIIIeA25G2R1HumanIndiaSC1309KJ210605Stensvoldetal.(2014)CryptosporidiummeleagridisIIIgA22G3R1HumanNepalSwec759KU852730J.Beser,unpublisheddataCryptosporidiummeleagridisIIIgA31G3R1HumanSwedenSC1029KJ210619Stensvoldetal.(2014)84 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China2.3.ResultsanddiscussionUsingthreeseparatePCRassays,the500individualfaecalsamplesfromchildrenwithdiarrhoeafromWuhanCitywerescreenedbyPCRforthepresenceofDNAofthethreeintestinalprotozoantaxa.Intotal,10,7and1faecalDNAsamplesweretest-positiveforCryptosporidiumspp.,GiardiaduodenalisandEnterocytozoonbieneusi,respectively.Nomixedinfectionsofthesepathogensweredetected.ThenucleotidesequencesofCryptosporidium,GiardiaandEnterocytozooninthisstudyweredepositedinGenBankunderaccessionnumbersKY575457toKY575459(seeTable2-2).2.3.1.CryptosporidiumOfthethreepathogenstestedforinthepresentstudy,theinfectionrateofCryptosporidiumspp.wasthehighest(2.0%)amongthe500children.ThisnumberwasconsistentwithpreviousmolecularepidemiologicalstudiesofCryptosporidiuminchildrenfromothercities/provincesofChina(1.6%,Fengetal.,2012;0.1%,Zhangetal.,2016a).TheageandgenderdistributionofentericpathogeninfectionsisshowninTable2-3.Cryptosporidiumwasfoundacrossallageandgendergroupsofdiarrhoeicchildren(Table2-3).Datashowedthat2.2%(6/271)ofmalesand1.7%(4/229)offemalesweretest-positiveforCryptosporidium;accordingtoage,2.4%(5/209)ofinfantsof<2yearsofage,1.7%(4/238)childrenof2to5years,and1.9%(1/53)ofchildrenofN5yearsweretest-positiveforthisprotist.Nevertheless,nosignificantdifferencesweredetectedamongdifferentgenderoragegroups(P=0.710or0.865,respectively).SequencingofampliconsfromCryptosporidium-positivesamples(n=10)revealedC.meleagridis,whichmainlyinfectsbirds(e.g.,turkeys,chickens,pigeons,parrots,cockatielsandotherpetbirds)(Ryan,2010;Qietal.,2011).C.meleagridis,asazoonoticCryptosporidiumspecies,hasbeenmainlyreportedfromchildrenandimmuno-compromisedor-suppressedpersonsaroundtheworld(Gateietal.,2002;Camaetal.,2003,2007;Koehleretal.,2013).Duetoitsrelativelylowhostspecificity,bothanthroponoticandzoonotictransmissionpathsofC.meleagridishavebeensuggested(Xiao,2010).85 HuazhongAgriculturalUniversityMaster’sDegree2018Table2-3:AgeandgenderdistributionsofCryptosporidiummeleagridis,GiardiaduodenalisandEnterocytozoonbieneusidetectedusingPCR-basedmethodsinfaecalsamplesfromdiarrhoeicchildrenfromWuhaninHubeiprovince(betweenJuneandAugust2016).GroupsTest-positive/totalno.Test-positive/totalTest-positive/totalno.tested(%)forno.tested(%)fortested(%)forCryptosporidiumGiardiaduodenalisEnterocytozoonmeleagridisbieneusiAge(years)<25/209(2.4)2/209(1.0)1/209(0.5)2–54/238(1.7)3/238(1.3)0/238(0)>51/53(1.9)2/53(3.8)0/53(0)GenderMale6/271(2.2)3/271(1.1)1/271(0.4)Female4/229(1.7)4/229(1.7)0/229(0)Totals10/500(2.0)7/500(1.4)1/500(0.2)InChina,C.meleagridishasbeendetectedpreviouslyinsixpediatricpatientsinthreehospitalsinShanghaiandfiveHIV-positiveindividualsinHenanprovince(Fengetal.,2012;Wangetal.,2013b).GiventhelargenumberofpetbirdsandmorefrequentcontactofpeoplewiththeirpetbirdsinChina,childrenandtheelderly,whicharethoughttobemoresusceptibletoC.meleagridis(seeQietal.,2011),couldbecomeinfected.Interestingly,thisisthefirststudytoshowadominanceofC.meleagridisinacohortofchildreninWuhan,whereaspreviousdatahaveindicatedthatC.hominisisthemostfrequentlyidentifiedspeciesinChina(Pengetal.,2001;Wangetal.,2011;Fengetal.,2012).Afurtheranalysisofpgp60nucleotidesequencedatashowedthatonlythesubtypeIIIb[IIIbA21G1R1(n=1),IIIbA22G1R1(n=8)andIIIbA26G1R1(n=1)]wasrepresentedbyC.meleagridis-positivesamples(cf.Fig.2-1).ThissubtypehasbeenfoundmainlyinAsiancountries,includingChina,India,IndonesiaandThailand(Stensvoldetal.,2014).Inthepresentstudy,itwasnotpossibletoacquiredetailedepidemiologicalinformationrelatingtothesamplestested,suchthatthesourceofC.meleagridisinfectioninchildrenispresentlyunclear.Therefore,detailedstudies(i.e.largersamplesizes,differentlocationsanddynamictimepoints)areneededtoestablishthesource(s)ofC.meleagridisinfectioninhumansinWuhan,andtoconductmoresurveysinHubeiprovincetoestablishwhetherdomesticorwildbirds(e.g.,pigeonsorsparrows)playaroleintransmission.86 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaFig.2-1:PhylogeneticrelationshipofCryptosporidiummeleagridisfromchildreninthepresentstudy(bold-type,withGenBankaccessionnumber)withotherCryptosporidiumtaxabasedonanalysisofpgp60sequencedatabyBayesianinference.ReferencesequencesfortheCryptosporidiumtaxaarelistedinTable2-2;CryptosporidiummeleagridissubtypeIIId(GenBankaccessionno.DQ067570.1)wasusedastheoutgroup.Posteriorprobabilitiesareindicatedatthenodes.2.3.2.GiardiaPCR(ptpi)screeningidentifiedsevensamplesthatweretest-positiveforGiardia.Thissmallpercentage(1.4%)ofG.duodenalisdeterminedhereisinaccordancewithfiguresreportedforoutpatients(1.4-9.5%)inotherpartsofChina(Wangetal.,2011).SimilartoresultsforCryptosporidium,G.duodenaliswasalsofoundacrossallageandgendergroupsofdiarrhoeicchildren(Table2-1).Datahereshowthat1.1%(3/271)ofmaleand1.7%(4/229)offemalechildrenweretest-positiveforGiardia,whereas1.0%(2/209)infantsof<2yearsofage,1.3%(3/238)ofchildrenagedbetween2and5yearsand3.8%(2/53)childrenof>5yearsofagewereidentifiedastest-positiveusingthePCR-coupledsequencingapproach.TheChi-squaretestdidnotshowanysignificantage-orgender-associateddifferenceintheprevalenceofG.duodenalisinfection(P=0.287and0.544,respectively).87 HuazhongAgriculturalUniversityMaster’sDegree2018AlthoughG.duodenalisassemblageBhasbeendescribedpreviouslyasbeingmorecommoninchildreninothercountries,suchasCambodia,Ethiopia,LebanonandMorocco(ElFatnietal.,2014;Loboetal.,2014;deLucioetal.,2016;Mooreetal.,2016;Osmanetal.,2016),allsevenptpisequences(490bp)determinedherewerethesameasthatwithGenBankaccessionno.GU564278,whichbelongstosub-assemblageA-II(Wangetal.,2011).AccordingtolimitedgenotypicstudiesofG.duodenalisinChina,exceptforarecentstudywhichidentified16canid-specificassemblageCfrom17Giardia-positivediarrhealoutpatientsinShanghai(Liuetal.,2014),bothsubtypesA-IandA-IIofassemblageA,aswellasassemblageB,werefoundpreviouslyinChineseoutpatientsandinpatients(Yongetal.,2000;Wangetal.,2011;Wangetal.,2013a).InAnhuiprovince,thefourtest-positivesamplesfromindividualswereeitherassemblageAorB(Yongetal.,2000);inHebeiprovince,threepatientswerediagnosedashavingG.duodenalissub-assemblageA-IIinfection(Chengetal.,2001);inHenanprovince,assemblagesAandBwereidentifiedin12andsixpatients,respectively(Wangetal.,2011);inShanghai,G.duodenalisassemblagesAandBwerereportedinsevenand11patients,respectively,duringacryptosporidiosisoutbreakinapediatrichospital(Wangetal.,2013a).BothassemblagesAandBtypicallyfollowananthroponoticratherthanazoonotictransmissionroute(RyanandCaccio,2013).ThisinformationsuggeststhathumansareprobablythemajorsourceforgiardiasisinWuhan,Hubeiprovince,butthisproposalrequirestesting.2.3.3.EnterocytozoonE.bieneusiwasidentifiedinonefaecalsamplefromaoneyear-oldboy.AnanalysisrevealedthatthesequencederivedfromthissamplewasthesameasthatwithGenBankaccessionno.JQ029731(Wangetal.,2013b).ThissequencerepresentsgenotypeD,whichispresentlythepredominatezoonoticgenotype(SantínandFayer,2009).Duetothefactthattherewasonlyonetest-positivesample,itwasnotpossibletoconductanystatisticalanalyses.AlthoughE.bieneusicauses~90%ofreportedhumancasesofmicrosporidiosis,thetransmissionroute/softhispathogenis/areunclear(Matosetal.,2012).ApreviousstudyindicatedthatthereleaseofsporesfromE.bieneusiintotheenvironmentviastoolandrespiratorysecretionsisprobablytheprincipalsourceofcontamination/infection(Mathisetal.,2005).InChina,E.bieneusiwasfirstreportedindiarrhoeicchildren(prevalence:9/40,22.5%),pigs(prevalence:10/61,16.4%),dogs(prevalence:2/26,7.8%)andcows88 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China(prevalence:35/93,37.6%)inJilinprovincein2011(Zhangetal.,2011).Subsequently,E.bieneusiinfectionwasreportedinhumansfromothercities/provinces(Wangetal.,2013a,2013b;Liuetal.,2014;Yangetal.,2014)aswellasinotherhostspecies,includingbuffaloes(Bubalusbubalis)(Maetal.,2015),cattle(Bostaurus)(Maetal.,2015;Lietal.,2016a),goats(Capraaegagrushircus)(Pengetal.,2016),horses(Equusferuscaballus)(Qietal.,2016),cats(Feliscatus)(Lietal.,2015),rabbits(Oryctolaguscuniculus)(Yangetal.,2016),birds(Anasplatyrhynchosdomesticus,Anseranserdomestica,Columbalivia,Gallusgallusdomesticus,Grusjaponensis,Gr.grus,Gr.vipioandGr.leucogeranus)(Lietal.,2014;Zhaoetal.,2016),bears(Helarctosmalayanus,UrsusarctospruinosusandU.thibetanus)(Lietal.,2016b),deer(Axisporcinus,Cervuselaphus,Ce.nipponandElaphurusdavidianus)(Zhangetal.,2015;Lietal.,2016b),foxes(Alopexlagopus,VulpesvulpesandV.lagopus)(Yangetal.,2015;Zhaoetal.,2015;Zhangetal.,2016b),non-humanprimates(i.e.Macacafascicularis,M.mulatta,M.fuscata,Papioanubis,PresbytisleucocephalusandRhinopithecusroxellana)(Karimetal.,2014a,2014b;Lietal.,2016b),raccoondogs(Nyctereutesprocyonoides)(Yangetal.,2015;Zhaoetal.,2015),reptiles(NajanajaandPtyasmucosus)(Karimetal.,2014c),rodents(Chinchillalanigera)(Qietal.,2015),squirrels(Callosciuruserythraeus)(Dengetal.,2016),redpandas(Ailurusfulgens)andgiantpandas(Ailuropodamelanoleuca)(Tianetal.,2015),inChina.Notably,highsequencediversityinITSisknowntoexistwithinE.bieneusiinChina(Wangetal.,2013b;Yangetal.,2014),indicatingthatmorein-depthstudiesareneededtoestablishthesub-structuringofE.bieneusipopulationsandtheepidemiologicalsignificanceofEnterocytozoongenotypes.2.4.ConcludingremarksInthepresentstudy,forthefirsttime,wehavereportedandcharacterizedgenotypesorassemblagesofCryptosporidium,GiardiaandEnterocytozooninhumansinHubeiprovince,China.Itisnoteworthythat,althoughnonewgenotypesorassemblagesofCryptosporidium,GiardiaorEnterocytozoonwerediscovered,C.meleagridis(mainlyinfectingavianhosts)wasfoundtodominateinthechildrenstudiedinWuhan.Clearly,thisfindingencouragesfuturedetailed,molecular-basedstudiesofhumansandanimals(especiallypetbirdsandpoultry)inanefforttobetterunderstandthetransmissionofthisentericpathogeninhumansinChina.89 HuazhongAgriculturalUniversityMaster’sDegree2018CHAPTER3-MOLECULARINVESTIGATIONOFCRYPTOSPORIDIUMANDGIARDIAFROMPRE-ANDPOST-WEANEDCALVESINHUBEIPROVINCEAbstractTheobjectiveofthestudywastoestimatetheprevalenceofCryptosporidiumandGiardiaduodenalisindairyandbeefcalvesfromHubeiprovinceofChina.Faecalsamplesfromcalvesaged2-12weekswerecollectedfrom5differentdairyfarms(n=309)and1beeffarm(n=30)andtestedmolecularlyforthepresenceofCryptosporidiumandGiardia.ForCryptosporidium,genomicDNAswereextractedfromfaecalsamplesandthesmallsubunitrRNAand60kDaglycoproteingenes(designatedpSSUandpgp60,respectively)amplifiedbynested-PCRanddirectlysequenced.Amplifiedusinganested-PCRandsequenced.ThreespeciesofCryptosporidiumwereidentified,namelyC.bovis(n=37),C.ryanae(n=14),C.andersoni(n=2),ForGiardia,geneticanalysiswasconductedusingnestedPCR-basedsequencingofpartofthe18S-rRNAgene,revealedthatall77test-positivesamplesofisolatesrepresentedassemblageE.Basedonthesefindings,theoverallprevalencewasestimatedat15.6%(53/339)and22.7%(77/339)forCryptosporidiumandGiardia,respectively.ThisisthefirsttoreporttheprevalenceofCryptosporidiumandGiardiainpre-andpost-weanedcalvesinHubeiProvince,andencourageslarge-scalemolecularstudiesofanimalsandhumans,inanefforttobetterunderstandtheepidemiologyoftheseentericpathogensinChina.Keywords:Cryptosporidium,Giardia,Calves,PCR-basedsequencing,NuclearribosomalRNAgenes,China90 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China3.1.IntroductionCryptosporidiumandGiardiaareprotistanpathogensthatcancauseintestinaldiseasesinanimalsandhumans(ThompsonandMonis,2004;XiaoandFeng,2008;Fayer,2014).Generally,infectionofthetwoparasitesareusuallyspreadthroughthefaecal-oralroute,followingtheingestionofinfectiveoocystsorcysts(Korichetal.,1990;Fayeretal.,2000).Diarrhoeaisthemainclinicalsymptomassociatedwithcryptosporidiosisandgiardiasis,aswellasdehydration,fever,inappetence,nausea,anorexiaanddeath(Siddiquietal.,2007).Theseparasitescaninfectarangeofvertebratesincludinghumans(Fayer,2010;RyanandCacciò,2013).Clinicalsymptomsareoftenself-limitinginimmuno-competentindividuals(O'Donoghue,1995;HomanandMank,2001).However,immuno-compromisedor-suppressedindividualsareatriskoflife-threateninginfections/disease(Zardietal.,2005;Petrietal.,2008).Themainriskfactorsforhumancryptosporidiosisandgiardiasisincludedirectcontactwithdiarrhoeicpersons,theingestionsofcontaminatedfoodorwateranddirectcontactwithinfectedanimals(Fayeretal.,2000;XiaoandRyan,2004;Xiao,2010).Cryptosporidiosisandgiardiasiscanbetransmittedviaanthroponoticorzoonotictransmission(XiaoandFayer,2008).Inthepast20years,infectedcattleareconsideredasthemaincontributorofhumancryptosporidiosis,particularlyinpre-weanedcalves,whicharelikelyakeyreservoirforzoonoticinfections(O'Handleyetal.,2000;Learmonthetal.,2003,Xiao,2010).Todate,molecularstudieshaveidentifiedC.andersoni,C.bovis,C.felis,C.hominis,C.parvum,C.ryanae(previouslycalleddeer-likegenotype)andC.suisandtwogenotypesof“CryptosporidiumpiggenotypeII”andanewC.suis-likegenotypeincattle(TroutandSantín,2008;Wangetal.,2011).ForGiardiaduodenalis,molecularepidemiologicalstudieshavereportedassemblagesA,B,E,withthelatterbeingthepredominantgenotypeofcattle,followedbyassemblagesAandB(CacciòandRyan,2008).Cattle,particularlycalves,infectedwiththeseprotistscanrepresentasignificantsourceofzoonoticinfectionsanddisease(O'Handleyetal.,2000;Xiao,2010).Nevertheless,cattlecanbecomeinfectedwithvariousspeciesofCryptosporidiumorGiardia(Ortega-Pierresetal.,2009;XiaoandFeng,2011).Thespeciesandgenotypesoftheseprotistsareknowntovaryamonghostsofdifferentagegroups(Xiao,2010;XiaoandFeng,2011)andgeographicallocation(Fengetal.,2007;Ngetal.,2011)andnew,previouslyundescribedgenotypesofCryptosporidiumarebeingdiscoveredinregionsof91 HuazhongAgriculturalUniversityMaster’sDegree2018theworldnotexploredpreviously(cf.JexandGasser,2010;Abeywardenaetal.,2015).Therefore,incountries,suchasChina,inwhichtherearerelativelylimitedmolecularepidemiologicaldata,establishingandcomparingthespecificand/orgenotypicidentityoftheseprotistsinanimalsandhumansarecentraltoassessingtheirzoonoticpotential.InChina,thediseasescausedbyCryptosporidiumandGiardiaarenotnotifiableinChina,butinfectedcattlearebelievedtoserveassignificantreservoirsforzoonoticinfections(Qietal.,2015).Althoughthereisapositiveassociationbetweenthedensityofdomesticlivestock,includingcattle,andtheprevalenceofCryptosporidiuminfectioninhumansinothercountries(e.g.,NewZealand)(Sneletal.,2009),therearepresentlylimitedepidemiologicaldataforChinatosupportthisobservation.MostpreviousstudiesdescribingthegeneticcharacterizationofCryptosporidiumfromcalveshavebeenconductedmainlyinthenortheast,northwestandcentralregionsofChina(Liuetal.,2009;Wangetal.,2011a;Wangetal.,2011b;Zhangetal.,2013;Cuietal.,2014;Huangetal.,2014;Qietal.,2015;Lietal.,2016).Similarly,limitedmoleculardataareavailableforGiardiaofcattleinChina,althoughG.duodenalisassemblagesAandBhavebeenidentifiedincalves(Huangetal.,2014;Wangetal.,2014;Liuetal.,2015;Lietal.,2016;Wangetal.,2016;Wangetal.,2017).InHubeiProvince,theonlystudiesofCryptosporidiumandGiardiaarethoseindiarrhoeicchildren(Wangetal.,2017)andgoats(Mietal.,2014).TheaimofthepresentstudywastoexplorethespeciesandgenotypesofCryptosporidiumandGiardiaincalvesonfarmsinHubeiprovinceusingmarkersinthelarge(LSU)orsmall(SSU)subunitsofnuclearribosomalRNAgenes,andtoassessthezoonoticpotentialoftheseparasites.3.2.MaterialsandmethodsAtotalof339freshfaecalsampleswerecollected(SeptembertoDecember2016)frompre-andpost-weanedcalves(1to12weeksold)fromonebeeffarmandfivedairyfarms(i.e.Suizhou,Chezhan,Guangming,Meijiadun,QiaonerandYangzijiang)inthenorth,eastandwestregionsofHubeiProvince,China(Fig.3-1).Faecalsampleswerecollectedrectallyfromindividualcalvesandkeptat4°Cfollowingsampling,andthenfrozenat-20°CforsubsequentDNAisolationandmoleculartesting.GenomicDNAwasextractedfrom0.2gofeachfaecalsampleusingthePowerSoilDNAIsolationKit(MoBio,Carlsbad,USA),accordingtothemanufacturer’sinstructions,andthenfrozenat-20ºC92 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,Chinauntiluse.Thiskitwasused,asitishighlyeffectiveatremovingcomponentsthatareinhibitorytoPCR(Pontirolietal.,2011;Koschetal.,2013;Nolanetal.,2013).NSuizhouChezhanYangzijiangQiaoniurGuangmingMeijiadun50KmFig.3-1:LocationsofthesixfarmsinHubeiprovincefromwhichfaecalsamples(numbersinparentheses)werecollectedfrompre-andpost-weanedcalvesandtestedusingPCR-basedmethods.ShortlyafterDNAextraction,Aliquots(2μl)ofindividualgenomicDNAsamplesweresubjectedtonestedPCR-basedamplificationandsequencing,employing(individually)fourdistinctlociofnuclearDNAinseparateassays.FordetectionofCryptosporidium,aportionofthelargesubunitofthenuclearribosomalRNAgene(pLSUc;~500bp)wasused(Koehleretal.,2017),andfurthergenotypic/subgenotypicclassificationwasachievedbyemployingaportionofthesmallsubunitofthenuclearribosomalRNAgene(pSSUc;~590bp)(Ryanetal.,2003).Forthespecificandassemblage-basedclassificationofGiardia,aportionoftheSSUgene(pSSUg;~290bp)wasemployed(Hopkinsetal.,1997;Appelbeeetal.,2003)(listedinTable3-1).Inbrief,nestedPCRswerecarriedoutin50μlusingastandardreactionbuffer,2.0-3.0mMofMgCl2(dependingonthelocus),200μMofeachdNTP,50pmolofeachprimerand1UofTaqpolymerase(MangoDNApolymerase,Bioline,London,UK)usingestablishedcyclingprotocols.Exceptfortheno-templatecontrols,2μlofgenomicDNAwereaddedtotheprimaryPCR,fromwhich1μlwascarriedovertothesecondaryPCR.Knowntest-positive,test-negativeandno-templatecontrolswereincludedineachPCRrun.93 