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分类号：密级：＃■ｖ＿ｎｍＷＨＵＡＺＨＯＮＧＡＧＲＩＣＵＬＴＵＲＡＬＵＮＩＶＥＲＳＩＴＹＩ士学位论文｜博ＰｈＤＤＩＳＳＥＲＴＡＴＩＯＮ湖北省部分地区山羊重要寄生蠕虫分子流行病学调查ＭＯＬＥＣＵＬＡＲＡＮＤＥＰＩＤＥＭＩＯＬＯＧＩＣＡＬＩＮＶＥＳＴＩＧＡＴＩＯＮＳＯＦＫＥＹＰＡＲＡＳＩＴＩＣＨＥＬＭＩＮＴＨＳＯＦＧＯＡＴＳＩＮＰＡＲＴＳＯＦＨＵＢＥＩＰＲＯＶＩＮＣＥ■…二＾２０１５３０２０１００１２ＴＪＤＥ＾ＴＮＯ．：专业：预防兽医学ＭＡＪＯＲ：ＰＲＥＶＥＮＴＩＶＥＶＥＴＥＲＩＮＡＲＹＭＥＤＩＣＩＮＥ导师：ＳＵＰＥＲＶＩＳＯＲ：ＰＲＯＦＥＳＳＯＲＲＯＢＩＮＢ．ＧＡＳＳＥＲ相武汉ＷＵＨＡＮ，ＣＨＩＮＡ二〇一八年六月｜ HUAZHONGAGRICULTURALUNIVERSITYPhDDISSERTATIONMolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincetudentID:Candidate：XinYangYangXinStudentID：2015302010012Supervisor：ProfessorRobinB.GasserStudentID:2015302010012Guidinggroup:ProfessorHuMinSupervisor:ProfessorRobinB.GasserDoctorWangTaoGuidinggroupProfessorMinHuAssociateProfessorFangRuiMAJOR:PREVENTIVEVETERINARYMEDICINEFIELD:MOLECULARPARASITOLOGYDEGREENAME: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如需保密，解密时间年月曰否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一，指导教师对此进行了审定。与我同工作的同志对本研究所做的任何贡献均已在论文中做了明确的说明，并表示了谢意。研究生签名：时间：年６月人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、并提供目录捡索和阅览服务，可以采用影印缩印或扫描等复制手段保存、汇编学位论文。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：导师签名：：月日签名日期：年６月ｙ日签名日期年＞！＾／ ThisworkwasfundedbythespecializedresearchfundfromSpecialFundforAgro-scientificResearchinthePublicInterest(Grantno.201303037)andHuazhongAgriculturalUniversityScientific&TechnologicalSelf-innovationFoundation(Grantno.2015RC005). MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTableofContentsAbstract.................................................................................................................................i中文摘要............................................................................................................................iiiChapter1-Literaturereview...............................................................................................11.1Introduction............................................................................................................11.2ThedevelopinggoatindustryinChinaandtheimportanceofparasites...............41.3Keyparasitichelminthsofgoatsandtheirbiology...............................................61.4Thepathogenesisofdiseasesandclinicalmanifestationofparasitichelminthsinfections/diseases.......................................................................................................71.4.1Haemonchuscontortus...............................................................................71.4.2Ostertagiaspecies.....................................................................................181.4.3Teladorsagiacircumcincta........................................................................181.4.4Trichostrongyluscolubriformis.................................................................191.4.5Cooperiaspecies.......................................................................................191.4.6Bunostomumtrigonocephalum.................................................................201.4.7Oesophagostomumcolumbianum.............................................................201.4.8Oesophagostomumasperum.....................................................................211.4.9Trichurisspecies.......................................................................................211.4.10Paramphistomumcervi...........................................................................211.4.11Fischoederiuselongatus.........................................................................221.4.12Gastrothylaxcrumenifer.........................................................................221.4.13Homalogasterpaloniae..........................................................................221.4.14Fasciolahepatica....................................................................................231.4.15Fasciolagigantica..................................................................................231.4.16Dicrocoeliumdendriticum......................................................................241.4.17Eurytremapancreaticum........................................................................241.4.18Taeniahydatigena...................................................................................241.4.19Monieziaexpansa...................................................................................251.5Diagnosisofparasitichelminthsinfections/diseasesofgoats.............................261.5.1Diagnosticmethodsbasedonclinicalsymptomsofanimals...................261.5.2Faecaleggcounts......................................................................................271.5.3Larvalculture............................................................................................281.5.4Immunologicalmethods...........................................................................30I HuazhongAgriculturalUniversityPhDDissertation20181.5.5Post-mortemdiagnosis.............................................................................311.5.6Molecularmethods...................................................................................311.5.6.1PCRandsequencing......................................................................321.5.6.2Isothermalamplificationtechniques..............................................341.5.6.3Characterisationofparasitichelminthsusingmitochondrialgenome.......................................................................................................351.6Epidemiologyofparasitichelminthsinfections/diseasesofgoats......................411.7CurrenttreatmentandcontrolstrategiesinChina...............................................431.7.1Vaccine......................................................................................................431.7.2Anthelmintics............................................................................................541.7.3Management.............................................................................................541.8DrugresistanceinparasitichelminthsofgoatsinChina....................................551.8.1Mechanismofdrugresistance..................................................................561.8.1.1Mechanismofbenzimidazoleresistance.......................................561.8.1.2Mechanismofimidazothiazolesresistance...................................561.8.1.3Mechanismofmacrocycliclactonesresistance.............................571.8.1.4Mechanismofamino-acetonitrilederivativesresistance...............581.8.1.5Mechanismofcyclooctadepsipeptidesresistance........................581.8.2Diagnosticmethodsfordetectingdrugresistance....................................581.8.2.1Faecaleggcountreductiontest.....................................................581.8.2.2Controlledtest................................................................................591.8.2.3Egghatchassay..............................................................................591.8.2.4Larvaldevelopmenttest.................................................................591.8.2.5Larvalparalysisandmigrationtest................................................601.8.2.6PCR-basedmethod........................................................................601.8.3CurrentdrugresistanceinChina..............................................................611.9Conclusionsfromtheliteraturereview,andaimsofthethesis...........................64Chapter2-SurveyofparasitichelminthsofgoatsalongtheHanRiverinHubeiProvince,China..................................................................................................................................662.1Introduction..........................................................................................................662.2Materialsandmethods.........................................................................................662.2.1Studyarea,animalsandsamplecollection...............................................662.2.2Conventionalfaecalexamination.............................................................672.2.3Moleculardiagnosis..................................................................................67II MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince2.3Results.................................................................................................................682.4Discussion............................................................................................................72Chapter3-InvestigationofhelminthsofgoatsalongtheDabieMountaininHubeiProvince.............................................................................................................................733.1Introduction..........................................................................................................733.2Materialsandmethods.........................................................................................743.2.1Studyarea.................................................................................................743.2.2Postmortemexamination.........................................................................753.2.3IsolationofgenomicDNAfromparasites................................................763.2.4Molecularidentificationofgastrointestinalnematodes............................763.2.5Molecularidentificationoftrematodes.....................................................763.3Results.................................................................................................................763.4Discussion............................................................................................................77Chapter4-GeneticalcharacterizationofFischoederiuselongatususingmitochondrialgenomesequencing............................................................................................................844.1Introduction..........................................................................................................844.2Materialsandmethods.........................................................................................854.2.1ParasitecollectionandDNAisolation......................................................854.2.2AmplificationandsequencingofF.elongatusmtgenome.......................854.2.3Sequenceanalyses....................................................................................874.2.4Nucleotidevariationanalysis....................................................................874.2.5Phylogeneticanalysis...............................................................................874.3Results.................................................................................................................884.3.1FeaturesofthemtgenomeofF.elongatus...............................................884.3.2Protein-codinggenes................................................................................894.3.3TransferRNAandribosomalRNAgenes.................................................914.3.4Non-codingregions..................................................................................914.3.5NucleotidevariabilitybetweenF.elongatusandP.cervi.........................924.3.6Geneticrelationships................................................................................934.4Discussion............................................................................................................94Chapter5-ComparativeanalysisofthefullmitochondrialgenomeofGastrothylaxcrumenifer..........................................................................................................................955.1Introduction..........................................................................................................955.2Materialsandmethods.........................................................................................96III HuazhongAgriculturalUniversityPhDDissertation20185.2.1ParasitesandDNAisolation.....................................................................965.2.2AmplificationandsequencingofG.crumenifermtgenome....................965.2.3Sequenceanalyses....................................................................................975.2.4Phylogeneticanalysis...............................................................................975.3Results.................................................................................................................995.3.1CharacteristicsoftheG.crumenifermtgenome......................................995.3.2Protein-codinggenes..............................................................................1025.3.3TransferRNAgenes,ribosomalRNAgenesandNon-codingregions...1025.3.4Nucleotidevariability.............................................................................1055.3.5Phylogeneticanalyses.............................................................................1055.4Discussion..........................................................................................................106Chapter6-CharacterizationofthecompletemitochondrialgenomesequenceofHomalogasterpaloniae...................................................................................................1086.1Introduction........................................................................................................1086.2Materialsandmethods.......................................................................................1096.2.1ParasitesandDNAextraction.................................................................1096.2.2PCR-coupledsequencingofH.paloniaemitochondrialgenome..........1096.2.3Assembly,annotationandbioinformaticsanalyses................................1106.2.4Slidingwindowanalysisofnucleotidevariation....................................1106.2.5Phylogeneticanalysis.............................................................................1106.3Results...............................................................................................................1116.3.1Genomecontentandorganization..........................................................1116.3.2AnnotationofH.paloniaemtgenome...................................................1116.3.3Comparativeanalysesofthemtgenomes..............................................1146.3.4Nucleotidevariability.............................................................................1156.3.5Phylogeneticanalyses.............................................................................1186.4Discussion..........................................................................................................119Chapter7-Developmentandevaluationofaloop-mediatedisothermalamplification(LAMP)assayforthedetectionofHaemonchuscontortusingoatfecalsamples..........1207.1Introduction........................................................................................................1207.2Materialsandmethods.......................................................................................1217.2.1ParasitesandgenomicDNAisolation....................................................1217.2.2LAMPprimerdesign..............................................................................1227.2.3LAMPreaction.......................................................................................123IV MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince7.2.4PCRassay...............................................................................................1247.2.5Assessmentofnonspecificamplificationandpotentialinhibition.........1247.2.6Statisticalanalysis...................................................................................1247.3Results...............................................................................................................1257.3.1Speciesidentification..............................................................................1257.3.2SensitivityofLAMPmethod..................................................................1257.3.3SpecificityofLAMPmethod..................................................................1267.3.4Assessmentofnonspecificamplificationandpotentialinhibition.........1