HuazhongAgriculturalUniversityMaster’sDegree2018ForCryptosporidium,theprimaryamplificationprotocolwas:94℃/5min(initialdenaturation),followedby30cyclesof94℃/45s(denaturation),45℃/2min(annealing),and72℃/1.5min(extension)andafinalextensionof72℃/10min.OneµlofprimaryampliconwascarriedovertothesecondaryPCR,whichwasperformedusingthefollowingconditions:acyclingprotocolof94℃/5min(initialdenaturation),followedby35cyclesof94℃/30s(denaturation),55℃/30s(annealing)and72℃/30s(extension),withafinalextensionof72℃/10min.ForGiardia,primaryamplificationofSSUutilisedthecyclingprotocol,94°Cfor4min(initialdenaturation),followedby35cyclesof94°Cfor45s(denaturation),55°Cfor30s(annealing)and72°Cfor45s(extension),withafinalextensionof72°Cfor4min.ForthesecondaryamplificationofSSU,weemployed94°Cfor2min,followedby35cyclesof94°Cfor20s,59°Cfor20s,and72°Cfor30s,withafinalextensionat72°Cfor7min.AllnestedPCRproductsweredetectedbyelectrophoresisinethidiumbromide-stained(1.5%)agarosegelsbeforesequencing.Forsequencing,aliquots(5μl)ofindividualamplicons(undigested)weretreatedwiththeenzymesExoIandathermosensitivealkalinephosphatase(FastAP,Thermofisher,Carlsbad,USA),accordingtothemanufacturer’sinstructions,andthensubjectedtodirect,automatedsequencing(BigDyeTerminatorv.3.1chemistry,AppliedBiosystems,USA)inbothdirectionsusingthesame,internalprimersasemployedinPCR.Thequalityofeachsequencewasassessedbasedonthecorrespondingchromatogram,andsequenceswerematchedtoreferencesequencesfromtheGenBankdata-base(listedinTable3-2)usingtheBasicLocalAlignmentSearchTool(BLAST;http://www.ncbi.nlm.nih.gov/BLAST).Chi-squaretestwasperformedusingSPSSStatistics24software(IBM,NewYork,USA).3.3.ResultsanddiscussionUsingtwoseparatePCRassays,the339individualfaecalDNAsamplesfromsixfarmsfromfourcounties/citiesofHubeiProvincewerescreenedbyPCRforthepresenceofCryptosporidiumspp.andGiardiaduodenalisDNA,respectively.Intotal,15.5%(n=53)and22.6%(n=77)faecalDNAsamplesweretest-positiveforCryptosporidiumandGiardia,respectively.Ampliconsweresequenced,andthenucleotidesequencesofCryptosporidiumandGiardiaweredepositedintheGenBankdatabaseunderaccessionnos.MF196907-MF196910.94 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China3.3.1.CryptosporidiumNestedPCR-basedsequencingofpLSUcidentified53samplesthatweretest-positiveforthreeCryptosporidiumspecies,includingCryptosporidiumandersoni(0.6%;2of339)ontwofarms,andC.bovis(10.9%;37of339)andC.ryanae(4.1%;14of339)onfivefarms(Table3-3).Nomixedinfectionsofthesespeciesweredetected.FarmMeijiadunshowedthehighestoverallinfectionrate(31.3%;5of16)ofCryptosporidium,whereasfarmSuizhouhadthelowestrate(3.3%;1of30).AtleastoneCryptosporidiumspecieswasdetectedoneachfarm.CryptosporidiumandersoniwasfoundonlyonfarmsQiaonerandYangzijiangintwopre-weanedcalvesof2and8weeksofage,respectively.CryptosporidiumboviswasthemajorspeciesonfarmsYangzijiang,Meijiadun,QiaonerandGuangming,whereasC.ryanaewasthedominantspeciesonfarmsChenzhanandShuizhou(Table1).TherewasnosignificantdifferenceinCryptosporidiumprevalencebetweenthebeeffarmandthedairyfarms(χ2=3.776,df=1,P=0.0520).FurtheranalysisofpSSUcnucleotidesequencedatashowedthatallsequencesobtainedwereidenticaltoreferencesequencesfromGenBank(Table3-2).ForC.andersoni,thesequencematchedthatrecordedpreviouslyinChongqing,China(calves;JX515549;unpublished);forC.bovis,threesequencesmatchedthoserecordedinQinghaiProvince(yak;KU052813;Lietal.,2016),SichuanProvince(cattle;KT884495;unpublished)orGansuProvince(cattle;KP994913;Zhangetal.,2015),China;forC.ryanae,twosequencesmatchedthoserecordedpreviouslyinGansuProvince(cattle;KP994915;Zhangetal.,2015)andXinjiangProvince(cattle;KP793013;Qietal.,2015),China.Cryptosporidiumwasfoundacrossbothgendersandallagegroupsofcalves(Table3-4).Theresultsshowthat8.2%(4of49)ofmaleand16.9%(49of290)offemalecalvesweretest-positiveforCryptosporidium,whereas15.8%(42of265)ofpre-weanedcalves(≤8weeks)and15.2%(11of74)ofpost-weanedcalves(>8weeks)wereidentifiedastest-positiveusingthePCR-coupledsequencingapproachforpSSUc(Table3-4).TheChi-squaretestdidnotshowanysignificantage-orgender-associateddifferenceintheprevalenceofCryptosporidiuminfection(P=0.838and0.119,respectively).95 HuazhongAgriculturalUniversityMaster’sDegree2018Table3-1:OligonucleotideprimersandPCRprotocolusedinthisstudy.GenusGeneregionSequenceofprimers(5'to3')AmpliconReferencesize(bp)CryptosporidiumpLSUcLSU2040F:CGAATAGCGTTATCTTTGCTATTT~500Koehleretal.(2017)LSU3020R:GTCTTCCGCGAAGATCAGLSU2065F:TTACCATGGAATYAGTTCAGCLSU2557R:AACACCATTTTCTGGCCATCCryptosporidiumpSSUc18SiCF2:GACATATCATTCAAGTTTCTGACC~590Ryanetal.(2003)18SiCR2:CTGAAGGAGTAAGGAACAACC18SiCF1:CCTATCAGCTTTAGACGGTAGG18SiCR1:TCTAAGAATTTCACCTCTGACTGGiardiapSSUgGia2029:AAGTGTGGTGCAGACGGACTC~290Appelbeeetal.(2003)Gia2150c:CTGCTGCCGTCCTTGGATGTRH11:CATCCGGTCGATCCTGCCHopkinsetal.(1997)RH4:AGTCGAACCCTGATTCTCCGCCAGG96 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaInChina,increasedattentionisnowbeingpaidtocryptosporidiosisoflivestock(Gongetal.,2017)dueitsclinicalsignificanceinyoungfarmanimals(Hollandetal.,1990;deGraafetal.,1999)andpotentialtospreadtohumans.PreviousstudieshaveindicatedthatCryptosporidiumhasarelativelywidedistributionincattleofdifferentbreedsandagesinChina[reviewedbyGongetal(2017)].SincetheinitialdetectionofCryptosporidiumindiarrhoeiccalves(prevalence:45.2%;19of42)inGansuProvince(Chenetal.,1986),atleastninespeciesofCryptosporidium[i.e.C.andersoni(Wangetal.,2011),C.bovis(Wangetal.,2011),C.parvum(Cuietal.,2015),C.ryanae(Liuetal.,2009),C.ubiquitum(Maetal.,2014),C.meleagridis(Zhangetal.,2013),C.xiaoi(Maetal.,2014),C.serpentis(Chenetal.,2012)andC.suis-like(Maetal.,2015)]havebeenreportedtodateincattleofdifferentbreedsandagesinChina(reviewedbyGongetal(2017)).SincetheinitialdetectionofCryptosporidiumindiarrhoeiccalves(prevalence:45.2%;19of42)inGansuProvince(Chenetal.,1986),atleastninespeciesofCryptosporidium(i.e.C.andersoni(Wangetal.,2011),C.bovis(Wangetal.,2011),C.parvum(Cuietal.,2015),C.ryanae(Liuetal.,2009),C.ubiquitum(Maetal.,2014),C.meleagridis(Zhangetal.,2013),C.xiaoi(Maetal.,2014),C.serpentis(Chenetal.,2012)andC.suis-like(Maetal.,2015))havebeenreportedtodateincattlein19regions/provincesofChina.OfthesenineCryptosporidiumspecies,C.andersoni,C.bovis,C.parvumandC.ryanaewerethepredominantspeciesincattleinChina(Gongetal.,2017).AlthoughC.parvumhasbeendescribedpreviouslyasbeingmorecommoninpre-weanedcalvesinnumerousindustrializedcountries(e.g.Australia,Belgium,JapanandtheUSA(Santínetal.,2004;Geurdenetal.,2007;Nolanetal.,2009;Karanisetal.,2010)aswellassomeprovinces/regionsofChina(Cuietal.,2014;Huangetal.,2014;Qietal.,2015;Lietal.,2016),thiswasnotthecaseinthepresentstudy,inwhichC.boviswasidentifiedasbeingthepredominantspeciesinpre-weanedcalvesinHubeiProvince.ThisfindingisconsistentwithpreviousobservationsintheprovincesGansu(Zhangetal.,2015),Henan(Wangetal.,2011),Helongjiang(Zhangetal.,2013),Shaanxi(Qietal.,2015)aswellasinShanghai(Caietal.,2017).Interestingly,thepredominanceofC.bovisinpre-weanedcalvesinChinacontraststhesituationinmanyothercountriesstudiedtodate,whereC.parvumtendstobethemajoragentdetected(Abeywardenaetal.,2014).Althoughthisdifferenceischallengingtoexplain,itmightrelatetodifferentfarmingpracticesorsimplythatC.parvumisnotwidelyestablishedinlivestockpopulationsinsomeregionsofChina.Nonetheless,theremaybeseasonaldifferencesinthepresenceofC.bovisandC.parvum.Indeed,Wangetal.proposedthataseasonalshiftmightberesponsibleforthedominance97 HuazhongAgriculturalUniversityMaster’sDegree2018ofoneormoreCryptosporidiumspeciesoverothersinpre-weanedcalves(Wangetal.,2011).Future,large-scaletemporalandspatialstudiesarerequiredtotestthishypothesis.Asseeninthisstudy,C.andersoni,C.bovisandC.ryanaeinfectionspredominatedinpost-weanedcalves,andarenormallynotassociatedwithobviousclinicalsignsinthecattle(Estebanetal.,1999;Fayeretal.,2005;Fayeretal.,2008;Wangetal.,2011).Nevertheless,suchsubclinicalCryptosporidiuminfectionsincattleshouldnotbeneglected,astheymayrelatetochronicinfectionsandcanleadtodecreasedfeedefficiency(Ralstonetal.,2003),impairedweightgainorweightloss(Andersonetal.,1987)andmilkproductionlossesindairycows(Estebanetal.,1995).GiventheexpansionofthedairyindustryinHubeiProvinceinChina,furtherstudiesmightexploretheepidemiologicalsignificanceofCryptosporidiumgenotypesandthesub-structuringofCryptosporidiumpopulationsindairycattle.Table3-2:SummaryofinformationonthereferencesequencesfromtheGenBankdatabaseusedinthepresentstudy.SpeciesHostLocalityGeneGenBankNo.ReferenceCryptosporidiumandersoniCattleChinapSSUcJX515549UnpublisheddataCryptosporidiumbovisYakChinapSSUcKU052813Lietal.(2016)CryptosporidiumbovisCattleChinapSSUcKT884495UnpublisheddataCryptosporidiumbovisCattleChinapSSUcKP994913Zhangetal.(2015)CryptosporidiumryanaeCattleChinapSSUcKP994915Zhangetal.(2015)CryptosporidiumryanaeCattleChinapSSUcKP793013Qietal.(2015)GiardiaduodenalisCattleChinapSSUgKF843921Wangetal.(2014)GiardiaduodenalisCattleBrazilpSSUgJF957620UnpublisheddataGiardiaduodenalisCattletheUSApSSUgJN375981Santínetal.(2012)GiardiaduodenalisCattletheUSApSSUgJN375981Troutetal.(2004)GiardiaduodenalisDeerChinapSSUgKX259145UnpublisheddataGiardiaduodenalisTakinChinapSSUgKR048491Zhaoetal.(2015)98 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaTable3-3:OccurrenceofCryptosporidiumandersoni,Cryptosporidiumbovis,CryptosporidiumryanaeandGiardiaduodenalisinfectioninpre-andpost-weanedcalves(n=339)fromsixfarmsinHubeiprovince.FarmNo.ofsamplesNo.ofsamplesCryptosporidiumspeciesNo.ofsamplestestedtest-positiveforC.andersoni(%)C.bovis(%)C.ryanae(%)test-positiveforCryptosporidiumGiardiaduodenalis(%)spp.(%)Suizhou(beeffarm)301(3.3)001(3.3)7(23.3)Chezhan(dairyfarm)205(25.0)01(5.0)4(20.0)12(60.0)Guangming(dairyfarm)15621(13.4)017(10.9)4(2.6)31(19.9)Meijiadun(dairyfarm)165(31.2)05(31.3)03(18.8)Qiaoner(dairyfarm)248(33.3)1(4.2)4(16.7)3(12.5)15(57.7)Yanzijiang(dairyfarm)9313(14.0)1(1.1)10(10.8)2(2.2)9(9.7)Totals33953(15.6)2(0.6)37(10.9)14(4.1)77(22.6)99 HuazhongAgriculturalUniversityMaster’sDegree2018Table3-4:AgeandgenderdistributionsofCryptosporidiumspp.andGiardiaduodenalisdetected,usingPCR-basedmethods,infaecalsamplesfrompre-andpost-weanedcalves(n=339)fromsixfarmsinHubeiprovince.GroupsNo.ofNo.test-positiveforCryptosporidiumspp.No.test-positiveforsamplesCryptosporidiumC.andersoni(%)C.bovis(%)C.ryanae(%)Giardiaduodenalistestedspp.(%)(%)Age(weeks)Pre-weaned(≤8)26542(15.8)2(0.8)32(12.1)8(3.0)49(18.5)Post-weaned(>8)7411(14.9)05(6.8)6(8.1)28(37.8)GenderFemale29049(16.9)2(0.7)34(11.7)13(4.5)66(22.8)Male494(8.2)03(6.1)1(2.0)11(22.4)100 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China3.3.2.GiardiaGiardiaDNAwasdetectedin77faecalDNAsamples.SimilartotheresultsforCryptosporidium,G.duodenaliswasalsofoundincalvesonallsixfarms,withanaverageprevalenceof22.6%(Table3-3).Thisnumberwasconsistentwithpreviousstudiesofpre-andpost-weanedcalvesfromothercities/provincesofChina(Qietal.,2016;Wangetal.,2016)aswellasothercountries(cf.(Abeywardenaetal.,2014)).TheprevalenceofGiardiavariedfromfarmtofarminHubeiProvince.FarmChezhanhadthehighestpercentage(60.0%;12of20)ofinfectedcalves,whereasfarmYangzijianghadthelowestpercentage(9.7%;9of93).TherewasnosignificantdifferenceintheprevalenceofG.duodenalisbetweenthebeefanddairyfarms(χ2=0.007,df=1,P=0.933).TheageandgenderdistributionsofG.duodenalisareshowninTable3-4.Giardiawasfoundacrossbothsexesandallagesofcalves(Table3-4).TheprevalencesofG.duodenaliswere18.5%(49of265)and37.8%(28of74)inpre-andpost-weanedcalves,respectively,withasignificantdifference(χ2=12.335,df=1,P=0.0004)betweenthetwogroups.Accordingtogender,theresultsshowedthat22.4%(11of49)ofmalesand22.8%(66of290)offemalesweretest-positiveforGiardia(Table3-4).Nevertheless,therewasnosignificantdifferencebetweenthesexes(χ2=0.002,df=1,P=0.964).ThesequencingofampliconsfromGiardia-positivesamples(n=77)revealedassemblageE.Thisassemblagehasbeencommonlydetectedinpreviousstudiesofpre-andpost-weanedcalvesfromotherregionsinChina,includingHeilongjiang(Liuetal.,2014;Liuetal.,2015),Shaanxi(Wangetal.,2016),Beijing(Lietal.,2016),Henan(Wangetal.,2014),Liaoning(Liuetal.,2015),Jinling(Lietal.,2016)andShanghai(Wangetal.,2017),aswellasothercountriesintheworld(Abeywardenaetal.,2014).ThepSSUgsequences(292bp)determinedherewerethesameasthoserepresentingG.duodenalisassemblageEisolatesfromcattlefromChina(GenBank:KF843921(Wangetal.,2014)),Brazil(GenBank:JF957620;unpublisheddata)andtheUSA(GenBank:JN375981(Santínetal.,2012));(GenBank:AY655701(Troutetal.,2004)),aswellasfromdeer(GenBank:KX259145;unpublisheddata)andtakin(Budorcastaxicolor)(GenBank:KR048491(Zhaoetal.,2015))fromChina.AlthoughcurrentpreliminaryresultsofG.duodenalisfromcalvesinHubeiProvince101 HuazhongAgriculturalUniversityMaster’sDegree2018suggestthatthepublichealthriskofzoonoticgiardiasisislowhere,previouslongitudinalinvestigationsofcattlegiardiasishaveindicatedthepossibilityofthetransientpresenceofzoonoticassemblages(Becheretal.,2004).AlthoughassemblageE,identifiedinthepresentstudy,hadnotbeenconsideredzoonotic(Xiao,2010;RyanandCacciò,2013),somerecentstudiesdescribetheoccurrenceofassemblageEinhumansinQueensland,Australia(Zahedietal.,2017),Brazil(Fantinattietal.,2016)andEgypt(Forondaetal.,2008;Abdel-Moeinetal.,2016;Helmyetal.,2014),indicatingthatthisassemblagecanindeedbezoonotic.TheserecentfindingscouldhaveimportantpublicimplicationsinruralregionsofcountriessuchasChina,wherethereisacloserelationshipbetweenlivestockandhumans.Therefore,large-scaletemporalandspatialstudiesshouldbeconductedinthefuturetoassessthemolecularepidemiologyandzoonoticpotentialofG.duodenalisassemblagesindifferentagegroupsandbreedsofcattleandinhumansinagriculturalregionsofHubeiProvince.3.4.ConclusionsInthepresentstudy,forthefirsttime,wehavereportedtheprevalenceofCryptosporidiumandGiardiainpre-andpost-weanedcalvesinHubeiProvince,China.Basedonthis‘snapshot’study,theprevalenceofG.duodenaliswasshowntobehigherthanthatofCryptosporidiumspp.incalvesinthisprovince.Cryptosporidiumboviswasidentifiedasthepredominantspeciesinpre-weanedcalves,consistentwithfindingsfrompreviousstudiesfromotherregionsinChina.InordertobetterunderstandthetransmissionoftheseentericpathogensinChina,furtherworkisneededtoevaluatetheprevalenceinlargenumbersofcattlefromdifferentagegroupsandbreedsindifferentregionsofChinaandatdifferenttimepointsthroughouttheyear,andalsotogainrelevantinformationregardingCryptosporidiumandGiardiainhumansandotheranimalsinoneofthefastestgrowingagriculturalandeconomicregionsinChina(Hubei).102 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaCHAPTER4-GENERALDISCUSSIONCryptosporidiumandGiardiaaretwosignificantzoonoticprotistswiththecapacitytoinfectandaffecthumans,domesticandwildanimals.Molecularmethodsareusedasepidemiologicaltoolstoconfirmtheoccurrenceofthesetwoprotistsinanimalsandhumansglobally,andzoonoticC.parvumandassemblageAofGiardiaarecommonlytransmittedfromcalvestohumans,whereasC.hominisisacquiredviaanthroponotictransmission.VerylittleisknownaboutthemolecularepidemiologyoftheseparasitesinHubeiProvince,China,includingthespecificand/orgenotypicidentityoftheseprotistsinanimalsandhumansandtheirzoonoticpotential.Inordertoaddressthisknowledgegap,studieswereundertakentogeneticallycharacteriseCryptosporidiumandGiardiaindiarrhoeicchildreninWuhan,andpre-andpost-weanedcalvesinHubeiProvince(Chapters2and3).Theaimsofthisthesiswereachieved.Inthisthesis,genotypesorassemblagesofCryptosporidiumandGiardiawerecharacterisedinhumansinHubeiProvince,China.Itisnoteworthythat,althoughnonovelgenotypesorassemblagesofCryptosporidiumandGiardiaweredetected,surprisingly,C.meleagridis(mainlyinfectingavianhosts)wasfoundinthechildrenstudiedinWuhan.Reportedwasalsotheprevalenceofthesetwoparasitesinpre-andpost-weanedcalvesinHubeiProvince,China.Overall,CryptosporidiumandGiardiaweredetectedinaconsiderablepercentageofsamplesfromfourlocations.ThreeCryptosporidiumspecies(i.e.C.andersoni,C.bovisandC.ryanae)andG.duodenalisassemblageEwereidentified.Basedonthisstudy,C.bovisandGiardiaassemblageEwereidentifiedaspredominantspeciesinpre-weanedcalves,consistentwithfindingsfrompreviousstudiesfromotherregionsofChina.InChina,thereislimitedinformationonthespecies/genotypesofCryptosporidiumandGiardia.InChapter1,bysearchingthePubMeddatabase,alistofallrecognisedspecies/genotypeswascompiled.ThereareonlyafewreportsfromChina(Table1-12),andalmostnothingforHubeiprovince,andnopreviousmolecularinformationfortheseparasitesfromhumans.Therefore,thefirstmolecularsurveywasconductedinHubei(Chapter2),andC.meleagridisandGiardiaassemblageAwereidentifiedinthissurvey.Interestingly,thisisthefirststudytorevealC.meleagridisin103 HuazhongAgriculturalUniversityMaster’sDegree2018diarrhoeicchildreninWuhan,whereaspreviousdatahadindicatedthatC.hominisiscommonestinChina(Pengetal.,2001;Wangetal.,2011;Fengetal.,2012).C.meleagridis,asazoonoticCryptosporidiumspecies,isthethirdcommonestspecies,causinginfectionsinhumans,afterC.parvumandC.hominis(seeFayer,2010).Accordingtomanyepidemiologicalsurveys,C.meleagridishasbeenrecordedprimarilyinchildrenandimmuno-suppressedpatients(Gateietal.,2002;Camaetal.,2003,2007;Koehleretal.,2013)butismainlyfoundinbirds(Ryan,2010;Qietal.,2011).Forexample,basedonamolecularstudyconductedbyWesołowskaetal.(2016),C.meleagridiswasidentifiedinanHIV-positivewomaninPoland.Similarly,C.meleagridiswasalsofoundinhumansincountriessuchasKenya,SouthAfrica,Australia,Ethiopia(Ng-Hublinetal.,2013;Adamuetal.,2014;Mbaeetal.,2015;Abaetal.,2016).Otherstudieshavealsoconfirmedthezoonoticrouteofinfection,particularlyinpeoplewhohaveintimatecontactwithpets,suchasinruralareas,suggestingthatcross-speciestransmissionofC.meleagridisbetweenbirdsorotheranimalsandhumansoccurs(Silverlåsetal.,2012;Widmeretal.,2015).Therefore,thedetectionofC.meleagridisinthisstudymaybeexplainedbythefactthatpetbirdscaninfecttheirowners(childrenandtheelderly)whomightbemoresusceptibletoinfectiousdiseases(seeQietal.,2011).Duetoalackofdetailedinformationonthesediarrhoeicchildren(Chapter2),wecouldnottracethesourceofinfection.Therefore,moresurveysshouldbeconductedtoestablishwhetherbirdsdoactuallyplayaroleinzoonotictransmission,thusaddressingapotentialpublichealthissue.VeryfewstudieshavebeenconductedonthemolecularepidemiologyofG.duodenalisinChina(Yongetal.,2000;Wangetal.,2011;Wangetal.,2013).InChapter2,allgenotypesofGiardiawereidentifiedasassemblageA.ThisfindingissimilartopreviousfindingsinChina,suchasinAnhui,HebeiandHenan,whereassemblagesAandBweredetected(Yongetal.,2000;Wangetal.,2011;Wangetal.,2013).Inaddition,duringacryptosporidiosisoutbreakinapediatrichospitalinShanghai,bothassemblagesAandBinnon-outbreakchildrenwereidentified(Wangetal.,2013).TheoccurrenceofassemblagesAandBcandifferconsiderablyamongcountries.Forexample,assemblageBhasbeendescribedasbeingrelativelydominantinchildrenincountriessuchasCambodia,Ethiopia,LebanonandMorocco(ElFatnietal.,2014;Loboetal.,2014;deLucioetal.,2016;Mooreetal.,2016;Osmanetal.,2016).AccordingtoreviewbyRyanandCaccio(2013),bothassemblagesAandBtypically104 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,Chinafollowananthroponoticratherthanazoonoticrouteoftransmission(RyanandCaccio,2013).Anotherinterestingfinding,insomestudies,isthatapotentialrelationshipmightexistbetweenassemblageAinfectionanddiarrhoea,whereasahigherinfectionratehasbeenobservedforassemblageB(Goñietal.,2010;Perézetal.,2008;Tagajdidetal.,2012).Clearly,moresurveysareneededtoilluminatetheroleassemblagesplayinGiardiainfectionsinsusceptiblehosts,particularlyinchildren,andtheirvirulenceandpathogenicity.CryptosporidiumandGiardiaarewidelydistributedincattleinChina(cf.Chapter1).ForCryptosporidium,thusfar,tenspecieshavebeenreported.Amongthem,C.andersoni,C.bovis,C.parvum,andC.ryanaearethecommonest.Molecularinvestigationhasshownthattherearedifferencesinprevalenceandspecies(Gongetal.,2017).InChina,thehighestprevalencewasfoundinpre-weanedcalves.Basedonpublishedevidence,areaswithhigherprevalenceofCryptosporidiumspeciesappeartobeineasternaswellasnorthernChina(Gongetal.,2017),andthecommonestspeciesincattlearereportedtobeC.andersoni,C.bovis,C.ryanae,andC.parvum,accordingtoallpublishedrecordsinChina(Table1-6).However,littleisknownaboutthespeciesorgenotypes/subtypesoftheseparasitesincattleinHubeiprovince,China.Therefore,asecondmolecularinvestigationofCryptosporidiumandGiardiawasundertakeninpre-andpost-weanedcalvesinHubeiprovince,China(Chapter3).Thezoonoticpotentialoftheseprotistswasassessed.Overall,theseparasitesweredetectedinaconsiderablepercentage(15.5%-22.6%)ofpre-andpost-weanedcalvesfromfourdistinctlocations.ThreeCryptosporidiumspecies(i.e.C.andersoni,C.bovisandC.ryanae)andGiardiaassemblageEwereidentified.ForCryptosporidium,C.boviswasrecordedasbeingthecommonestinpre-weanedcalves.Interestingly,thisfindingisconsistentwithpreviousobservationsincattle,inChina,suchasintheprovincesGansu(Zhangetal.,2015),Henan(Wangetal.,2011),Helongjiang(Zhangetal.,2013),Shaanxi(Qietal.,2015),andinShanghai(Caietal.,2017).Itneedstoberemarked,however,thattheabsenceofC.parvumfromsamplescollectedfromcalvesinthisstudywasunexpected,becauseitisusuallythecommonestspeciesofCryptosporidiuminpre-weanedcalvesglobally(XiaoandFeng,2008;Xiao,2010).Accordingtoepidemiologicalsurveys,C.parvumisrecognisedasthemajorspeciesinpre-weanedcalvesreportedinNorthAmericaandEuropeancountriesincludingSpain,England,GermanyandBelgium(seeSantinetal.,2004;Geurdenetal.,2007;Brogliaetal.,2008;Quilezetal.,2008;Brooketal.,2009),andismost105 HuazhongAgriculturalUniversityMaster’sDegree2018commonlydetectedincalvesoflessthan3monthsofage.Forexample,IntheUSA,basedonastudyconductedbySantínetal(2004),C.parvumwasfoundin85%of503pre-weanedcalves,whereasonly1%wasrelatedtopost-weanedcalves.Onthisaccount,pre-weanedcalvesareconsideredasthemainreservoirhostforinfectioninhumans(Jexetal.,2011).Ontheotherhand,recently,severalmolecularsurveyshavereportedanabundanceofC.bovisandalimitedexistenceofC.parvumincalvesof1-3monthsoldinChina(Fengetal.,2007;Wangetal.,2011;Zhangetal.,2013),India(Fengetal.,2007),Nigeria(Maikaietal.,2011)andSweden(Silverlåsetal.,2010).AccordingtoasurveyconductedbyZhangetal.(2013)inChina,C.boviswasdetectedin22.5%(n=34)of151pre-weanedcalves,whereasC.parvumwasfoundinonly1.3%(n=2)ofcalvesintheirstudy(Zhangetal.,2013).Interestingly,otherstudiesconductedinotherregions,suchasHenan,Gansu,Shaanxi,andHeilongjiang,havealsoreportedasimilarfinding(Wangetal.,2011;Zhangetal.,2013;Qietal.,2015;Zhangetal.,2015).Althoughthisdifferenceischallengingtoreconcile,itmaybeexplainedbythefactthatarelativelysmallamountofspecimenwereexaminedinthisstudy(Chapter3),orsimplythatthisspeciesdidordoesnotoccur.Ontheotherhand,theremaybeseasonaldifferencesinthepresenceofC.bovisandC.parvum.Wangetal(2011)proposedthataseasonalshiftmightberesponsibleforthedominanceofoneormoreCryptosporidiumspeciesoverothersinpre-weanedcalves(Wangetal.,2011).Inanycase,large-scaletemporalandspatialsurveysareneededtotestthehypothesisinthefuture.Previoussurveysfromvariouscountrieshavereportedthat80-100%oftheGiardiaisolatedfromcattleareassemblageE(Appelbeeetal.,2003;Becheretal.,2004;Troutetal.,2004;Troutetal.,2006;Ngetal.,2011;Abeywardenaetal.,2013),whichisrelativelyconsistentwiththepresentfindingsforHubei(Chapter3)andotherpartsofChina,suchasHeilongjiang,Shaanxi,Beijing,Henan,Liaoning,JinlingandShanghai(Liuetal.,2014;Wangetal.,2014;Liuetal.,2015;Lietal.,2016;Wangetal.,2016,2017).Incontrast,assemblagesAandB,whicharerecognisedasthemainzoonoticgenotypes,werenotdetectedinthisstudyinHubei,suggestingthatthepublichealthriskofzoonoticgiardiasismightbelowinthisprovince.Interestingly,Zahedietal(2017)examined88humansamplesforG.duodenalisinQueensland,Australia,andidentifiedsixsamplestocontainassemblageE.ThisisthefirstreportofassemblageEinhumansinAustralia,indicatingthatitmightbezoonotic106 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China(Zahedietal.,2017).Inaddition,Abdel-MoeinandSaeed(2016)foundthat62.5%of40Giardiatest-positivesamplesfromhumansrepresentedassemblageEofG.duodenalis.Inaddition,allisolatesfromdiarrhoeiccattlerepresentedthisassemblage(Abdel-MoeinandSaeed,2016).OtherstudiesalsoindicatethelikelyzoonoticimportanceofassemblageEinruralsettingsincountriesincludingBrazilandEgypt(Forondaetal.,2008;Helmyetal.,2014;Fantinattietal.,2016).Therefore,assemblageEmaybeemergingasazoonoticassemblage.Futurestudiesshouldpayincreasedattentiontothisaspect.Manyfactors,suchasexaminationtechniquesandsamplesizes,sanitationconditions,rearingconditions,andclimate,mayrelatetotheprevalenceofcryptosporidiosis(Gongetal.,2017).Todate,thereisalackofeffectivetreatmentsapprovedforCryptosporidium.Therefore,cattleownersshouldimplementpropermanagement,husbandryandsanitationpracticestopreventorcontrolcryptosporidiosis.Inordertodecreasetheriskoftransmissiontohumans,an‘all-in-all-out’managementstrategy(Silverlåsetal.,2009)wouldbeusefulandmightbeeffective.Inaddition,thenutritionalstatusofanimalsneedstobemaintainedorimproved,andthegovernmentshouldfocusonincreasingpublicawarenessofthesignificanceofhygienemeasuresandprovidefundingtosupportresearchinthisarea(Gongetal.,2017).Inaddition,thedevelopmentofvaccinesagainsttheseparasitesshouldimprovetheoutlookforcontrol.ConclusionThedetectionofzoonoticspecies/genotypesofCryptosporidiumand/orGiardiaindiarrhoeicchildrenandincattle(dairyandbeefcattle)reinforcestheimportanceofstudyingtheirprevalenceandtransmissiondynamicsindifferentregionsofChina.AlthoughCryptosporidiumandGiardiahavebeendetectedinhumansandcattleinmanystudies,theirfateandtransportintheenvironmentandwaterarenotwellunderstood(Abeywardenaetal.,2015).Therefore,futureresearchmightfocusoninvestigatingthefateofoocystsorcystsofCryptosporidiumandGiardiareleasedbycattleandotheranimalsintotheenvironment.Inaddition,researchinthefuturecouldfocusonvariouswildanimals,toassesstheirimportanceasvehiclesorreservoirsforzoonotictransmission.Additionally,thereisalsoaneedtoexpandfuturesurveysindevelopingcountries,wherecryptosporidiosis107 HuazhongAgriculturalUniversityMaster’sDegree2018andgiardiasisareparticularlyprevalentinhumans,butlittleisknownabouttransmissionpatternsandsourcesofinfection.Inthefuture,advancedmoleculardiagnostictools,incombinationwithtraditionalmethods,shouldbeusedtoaddressquestionsregardingtotransmissiondynamicsofCryptosporidiumandGiardiaindifferentgeographicalregions,andtheroleoftheseparasitesinzoonotictransmission.Addressingthesequestionsshouldleadtoabetterunderstandingoftheepidemiologyoftheseprotistsinhumansandanimals.Finally,vaccinesshouldbedevelopedforthepreventionofcryptosporidiosisandgiardiasis,which,ifeffective,wouldassistinthecontroloftheseimportantdiseases.108 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaReferences1.AbeN,MatsubayashiM,KimataI,IsekiM.SubgenotypeanalysisofCryptosporidiumparvumisolatesfromhumansandanimalsinJapanusingthe60-kDaglycoproteingenesequences.ParasitologyResearch,2006,99(3):303-305.2.AbeN,ReadC,ThompsonRC,etal.ZoonoticgenotypeofGiardiaintestinalisdetectedinaferret.JournalofParasitology,2005,91(1):179-182.3.AbeywardenaH,JexAR,GasserRB.AperspectiveonCryptosporidiumandGiardia,withanemphasisonbovinesandrecentepidemiologicalfindings.AdvancesinParasitology,2015,88:243-301.4.AbrahamsenMS,LanctoCA,WalcheckB,etal.Localizationofa/bandg/dlymphocytesinCryptosporidiumparvum-infectedtissuesinnaiveandimmunecalves.InfectionandImmunity,1997,65(6):2428-2433.5.AmadiB,KellyP,MwiyaM,etal.Intestinalandsystemicinfection,HIV,andmortalityinZambianchildrenwithpersistentdiarrheaandmalnutrition.JournalofPediatricGastroenterologyNutrition,2001,32(5):550-554.6.AdamsRB,GuerrantRL,ZuS,etal.Cryptosporidiumparvuminfectionofintestinalepithelium:morphologicandfunctionalstudiesinaninvitromodel.JournalofInfectiousDiseases,1994,169(1):170-177.7.AdamRD.ThebiologyofGiardiaspp.MicrobiologicalReviews,1991,55(4):706-732.8.AdamRD.TheGiardialambliagenome.InternationalJournalParasitology,2000,30(4):475-484.9.AkiyoshiDE,DiloJ,PearsonC,etal.CharacterizationofCryptosporidiummeleagridisofhumanoriginpassagedthroughdifferenthostspecies.InfectionandImmunology,2003,71(4):1828-1832.10.AkiyoshiDE,TumwineJK,Bakeera-KitakaS,etal.SubtypeanalysisofCryptosporidiumisolatesfromchildreninUganda.JournalofParasitology,2006,92(5):1097-1100.11.AmarCF,DearP,Pedraza-DiazSP,etal.SensitivePCR-restrictionfragmentlengthpolymorphismassayfordetectionandgenotypingofGiardiaduodenalisinhumanfeces.JournalofClinicalMicrobiology,2002,40(2):446-452.12.AmerS,HonmaH,IkarashiM,etal.CryptosporidiumgenotypesandsubtypesindairycalvesinEgypt.VeterinaryParasitology,2010,169(3-4):382-386.13.AmerS,ZidanS,FengY,etal.IdentityandpublichealthpotentialofCryptosporidiumspp.inwaterbuffalocalvesinEgypt.VeterinaryParasitology,2012,191(1-2):123-127.14.AloisioF,FilippiniG,AntenucciP,etal.SevereweightlossinlambsinfectedwithGiardiaduodenalisassemblageB.VeterinaryParasitology,2006,142(1-2):154-158.15.Alvarez-PelliteroP,PerezA,QuirogaMI,etal.HostandenvironmentalriskfactorsassociatedwithCryptosporidiumscophthalmi(Apicomplexa)infectioninculturedturbot,Psettamaxima(L.)(Pisces,Teleostei).VeterinaryParasitology,2009,165(3-4):207-215.16.Alvarez-PelliteroP,Quiroga,MI,Sitjà-BobadillaA,etal.Cryptosporidiumscophthalmin.sp.(Apicomplexa:Cryptosporidiidae)fromculturedturbotScophthalmusmaximus.Lightandelectronmicroscopedescriptionandhistopathologicalstudy.DiseasesofAquaticOrganisms,2004,62(1-2):133-145.109 HuazhongAgriculturalUniversityMaster’sDegree201817.Alvarez-PelliteroP,Sitjà-BobadillaA.Cryptosporidiummolnarin.sp.(Apicomplexa:Cryptosporidiidae)infectingtwomarinefishspecies,SparusaurataL.andDicentrarchuslabraxL.InternationalJournalforParasitology,2002,32(8):1007-1021.18.AlvesM,XiaoL,AntunesF,etal.DistributionofCryptosporidiumsubtypesinhumansanddomesticandwildruminantsinPortugal.ParasitologyResearch,2006,99(3):287-292.19.AlvesM,XiaoL,SulaimanI,etal.SubgenotypeanalysisofCryptosporidiumisolatesfromhumans,cattle,andzooruminantsinPortugal.JournalofClinicalMicrobiology,2003,41(6):2744-2747.20.AndersonBC.PrevalenceofCryptosporidiummuris-likeoocystsamongcattlepopulationsoftheUnitedStates:preliminaryreport.JournalofProtozoology,1991,38(s6):14s-15s.21.AntunesRG,SimoesDC,NakamuraAA,etal.NaturalinfectionwithCryptosporidiumgalliincanaries(Serinuscanaria),inacockatiel(Nymphicushollandicus),andinlesserseed-finches(Oryzoborusangolensis)fromBrazil.AvianDiseases,2008,52(4):702-705.22.AppelbeeA,ThorlaksonC,OlsonME.GenotypiccharacterizationofGiardiacystsisolatedfromwildbeaverinsouthernAlberta,Canada.In:OlsonBE,OlsonME,WallisPM.(Eds.),Giardia:TheCosmopolitanParasite.CABInternational,Wallingford,UnitedKingdom,2002,pp.299-300.23.ArrowoodMJ.Diagnosis.In:CryptosporidiumandCryptosporidiosis.FayerR(Ed.),CRCPress,NewYork,LondonandTokyo,1997,43-64.24.ArtiedaJ,BasterrecheaM,ArriolaL,etal.Outbreakofcryptosporidiosisinachildday-carecentreinGipuzkoa,Spain,OctobertoDecember2011.EuroSurveillance,2012,17(5):23-25.25.AsahiH,KoyamaT,AraiH,etal.,1991.BiologicalnatureofCryptosporidiumsp.isolatedfromacat.ParasitologyResearch,1991,77(3):237-240.26.BaiYH,JiaGY,XingJS.InvestigationofcryptosporidiosisofyakcalvesinTongdeCounty.SichuanAnimalandVeterinarySciences,2001,28(7):22(InChinese).27.BaldurssonS,KaranisP.Waterbornetransmissionofprotozoanparasites:reviewofworldwideoutbreakseanupdate2004-2010.WaterReseach,2011,45(20):6603-6614.28.BallweberLR,XiaoL,BowmanDD,etal.Giardiasisindogsandcats:updateonepidemiologyandpublichealthsignificance.TrendsParasitology,2010,26(4):180-189.29.BarkerIK,CarbonellPL.Cryptosporidiumagnisp.n.fromlambs,andCryptosporidiumbovissp.n.fromacalf,withobservationsontheoocyst.ParasitologyResearch,1974,44(4):289-298.30.BarigyeR,DyerNW,NewellTK,etal.MolecularandimmunohistochemicaldetectionofassemblageE,GiardiaduodenalisinscouringNorthDakotacalves.VeterinaryParasitology,2008,157(3-4):196-202.31.BaoJM,ChenTH,YangJM.ApreliminarysurveyofCryptosporidiuminfectionindairycattleinYinchuanregion.ChineseJournalofVeterinaryParasitology,1997,5(2):25-26(InChinese).32.BirkyCWJr.Sex:isGiardiadoingitinthedark?CurrentBiology,2005,15(2):R56-R58.33.BlagburnBL,LindsayDS,HoerrFJ,etal.CryptosporidiumspinfectionintheproventriculusofanAustraliandiamondfiretailfinch(Staganoplurabella:Passeriformes,Estrildidae).AvianDiseases,1990,34(4):1027-1030.34.Bornay-LlinaresFJ,daSilvaAJ,MouraIN,etal.,IdentificationofCryptosporidiumfelisina110 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,Chinacowbymorphologicandmolecularmethods.AppliedandEnvironmentalMicrobiology,1999,65(4):1455-1458.35.BouzidM,TylerKM,ChristenR,etal.Multi-locusanalysisofhumaninfectiveCryptosporidiumspeciesandsubtypesusingtennovelgeneticloci.BMCMicrobiology,2010,10:213.36.BrogliaA,ReckingerS,CacciòSM,etal.DistributionofCryptosporidiumparvumsubtypesincalvesinGermany.VeterinaryParasitology,2008,154(1-2):8-13.37.BrownsteinDG,StrandbergJD,MontaliRJ,etal.Cryptosporidiuminsnakeswithhypertrophicgastritis.VeterinaryPathology,1977,14(6):606-617.38.BuckholtMA,LeeJH,TziporiS.PrevalenceofEnterocytozoonbieneusiinswine:an18-monthsurveyataslaughterhouseinMassachusetts.AppliedEnvironmentalMicrobiology,2002,68(5):2595-2599.39.Budu-AmoakoE,GreenwoodSJ,DixonBR,etal.GiardiaandCryptosporidiumondairyfarmsandtherolethesefarmsmayplayincontaminatingwatersourcesinPrinceEdwardIsland,Canada.JournalofVeterinaryInternalMedicine,2012,26(3):668-673.40.BukhariZ,SmithHV.DetectionofCryptosporidiummurisoocystsinthefaecesofadultdairycattleinScotland.VeterinaryRecord,1996,138(9):207-208.41.BurtonAJ,NydamDV,JonesG,etal.AntibodyresponsesfollowingadministrationofaCryptosporidiumparvumrCP15/60vaccinetopregnantcattle.VeterinaryParasitology,2011,175(1-2):178-181.42.BushenOY,KohliA,PinkertonRC,etal.HeavycryptosporidialinfectionsinchildreninnortheastBrazil:comparisonofCryptosporidiumhominisandCryptosporidiumparvum.TransactionsoftheRoyalSocietyofTropicalMedicineHygiene,2007,101(4):378-384.43.CacciòSM,BeckR,LalleM,etal.MultilocusgenotypingofGiardiaduodenalisrevealsstrikingdifferencesbetweenassemblagesAandB.InternationalJournalforParasitology,2008,38(13):1523-1531.44.CacciòSM,GiacomoMD,PozioE.Sequenceanalysisoftheβ-giardingeneanddevelopmentofaPCR-RFLPassaytogenotypeGiardiaduodenaliscystsfromhumanfaecalsamples.InternationalJournalforParasitology,2002,32(8):1023-1030.45.CacciòS,PinterE,FantiniR,etal.HumaninfectionwithCryptosporidiumfelis:casereportandliteraturereview.EmergingInfectiousDiseases,2002,8(1):85-86.46.CacciòSM,PozioE.Advancesintheepidemiology,diagnosisandtreatmentofcryptosporidiosis.Expertreviewofanti-infectivetherapy,2014,4(3):429-443.47.CacciòSM,RyanU.Molecularepidemiologyofgiardiasis.MolecularandBiochemicalParasitology,2008,160(2):75-80.48.CacciòSM,ThompsonRC,McLauchlinJ,etal.UnravellingCryptosporidiumandGiardiaepidemiology.TrendsinParasitology,2005,21(9):430-437.49.CaiR,LiCP,WangJ,etal.AnepidemiologicalsurveyofcryptosporidiosiswithdiarrheainHuainanarea.JournalofTropicalDiseasesandParasitology,2003,1(1):26-28(InChinese).50.CamaVA,BernC,RobertsJ,etal.Cryptosporidiumspeciesandsubtypesandclinicalmanifestationsinchildren,Peru.EmergingInfectiousDiseases,2008,14(10):1567-1574.51.CamaVA,BernC,SulaimanIM,etal.CryptosporidiumspeciesandgenotypesinHIV-positivepatientsinLima,Peru.JournalofEukaryoticMicrobiology,2003,50(1):531-533.111 HuazhongAgriculturalUniversityMaster’sDegree201852.CamaVA,RossJM,CrawfordS,etal.DifferenceinclinicalmanifestationsamongCryptosporidiumspeciesandgenotypesinHIV-infectedpersons.JournalofInfectiousDiseases.2007,196(5):684-691.53.CasemoreDP,JacksonFB.Hypothesis:cryptosporidiosisinhumanbeingsisnotprimarilyazoonosis.JournalofInfection,1984,9(2):153-156.54.CasemoreDP,SandsRL,CurryA.Cryptosporidiumspeciesa“newhuamnpathogen”.JournalofClinicalPathology,1985,38(12):1321-1336.55.ChalmersRM,DaviesAP.Minireview:clinicalcryptosporidiosis.ExperimentalParasitology,2010,124(1):138-146.56.ChalmersRM,ElwinK,ThomasALetal.InfectionwithunusualtypesofCryptosporidiumisnotrestrictedtoimmunocompromisedpatients.JournalofInfectiousDiseases,2002,185(2):270-271.57.ChalmersRM,ElwinK,ThomasAL,etal.Long-termCryptosporidiumtypingrevealstheaetiologyandspecies-specificepidemiologyofhumancryptosporidiosisinEnglandandWales,2000to2003.EuroSurveillance,2009,14(2):785-79458.ChalmersRM,FergusonC,CaccioSetal.DirectcomparisonofselectedmethodsforgeneticcategorisationofCryptosporidiumparvumandCryptosporidiumhominisspecies.InternationalJournalParasitology,2005,35(4):397-410.59.Chalmers,R.M,RobinsonG,ElwinK,etal.DetectionofCryptosporidiumspeciesandsourcesofcontaminationwithCryptosporidiumhominisduringawaterborneoutbreakinNorthWestWales.JournalofWaterandHealth,2010,8(2):311-325.60.ChalmersRM,SmithR,ElwinK,etal.EpidemiologyofanthroponoticandzoonotichumancryptosporidiosisinEnglandandWales,2004-2006.EpidemiologyandInfection,2011,139(5):700-712.61.ChalmersRM,SturdeeAP,BullSA,etal.TheprevalenceofCryptosporidiumparvumandC.murisinMusdomesticus,ApodemussylvaticusandClethrionomysglareolusinanagriculturalsystem.ParasitologyResearch,1997,83(5):478-482.62.ChappellCL,OkhuysenPC,Langer-CurryRC,etal.Cryptosporidiummeleagridis:infectivityinhealthyadultvolunteers.AmericanJournalofTropicalMedicineandHygiene,2011,85(2):238-242.63.ChalmersRM,ElwinK,HadfieldSJ,etal.SporadichumancryptosporidiosiscausedbyCryptosporidiumcuniculus,UnitedKingdom,2007-2008.EmergingInfectiousDiseases,2011,17(3):536-538.64.ChalmersRM,RobinsonG,ElwinK,etal.Cryptosporidiumsp.rabbitgenotype,anewlyidentifiedhumanpathogen.EmergingInfectiousDiseases,2009,15(5):829-830.65.CheadleMA,Toivio-KinnucanM,BlagburnBL.TheultrastructureofgametogenesisofCryptosporidiumbaileyi(Eimeriorina;Cryptosporidiidae)intherespiratorytractofbroilerchickens(Gallusdomesticus).JournalofParasitology,1999,85(4):609-615.66.CheckleyW,WhiteACJr,JaganathD,etal.Areviewoftheglobalburden,noveldiagnostics,therapeutics,andvaccinetargetsforCryptosporidium.LancetInfectiousDiseases,2015,15(1):85-94.67.ChenF,QiuH.IdentificationandcharacterizationofaChineseisolateofCryptosporidiumserpentisfromdairycattle.ParasitologyResearch,2012,111(4):1785-1791.68.ChenG,ShaoLH,MoCC,etal.EpidemiologicalinvestigationofCryptosporidiuminyaks112 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinafromapastureinHaiyancounty.ChineseQinghaiJournalofAnimalandVeterinarySciences,1991,3:43(InChinese).69.ChenG,WangHS,LuoJZ,etal.EpidemiologicalinvestigationonCryptosporidiumindairycattlefromXiningarea.ChineseQinghaiJournalofAnimalandVeterinarySciences,1989,6:23-24(InChinese).70.ChenG,YangZ,SunXH,etal.EpidemiologicalinvestigationonCryptosporidiuminpre-weanedcattleinMinhecounty,Chuanhedistrict.JournalofQinghaiAnimalHusbandryandVeterinaryMedicineCollege,1991,8(1):32-34(InChinese).71.ChenGY,LiAX,LiL,etal.EpidemiologicalinvestigationandgenotypesidentificationofCryptosporidiumandGiardiainZhongmouCounty,Henanprovince.DairyHealth,2015,4(5):35-38(InChinese).72.ChenL,XuSZ,ShanH,etal.InvestigationoncowcryptosporidiosisinsomeregionsofYantai.ProgressinVeterinaryMedicine,2011,32(1):114-117(InChinese).73.ChenSH,ZhangYN,LiH,etal.AnalysisonparasiticinfectionofclinicalsamplesfromhospitalsinShanghaiduring2011-2013.ChinaJournalParasitologyParasiticDisease,2014,32(6):446-451(InChinese).74.ChenXC,SuLY,SaiSY,etal.InvestigationsurveyontheinfectionofhumancryptosporidiosisinCentral-southofShandong.ChineseJournalofParasiticDiseaseControl,1991,4(4):311(InChinese).75.ChenXM,GoresGJ,PayaCV,etal.CryptosporidiumparvuminducesapoptosisinbiliaryepitheliabyaFas/Fasligand-dependentmechanism.AmericanJournalofPhysiology,1999,277(1):599-608.76.ChenYG,DaiMX,YaoFB.InvestigationonCryptosporidiuminfectioninZhoupengvillageinTongshanCounty.ActaAcademiaeMedicinaeXuzhou,1990,1,40-41(InChinese).77.ChenYG,LiHS,DaiMX,etal.EpidemiologicalstudiesonhumancryptosporidiosisinruralandurbanareasofJiangsu.ChineseJournalofParasitologyandParasiticDiseases,1991,11(3):207-210(InChinese).78.ChenYG,YaoFB.Areportof14casesofhumancryptosporidiosisinXuzhouarea.ChineseJournalofZoonoses,1990,6(3):28,15(InChinese).79.ChenYM,LiGD.CalfcryptosporidiosisinLanzhou.ChineseVeterinaryScience,1986,12(12):41-42(InChinese).80.ChengXN,LuSQ,LiJH,etal.IsolationofHebeiisolatesofGiardialambliaandinvestigationontheirgenotypes.ChineseJournalofParasiticDiseaseControl,2001,14(2):100-102.81.ChengYZ,ChenXJ,LinAF,etal.HumancryptosporidiosisinHangzhou:26CasesReport.LaboratoryMedicine,2000,15(5):307(InChinese).82.CottonJA,BeattyJK,BuretAG.HostparasiteinteractionsandpathophysiologyinGiardiainfections.InternationalJournalParasitology,2011,41(9):925-933.83.CoupeS,SarfatiC,HamaneS,etal.DetectionofCryptosporidiumandidentificationtothe113 HuazhongAgriculturalUniversityMaster’sDegree2018specieslevelbynestedPCRandrestrictionfragmentlengthpolymorphism.JournalofClinicalMicrobiology,2005,43(3):1017-1023.84.CranfieldMR,GraczykTK.ExperimentalinfectionofelaphidsnakeswithCryptosporidiumserpentis(Apicomplexa:Cryptosporidiidae).JournalofParasitology,1994,80(5):823-826.85.CurrentWL,UptonSJ,HaynesTB.ThelifecycleofCryptosporidiumbaileyin.sp.(Apicomplexa,Cryptosporidiidae)infectingchickens.JournalofProtozoology,1986,33(2):289-296.86.D'AntonioRG,WinnRE,TaylorJP,etal.Awaterborneoutbreakofcryptosporidiosisinnormalhosts.AnnalsofInternalMedicine,1985,103(6):886-888.87.DarribaD,TaboadaGL,DoallaR,etal.jModelTest2:moremodels,newheuristicsandparallelcomputing.NatureMethods,2012,9(8):772.88.DasP,RoySS,MitradharK,etal.MolecularcharacterizationofCryptosporidiumspp.fromchildreninKolkata,India.JournalofClinicalMicrobiology,2006,44(11):4246-4249.89.deGraafDC,VanopdenboschE,Ortega-MoraLM,etal.Areviewoftheimportanceofcryptosporidiosisinfarmanimals.InternationalJournalforParasitology,1999,29(8):1269-1287.90.deLucioA,Amor-AramendíaA,BailoB,etal.PrevalenceandgeneticdiversityofGiardiaduodenalisandCryptosporidiumspp.amongschoolchildreninaruralareaoftheAmhararegion,North-WestEthiopia.PLoSOne,2016,11(7):e0159992.91.DengL,LiW,YuX,etal.Firstreportofthehuman-pathogenicEnterocytozoonbieneusifromred-belliedtreesquirrels(Callosciuruserythraeus)inSichuan,China.PLoSOne,2016,11(9):e0163605.92.DobellC.TheDiscoveryoftheIntestinalProtozoaofMan.ProceedingsoftheRoyalSocietyofMedicine,1920,13(1):1-15.93.DouCX.InvestigationofcryptosporidiosisofyakcalvesinHuangyuanCounty.AnimalHusbandryandFeedScience,2007,3(11):14(InChinese).94.DuXL,GeJJ,QinYF,etal.TheepidemiologicalstudyoncryptosporidiosisinoutpatientsofNanjingChildren'shospital.JournalofTropicalMedicine,2009,9(4):382-385(InChinese).95.DubeyJP,MarkovitsJE,KillaryKA.Cryptosporidiummuris-likeinfectioninstomachofcynomolgusmonkeys(Macacafascicularis).VeterinaryPathology,2002,39(3):363-371.96.EbeidM,MathisA,PospischilA,etal.InfectivityofCryptosporidiumparvumgenotypeIinconventionallyrearedpigletsandlambs.Parasitology.Research,2003,90(3):232-235.97.ElFatniC,OlmoF,ElFatniH,etal.FirstgenotypingofGiardiaduodenalisandprevalenceofenteroparasitesinchildrenfromTetouan(Morocco).Parasite,2014,21(5):48.98.ElwinK,ChalmersRM.ContempomporaryidentificationofpreviouslyreportednovelCryptosporidiumisolatesrevealsmueCryptosporidiumbovisandthecervinegenotypeinsheep(Ovisaries).ParasitologyResearch,2008,102(5):1103-1105.99.ElwinK,HadfieldSJ,RobinsonG,etal.TheepidemiologyofsporadichumaninfectionswithunusualcryptosporidiadetectedduringroutinetypinginEnglandandWales,2000-2008.114 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaEpidemiologyandInfection,2012,140(4):673-683.100.EnemarkHL,AhrensP,Bille-Hansenetal.Cryptosporidiumparvuminfectivityandpathogenicityoftheporcinegenotype.Parasitology,2003,126(5):407-416.101.EnemarkHL,AhrensP,LoweryGJ,etal.CryptosporidiumandersonifromaDanishcattleherd:identificationandpreliminarycharacterization.VeterinaryParasitology.2002,107(1-2):37-49.102.EscobedoAA,AlmirallP,RobertsonLJ,etal.Giardiasis:theever-presentthreatofaneglecteddisease.InfectiousDisordDrugTargets,2010,10(5):329-348.103.EthelbergS,LisbyM,VestergaardLS,etal.AfoodborneoutbreakofCryptosporidiumhominisinfection.EpidemiologyandInfection,2009,137(3):348-356.104.EyPL,MansouriM,KuldaJ,etal.GeneticanalysisofGiardiafromhoofedfarmanimalsrevealsartiodactyl-specificandpotentiallyzoonoticgenotypes.JournalofEukaryoticMicrobiology,1997,44(6):626-635.105.FarthingMJ.Treatmentoptionsfortheeradicationofintestinalprotozoa.NatureClinicalPracticeGastroenterologyandHepatology,2006,3(8):436-445.106.FarzanA,ParringtonL,CoklinT,etal.DetectionandcharacterizationofGiardiaduodenalisandCryptosporidiumspp.onswinefarmsinOntario,Canada.FoodbornePathogensandDisease,2011,8(11):1207-1213.107.FayerR.Cryptosporidium:awater-bornezoonoticparasite.VeterinaryParasitology,2004,126(1-2):37-56.108.FayerR.TaxonomyandspeciesdelimitationinCryptosporidium.ExperimentalParasitology,2010,124(1):90-97.109.FayerR,GasbarreL,PasqualiP,etal.Cryptosporidiumparvuminfectioninbovineneonates:dynamicclinical,parasiticandimmunologicpatterns.InternationalJournalforParasitology,1998,28(1):49-56.110.FayerR,MorganU,UptonSJ.EpidemiologyofCryptosporidium:transmission,detectionandidentification.InternationalJournalforParasitology,2000,30(12-13):1305-1322.111.FayerR,SantínM.Cryptosporidiumxiaoin.sp.(Apicomplexa:Cryptosporidiidae)insheep(Ovisaries).VeterinaryParasitology,2009,164(2-4):192-200.112.FayerR,SantínM,TroutJM.Cryptosporidiumryanaen.sp.(Apicomplexa:Cryptosporidiidae)incattle(Bostaurus).VeteninaryParasitology,2008,156(3-4):191-198.113.FayerR,SantínM,TroutJM,etal.PrevalenceofspeciesandgenotypesofCryptosporidiumfoundin1-2-year-olddairycattleintheeasternUnitedStates.VeterinaryParasitology,2006,135(2):105-112.114.FayerR,SantinM,MacarisinD.Cryptosporidiumubiquitumn.sp.inanimalsandhumans.VeterinaryParasitology,2010,172(1-2):23-32.115.FayerR,SantinM,MacarisinD.DetectionofconcurrentinfectionofdairycattlewithBlastocystis,Cryptosporidium,Giardia,andEnterocytozoonbymolecularandmicroscopicmethods.ParasitologyResearch,2012,111(3):1349-1355.115 HuazhongAgriculturalUniversityMaster’sDegree2018116.FayerR,SantínM,XiaoL.Cryptosporidiumbovisn.sp.(Apicomplexa:Cryptosporidiidae)incattle(Bostaurus).JournalofParasitology.2005,91(3):624-629.117.FayerR,SpeerCA,DubeyJP.ThegeneralbiologyofCryptosporidium.In:FayerR(Eds).CryptosporidiumandCryptosporidiosis,CRCPress,BocaRaton,FL,1997,pp.1-42.118.FayerR,TroutJM,XiaoL,etal.Cryptosporidiumcanisn.sp.fromdomesticdogs.JournalofParasitology,2001,87(6):1415-1422.119.FayerR,XiaoL.CryptosporidiumandCryptosporidiosis,seconded.CRCPress,BocaRaton,Florida.c2008.120.FengY,LiN,DuanL,etal.CryptosporidiumgenotypeandsubtypedistributioninrawwastewaterinShanghai,China:EvidenceforpossibleuniqueCryptosporidiumhominisTransmission.JournalofClinicalMicrobiology,2009,47(1):153-157.121.FengY,OrtegaY,CamaV,etal.HighintragenotypicdiversityofGiardiaduodenalisindairycattleonthreefarms.ParasitologyResearch,2008,103(1):87-92.122.FengY,OrtegaY,HeG,etal.WidegeographicdistributionofCryptosporidiumbovisandthedeer-likegenotypeinbovines.VeterinaryParasitology,2007,144(1):1-9.123.FengY,WangL,DuanL,etal.Extendedoutbreakofcryptosporidiosisinapediatrichospital,China.EmergingInfectiousDiseases,2012,18(2):312-314.124.FengY,XiaoL.ZoonoticpotentialandmolecularepidemiologyofGiardiaspeciesandgiardiasis.ClinicalMicrobiologyReviews,2011,24(1):110-140.125.FengY,YangW,RyanU,etal.DevelopmentofamultilocussequencetoolfortypingCryptosporidiummurisandCryptosporidiumandersoni.JournalofClinicalMicrobiology,2011,49(1):34-41.126.FeltusDC,GiddingsCW,SchneckBL,etal.EvidencesupportingzoonotictransmissionofCryptosporidiuminWisconsin.JournalofClinicalMicrobiology,2006,44(12):4303-4308.127.FiliceFP.StudiesonthecytologyandlifehistoryofaGiardiafromthelaboratoryrat.UniversityCaliforniaPublicationZoology.1952,57:53-146.128.FlaniganT,WhalenC,TurnerJ,etal.CryptosporidiuminfectionandCD4counts.AnnalsofInternalMedicine,1992,116(10):840-842.129.GaitR,SoutarRH,HansonM,etal.Outbreakofcryptosporidiosisamongveterinarystudents.VeterinaryRecord,2008,28,162(26):843-845.130.GaluppiR,PivaS,CastagnettiC,etal.Cryptosporidiumparvum:Fromfoaltoveterinarystudents.VeterinaryParasitology,2016,219:53-56.131.GasserRB.Moleculartools-advances,opportunitiesandprospects.VeterinaryParasitology,2006,136(2):69-89.132.GarciaLS,BrewerTC,BrucknerDA.FluorescencedetectionofCryptosporidiumoocystsinhumanfecalspecimensbyusingmonoclonalantibodies.JournalofClinicalMicrobiology,1987,25(1):119-121.133.GarciaLS,BrewerTC,BrucknerDA.IncidenceofCryptosporidiuminallpatientssubmittingstoolspecimensforovaandparasiteexamination:monoclonalantibodyIFA116 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,Chinamethod.DiagnosticMicrobiologyandInfectiousDisease,1988,11(1):25-27.134.GarciaLS,ShimizuRY.Evaluationofnineimmunoassaykits(enzymeimmunoassayanddirectfluorescence)fordetectionofGiardialambliaandCryptosporidiumparvuminhumanfecalspecimens.JournalofClinicalMicrobiology,1997,35(6):1526-1529.135.GasserRB,HuM,ChiltonNB,etal.Single-strandconformationpolymorphism(SSCP)fortheanalysisofgeneticvariation.NatureProtocols,2006,1(6):3121-3128.136.GateiW,AshfordRW,BeechingNJ,etal.CryptosporidiummurisinfectioninanHIV-infectedadult,Kenya.EmergingInfectiousDiseases,2002,8(2):204-206.137.GateiW,BarrettD,LindoJFetal.UniqueCryptosporidiumpopulationinHIV-infectedpersons,Jamaica.EmergingInfectiousDiseases,2008,14(5):841-843.138.GateiW,DasP,Dutta,P,etal.MultilocussequencetypingandgeneticstructureofCryptosporidiumhominisfromchildreninKolkata,India.InfectionGeneticsandEvolution,2007,7(2):197-205.139.GateiW,GreensillJ,AshfordRW,etal.Molecularanalysisofthe18SrRNAgeneofCryptosporidiumparasitesfrompatientswithorwithouthumanimmunodeficiencyvirusinfectionslivinginKenya,Malawi,Brazil,theUK,andVietnam.JournalofClinicalMicrobiology,2003,41(4):1458-1462.140.GateiW,SuputtamongkolY,WaywaD,etal.ZoonoticspeciesofCryptosporidiumareasprevalentastheanthroponoticinHIV-infectedpatientsinThailand.AnnalsofTropicalMedicineandParasitology,2002,96(8):797-802.141.GelletlieR,StuartJ,SoltanpoorN,etal.Cryptosporidiosisassociatedwithschoolmilk.Lancet.1997,350(9083):1005-1006.142.GeurdenT,BerkvensD,CasaertS,etal.ABayesianevaluationofthreediagnosticassaysforthedetectionofGiardiaduodenalisinsymptomaticandasymptomaticdogs.VeterinaryParasitology,2008,157(1-2):14-20.143.GeurdenT,ClaereboutE,DursinL,etal.TheefficacyofanoraltreatmentwithparomomycinagainstanexperimentalinfectionwithGiardiaincalves.VeterinaryParasitology,2006a,135(3-4):241-247.144.GeurdenT,GomaFY,SiwilaJ,etal.PrevalenceandgenotypingofCryptosporidiuminthreecattleandhusbandrysystemsinZambia.VeterinaryParasitology,2006b,138(3-4):217-222.145.GeurdenT,ThomasP,CasaertS,etal.PrevalenceandmolecularcharacterisationofCryptosporidiumandGiardiainlambsandgoatkidsinBelgium.VeterinaryParasitology,2008,155(1-2):142-145.146.GeurdenT,VanderstichelR,PohleH,etal.AmulticentreprevalencestudyinEuropeonGiardiaduodenalisincalves,withmolecularidentificationandriskfactoranalysis.VeterinaryParasitology,2012,190(3-4):383-390.147.GherasimA,LebbadM,InsulanderM,etal.Twogeographicallyseparatedfood-borneoutbreaksinSwedenlinkedbyanunusualCryptosporidiumparvumsubtype,October2010.EuroSurveillance,2012,17(46):29-36.117 HuazhongAgriculturalUniversityMaster’sDegree2018148.GibbonsCL,Awad-El-KariemFM.NestedPCRforthedetectionofCryptosporidiumparvum.ParasitologyToday,1999,15(8):345.149.GilesM,WebsterAK,MarshallAJ,etal.ExperimentalinfectionofalambwithCryptosporidiumparvumgenotype1.VeterinaryRecord,2001,149(17):523-525.150.GlabermanS,MooreJE,LoweryCJ,etal.Threedrinking-water-associatedcryptosporidiosisoutbreaks,NorthernIreland.EmergingInfectiousDiseases,2002,8(6):631-633.151.GlabermanS,SulaimanIM,BernC,etal.AmultilocusgenotypicanalysisofCryptosporidiummeleagridis.JournalofEukaryoticMicrobiology,2001,48(s1):19s-22s.152.GohS,ReacherM,CasemoreDP,etal.Sporadiccryptosporidiosis,NorthCumbria,England,1996-2000.EmergingInfectiousDiseases,2004,10(6):1007-1015.153.GomezMoralesMA,PozioE,CroppoGP.SerodiagnosisofcryptosporidiosisinItalianHIV-positivepatientsbymeansofanoocystsolubleantigeninanELISA.JournalofInfection,1992,25(2):229-236.154.GoodgameRW,GentaRM,WhiteAC,etal.IntensityofinfectioninAIDS-associatedcryptosporidiosis.JournalofInfectiousDiseases,1993,167(3):704-709.155.GoodwinMA.Cryptosporidiosisinbirds-areview.AvianPathology,1989,18(3):365-384.156.GraczykTK,CranfieldMR,GeitnerME.MultipleCryptosporidiumserpentisoocystisolatesfromcaptivesnakesarenottransmissibletoamphibians.JournalofParasitology,1998,84(6):1298-300.157.GraczykTK,FayerR,CranfieldMR.Cryptosporidiumparvumisnottransmissibletofish,amphibians,orreptiles.JournalofParasitology,1996,82(5):748-751.158.GraczykTK,LewisEJ,GlassG,etal.QuantitativeassessmentofviableCryptosporidiumparvumloadincommercialoysters(Crassostreavirginica)intheChesapeakeBay.ParasitologyResarch.2007,100(2):247-253.159.GreenSL,BouleyDM,JoslingCA,etal.Cryptosporidiosisassociatedwithemaciationandproliferativegastritisinalaboratory-rearedSouthAfricanclawedfrog(Xenopuslaevis).ComparativeMedicine,2003,53(1):81-84.160.GiangasperoA,PaolettiB,IorioR,etal.PrevalenceandmolecularcharacterizationofGiardiaduodenalisfromsheepincentralItaly.ParasitologyResearch,2005,96(1):32-37.161.GrinbergA,PomroyWE,SquiresRA,etal.RetrospectivecohortstudyofanoutbreakofcryptosporidiosiscausedbyarareCryptosporidiumparvumsubgenotype.EpidemiologyandInfection,2011,139(10):1542-1550.162.GuoBP,LianDR.EpidemiologicalinvestigationonCryptosporidiumindairycattlefromGuangzhouregion.ChineseJournalofZoonoses,1999,15(3):10(InChinese).163.GuoSL,YuanFS,YuH,etal.EpidemiologicalinvestigationonCryptosporidiumincattleinJinanregion.ChineseJournalofPreventiveVeterinaryMedicine,1993,1:34-35(InChinese).164.GuyotK,Follet-DumoulinA,LelièvreE,etal.MolecularcharacterizationofCryptosporidiumisolatesobtainedfromhumansinFrance.JournalofClinicalMicrobiology,118 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China2001,39(10):3472-3480.165.HallRL,JonesJL,HurdS,etal.Population-basedactivesurveillanceforCyclosporainfection-UnitedStates,FoodborneDiseasesActiveSurveillanceNetwork(FoodNet),1997-2009.ClinicalInfectiousDiseases.2012,54(s5):s411-s417.166.HanF,WangL,WangRZ,etal.ThecryptosporidiosiswasfoundinNanjingregion.ChineseJournalofZoonoses,1989,5(5):51-23(InChinese).167.HajdusekO.DitrichO,SlapetaJ.MolecularidentificationofCryptosporidiumspp.inanimalandhumanhostsfromtheCzechRepublic.VeterinaryParasitology,2004,122(3):183-192.168.HaoLL,LiR,DuanL,etal.PrevalenceandmolecularidentificationofCryptosporidiumspp.inyaksinHongyuanCountyofSichuanProvince.ActaAgriculturaeZhejiangensis,2016,28(11):1842-1846(InChinese).169.HaqueR,RoyS,KabirM,etal.GiardiaassemblageAinfectionanddiarrheainBangladesh.JournalofInfectiousDiseases,2005,192(12):2171-2173.170.HayesEB,MatteTD,O'BrienTR,etal.Largecommunityoutbreakofcryptosporidiosisduetocontaminationofafilteredpublicwatersupply.TheNewEnglandJournalofMedicine,1989,320(21):1372-1376.171.HeHX,WangXH,ZhaoK,etal.TheinvestigationofCryptosporidiumindiarrhoeiccattlefromXinxiangdistrictofHenan.ChinaDairyCattle,2001,2:22-23(InChinese).172.HeZA,FuLX,ZhouWS,etal.InvestigationonCryptosporidiuminfectionofchildreninruralareaofChibicity,Hubei.JournalofPublicHealthandPreventiveMedicine,2013,24(6):72-74(InChinese).173.HopkinsRM,MeloniBP,GrothDM,etal.RibosomalRNAsequencingrevealsdifferencesbetweenthegenotypesofGiardiaisolatesrecoveredfromhumansanddogslivinginthesamelocality.JournalofParasitology,1997,83(1):44-51.174.HolubováN,SakB,HorčičkováM,etal.Cryptosporidiumaviumn.sp.(Apicomplexa:Cryptosporidiidae)inbirds.ParasitologyResearch,2016,115:2243-2251.175.HomanWL,GilsingM,BentalaH,etal.CharacterizationofGiardiaduodenalisbypolymerase-chain-reactionfingerprinting.ParasitologyResearch,1998,84(9):707-714.176.HouX,ChenJM,LiuW.InfectionstatusandepidemiologicalcharacteristicsofCryptosporidiuminXi'anChildren'sHospital.ContemporaryMedicine,2011,17(24):53(InChinese).177.HuangJ,YueD,QiM,etal.PrevalenceandmolecularcharacterizationofCryptosporidiumspp.andGiardiaduodenalisindairycattleinNingxia,northwesternChina.VeterinaryResearch,2014,10(1):292.178.HuangK,AkiyoshiDE,FengXC.DevelopmentofpatentinfectioninimmunosuppressedC57BL/6micewithasingleCryptosporidiummeleagridisoocyst.JournalofParasitology,2003,89(3):620-622.179.HuangDB,ChappellC,OkhuysenPC.Cryptosporidiosisinchildren.SeminarsinPediatricInfectionDiseases,2004,15(4):253-259.119 HuazhongAgriculturalUniversityMaster’sDegree2018180.HuangMZ,GuanL,XieMZ,etal.StudyonconditionofCryptosporidiuminfectionamongmaledrugusersindetoxificationinstituteinChangshacity.ChineseJournalofPublicHealth,2003,19(3):301-303(InChinese).181.HuangMZ,GuanL,ZhouCX.EpidemiologicalsurveyofCryptosporidiumindiarrhoeicchildrenfromChangsha.BulletinofHunanMedicalUniversity,1998,23(3):255-256(InChinese).182.HuangMZ,ZhouCX,GuanL,etal.StudyonCryptosporidiuminfectioninadultdiarrhoeicpatients.CurrentPhysician,1998,10(3):42-43(InChinese).183.HunterPR,ChalmersRM,SyedQ,etal.Footandmouthdiseaseandcryptosporidiosis:possibleinteractionbetweentwoemerginginfectiousdiseases.EmergingInfectiousDiseases,2003,9(1):109-112.184.HunterPR,HughesS,WoodhouseS,etal.Sporadiccryptosporidiosiscase-controlstudywithgenotyping.EmergingInfectiousDiseases,2004,10(7):1241-1249.185.HunterPR,HadfieldSJ,WilkinsonD,etal.SubtypesofCryptosporidiumparvuminhumansanddiseaserisk.EmergingInfectiousDiseases,2007,13(1):82-88.186.HunterPR,NicholsG.EpidemiologyandclinicalfeaturesofCryptosporidiuminfectioninimmunocompromisedpatients.ClinicalMicrobiologyReviews.2002,15(1):145-154.187.InmanLR,TakeuchiA.Spontaneouscryptosporidiosisinanadultfemalerabbit.VeterinaryPathology,1979,16(1):89-95.188.InpankaewT,TraubR,ThompsonRC,etal.CanineparasiticzoonosesinBangkoktemples.SoutheastAsianJournalofTropicMedicineandPublicHealth,2007,38(2):247-255.189.InsulanderM,SilverlåsC,LebbadM,etal.MolecularepidemiologyandclinicalmanifestationsofhumancryptosporidiosisinSweden.EpidemiologyandInfection,2013,141(5):1009-1020.190.IqbalA,LimYA,SurinJ,etal.HighdiversityofCryptosporidiumsubgenotypesidentifiedinMalaysianHIV/AIDSindividualstargetinggp60gene.PLoSOne,2012,7(2):e31139.191.IsekiM.Cryptosporidiumfelissp.n.(Protozoa:Eimeriorina)fromthedomesticcat.JapaneseJournalofParasitology,1979,28,285-307.192.IsekiM,MaekawaT,MoriyaK,etal.InfectivityofCryptosporidiummuris(strainRN66)invariouslaboratoryanimals.ParasitologyResearch,1989,75(3):218-222.193.ItagakiT,KinoshitaS,AokiM,etal.GenotypingofGiardiaintestinalisfromdomesticandwildanimalsinJapanusingglutametedehydrogenasegenesequencing.VeterinaryParasitology,2005,133(4):283-287.194.IzadiM,Jonaidi-JafariN,SaburiA,etal.CryptosporidiosisinIranianfarmworkersandtheirhouseholdmembers:Ahypothesisaboutpossiblezoonotictransmission.JournalofTropicalMedicine,2014,2:1-7.195.JarosD,ZygnerW,JarosS,etal.DetectionofGiardiaintestinalisassemblagesA,BandDindomesticcatsfromWarsaw,Poland.PolishJournalofMicrobiology,2011,60(3):259-263.120 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China196.JarvieBD,Trotz-WilliamsLA,McKnightDR,etal.EffectofhalofuginonelactateontheoccurrenceofCryptosporidiumparvumandgrowthofneonataldairycalves.JournalofDairyScience,2005,88(5):1801-1806.197.JervisHR,MerrillTG,SprinzH.CoccidiosisintheguineapigsmallintestinalduetoaCryptosporidium.AmericanJournalofVeterinaryResearch,1966,27(117):408-414.198.JexAR,ChalmersRM,SmithHV,etal.Cryptosporidiosis.In:PalmerSR,SoulsbyL,TorgersonPR,BrownDWG.(Eds.),OxfordTextbookofZoonoses,OxfordUniversityPress,2011,pp.536-568.199.JexAR,GasserRB.AnalysisofthegeneticdiversitywithinCryptosporidiumhominisandCryptosporidiumparvumfromimportedandautochtonouscasesofhumancryptosporidiosisbymutationscanning.Electrophoresis,2008,29(20):4119-4129.200.JexAR,PangasaA,CampbellBE,etal.ClassificationofCryptosporidiumspeciesfrompatientswithsporadiccryptosporidiosisbyuseofsequence-basedmultilocusanalysisfollowingmutationscanning.JournalofClinicalMicrobiology,2008,46(7):2252-2262.201.JexAR,StanleyKK,LoW,etal.Detectionofdiarrhoealpathogensinhumanfaecesusinganautomated,roboticplatform.MolecularandCellularProbes,2012,26:11-15.202.JiR,CuiW,LiangRW,etal.AnalysisofinfectionstatusandfactorsofCryptosporidiumamongmalignanttumorpatient.ActaAcademiaeMedicinaeWeifang.2008,30(2):130-141(InChinese).203.JianF,SunM,YanW,etal.EpidemiologicalsurveyofCryptosporidiuminfectionincowsandartificialinfectionofCryptosporidiumparvuminneonatalcalves.ChineseJournalofVeterinaryMedicine,2005,41(8):3-5(InChinese).204.JiangH,HongMZ,DanL.InvestigationoncowinfectionwithCryptosporidiumparvuminsouthareasofGuangxi.ChinaTropicalMedicine,2008,8(12):2210-2211(InChinese).205.JiangXS,ZhuHQ.EpidemiologicalinvestigationofCryptosporidiuminyaksfromNorthwestmeadowofSichuan.JournalofSouthwestUniversityforNationalities(NaturalScienceEdition),1989,3:18-20(InChinese).206.JiangY,RenJ,YuanZ,etal.CryptosporidiumandersoniasanovelpredominantCryptosporidiumspeciesinoutpatientswithdiarrheainJiangsuProvince,China.BMCInfectiousDiseases,2014,14(1):555.207.JirkuM,ValigurováA,KoudelaB,etal.NewspeciesofCryptosporidiumTyzzer,1907(Apicomplexa)fromamphibianhost:morphology,biologyandphylogeny.FoliaParasitology.2008,55(2):81-94.208.JohnstonSP,BallardMM,BeachMJ,etal.EvaluationofthreecommercialassaysfordetectionofGiardiaandCryptosporidiumorganismsinfecalspecimens.JournalofClinicalMicrobiology,2003,41(2):623-626.209.KanetaY,NakaiY.SurveyofCryptosporidiumoocystsfromadultcattleinaslaughterhouse.JournalofVeterinaryMedicalScience,1998,60(5):585-588.210.KaranisP,PlutzerJ,HalimNA,etal.MolecularcharacterizationofCryptosporidiumfrom121 HuazhongAgriculturalUniversityMaster’sDegree2018animalsourcesinQinghaiprovinceofChina.ParasitologyResearch,2007,101(6):1575-1580.211.KarimMR,WangR,DongH,etal.GeneticpolymorphismandzoonoticpotentialofEnterocytozoonbieneusifromnonhumanprimatesinChina.AppliedandEnvironmentalMicrobiology,2014a,80(6):1893-1898.212.KarimMR,WangR,HeX,etal.MultilocussequencetypingofEnterocytozoonbieneusiinnonhumanprimatesinChina.VeterinaryParasitology,2014b,200(1-2):13-23.213.KarimMR,YuF,LiJ,etal.Firstmolecularcharacterizationofentericprotozoaandthehumanpathogenicmicrosporidian,Enterocytozoonbieneusi,incaptivesnakesinChina.ParasitologyResearch,2014c,113(8):3041-3048.214.KeatingJP.Chronicdiarrhea.PediatricinReview2005,26(1):5-14.215.KhanSM,DebnathC,PramanikAK,etal.MolecularevidenceforzoonotictransmissionofGiardiaduodenalisamongdairyfarmworkersinWestBengal,India.VeterinaryParasitology,2011,178:342-345.216.KiangKM,ScheftelJM,LeanoFT,etal.Recurrentoutbreaksofcryptosporidiosisassociatedwithcalvesamongstudentsataneducationalfarmprogramme,Minnesota,2003.EpidemiologyandInfection,2006,134(4):878-886.217.KoehlerAV,BradburyRS,StevensMA.Geneticcharacterizationofselectedparasitesfrompeoplewithhistoriesofgastrointestinaldisordersusingamutationscanning-coupledapproach.Electrophoresis,2013,34(12):1720-1728.218.KoehlerAV,KorhonenPK,HallRS,etal.Useofabioinformatics-assistedprimerdesignstrategytoestablishanewnestedPCR-basedmethodforCryptosporidium.ParasitesandVectors,2017,10(1):509.219.KoehlerAV,JexAR,HaydonSR,etal.Giardia/giardiasis-aperspectiveondiagnosticandanalyticaltools.BiotechnologyAdvances,2014,32(2):280-289.220.KoinariM,KarlS,Ng-HublinJ,etal.IdentificationofnovelandzoonoticCryptosporidiumspeciesinfishfromPapuaNewGuinea.VeterinaryParasitology,2013,198(1-2):1-9.221.KorichDG,MeadJR,MadoreMS,etal.Effectsofozone,chlorinedioxide,chlorine,andmonochloramineonCryptosporidiumparvumoocystviability.AppliedandEnvironmentalMicrobiology,1990,56(5):1423-1428.222.KoschTA,SummersK.TechniquesforminimizingtheeffectsofPCRinhibitorsinthechytridiomycosisassay.MolecularEcologyResources,2013,13(2):230-236.223.KosekM,AlcantaraC,LimaAA,etal.Cryptosporidiosis:anupdate.TheLancetInfectiousDiseases,2001,1(4):262-269.224.KotloffKL,NataroJP,BlackwelderWC,etal.Burdenandaetiologyofdiarrhoealdiseaseininfantsandyoungchildrenindevelopingcountries(theGlobalEntericMulticenterStudy,GEMS):aprospective,case-controlstudy.Lancet,2013,382(9888):209-222.225.KoudelaB,ModryD.NewspeciesofCryptosporidium(Apicomplexa,Cryptosporidiidae)fromlizards.FoliaParasitology,1998,45(2):93-100.122 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China226.KoudelaB,ModryD,VitovecJ.InfectivityofCryptosporidiummurisisolatedfromcattle.VeterinaryParasitology,1998,76(3):181-188.227.KongYX,CaoSQ,ShiXZ,etal.InvestigationsurveyofcryptosporidiosisindiarrhoeicpatientsfromQinghai.ChineseJournalofParasiticDiseaseControl,1993,6(1):63(InChinese).228.KoyamaY,SatohM,MaekawaK,etal.IsolationofCryptosporidiumandersonikawatabitypeinaslaughterhouseinthenorthernislandofJapan.VeterinaryParasitology.2005,130(3):323-326.229.KváčM,HavrdováN,HláskováL,etal.Cryptosporidiumproliferansn.sp.(Apicomplexa:Cryptosporidiidae):molecularandbiologicalevidenceofcrypticspecieswithingastricCryptosporidiumofmammals.PLoSOne.2016,11(1):e0147090.230.KváčM,KestřánováM,PinkováM,etal.Cryptosporidiumscrofarumn.sp.(Apicomplexa:Cryptosporidiidae)indomesticpigs(Susscrofa).VeterinaryParasitology,2013,191(3-4),218-227.231.KváčM,KoubaM,VitovecJ.Aged-relatedandhousing-dependenceofCryptosporidiuminfectionofcalvesfromcattleandbeefherdsinSouthBohemia,CzechRepublic.VeterinaryParasitology,2006,137(3-4):202-209.232.KváčM,KestřánováD,SakB,etal.CryptosporidiumpiggenotypeIIinimmunocompetentman.EmergingInfectiousDiseases,2009,15(6):982-983.233.LangeH,JohansenOH,VoldL,etal.SecondoutbreakofinfectionwitharareCryptosporidiumparvumgenotypeinschoolchildrenassociatedwithcontactwithlambs/goatkidsataholidayfarminNorway.EpidemiologyandInfection,2014,142(10):2105-2113.234.LalA,HalesS,FrenchN,etal.SeasonalityinHumanZoonoticEntericDiseases:ASystematicReview.PLoSOne,2012,7(4):e31883.235.LalleM,Jimenez-CardosaE,CaccioSM,etal.GenotypingofGiardiaduodenalisfromhumansanddogsfromMexicousingabeta-giardinnestedpolymerasechainreactionassay.JournalofParasitology.2005a,91(1):203-205.236.LamelW.MikroskopischeuntersuchungenderDarmexcrete.VierteljahrsschrPrakstHeilkunde1859,61:1-58.237.LaneS,LloydD.CurrenttrendsinresearchintothewaterborneparasiteGiardia.CriticalReviewsinMicrobiology,2002,28(2):123-147.238.LearmonthJJ,IonasG,EbbettKA.GeneticcharacterizationandtransmissioncyclesofCryptosporidiumspeciesisolatedfromhumansinNewZealand.AppliedandEnvironmentalMicrobiology,2004,70(7):3973-3978.239.LebbadM,MattssonJG,ChristenssonB,etal.Frommousetomoose:multilocusgenotypingofGiardiaisolatesfromvariousanimalspecies.VeterinaryParasitology,2010,168(3-4):231-239.240.LederK.Intestinalprotozoa:Giardia,Amebiasis,Cyclospora,Blastocystishominis,Dientamoebafragilis,andCryptosporidiumparvum.In:Schwartz,E.(Ed.),Tropical123 HuazhongAgriculturalUniversityMaster’sDegree2018DiseasesinTravelers.Wiley-Blackwell,Chichester;UK,2009,pp.294-302.241.LefayD,NaciriM,PoirierP,etal.Efficacyofhalofuginonelactateinthepreventionofcryptosporidiosisinsucklingcalves.VeterinaryRecord.2001,148(4):108-112.242.LemosV,GraczykTK,AlvesM,etal.IdentificationanddeterminationoftheviabilityofGiardialambliacystsandCryptosporidiumparvumandCryptosporidiumhominisoocystsinhumanfecalandwatersupplysamplesbyfluorescentinsituhybridization(FISH)andmonoclonalantibodies.ParasitologyResearch,2005,98(1):48-53.243.LeonhardS,PfisterK,BeelitzP,etal.ThemolecularcharacterisationofGiardiafromdogsinsouthernGermany.VeterinaryParasitology,2007,150(1-2):33-38.244.LeoniF,MallonME,SmithHV,MultilocusanalysisofCryptosporidiumhominisandCryptosporidiumparvumisolatesfromsporadicandoutbreak-relatedhumancasesandC.parvumisolatesfromsporadiclivestockcasesintheUnitedKingdom.JournalofClinicalMicrobiology,2007,45(10):3286-3294.245.LeavBA,MackayMR,AnyanwuA,etal.AnalysisofsequencediversityatthehighlypolymorphicCpgp40/15locusamongCryptosporidiumisolatesfromhumanimmunodeficiencyvirus-infectedchildreninSouthAfrica.InfectionandImmunity,2002,70(7):3881-3890.246.LeoniF,AmarC,NicholsG,etal.GeneticanalysisofCryptosporidiumfrom2414humanswithdiarrheainEnglandbetween1985and2000.JournalofMedicalMicrobiology,2006,55(Pt6):703-707.247.LevineND.Somecorrectionsofcoccidian(Apicomplexa:Protozoa)nomenclature.JournalParasitology,1980,66(5):830-834.248.LiB,PangZC,FanBJ.InvestigationsurveyofCryptosporidiuminfectionindairycattleinLiuzhouarea.GuangxiAgriculturalSciences,1992,6:281(InChinese).249.LiCP,WangKX,WangJ,etal.EpidemiologicalsurveyofcryptosporidiosisinAnhuiprovince.JournaloftheFourthMilitaryMedicalUniversity,2004,25(2):125-127(InChinese).250.LiF,ZhangXH,WuFQ,etal.InvestigationofCryptosporidiuminfectioninpatientswithchronichepatitisB.NewChineseMedicine,2007,38(2):81-83(InChinese).251.LiF,WangH,ZhangZ,etal.PrevalenceandmolecularcharacterizationofCryptosporidiumspp.andGiardiaduodenalisindairycattleinBeijing,China.VeterinaryParasitology,2016,219,61-65.252.LiJ,LuoN,WangC,etal.Occurrence,molecularcharacterizationandpredominantgenotypesofEnterocytozoonbieneusiindairycattleinHenanandNingxia,China.ParasiteVectors,2016a,9(1):142.253.LiN,XiaoL,WangL,etal.MolecularsurveillanceofCryptosporidiumspp.,Giardiaduodenalis,andEnterocytozoonbieneusibygenotypingandsubtypingparasitesinwastewater.PLoSNeglectedTropicalDiseases,2012,6(9):e1809.254.LiPY,LiaoSF,LuFL.Epidemiologicalsurveyofbovinecryptosporidiosiscattlefrom124 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaHuaibeiareaofAnhui.ChineseJournalofVeterinaryScience,1991,18(5):473-474(InChinese).255.LiPY,LiaoSF,LuFL,etal.CryptosporidiuminfectionofcowanditsregionaldistributioninAnhuiProvince.ChineseJournalofVeterinaryScience,1999,19(3):275-277(InChinese).256.LiPY,LuFL,LiJN,etal.EpidemiologicalsurveyofCryptosporidiuminbeefcattleinAnhuiprovince.AnimalHusbandryandVeterinaryMedicine,2003,35(3):13-15(InChinese).257.LiW,DengL,YuX,etal.Multilocusgenotypesandbroadhost-rangeofEnterocytozoonbieneusiincaptivewildlifeatzoologicalgardensinChina.ParasiteVectors,2016b,9(1):395.258.LiW,LiY,SongM,etal.PrevalenceandgeneticcharacteristicsofCryptosporidium,EnterocytozoonbieneusiandGiardiaduodenalisincatsanddogsinHeilongjiangprovince,China.VeterinaryParasitology,2015,208(3-4):125-134.259.LiW,TaoW,JiangY,etal.GenotypicdistributionandphylogeneticcharacterizationofEnterocytozoonbieneusiindiarrheicchickensandpigsinmultiplecities,China:potentialzoonotictransmission.PLoSOne,2014,9(9):e108279.260.LiX,PereiraMd,LarsenR,etal.Cryptosporidiumrubeyin.sp.(Apicomplexa:Cryptosporidiidae)inmultipleSpermophilusgroundsquirrelspecies.InternationalJournalforParasitology:ParasitesandWildlife,2015,4(3):343-350.261.LiY,BingYY,CongF,etal.InvestigationofhumanCryptosporidiuminfectioninShenyangarea.ChineseJournalofZoonoses,2006,22(5):473-475(InChinese).262.LiYQ,HouYS,LiuJZ,etal.ApreliminarysurveyofCryptosporidiuminfectioninpre-weaneddairycattleinXi'anregion.ChineseJournalofZoonoses,1991,7(6):30(InChinese).263.LiangN,JianFC,LiJC,etal.SpeciesidentificationofCryptosporidiumisolatedfromdairycattlebyPCR-RFLPandphylogeneticanalysis.ChineseVeterinaryScience,2009,39(1):22-28(InChinese).264.LindsayD,BlagburnB.Cryptosporidiosisinbirds,In:DubeyJP,SpeerCA,andR.Fayer(Ed.),cryptosporidiosisinmanandanimals,CRCPress,Inc.,BocaRaton,FL,1990,pp.133-148.265.LindsayDS,BlagburnBL,HoerrFJ,etal.ExperimentalCryptosporidiumbaileyiinfectionsinchickensandturkeysproducedbyocularinoculationofoocysts.AvianDiseases,1987,31(2):355-357.266.LindsayDS.MorphometriccomparisonoftheoocystsofCryptosporidiummeleagridisandCryptosporidiumbaileyifrombirds.ProceedingsoftheHelminthologicalSociety,1989,56(1):91-92.267.LindsayDS,BlagburnBL,SundermannCA,etal.ExperimentalinfectionsindomesticduckswithCryptosporidiumbaileyiisolatedfromchickens.AvianDiseases,1989,33(1):69-73.125 HuazhongAgriculturalUniversityMaster’sDegree2018268.LindsayDS,UptonSJ,OwensDS,etal.Cryptosporidiumandersonisp.(Apicomplexa:cryptosporiidae)fromcattle,BosTaurus.JournalofEukaryoticMicrobiology,2000,47(1):91-95.269.LiuH,ShenY,YinJ,etal.PrevalenceandgeneticcharacterizationofCryptosporidium,Enterocytozoon,GiardiaandCyclosporaindiarrhealoutpatientsinChina.BMCInfectiousDiseases,2014,14(1):25.270.LiuY,CaoXM,YinLY,etal.EpidemiologicalinvestigationofCryptosporidiuminwaterbuffaloinHumanprovinceandmouseinfectionexperiment.ChineseJournalofVeterinaryScienceandTechnology,1994,24(11):14-15(InChinese).271.LiuYX,CuiHP,ZhouYH,etal.PreliminaryinvestigationonCryptosporidiuminfectionofmilchcowsinWuxiareas.ChineseJournalofSchistosomiasisControl.2005,17(3):240(InChinese).272.LoboML,AugustoJ,AntunesF,etal.Cryptosporidiumspp.,Giardiaduodenalis,EnterocytozoonbieneusiandotherintestinalparasitesinyoungchildreninLobataprovince,DemocraticRepublicofSaoTomeandPrincipe.PLoSOne,2014,9(5):e97708.273.LoweryCJ,MillarBC,MooreJEetal.MoleculargenotypingofhumancryptosporidiosisinNorthernIreland:epidemiologicalaspectsandreview.IrishJournalofMedicalScience,2001,170(4):246-250.274.LuJ,LiCP,YangZH,etal.AnepidemiologicalsurveyofcryptosporidiosisinHuainandistrict.JournalofHuainanInstituteofTechnology,2001,21(4):71-72(InChinese).275.LuLA,ZhangWS,LiBB,etal.Etiologicaldiagnosisandcasecontrolstudyofcryptosporidiosis.ContemporaryMedicine,2009,15(13):150-151(InChinese).276.LuSH,LinAF,ChenR,etal.ThediagnosisandanalysisofCryptosporidiuminfectioninchildrenofZhejiang.ChineseJournalofZoonoses,2000,16(1):42-44(InChinese).277.LuQB,QiuSX,RuBR,etal.EpidemiologicalinvestigationofcryptosporidiosisindairycalvesinsomeprefecturesofHenanProvince.ChineseVeterinaryScience,2008,38(3):261-267(InChinese).278.LupoPJ,Langer-CurryRC,RobinsonM,etal.CryptosporidiummurisinaTexascaninepopulation.AmericanJournalTropicalMedicineandHygiene,2008,78(6):917-921.279.LvC,ZhangL,WangR,etal.Cryptosporidiumspp.inwild,laboratory,andpetrodentsinChina:prevalenceandmolecularcharacterization.AppliedEnvironmentalMicrobiology,2009,75(24):7692-7699.280.MaJ,LiP,ZhaoX,etal.OccurrenceandmolecularcharacterizationofCryptosporidiumspp.andEnterocytozoonbieneusiindairycattle,beefcattleandwaterbuffaloesinChina.VeterinaryParasitology,2015,207(3-4):220-227.281.MaL,LuY,CaiQ,etal.SerologicalinvestigationofyakcryptosporidiosisinQinghaiProvince.JournalofDomesticAnimalEcology,2011,32(2):47-49(InChinese).282.MacKenzieWR,HoxieNJ,ProctorME,etal.AmassiveoutbreakinMilwaukeeofCryptosporidiuminfectiontransmittedthroughthepublicwatersupply.TheNewEngland126 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaJournalofMedicine,1994,331(3):161-167.283.MagninoS,ColinP,Dei-CasEetal.Biologicalrisksassociatedwithconsumptionofreptileproducts.InternationalJournalofFoodMicrobiology.2009,134(3):163-175.284.MarangiM,BerrilliF,OtrantoD,etal.GenotypingofGiardiaduodenalisamongchildrenanddogsinaclosedsociallydeprivedcommunityfromItaly.ZoonosesandPublicHealth,2010,57(7-8):e54-e58.285.ManquePA,TenjoF,WoehlbierU,etal.IdentificationandimmunologicalcharacterizationofthreepotentialvaccinogensagainstCryptosporidiumspecies.ClinicalandVaccineImmunology,2011,18(11):1796-1802.286.MathisA,WeberR,DeplazesP.Zoonoticpotentialofthemicrosporidia.ClinicalMicrobiologyReviews,2005,18(3):423-445.287.MatosO,LoboML,XiaoL.EpidemiologyofEnterocytozoonbieneusiinfectioninhumans.JournalofParasitologyResearch,2012,2012(4):981424.288.MatsubayashiM,KimataI,IsekiM,etal.InfectivityofanoveltypeofCryptosporidiumandersonitolaboratorymice.VeterinaryParasitology,2005,129(1-2):165-168.289.MayerCL,PalmerCJ.EvaluationofPCR,nestedPCR,andfluorescentantibodiesfordetectionofGiardiaandCryptosporidiumspeciesinwastewater.AppliedandEnvironmentalMicrobiology.1996,62(6):2081-2085.290.MayrhoferG,AndrewsRH,EyPL,etal.DivisionofGiardiaisolatesfromhumansintotwogeneticallydistinctassemblagesbyelectrophoreticanalysisofenzymesencodedat27lociandcomparisonwithGiardiamuris.Parasitology,1995,111(Pt.1):11-17.291.McCarthyS,NgJ,GordonC,etal.PrevalenceofCryptosporidiumandGiardiaspeciesinanimalsinirrigationcatchmentsinthesouthwestofAustralia.ExperimentalParasitology,2008,118(4):596-599.292.McColeDF,EckmannL,LaurentF,etal.IntestinalepithelialcellapoptosisfollowingCryptosporidiumparvuminfection.InfectionandImmunity,2000,68(3):1710-1713.293.McDowallRM,PeregrineAS,LeonardE.K,etal.EvaluationofthezoonoticpotentialofGiardiaduodenalisinfecalamplesfromdogsandcatsinOntario.CanadianVeterinaryJournal,2011,52(12):1329-1333.294.McGladeTR,RobertsonID,ElliotAD,etal.HighPrevalenceofGiardiadetectedincatsbyPCR.VeterenaryParasitology,2003,110(3-4):197-205.295.McLauchlinJ,AmarC,Pedraza-DíazS,etal.MolecularepidemiologicalanalysisofCryptosporidiumspp.intheUnitedKingdom:resultsofgenotypingCryptosporidiumspp.in1,705fecalsamplesfromhumansand105fecalsamplesfromlivestockanimals,JournalofClinicalMicrobiology,2000,38(11):3984-3990.296.McLauchlinJ,Pedraza-DiazS,Amar-HoetzenederC,etal.GeneticcharacterizationofCryptosporidiumstrainsfrom218patientswithdiarrheadiagnosedashavingsporadiccryptosporidiosis.JournalofClinicalMicrobiology,1999,37(10):3153-3158.297.MeirelesMV,SoaresRM,BonelloF,etal.Naturalinfectionwithzoonoticsubtypeof127 HuazhongAgriculturalUniversityMaster’sDegree2018CryptosporidiumparvuminCapybara(Hydrochoerushydrochaeris)fromBrazil.VeterinaryParasitology,2007,147(1-2):166-170.298.MeiselJL,PereraDR,MeligroC,etal.OverwhelmingwaterydiarrheaassociatedwithaCryptosporidiuminanimmunosuppressedpatient.Gastroenterology,1976,70(6):1156-1160.299.MekaruSR,MarksSL,FelleyAJ,etal.Comparisonofdirectimmunofluorescence,immunoassays,andfecalflotationfordetectionofCryptosporidiumspp.andGiardiaspp.innaturallyexposedcatsin4NorthernCaliforniaanimalshelters.JournalofVeterinaryInternalMedicine,2007,21(5):959-65.300.MendoncaC,AlmeidaA,CastroA,etal.MolecularcharacterizationofCryptosporidiumandGiardiaisolatesfromcattlefromPortugal.VeterinaryParasitology,2007,147(1):47-50.301.MengR,WangX,DongM,etal.InvestigationonCryptosporidiuminfectionamongdairycattleinTianjinarea,China.JournalofPathogenBiology,2007,2(3):225-227.302.MinHL,ChenSM,HuJX,etal.InvestigationsurveyofcryptosporidiosisinXianning,city,Xian'anDistrict.JournalofPublicHealthandPreventiveMedicine.2014,25(6):111-112(InChinese).303.MinvielleMC,MolinaNB,PolverinoD,etal.FirstgenotypingofGiardialambliafromhumanandanimalfecesinArgentina,SouthAmerica.MemóriasInstitutoOswaldoCruz,2008,103(1):98-103.304.MisicZ,AbeN.SubtypeanalysisofCryptosporidiumparvumisolatesfromcalvesonfarmsaroundBelgrade,SerbiaandMontenegro,usingthe60kDaglycoproteingenesequences.Parasitology,2007,134(Pt3):351-358.305.MiR,WangX,LiC,etal.PrevalenceandgeneticcharacterizationofCryptosporidiuminyaksinQinghaiProvinceofChina.PLoSOne,2013,8(9):e74985.306.MMWR.Cryptosporidiosis:assessmentofchemotherapyofmaleswithacquiredimmunedeficiencysyndrome(AIDS).MorbidityMortalityWeekklyReport,1982,31(44):589-592.307.MonisPT,AndrewsRH,MayrhoferG,etal.MolecularsystematicsoftheparasiticprotozoanGiardiaintestinalis.MolecularBiologyandEvolution,1999,16(9):1135-1144.308.MonisPT,AndrewsRH,MayrhoferG,etal.GeneticdiversitywithinthemorphologicalspeciesGiardiaintestinalisanditsrelationshiptohostorigin.InfectionGeneticsEvolution,2003,3(1):29-38.309.MonisPT,MayrhoferG,AndrewsRH,etal.MoleculargeneticanalysisofGiardiaintestinalisisolatesattheglutamatedehydrogenaselocus.Parasitology,1996,112(1):1-12.310.MooreCE,ElwinK,PhotN,etal.MolecularcharacterizationofCryptosporidiumspeciesandGiardiaduodenalisfromsymptomaticCambodianchildren.PLoSNeglectedTropicalDiseases,2016,10(7):e0004822.311.MorganUM,BuddleJR,ArmsonA,etal.MolecularofbiologicalcharacterisationofCryptosporidiuminpigs.AustralianVeterinaryJournal,1999a,77(1):44-47.312.MorganUM,DeplazesP,ForbesDA.SequenceandPCR-RFLPanalysisoftheinternal128 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinatranscribedspacersoftherDNArepeatunitinisolatesofCryptosporidiumfromdifferenthosts.Parasitology,1999b,118(1):49-58.313.MorganUM,MonisPT,FayerR,etal.PhylogeneticrelationshipsamongisolatedofCryptosporidium:evidenceforseveralnewspecies.JournalofParasitology,1999c,85(6):1126-1133.314.MorganUM,SturdeeAP,SingletonG,etal.TheCryptosporidium"mouse"genotypeisconservedacrossgeographicareas.JournalofClinicalMicrobiology,1999d,37(5):1302-1305.315.MorganUM,XiaoL,MonisP,etal.Cryptosporidiumspp.indomesticdogs:the"dog"genotype.AppliedandEnvironmentalMicrobiology,2000d,66(5):2220-2223.316.MorganUM,XiaoL,FayerR,etal.VariationinCryptosporidium:towardsataxonomicrevisionofthegenus.InternationalJournalofParasitology,1999e,29(11):1733-1751.317.MorganUM,PallantL,DwyerBW,etal.ComparisonofPCRandmicroscopyfordetectionofCryptosporidiumparvuminhumanfecalspecimens:clinicaltrial.JournalofClinicalMicrobiology,1998,36(4):995-998.318.MorganUM,XiaoL,HillBD,etal.DetectionoftheCryptosporidiumparvum“human”genotypeinadugong(Dugongdugon).JournalofParasitology,2000a,86(6):1352-1354.319.MorganUM,XiaoL,LimorJ,etal.CryptosporidiummeleagridisinanIndianring-neckedparrot(Psittaculakrameri).AustralianVeterinaryJournal,2000b,78(3):182-183.320.MorganUM,XiaoL,MonisP,etal.MolecualrphylogeneticanalysisofCryptosporidiummurisfromvarioushosts.Parasitology,2000c,120(5):457-464.321.MorseTD,NicholsRA,GrimasonAM,etal.IncidenceofcryptosporidiosisspeciesinpaediatricpatientsinMalawi.EpidemiologyandInfection,2007,135(8):1307-1315.322.MooreR,TziporiS,GriffithsJK,etal.,TemporalchangesinpermeabilityandstructureofpigletileumaftersitespecificinfectionbyCryptosporidiumparvum.Gastroenterology,1995,108(4):1030-1039.323.Mueller-DobliesD,GilesM,ElwinK,etal.DistributionofCryptosporidiumspeciesinsheepintheUK.VeterinaryParasitology,2008,154(3):214-219.324.MuthusamyD,RaoSS,RamaniS,etal.MultilocusgenotypingofCryptosporidiumsp.isolatesfromhumanimmunodeficiencyvirus-infectedindividualsinSouthIndia.JournalClinicalMicrobiology,2006,44(2):632-634.325.NascimentoWR,CavalcantiIM,IrmãoJI,etal.PresenceofCryptosporidiumsppinchildrenwithacutediarrheainapublicdaycarecenterinRecife,StateofPernambuco.RevistadaSociedateBrasileiraMedicinaTropical,2009,42(2):175-178.326.Navarro-i-MartinezL,daSilvaAJ,Bornay-LlinaresFJ,etal.DetectionandmolecularcharacterizationofCryptosporidiumbovis-likeisolatefromanewbornlambinSpain.JournalofParasitology,2007,93(6):1536-1538.327.Nazemalhosseini-MojaradE,FengY,XiaoL.TheimportanceofsubtypeanalysisofCryptosporidiumspp.inepidemiologicalinvestigationsofhumancryptosporidiosisinIran129 HuazhongAgriculturalUniversityMaster’sDegree2018andotherMideastcountries.GastroenterologyandHepatologyfromBedtoBench,2012,5(2):67-70.328.NgJ,EastwoodK,DurrheimD,etal.EvidencesupportingzoonotictransmissionofCryptosporidiuminruralNewSouthWales.ExperimentalParasitology,2008,119(1):192-195.329.NgJ,MacKenzieB,RyanU.Longitudinalmulti-locusmolecularcharacterisationofsporadicAustralianhumanclinicalcasesofcryptosporidiosisfrom2005to2008.ExperimentalParasitology,2010a,125(4):348-356.330.NgJ,PingaultN,GibbsR,etal.MolecularcharacterisationofCryptosporidiumoutbreaksinWesternandSouthAustralia.ExperimentalParasitology,2010b,125(4):325-328.331.NgJ,YangR,McCarthyS,etal.MolecularcharacterizationofCryptosporidiumandGiardiainpre-weanedcalvesinWesternAustraliaandNewSouthWales.VeterinaryParasitology,2011,176(2-3):145-150.332.NgouanesavanhT,GuyotK,CertadGetal.Cryptosporidiumpopulationgenetics:evidenceofclonalityinisolatesfromFranceandHaiti.JournalofEukaryoticMicrobiology,2006,53(1):S33-S36.333.NimeFA,BurekJD,PageDL,etal.AcuteenterocolitisinahumanbeinginfectedwiththeprotozoanCryptosporidium.Gastroenterology,1976,70(4):592-598.334.NiuY,LiYH,YuXH,etal.ApreliminaryinvestigationofCryptosporidiuminfectionindiarrhoeicpatientsinQiqihar.JournalofQiqiharMedicalColleges,2006,27(2):184(InChinese).335.NolanMJ,JexAR,HaydonSR,etal.MoleculardetectionofCryptosporidiumcuniculusinrabbitsinAustralia.InfectionGeneticsandEvolution,2010,10(8):1179-1187.336.NolanMJ,JexAR,KoehlerAV,etal.Molecular-basedinvestigationofCryptosporidiumandGiardiafromanimalsinwatercatchmentsinsoutheasternAustralia.WaterResearch,2013,47(5):1726-1740.337.NolanMJ,JexAR,MansellPD,etal.GeneticcharacterizationofCryptosporidiumparvumfromcalvesbymutationscanningandtargetedsequencing-zoonoticimplications.Electrophoresis.2009,30(15):2640-2647.338.NolanMJ,JexAR,PangasaA,etal.,Analysisofnucleotidevariationwithinthetriose-phosphateisomerasegeneofGiardiaduodenalisfromsheepanditszoonoticimplications.Electrophororesis,2010,31(2):287-298.339.NouriM.ACryptosporidiummuris-likeparasiteincattleandcamelsinIran.IndianVeterinaryJournal,2002,79(6):558-559.340.O'BrienE,McInnesL,RyanU.CryptosporidiumGP60genotypesfromhumansanddomesticatedanimalsinAustralia,NorthAmericaandEurope.ExperimentalParasitology,2008,118(1):118-121.341.O’DonoghuePJ.Cryptosporidiumandcryptosporidiosisinmanandanimals.InternationalJournalforParasitology,1995,25(2):139-195.130 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China342.O’HandleyRM,CockwillCL,McAllisterTA,etal.Durationofnaturallyacquiredgiardiasisandcryptosporidiosisindairycalvesandtheirassociationwithdiarrhea.JournaloftheAmericanVeterinaryMedicalAssociation,1999,214(3):391-396.343.O’HandleyRM,OlsonME.Giardiasisandcryptosporidiosisinruminants.VeterinaryClinicsofNorthAmericaFoodAnimalPractice,2006,22(3):623-643.344.OkhuysenPC,ChappellCL,CrabbJH,etal.VirulenceofthreedistinctCryptosporidiumparvumisolatesforhealthyadults.JournalofInfectiousDiseases,1999,180(4):1275-1281.345.OlsonME,MorckDW,CeriH.TheefficacyofaGiardialambliavaccineinkittens.CanadianJournalofVeterinaryResearch,1996,60(4):249-256.346.OlsonME,MorckDW,CeriH.PreliminarydataontheefficacyofaGiardiavaccineinpuppies.CanadianVeterinaryJournal,1997,38(12):777-779.347.OlsonME,CeriH,MorckDW.Giardiavaccination.ParasitologyToday,2000,16(5):213-217.348.OnS,LimE,LopezL,etal.Annualreportconcerningfood-bornediseaseinNewZealand.EnvironmentalScienceandResearchLimited(ESR),Christchurch.2011.349.OngCS,EislerDL,AlikhaniA,etal.NovelCryptosporidiumgenotypesinsporadiccryptosporidiosiscases:firstreportofhumaninfectionswithacervinegenotype.EmergingInfectiousDiseases,2002,8(3):263-268.350.OngCS,EislerDL,GohSHetal.MolecularepidemiologyofcryptosporidiosisoutbreaksandtransmissioninBritishColumbia,Canada.AmericanJournalofTropicalMedicineandHygiene,1999,61(1):63-69.351.OsmanM,ElSafadiD,CianA,etal.PrevalenceandriskfactorsforintestinalprotozoaninfectionswithCryptosporidium,Giardia,BlastocystisandDientamoebaamongschoolchildreninTripoli,Lebanon.PLoSNeglectedTropicalDiseases,2016,10(3):e0004496.352.OvergaauwPA,vanZutphenL,HoekD,etal.ZoonoticparasitesinfecalsamplesandfurfromdogsandcatsinTheNetherlands.VeterinaryParasitology,2009,163(1-2):115-122.353.Pagès-MantéA,Pagès-BoschM,Majó-MasferrerNetal.Anoutbreakofdiseaseassociatedwithcryptosporidiaonared-leggedpartridge(Alectorisrufa)gamefarm.AvianPathology,2007,36(4):275-278.354.PalenzuelaO,Alvarez-PelliteroP,Sitjà-BobadillaA.MolecularcharacterizationofCryptosporidiummolnarirevealsadistinctpiscineclade.AppliedandEnvironmentalMicrobiology,2010,76(22):7646-7649.355.PalmerCS,TraubRJ,RobertsonID,etal.DeterminingthezoonoticsignificanceofGiardiaandCryptosporidiuminAustraliandogsandcats.VeterinaryParasitology,2008,154(1-2):142-147.356.PalmerCJ,XiaoL,TerashimaAetal.Cryptosporidiummuris,arodentpathogen,recoveredfromahumaninPerú.EmergingInfectiousDiseases,2003,9(9):1174-1176.357.PancieraRJ,ThomassenRW,GarnerFM.Cryptosporidialinfectioninacalf.VeterinaryPathology,1971,8(5-6):479-484.131 HuazhongAgriculturalUniversityMaster’sDegree2018358.PangasaA,JexAR,NolanMJ,etal.Highlysensitivenon-isotopicrestrictionendonucleasefingerprintingofnucleotidevariabilityinthegp60genewithinCryptosporidiumspecies,genotypesandsubgenotypesinfectivetohumans,anditsimplications.Electrophoresis.2010,31(10):1637-1647.359.PantenburgB,DannSM,WangHC,etal.IntestinalimmuneresponsetohumanCryptosporidiumsp.infection.InfectionandImmunity,2008,76(1):23-29.360.ParkJH,GukSM,HanET,etal.GenotypeanalysisofCryptosporidiumspp.prevalentinaruralvillageinHwasun-gun,RepublicofKorea.KoreanJournalofParasitology,2006,44(1):27-33.361.PavlásekI.Blackheadedgull(LarusridibundusL),anewhostofCryptosporidiumbaileyi(Apicomplexa,Cryptosporidiidae).VeterinaryMedicine,1993,38(10):629-638.362.PavlásekI.Cryptosporidia:biology,diagnosis,hostspectrum,specificity,andtheenvironment.RemediaKlinickaMikrobiologie,1999,3:290-301.363.PavlásekI.FindingsofCryptosporidiainthestomachofchickensandofexoticandwildbirds.Veterinárství,2001,51:103-108.364.PavlásekI,LavickovaM,HorakP,etal.Cryptosporidiumvaraniin.sp.(Apicomplexa:Cryptosporidiidae)inEmeraldmonitor(VaranusprasinusSchegel,1893)incaptivityatPraguezoo.Gazella,1995,22:99-108.365.PavlásekI,RyanU.CryptosporidiumvaraniitakesprecedenceoverC.saurophilum.ExperimentalParasitology,2008,118(3):434-437.366.PenaHF,KasaiN,GennariSM.CryptosporidiummurisindairycattleinBrazil.VeterinaryParasitology,1997,73(3-4):353-355.367.PengMM,MatosO,GateiW,etal.AcomparisonofCryptosporidiumsubgenotypesfromseveralgeographicregions.JournalofEukaryoticMicrobiology,2001,48(S1):28S-31S.368.PengMM,MeshnickSR,CunliffeNA,etal.MolecularepidemiologyofcryptosporidiosisinchildreninMalawi.JournalofEukaryoticMicrobiology,2003a,50(S1):557-559.369.PengMM,WilsonML,HollandRE,etal.GeneticdiversityofCryptosporidiumspp.incattleinMichigan:implicationsforunderstandingthetransmissiondynamics.ParasitologyResearch,2003b,90(3):175-180.370.PengMM,XiaoL,FreemanARetal.GeneticpolymorphismamongCryptosporidiumparvumisolates:evidenceoftwodistincthumantransmissioncycles.EmergingInfectiousDiseases,1997,3(4):567-573.371.PengXQ,TianGR,RenGJ,etal.InfectionrateofGiardiaduodenalis,Cryptosporidiumspp.andEnterocytozoonbieneusiincashmere,dairyandmeatgoatsinChina.InfectionGeneticsandEvolution,2016,41:26-31.372.PereiraSJ,RamirezNE,LXiao,etal.PathogenesisofhumanandbovineCryptosporidiumparvumingnotobioticpigs.JournalofInfectiousDiseases,2002,186(5):715-718.373.PerzJF,LeBlancqSM.CryptosporidiumparvuminfectioninvolvingnovelgenotypesinwildlifefromlowerNewYorkState.AppliedandEnvironmentalMicrobiology.2001,67(3):132 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China1154-1161.374.PickeringLK,EngelkirkPG.Giardiaamongchildrenindaycare.In:HumanParasiticDiseases,Vol.3,Giardiasis(MeyerEA,ed.),Elsevier,Amsterdam,1990,pp,235-266.375.PlutzerJ,KaranisP.GenotypeandsubtypeanalysesofCryptosporidiumisolatesfromcattleinHungary.VeterinaryParasitology,2007,146(3-4):357-362.376.PohjolaS,OksanenH,JokipiiL,etal.Outbreakofcryptosporidiosisamongveterinarystudents.ScandinavianJournalofInfectiousDiseases,1986,18(2):173-178.377.PönkaA,KotilainenH,Rimhanen-FinneR,etal.AfoodborneoutbreakduetoCryptosporidiumparvuminHelsinki,November2008.EuroSurveillance,2009,14(28):1-8.378.PontiroliA,TravisER,SweeneyFP,etal.Pathogenquantitationincomplexmatrices:amulti-operatorcomparisonofDNAextractionmethodswithanovelassessmentofPCRinhibition.PLoSOne,2011,6(3):e17916.379.PowerML,RyanUM.AnewspeciesofCryptosporidium(Apicomplexa:Cryptosporidiidae)fromeasterngreykangaroos(Macropusgiganteus).JournalofParasitology,2008,94(5):1114-1117.380.PowerML,SangsterNC,SladeMB,etal.PatternsofCryptosporidiumoocystssheddingbyeasterngreykangaroosinhabitinganAustralianwatershed.AppliedandEnvironmentalMicrobiology,2005,71(10):6159-6164.381.PowerML,SladeMB,SangsterNC,etal.GeneticcharacterisationofCryptosporidiumfromawildpopulationofeasterngreykangaroosMacropusgiganteusinhabitingawatercatchment.InfectionGeneticsandEvolution,2004,4(1):59-67.382.PreiserG,PreiserL,MadeoL.Anoutbreakofcryptosporidiosisamongveterinarysciencestudentswhoworkwithcalves.JournalofAmericanCollegeHealth,2003,51(5):213-215.383.ProctorSJ,KempRL.Cryptosporidiumanserinumsp.N.(Sporozoa)inadomesticgooseAnseranserL.,fromIowa.TheJournalofProtozoology,1974,21(5):664-666.384.QadripirSA,KloseL.CryptosporidiumenteritidisoccurrenceinsouthernLowerSaxony.Studyofurticariaasanexample.DermatocogischeMonetsschrift,1985,171(7):438-442.385.QiM,WangH,JingB,etal.OccurrenceandmolecularidentificationofCryptosporidiumspp.indairycalvesinXinjiang,NorthwesternChina.VeterinaryParasitology,2015,212(3-4):404-407.386.QiM,CaiJZ,WangRJ,etal.MolecularcharacterizationofCryptosporidiumspp.andGiardiaduodenalisfromyaksinthecentralwesternregionofChina.BMCMicrobiology,2015,15(1):1-7.387.QiM,LuoN,WangH,etal.ZoonoticCryptosporidiumspp.andEnterocytozoonbieneusiinpetchinchillas(Chinchillalanigera)inChina.ParasitologyInternational,2015,64(5):339-341.388.QiM,WangR,NingC,etal.Cryptosporidiumspp.inpetbirds:geneticdiversityandpotentialpublichealthsignificance.ExperimentalParasitology,2011,128(4):336-340.389.QiM,WangR,WangH,etal.EnterocytozoonbieneusigenotypesingrazinghorsesinChina133 HuazhongAgriculturalUniversityMaster’sDegree2018andtheirzoonotictransmissionpotential.JournalofEukaryoticMicrobiology,2016,63(5):591-597.390.QianFX,ShiM,ShenSL,etal.InvestigationofCryptosporidiuminfectionamongnon-AIDSandHIVdiarrhoeicadultpatients.ChineseJournalofMisdiagnostics,2011,11(3):752(InChinese).391.QinJH,ZhaoYL,HaoCW,etal.InvestigationsurveyofCryptosporidiumindiarrhoeiccattlefromZhangjiakou.ChineseJournalofVeterinaryScience,1994,2:150.(InChinese)392.QuilezJ,TorresE,ChalmersRM,etal.CryptosporidiumspeciesandsubtypeanalysisfromdairycalvesinSpain.Parasitology,2008a,135(14):1613-1620.393.QuilezJ,TorresE,ChalmersRM,etal.GenotypeandsubtypecharacterizationofCryptosporidiuminlambsandgoatkidsinSpain.AppliedandEnvironmentalMicrobiology,2008,74(19):6026-6031.394.RamirezNE,WardLA,SreevatsanS.Areviewofthebiologyandepidemiologyofcryptosporidiosisinhumansandanimals.MicrobesandInfection,2004,6(8):773-785.395.RaccurtCP.WorldwidehumanzoonoticcryptosporidiosiscausedbyCryptosporidiumfelis.Parasite,2007,14(1):15-20.396.ReadCM,MonisPT,ThompsonRC.DiscriminationofallgenotypesofGiardiaduodenalisattheglutamatedehydrogenaselocususingPCR-RFLP.InfectionGeneticsandEvolution,2004,4(2):125-130.397.ReadC,WaltersJ,RobertsonID,etal.CorrelationbetweengenotypeofGiardiaduodenalisanddiarrhoea.InternationalJournalParasitology,2002,32(2):229-231.398.RendtorffRC.Theexperimentaltransmissionofhumanintestinalprotozoanparasites.II.Giardialambliacystsgivenincapsules.AmericanJournalofHygiene,1954,59(2):209-220.399.ReifJS,WimmerL,SmithJA,etal.Humancryptosporidiosisassociatedwithanepizooticincalves.AmericanJournalofPublicHealth,1989,79(11):1528-1530.400.RichardsonAJ,FrankenbergRA,BuckAC,etal.AnoutbreakofwaterbornecryptosporidiosisinSwindonandOxfordshire.EpidemiologyandInfection,1991,107(3):485-495.401.RiggsMW.Recentadvancesincryptosporidiosis:theimmuneresponse.MicrobesandInfection,2002,4(10):1067-1080.402.RiggsJL,DupuisKW,NakamuraK,etal.DetectionofGiardialambliabyimmuneflu-orescence.AppliedandEnvironmentalMicrobiology,1983,45(2):698-700.403.Rimhanen-FinneR,EnemarkHL,KolehmainenJ,etal.Evaluationofimmunofluorescencemicroscopyandenzyme-linkedimmunosorbentassayindetectionofCryptosporidiumandGiardiainfectionsinasymptomaticdogs.VeterinaryParasitology,2007,145(3-4):345-348.404.RimšelienėG,VoldL,RobertsonL,etal.Anoutbreakofgastroenteritisamongschoolchildrenstayinginawildlifereserve:thoroughinvestigationrevealsNorway'slargestcryptosporidiosisoutbreak.ScandinavianJournalofPublicHealth,2011,39(3):287-295.405.RobertsonLJ,GjerdeBK,Furusethetal.ThezoonoticpotentialofGiardiaand134 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaCryptosporidiuminNorwegiansheep:alongitudinalinvestigationof6flocksoflambs.VeterinaryParasitology,2010,171(1-2):140-145.406.RobertsonLJ,ChalmersRM.Foodbornecryptosporidiosis:istherereallymoreinNordiccountries?TrendsParasitology,2013,29(1):3-9.407.RobinsonG,ThomasAL,DanielRG,etal.SampleprevalenceandmolecularcharacterizationofCryptosporidiumandersoniwithinadairyherdintheUnitedKingdom.VeterinaryParasitology2006,142(1-2):163-167.408.RobinsonG,WrightS,ElwinK,etal.Re-descriptionofCryptosporidiumcuniculusInmanandTakeuchi,1979(Apicomplexa:Cryptosporidiidae):morphology,biologyandphylogeny.InternationalJournalforParasitology,2010,40(13):1539-1548.409.RongJQ,WuGP,ChenLH,etal.EpidemiologysurveyofhumancryptosporidiosisinQiannanarea.JournalofQiannanMedicalCollegeforNationalMinorities,1999,12(1):52-54(InChinese).410.Roxstrom-LindquistK,PalmD,ReinerD,etal.Giardiaimmunity-anupdate.TrendsParasitology,2006,22(1):26-31.411.RossignolJF.CryptosporidiumandGiardia:treatmentoptionsandprospectsfornewdrugs.ExperimentalParasitology,2010,124(1):45-53.412.RuizA,ForondaP,GonzálezJF,etal.OccurrenceandgenotypecharacterizationofGiardiaduodenalisingoatkidsfromtheCanaryIslands,Spain.VeterinaryParasitology,2008,154(1-2):137-141.413.RyanU.Cryptosporidiuminbirds,fishandamphibians.ExperimentalParasitology,2010,124(1):113-120.414.RyanUM,BathC,RobertsonI,etal.SheepmaynotbeanimportantzoonoticreservoirforCryptosporidiumandGiardiaparasites.AppliedandEnvironmentalMicrobiology,2005,71(9):4992-4997.415.RyanU,CaccioSM.ZoonoticpotentialofGiardia.InternationalJournalforParasitology,2013,43(12-13):943-956.416.RyanU,FayerR,XiaoL.Cryptosporidiumspeciesinhumansandanimals:currentunderstandingandresearchneeds.Parasitology,2014,141(13):1667-1685.417.RyanU,HijjawiN.NewdevelopmentsinCryptosporidiumresearch.InternationalJournalforParasitology,2015,45(6):367-373.418.RyanUM,MonisP,EnemarkHL,etal.Cryptosporidiumsuisn.sp.(Apicomplexa:Cryptosporidiidae)inpigs(Susscrofa).JournalofParasitology,2004b,90(4):769-773.419.RyanU,PapariniA,TongK,etal.Cryptosporidiumhuwin.sp.(Apicomplexa:Eimeriidae)fromtheguppy(Poeciliareticulata).ExperimentalParasitology,2015,150:31-35.420.RyanUM,PowerM,XiaoL.Cryptosporidiumfayerin.sp.(Apicomplexa:Cryptosporidiidae)fromtheRedKangaroo(Macropusrufus).JournalofEukaryoticMicrobiology,2008,55(1):22-26.421.RyanUM,XiaoL.Birds.In:FayerR,XiaoL.(Eds.),Cryptosporidiumandcryptosporidiosis,135 HuazhongAgriculturalUniversityMaster’sDegree2018seconded.CRCpress,BocaRaton,FL,2008,pp.395-418.422.RyanUM,SamarasingheB,ReadC,etal.IdentificationofanovelCryptosporidiumgenotypeinpigs.AppliedEnvironmentalMicrobiology,2003,69(7):3970-3974.423.RyanUM,XiaoL.TaxonomyandMolecularTaxonomy.InCryptosporidium:ParasiteandDisease(ed.CacciòSMandWidmerG).SpringerVienna,NewYork,NY,USA,2014,3-41.424.RyanU,XiaoL,ReadC,etal.IdentificationofnovelCryptosporidiumgenotypesfromtheCzechRepublic.AppliedandEnvironmentalMicrobiology,2003a,69(7):4302-4307.425.RyanUM,XiaoL,ReadC,etal.AredescriptionofCryptosporidiumgalliPavlasek,1999(Apicomplexa:Cryptosporidiidae)frombirds.JournalofParasitology,2003b,89(4):809-813.426.SantínM,FayerR.Enterocytozoonbieneusigenotypenomenclaturebasedontheinternaltranscribedspacersequence:aconsensus.JournalofEukaryoticMicrobiology,2009,56(1):34-38.427.SantínM,TroutJM.Companionanimals.In:FayerR,XiaoL(Eds).Cryptosporidiumandcryptosporidiosis.CRCPress,BocaRaton,FL,USA,2008,pp.437-450.428.SantínM,TroutJM,FayerR.PrevalenceandmolecularcharacterizationofCryptosporidiumandGiardiaspeciesandgenotypesinsheepinMaryland.VeterinaryParasitology,2007,146(1-2):17-24.429.SantínM,TroutJM,FayerR.Alongitudinalstudyofcryptosporidiosisindairycattlefrombirthto2yearsofage.VeterinaryParasitology,2008,155(1-2):15-23.430.SantínM,TroutJM,FayerR.AlongitudinalstudyofGiardiaduodenalisgenotypesindairycowsfrombirthto2yearsofage.VeterinaryParasitology,2009,162(1-2):40-45.431.SatohM,MatsubaraY,SasakiT.CharacterizationofCryptosporidiumcanisisolatedinJapan.ParasitologyResearch,2006,99(6):746-748.432.SchunkM,JelinekT,WetzelK,etal.DetectionofGiardialambliaandEntamoebahistolyticainstoolsamplesbytwoenzymeimmunoassays.EuropeanJournalofClinicalMicrobiologyInfectiousDiseases,2001,20(6):389-391.433.SchuurmanT,LankampP,VanBelkumA,etal.Comparisonofmicroscopy,real-timePCRandarapidimmunoassayforthedetectionofGiardialambliainhumanstoolspecimens.ClinicalMicrobiologyandInfection,2007,13(12):1186-1191.434.ShahiduzzamanM,DaugschiesA.Therapyandpreventionofcryptosporidiosisinanimals.VeterinaryParasitology,2012,188(3-4):203-214.435.ShenJP,GeJJ.Investigationandanalysisoftheinfectionrateof2018infantsinthekindergarten.ChineseJournalofPracticalPediatrics,1991,3:132(InChinese).436.SilverlåsC,EmanuelsonU,deVerdierK,etal.PrevalenceandassociatedmanagementfactorsofCryptosporidiumsheddingin50Swedishdairyherds.PreventiveVeterinaryMedicine,2009,90(3-4):242-253.437.SilverlåsC,MattssonJG,InsulanderM,etal.ZoonotictransmissionofCryptosporidiummeleagridisonanorganicSwedishfarm.InternationalJournalforParasitology,2012,136 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China42(11):963-967.438.SilverlåsC,NaslundK,BjorkmanC,etal.MolecularcharacterisationofCryptosporidiumisolatesfromSwedishdairycattleinrelationtoage,diarrhoea,andregion.VeterinaryParasitology,2010,169(3-4):289-295.439.Sitjà-Bobadilla,A,Alvarez-PelliteroP.ExperimentaltransmissionofCryptosporidiummolnari(Apicomplexa:Coccidia)togiltheadseabream(SparusaurataL.)andEuropeanseabass(DicentrarchuslabraxL.).ParasitologyResearch,2003,91(3):209-214.440.Sitjà-BobadillaA,PadrósF,AguileraC,etal.EpidemiologyofCryptosporidiummolnariinSpanishgiltheadseabream(SparusaurataL.)andEuropeanseabass(DicentrarchuslabraxL.)cultures:fromhatcherytomarketsize.AppliedandEnvironmentalMicrobiology,2005,71(1):131-139.441.ŠlapetaJ.Cryptosporidiumspeciesfoundincattle:aproposalforanewspecies.TrendsinParasitology,2006,22(10):469-474.442.ŠlapetaJ.ThenameCryptosporidiumtyzzeriRen,Zhao,Zhang,Ning,Jian,Wang,Lv,Wang,ArrowoodandXiao,2012ispermanentlyinvalid.ExperimentalParasitology,2012,130(3):306-307.443.ŠlapetaJ.CryptosporidiosisandCryptosporidiumspeciesinanimalsandhumans:athirtycolourrainbow?InternationalJournalforParasitology,2013,43(12-13):957-970.444.SlavinD.Cryptosporidiummeleagridis(sp.nov.).JournalofComparativePathology,1955,65(3):262-266.445.SmerdonWJ,NicholsT,ChalmersRM,etal.Footandmouthdiseaseinlivestockandreducedcryptosporidiosisinhumans,Englandandwales.EmergingInfectiousDiseases,2003,9(1):22-28.446.SmithHV,CacciòSM,CookN,etal.CryptosporidiumandGiardiaasfoodbornezoonoses.VeterinaryParasitology,2007,149(1-2):29-40.447.SmithHV,CacciòSM,TaitA,etal.ToolsforinvestigatingtheenvironmentaltransmissionofCryptosporidiumandGiardiainfectionsinhumans.TrendsParasitology,2006,22(4):160-167.448.SmithHV,CorcoranGD.Newdrugsandtreatmentforcryptosporidiosis.CurrentOpinioninInfectiousDiseases,2004,17(6):557-564.449.SmithHV,NicholsRA,MallonM,etal.NaturalCryptosporidiumhominisinfectionsinScottishcattle.VeterinaryRecords,2005,156(22):710-711.450.SobaB,LogarJ.GeneticclassificationofCryptosporidiumisolatesfromhumansandcalvesinSlovenia.Parasitology,2008,135(11):1263-1270.451.SprongH,CacciòSM,vanderGiessen,etal.IdentificationofzoonoticgenotypesofGiardiaduodenalis.PLoSNeglectedTropicalDiseases,2009,3(12):e558.452.SréterT,KovácsG,SilvaAJ,etal.Morphologic,hostspecificity,andmolecularcharacterizationofaHungarianCryptosporidiummeleagridisisolate.AppliedEnvironmentalMicrobiology,2000,66(2):735-738.137 HuazhongAgriculturalUniversityMaster’sDegree2018453.StensvoldCR,BeserJ,AxenC,etal.Highapplicabilityofanovelmethodforgp60-basedsubtypingofCryptosporidiummeleagridis.JournalofClinicalMicrobiology,2014,52(7):2311-2319.454.Stantic-PavlinicM,XiaoL,GlabermanSetal.Cryptosporidiosisassociatedwithanimalcontacts.WienerKlinischeWochenschrift,2003,115(3-4):125-127.455.StriepenB.Parasiticinfections:timetotacklecryptosporidiosis.Nature,2013,503(7475):189-191.456.StibbsHH,SamadpourM,ManningJF.EnzymeimmunoassayfordetectionofGiardialambliacystantigensinformalin-fixedandunfixedhumanstool.JournalClinicalMicrobiology,1988,26(9):1665-1669.457.SuDP,ChenDG,HuaXL,etal.CryptosporidiosisfrominfantsinFuzhouregion.ChineseJournalofZoonoses,1989,5(5):35-36(InChinese).458.SuSL,ChenGF,WuGH,etal.InvestigationsurveyofcryptosporidiosisindiarrhoeicchildrenfromGanzhou.ChineseJournalofParasiticDiseaseControl,1993,6(1):63(InChinese).459.SuQP,HuangMH,GaoQF,etal.Thereportof8casesofhumancryptosporidiosisinZhangzhou.JournalofFujianMedicalUniversity,1991,25(2):153(InChinese).460.SuQP,ZhengCR,ZhouBX,etal.InvestigationofCryptosporidiuminfectioninchildrenandcowsinShunchangCounty.ChineseJournalofZoonoses,1991,7(3):65(InChinese).461.SuYP,HeLJ,SongJD,etal.InvestigationsurveyofcryptosporidiosisindiarrhoeicinfantsinHenanProvince.HenanJournalofPreventiveMedicine,1991,2(1):533-535(InChinese).462.SulaimanIM,FayerR,BernC,etal.TriosephosphateisomerasegenecharacterizationandpotentialzoonotictransmissionofGiardiaduodenalis.EmergingInfectiousDiseases,2003,9(11):1444-1452.463.SulaimanIM,HiraPR,ZhouL,etal.UniqueendemicityofcryptosporidiosisinchildreninKuwait.JournalofClinicalMicrobiology,2005,43(6):2805-2809.464.SulaimanIM,LalAA,XiaoL.MolecularphylogenyandevolutionaryrelationshipsofCryptosporidiumparasitesattheactinlocus.JournalofParasitology,2002,88(2):388-394.465.SulaimanIM,XiaoL,YangCetal.DifferentiatinghumanfromanimalisolatesofCryptosporidiumparvum.EmergingInfectiousDiseases,1998,4(4):681-685.466.SunXi,GuGM,GuGS,etal.Investigationsurveyof341casesindiarrhoeicpatients.ChineseJournalofParasiticDiseaseControl,1993,6(1):64(InChinese).467.SunYR.InvestigationoftheinfectionsituationandtheriskfactorsinchildrenofCryptosporidium.ChinaHealthIndustry,2015(8):17-18(InChinese).468.SuzukiJ,MurataR,KobayashiS,etal.RiskofhumaninfectionwithGiardiaduodenalisfromcatsinJapanandgenotypingoftheisolatestoassesstherouteofinfectionincats.Parasitology,2011,138(4):493-500.469.TanriverdiS,ArslanMO,AkiyoshiDE,etal.IdentificationofgenotypicallymixedCryptosporidiumparvumpopulationsinhumansandcalves.MolecularandBiochemical138 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaParasitology,2003,130(1):13-22.470.TaoLi,ChenZ,WeiZ,etal.EpidemiologicalinvestigationofcryptosporidiosisdairycattleinGuangxi,China.ChineseVeterinaryScience,2012,42(7):742-746(InChinese).471.TaylorMA,CoopRL,WallRL.VeterinaryParasitology.4rdEdition.London,BlackwellPublishingLtd;2016.472.TerrellSP,UhlEW,FunkRS.Proliferativeenteritisinleopardgeckos(Eublepharismacularius)associatedwithCryptosporidiumsp.infection.JournalofZooandWildlifeMedicine,2003,34(1):69-75.473.ThompsonRC.GiardiaInfections.OxfordUniversityPressInc,2011,NewYork.474.ThompsonRCA.GiardiaInfections.Inc,NewYork.In:PalmerSR,LordSoulsby,TorgersonP,andBrown,DWG(Eds.),Biology,ClinicalPractice,andPublicHealthControl.OxfordUniversityPressOxford,UK,2011,pp.523-535.475.ThompsonHP,DooleyJS,KennyJ,etal.GenotypesandsubtypesofCryptosporidiumspp.inneonatalcalvesinNorthernIreland.ParasitologyResearch,2007,100(3):619-624.476.ThompsonRC,HopkinsRM,HomanWL.NomenclatureandgeneticgroupingsofGiardiainfectingmammals.ParasitologyToday,2000,16(5):210-213.477.ThompsonRC,MonisPT.VariationinGiardia:implicationsfortaxonomyandepidemiology.AdvancesinParasitology,2004,58(2):69-137.478.ThompsonRC,PalmerCS,O'HandleyR.ThepublichealthandclinicalsignificanceofGiardiaandCryptosporidiumindomesticanimals.VeterinaryJournal,2008,177(1):18-25.479.TianGR,ZhaoGH,DuSZ,etal.FirstreportofEnterocytozoonbieneusifromgiantpandas(Ailuropodamelanoleuca)andredpandas(Ailurusfulgens)inChina.InfectionGeneticsandEvolution,2015,34:32-35.480.TiangtipR,JongwutiwesS.MolecularanalysisofCryptosporidiumspeciesisolatedfromHIV-infectedpatientsinThailand.TropicalMedicineandInternationalHealth.2002,7(4):357-364.481.TilleyM,UptonSJ,FreedPS.AcomparativestudyofthebiologyofCryptosporidiumserpentisandCryptosporidiumparvum(Apicomplexa:Cryptosporidiidae.JournalofZooandWildlifeMedicine,1990,21(4):463-467.482.TorresJ,GraceneaM,GómezMS,etal.TheoccurrenceofCryptosporidiumparvumandC.murisinwildrodentsandinsectivoresinSpain.VeterinaryParasitology,2000,92(4):253-260.483.TraubRJ,MonisPT,RobertsonI,etal.EpidemiologicalandmolecularevidencesupportsthezoonotictransmissionofGiardiaamonghumansanddogslivinginthesamecommunity.Parasitology,2004,128(3):25-262.484.TraubRJ,InpankaewT,ReidSA,etal.TransmissioncyclesofGiardiaduodenalisindogsandhumansinTemplecommunitiesinBangkok-acriticalevaluationofitsprevalenceusingthreediagnostictestsinthefieldintheabsenceofagoldstandard.ActaTropica,2009,111(2):125-132.139 HuazhongAgriculturalUniversityMaster’sDegree2018485.Trotz-WilliamsLA,MartinDS,GateiWetal.GenotypeandsubtypeanalysesofCryptosporidiumisolatesfromdairycalvesandhumansinOntario.ParasitologyResearch,2006,99(4):346-352.486.TroutJM,SantínM,FayerR.GiardiaandCryptosporidiumspeciesandgenotypesincoyotes(Canislatrans).JournalZooWildlifeMedicine.2006,37(2):141-144.487.TumwineJK,KekitiinwaA,NabukeeraN,etal.CryptosporidiumparvuminchildrenwithdiarrheainMulagoHospital,Kampala,Uganda.AmericanJournalofTropicalMedicineandHygiene,2003,68(6):710-715.488.TyzzerE.Coccidiosisingallinaceousbirds.AmericanJournalofHygiene,1929,10(2):269.489.TyzzerEE.Asporozoanfoundinthepepticglandsofthecommonmouse.ExperimentalBiologyandMedicine,1907,5(1):12-13.490.TyzzerEE.AnextracellularCoccidium,Cryptosporidiummuris(Gen.EtSp.Nov.),ofthegastricglandsofthecommonmouse.JournalofMedicalResearch,1910,23(3):487-510.491.TyzzerEE.Cryptosporidiumparvum(sp.nov.),acoccidiumfoundinthesmallintestineofthecommonmouse.ArchProtistenkd,1912,26:394-412.492.TziporiS,AngusKW,CampbellI,etal.Cryptosporidiumevidenceforasingle-speciesgenus.InfectionandImmunity,1980,30(3):884-886.493.TziporiS,RandW,GriffithsJ,etal.Evaluationofananimalmodelsystemforcryptosporidiosis:therapeuticefficacyofparomomycinandhyperimmunebovinecolostrum-immunoglobulin.ClinicalandDiagnosticLaboratoryImmunology,1994,1(4):450-463.494.UehlingerFD,BarkemaHW,DixonBR,etal.GiardiaduodenalisandCryptosporidiumspp.inaveterinarycollegebovineteachingherd.VeterinaryParasitology,2006,142:231-237.495.UngarBLP,YolkenRH,NashTE,etal.Enzyme-linkedimmunosorbentassayforthedetectionofGiardialambliainfecalspecimens.JournalofInfectiousDiseases,1984,149(1):90-97.496.UptonSJ,McAllisterCT,FreedPS,etal.Cryptosporidiumspp.inwildandcaptivereptiles.JournalofWildlifeDiseases,1989,25(1):20-30.497.Upton,Zien.DescriptionofaGiardiavarani-likeflagellatefromawatermonitor,Varanussalvator,fromMalaysia.JournalofParasitology,1997,83(5):970-971.498.VakilNB,SchwartzSM,BuggyBP,etal.BiliarycryptosporidiosisinHIV-infectedpeopleafterthewaterborneoutbreakofcryptosporidiosisinMilwaukee.NewEnglandJournalofMedicine,1996,334(1):19-23.499.ValigurováA,JirkuM,KoudelaB,etal.Cryptosporidia:epicellularparasitesembracedbythehostcellmembrane.InternationalJournalParasitology,2008,38(8-9):913-922.500.VetterlingJM,JervisHR,MerrillTG.Cryptosporidiumwrairisp.n.fromtheguineapigCaviaporcellus,withanemendationofthegenus.JournalProtozool,1971a,18(2):243-247.501.VetterlingJM,TakeuchiA,MaddenPA.UltrastructureofCryptosporidiumwrairifromtheguineapig.JournalofEukaryoticMicrobiology,1971b,18(2):248-260.140 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China502.VolotaoAC,Costa-MacedoLM,HaddadFS,etal.GenotypingofGiardiaduodenalisfromhumanandanimalsamplesfromBrazilusingbeta-giardingene:aphylogeneticanalysis.ActaTropica,2007,102(1):10-19.503.WaldronLS,Cheung-Kwok-SangC,etal.Wildlife-associatedCryptosporidiumfayeriinhuman,Australia.EmergingInfectiousDiseases,2010,16(12):2006-2007.504.WaldronLS,DimeskiB,BeggsPJ,etal.Molecularepidemiology,spatiotemporalanalysis,andecologyofsporadichumancryptosporidiosisinAustralia.AppliedandEnvironmentalMicrobiology,2011,77(21):7757-7765.505.WaldronLS,FerrariBC,PowerML.Glycoprotein60diversityinC.hominisandC.parvumcausinghumancryptosporidiosisinNSWAustralia.ExperimentalParasitology,2009,122(2):124-127.506.WangCJ,LiuHM.InvestigationofcryptosporidiosisofyakcalvesinHualongCounty.ChineseJournalofVeterinaryMedicine,2007,43(5):31(InChinese).507.WangHY,RongJQ,WuGP.InvestigationoncryptosporidiosisinsomesouthernareasofGuizhouprovince.JournalofTropicalMedicine,2006,6(6):717-718(InChinese).508.WangHZ,JiaoBX,TianJH,etal.DetectionofCryptosporidiuminfectionamongHIV/AIDSpatientswithchronicdiarrheainBeijing,HenanandXinjiangProvinces.ChineseJournalofEpidemiology,2011,32(9):927-929(InChinese).509.WangL,PuD.InvestigationofCryptosporidiuminfectionamongAIDSpatientsinKunmingarea.JournalofPracticalMedicalTechniques,2011,18(4):360-361(InChinese).510.WangL,TianXH,HeZA,etal.CryptosporidiuminfectionofchildreninsomeruralareasofChina.JournalofEnvironmentalHygiene.2015,5(4):328-330(InChinese).511.WangL,XiaoL,DuanL,etal.ConcurrentinfectionsofGiardiaduodenalis,Enterocytozoonbieneusi,andClostridiumdifficileinchildrenduringacryptosporidiosisoutbreakinapediatrichospitalinChina.PLoSNeglectedTropicalDiseases,2013a,7(9):e2437.512.WangL,ZhangH,ZhaoX,etal.ZoonoticCryptosporidiumspeciesandEnterocytozoonbieneusigenotypesinHIV-positivepatientsonantiretroviraltherapy.JournalofClinicalMicrobiology,2013b,51(2):557-563.513.WangQQ,GuoJD,CaoZG,etal.InvestigationonhumanCryptosporidiuminfectioninlocalareaofAnhuiProvince.ChineseJournalofSchistosomiasisControl,2015,27(3):263-267(InChinese).514.WangR,WangH,SunY,etal.CharacteristicsofCryptosporidiumtransmissioninpre-weaneddairycattleinHenan,China.JournalofClinicalMicrobiology,2011,49(3):1077-1082.515.WangL,ZhangH,ZhaoX,etal.ZoonoticCryptosporidiumspeciesandEnterocytozoonbieneusigenotypesinHIV-positivepatientsonantiretroviraltherapy.JournalofClinicalMicrobiology,2013b,51(2):557-563.516.WangR,ZhangL,AxénC,etal.CryptosporidiumparvumIIdfamily:clonalpopulationanddispersalfromWesternAsiatoothergeographicalregions.ScientificReports,2014,4(7489):141 HuazhongAgriculturalUniversityMaster’sDegree20184208.517.WangR,ZhangL,NingC,etal.MultilocusphylogeneticanalysisofCryptosporidiumandersoni(Apicomplexa)isolatedfromabactriancamel(Camelusbactrianus)inChina.ParasitologyResearch,2008,102(5):915-920.518.WangR,ZhangX,ZhuH,etal.GeneticcharacterizationsofCryptosporidiumspp.andGiardiaduodenalisinhumansinHenan,China.ExperimentalParasitology,2011,127(1):42-45.519.WangYN,DongTT,WangAX,etal.EpidemiologicalsurveyofCryptosporidiumandEntamoebahistolyticainfectionamongpatientswithachronicconditioninLanzhou.JournalofPathogenBiology,2013,8(10):934-936(InChinese).520.WarrenKS,SwanRA,Morgan-RyanUM,etal.Cryptosporidiummurisinfectioninbilbies(Macrotislagotis).AustralianVeterinaryJournal,2003,81(12):739-741.521.WatanabeY,YangCH,OoiHK.CryptosporidiuminfectioninlivestockandfirstidentificationofCryptosporidiumparvumgenotypeincattlefecesinTaiwan.ParasitologyResearch,2005,97(3):238-241.522.WeberR,BryanRT,BishopHS,etal.ThresholdofdetectionofCryptosporidiumoocystsinhumanstoolspecimens:evidenceforlowsensitivityofcurrentdiagnosticmethods.JournalofClinicalMicrobiology,1991,29(7):1323-1327.523.WeiXR,TaoL,LiJ,etal.InvestigationsurveyofCryptosporidiumwithin2monthscalvesinGuangxiprovince.ChineseJournalofVeterinaryMedicine,2014,50(3):36-37(InChinese).524.WeitzelT,DittrichS,MöhlI,etal.EvaluationofsevencommercialantigendetectiontestsforGiardiaandCryptosporidiuminstoolsamples.ClinicalMicrobiologyandInfection,2006,12(7):656-659.525.WHO.ParasiticZoonoses.ReportofaWHOExpertCommitteewiththeparticipationofFAO.TechnicalReportSeries,WorldHealthOrganization,Geneva.1979,637.526.WHO.FromWorldHealthReport1996(WorldHealthOrganisation,Geneva).JournalCommunityDiseases,1996,28(3):215-219.527.WiderströmM,SchönningC,LiljaM,etal.LargeoutbreakofCryptosporidiumhominisinfectiontransmittedthroughthepublicwatersupply,Sweden.EmergingInfectiousDiseases,2014,20(4):581-589.528.WielingaCM,ThompsonRC.ComparativeevaluationofGiardiaduodenalissequencedata.Parasitology,2007,134(12):1795-1821.529.WielingaPR,deVriesA,vanderGootTH,etal.MolecularepidemiologyofCryptosporidiuminhumansandcattleintheNetherlands.InternationalJournalforParasitology,2008,38(7):809-817.530.WillocksL,CrampinA,MilneL,etal.Alargeoutbreakofcryptosporidiosisassociatedwithapublicwatersupplyfromadeepchalkborehole.OutbreakInvestigationTeam.CommunicableDiseasesPublicHealth,1998,1(4):239-243.142 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,China531.WuL,LiPY,LiuQS,etal.InvestigationofcryptosporidiosisofdairycalvesinHefeicity.ChinaDairyCattle,2006,4:40-41(InChinese).532.XiangYF,LiGQ,XiaoSM,etal.PrevalenceofCryptosporidiuminfectionincowsinGuangdong.ChineseJournalofVeterinaryScienceandTechnology,2004,34(1):32-36(InChinese).533.XiaoL.Molecularepidemiologyofcryptosporidiosis:Anupdate.ExperimentalParasitology,2010,124(1):80-89.534.XiaoL,BernC,LimorJ,etal.Identificationof5typesofCryptosporidiumparasitesinchildreninLima,Peru.JournalofInfectiousDiseases,2001,183(3):492-497.535.XiaoL,BernC,SulaimanIMetal.Molecularepidemiologyofhumancryptosporidiosis.Cryptosporidium:FromMoleculestoDisease(ThompsonRCA,ArmsonAandRyanUM,eds),Elsevier,Amsterdam,2004a,pp.121-146.536.XiaoL,EscalanteL,YangC,etal.PhylogeneticanalysisofCryptosporidiumparasitesbasedonthesmall-subunitrRNAgenelocus.AppliedandEnvironmentalMicrobiology,1999a,65(4):1578-1583.537.XiaoL,FayerRonald,RyanUna,etal.CryptosporidiumTaxonomy:RecentAdvancesandImplicationsforPublicHealth.ClinicalMicrobiologyReviews,2004,17(1):72-97.538.XiaoL,FayerR.MolecularcharacterisationofspeciesandgenotypesofCryptosporidiumandGiardiaandassessmentofzoonotictransmission.InternationalJournalforParasitology,2008,38(11):1239-1255.539.XiaoL,FayerR,RyanU,etal.Cryptosporidiumtaxonomy:recentadvancesandimplicationsforpublichealth.ClinicalMicrobiologyReviews,2004b,17(1):72-97.540.XiaoL,FengY.Zoonoticcryptosporidiosis.FemsImmunologyandMedicalMicrobiology,2008,52(3):309-323.541.XiaoL,LimorJ,MorganUM,etal.SequencedifferencesinthediagnostictargetregionoftheoocystwallproteingeneofCryptosporidiumparasites.AppliedEnvironmentalMicrobiology,2000,66(12):5499-5502.542.XiaoL,MorganUM,LimorJ,etal.GeneticdiversitywithinCryptosporidiumparvumandrelatedCryptosporidiumspecies.AppliedandEnvironmentalMicrobiology,1999b,65(8):3386-3391.543.XiaoL,RyanUM.Molecularepidemilogy.InFayerR,XiaoL(Eds).Cryptosporidiumandcryptosporidiosis,seconded.CRCpress,BocaRaton,FL,2008,119-172.544.XiaoL,RyanUM,GraczykTK,etal.GeneticdiversityofCryptosporidiumspp.incaptivereptiles.AppliedandEnvironmentalMicrobiology,2004b,70(2):891-899.545.XiaoL,SulaimanIM,RyanUM,etal.Hostadaptationandhost-parasiteco-evolutioninCryptosporidium:implicationsfortaxonomyandpublichealth.InternationalJournalforParasitology,32,2002,32(4):1773-1785.546.XiaoL,ZhouL,SantinM,etal.DistributionofCryptosporidiumparvumsubtypesincalvesineasternUnitedStates.ParasitologyResearch,2007,100(4):701-706.143 HuazhongAgriculturalUniversityMaster’sDegree2018547.XieJZ,LiuSQ,YanHetal.EpidemiologicalinvestigationofCryptosporidiuminwaterbuffaloandinfectionstudy.JiangxiJournalofAnimalHusbandryandVeterinaryMedicine,2002,6:14(InChinese).548.XieYP,WeiXR,LiJ,etal.InvestigationofCryptosporidiuminfectioninmilkbuffaloinGuangxiprovince.ProgressinVeterinaryMedicine,2013,34(6):88-91(InChinese).549.XinL,CuiW,LiangRW,etal.InvestigationontheinfectionofCryptosporidiumamongmalignanttumorpatients.JournalofPathogenBiology,2007,2(4):307-308(InChinese).550.XingWL,YangL,LiangSH,etal.EpidemiologicalsurveyofCryptosporidiumindiarrheachildrenfrompartsofWenzhou.JournalofWenzhouMedicalCollege,1999,29(2):117-118(InChinese).551.XuLF,LiCP,ZhangRB,etal.StudyoncharacterisiticofCryptosporidiuminfectionamongstudentsinAnhuiProvince.ChineseJournalofParasiticDiseaseControl,2005,18(4):265-267(InChinese).552.XuLF,WangJ,WangKX,etal.TheinvestigationofCryptosporidiuminfectionamongstudentsinAnhuiProvince.ChineseJournalofSchoolHealth,2003,24(4):347-348(InChinese).553.XuMQ,ZhuSH,HuangY,etal.InvestigationsurveyofCryptosporidiuminfectioninShanghai.ShanghaiJournalofAnimalHusbandryandVeterinaryMedicine,2007,3:36.554.XuWL,LiPY,GuYF,etal.EpidemiologicalsurveyofcowcryptosporidiosisinAnhuiProvince.JournalofAnhuiScienceandTechnologyUniversity,2007,21(6):9-11(InChinese).555.XuYT,ZhangSF,SongJC,etal.ApreliminarysurveyofCryptosporidiuminfectionincattleinYanbianarea.ChineseJournalofVeterinaryMedicine,2002,38(11):24(InChinese).556.YanGB,WangL,WangHX,etal.SurveyofCryptosporidiuminfectioninruralchildreninBeijing,ChangpingDistrict.ChineseJournalofSchoolHealth,2014,35(7):1107-1108(InChinese).557.YanRF,LiPY,ZhengTH.Epidemiologicalinvestigationofcryptosporidiosisinbeefcattle.ChineseJournalofVeterinaryMedicine,2002,38(6):28(InChinese).558.YangJ,SongM,WanQ,etal.EnterocytozoonbieneusigenotypesinchildreninNortheastChinaandassessmentofriskofzoonotictransmission.JournalofClinicalMicrobiology,2014,52(12):4363-4367.559.YangX,LiuZ,YangLR,etal.PreliminaryinvestigationofbovineCryptosporidiuminfestationinHuhhotRegionofInnerMongolia,China.NeimengguNongyeDaxueXuebao,2004,25(1):40-42(InChinese).560.YangY,LinY,LiQ,etal.Widespreadpresenceofhuman-pathogenicEnterocytozoonbieneusigenotypeDinfarmedfoxes(Vulpesvulpes)andraccoondogs(Nyctereutesprocyonoides)inChina:firstidentificationandzoonoticconcern.ParasitologyResearch,2015,114(11):4341-4348.561.YangZ,ZhaoW,ShenY,etal.SubtypingofCryptosporidiumcuniculusandgenotypingof144 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaEnterocytozoonbieneusiinrabbitsintwofarmsinHeilongjiangProvince,China.Parasite,2016,23:52.562.YaoXH,WangH,GouJZ,etal.DetectionofCryptosporidiuminfectionamongAIDSpatientsinGuangdongandYunnan.ChineseJournalofExperimentalandClinicalVirology,2008,22(5):339-341(InChinese).563.YaoYX,ChenHF,LiuX,etal.MolecularepidemiologicalstudiesofcryptosporidiosisindiarrhoeicchildrenofGuangzhousentinelhospital.JournalofTropicalMedicine.2014,14(1):60-63(InChinese).564.YasonJA,RiveraWL.GenotypingofGiardiaduodenalisisolatesamongresidentsofslumareainManila,Philippines.ParasitologyResearch,2007,101(3):681-687.565.YoshidaH,MatsuoM,MiyoshiT,etal.Anoutbreakofcryptosporidiosissuspectedtoberelatedtocontaminatedfood,October2006,SakaiCity,Japan.JapaneseJournalInfectiousDiseases,2007,60(6):405-407.566.ZardiEM,PicardiA,AfeltraA.Treatmentofcryptosporidiosisinimmunocompromisedhosts.Chemotherapy,2005,51(4):193-196.567.ZhangBY,YuH,ZhangLL,etal.PrevalencesurveyonCyclosporacayetanensisandCryptosporidiumspp.indiarrhoeiccasesinYunnanprovince.ChineseJournalofParasitologyandParasiticDisease.2002,20(2):106-108(InChinese).568.ZhangJ,XuJF,ShenXY.InvestigationofCryptosporidiuminfectionincattleinQinghaiProvince.ChineseQinghaiJournalofAnimalandVeterinarySciences,36(1):16-17(InChinese).569.ZhangRP,LiXY,LiH,etal.CryptosporidiuminfectioninEasternChongqing.ParasitosesandInfectiousDiseases,2012,10(2):72-74(InChinese).570.ZhangW,WangR,YangF,etal.DistributionandgeneticcharacterizationsofCryptosporidiumspp.inpre-weaneddairycalvesinNortheasternChina’sHeilongjiangProvince.PLoSOne,2013,8(1):e54857.571.ZhangXP,HeYY,WangZY,etal.InvestigationonCryptosporidiuminfectionsindiarrhoeicpatientsfromthegeneralhospitalsinShanghai.ChinaTropicalMedicine,2016,16(12):1183-1186(InChinese).572.ZhangXX,TanQD,ZhouDH,etal.PrevalenceandmolecularcharacterizationofCryptosporidiumspp.indairycattle,northwestChina.ParasitologyResearch,2015,114(7):2781-2787.573.ZhangYJ,LuoP,GaoR.InvestigationofCryptosporidiuminfectionindiarrhoeicchildreninaPediatricHospitalinChengduCity.JournalofPracticalParasiticDiseases,2001,9(2):72-73(InChinese).574.ZhangW,ZhangX,WangR,etal.GeneticcharacterizationsofGiardiaduodenalisinsheepandgoatsinHeilongjiangProvince,Chinaandpossibilityofzoonotictransmission.PLoSNeglectedTropicalDiseases,2012,6(9):e1826.575.ZhangYS,ZhaoXJ,QuanZH,etal.EpidemiologicalinvestigationonCryptosporidiumin145 HuazhongAgriculturalUniversityMaster’sDegree2018cattle.ChineseJournalofVeterinaryScienceandTechnology,1991,21(5):14-16(InChinese).576.ZhaoXN,ZhangDM,ZhangLX,etal.InvestigationsurveyofCryptosporidiuminfectionininfantsfromHarbin.ChineseJournalofParasiticDiseaseControl,1997,10(3):231(InChinese).577.ZhaoXN,ZhangDM,ZhangLX,etal.EpidemiologicalsurveyofCryptosporidiumindiarrhoeicchildrenfromHeilongjiang.ChineseJournalofZoonoses,1998,14(1):73-74(InChinese).578.ZhengCR,XiaoZL.InvestigationsurveyofcryptosporidiosisininfantsandchildreninNanpingarea.ChineseJournalofParasiticDiseaseControl,1993,6(1):64(InChinese).579.ZhouC,HeG,ZhangL,InvestigationonCryptosporidiuminfectioninyaks.ChineseJournalofZoonoses,2009,25(4):389-390(InChinese).580.ZhouHF,ZhuM,YuanJL,etal.AsurveyofCryptosporidiuminfectionamongdifferentpopulations,inShanghai,LuwanDistrict.ShanghaiJournalofPreventiveMedicine,2005,17(9):430-432(InChinese).581.ZhouL,FayerR,TroutJM,etal.GenotypesofCryptosporidiumspeciesinfectingfur-bearingmammalsdifferfromthoseofspeciesinfectinghumans.AppliedandEnvironmentalMicrobiology.2004,70(12):7574-7577.582.ZhouP,LiE,ZhuN,etal.Roleofinterleukin-6inthecontrolofacuteandchronicGiardialambliainfectionsinmice.InfectionandImmunity,2003a,71(3):1566-1568.583.ZhouL,SinghA,JiangJetal.MolecularsurveillanceofCryptosporidiumspp.inrawwastewaterinMilwaukee:Implicationsforunderstandingoutbreakoccurrenceandtransmissiondynamics.JournalofClinicalMicrobiology,2003b,41(11):5254-5257.584.ZhouQ,LiQ,LiP,etal.EpidemiologyinvestigationofcowcryptosporidiosisatacowdairyfarmonHefei.JournalofHebeiAgriculturalSciences,2008,12(1):122-124.585.ZhuHS,DuXL,YuRB,etal.SerumimmunologyinvestigationofCryptosporidiuminfectionamonginjectiondrugabusers.ChineseJournalofSchistosomiasisControl.2008,20(5):364-366(InChinese).586.ZhuMS,SongMH.TheCryptosporidiuminfectionamongsomeprimaryandmiddleschoolstudentsinShiyanarea.ChineseJournalofSchoolHealth.2007,28(6):549(InChinese).587.ZhuMS,ZhuJ,WangSJ,etal.AsurveyofCryptosporidiuminfectionamonghumansbeinginShiyan,China.JournalofPathogenBiology.2009,4(9):685-686(InChinese).588.ZimmermanSK,NeedhamCA.Comparisonofconventionalstoolconcentrationandpreserved-smearmethodswithMerifluorCryptosporidium/Giardiadirectimmunoflu-orescenceassayandProSpecTGiardiaEZmicroplateassayfordetectionofGiardialamblia.JournalofClinicalMicrobiology,1995,33(7):1942-1943.589.ZintlA,NevilleD,MaguireD,etal.PrevalenceofCryptosporidiumspeciesinintensivelyfarmedpigsinIreland.Parasitology,2007,134(Pt11):1575-1582.590.ZintlA,ProctorAF,ReadC,etal.TheprevalenceofCryptosporidiumspeciesandsubtypesin146 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinahumanfaecalsamplesinIreland.EpidemiologyandInfection,2009,137(2):270-277.591.ZuoYX,ChenFQ,FangLi.FindingofhumanandcalfcryptosporidiosisinYunnanprovinceandexperimentalinfections.ChineseJournalofZoonoses,1990,6(3):34-36(InChinese).147 HuazhongAgriculturalUniversityMaster’sDegree2018ACKNOWLEDGEMENTSThisdissertationisanaccumulationofmyworkinagraduatestudythatwouldnothavebeenpossiblewithouttheassistanceandguidanceofseveralpeopleduringthislongjourney.Firstandforemost,IwouldliketopresentmydeepssenseofgratitudeformySupervisors,Prof.RobinB.GasserandProf.MinHufortheirinvaluablesupportandkindnessinguidingmethroughthiswholejourney,bothacademicandliving,throughoutmymasterprogram.Withoutthem,Iwouldnothavegainedtheinvaluableknowledgeandexperiencevitaltomycareer.Inaddition,IwouldparticularlyliketoexpressmyappreciationtomyAdvisor,DrTaoWang(UM,Australia)forhisinvaluableassistance,auspiciousguidanceanddynamicsupervisiontomakeafoggypathclearer.Also,IwouldliketothankDrXinYang,whokeptonsupportingandencouragingmewhenthingsseemedhopelessandboostingupmymoraleduringthecourseofmylife.Mymasterstudyanddissertationwouldnothavebeenpossiblewithoutallofthemmentionedabove.Thisprojectwouldnothavebeenpossiblewithoutthefinancialsupportfrom“HuazhongAgriculturalUniversityScientific&TechnologicalSelf-innovationFoundation(ProgramNo.2015RC005)”fundedbyHuazhongAgriculturalUniversity.Iamalsogratefultomybelovedlab-matestoCongLiao,TingZhang,CaixianZhou,WendaDi,XingrunYanfortheirhelpinthecollectionoffaecalsamplesforthisstudy,also,Iwouldliketoacknowledgetheownersandtheiranimalsthatparticipatedinmystudy.Withouttheirsupport,Iwouldnothavehadthematerialsforthisproject.ItisworthpleasureformetothanktheotherteachersofthelaboratorytoProf.JunLongZhao,Prof.BangShen,YanqinZhou,RuiFang,andLanHefortheirskillfulguidance,scholarlycriticism,andsuggestionsduringthisproject.Iwanttothankallthewonderfulmembersinthelaboratory,theymademylifeinthelabsowonderfulbynotonlyteachingandtrainingmeonthelaboratorytechniques,butalsobybeingsuchgreatsister-likefriends.Lastbutnotleast,IfindnowordstoexpresswhileacknowledgingthesupportofmyParentsandSistersfortheirlove,understanding,encouragement,andunfailingsupportthroughthegoodtimesaswellasthebad.148 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaThisdissertationwouldnothavebeenpossiblewithoutmanypeoplewhoprovidedtechnicalassistanceinthelaboratoryincludingmybelovedlab-mates,thankallofyou.149 HuazhongAgriculturalUniversityMaster’sDegree2018CURRICULUMVITAEEducationalProfile:2015-2018MasterofPhilosophy(M.Philo)Major:PreventionVeterinaryMedicineField:MolecularParasitologyHuazhongAgriculturalUniversity,Wuhan,P.R.China.(GraduationCeremonyinJune,2018)2011-2015DoctorofVeterinaryMedicineHenanAgriculturalUniversity,Zhengzhou,China.Publications:1.YingyingFan,TaoWang,AnsonV.Koehler,MinHu,RobinB.Gasser.(2017)MolecularinvestigationofCryptosporidiumandGiardiainpre-andpost-weanedcalvesinHubeiprovince,China.Parasites&Vector,10:519.(FirstAuthor,IF=3.08)2.TaoWang,YingyingFan,AnsonV.Koehler,GuangxuMa,TaoLi,MinHu,RobinB.Gasser.(2017).FirstsurveyofCryptosporidium,GiardiaandEnterocytozoonindiarrhoeicchildrenfromWuhan,China.Infection,GeneticsandEvolution,51:127-131.(Co-FirstAuthor,IF=2.89)ExposuretoInternationalForums:The7thInternationalSymposiumonParasitology/the16thBiennialMeetingoftheChineseSocietyofParasitology(CSP);OrganizedbyChinaZoologicalSocietyofChineseSocietyofParasitology(CZS-CSP)onOctober13-16,2017inJiujiangCity,JiangxiProvince,China.ConferencePapers:YingyingFan,TaoWang,MinHu,RobinB.Gasser.GeneticcharacterizationofCryptosporidiumandGiardiainpre-andpost-weanedcalvesinHubeiprovince,China.TheAbstractsofthe7thInternationalSymposiumonParasitology.2017.150 MolecularinvestigationsofCryptosporidiumandGiardiainchildrenandcalvesinHubeiProvince,ChinaHonors:1.“ExcellentGraduatedStudent”inHZAUin2018;2.“ModelofAcademic”inHuanchunFundingin2018;3.“AcademicandInnovationAward”in2018;4.TheThirdPrizeof“AcademicPosters”in2018.5.“NationalScholarship”ofMasterstudentsin2017;6.“MeritMasterStudent”inHZAUin2017;7.TheThirdPrizeof“Excellentexperimentrecordbook”in2015;151
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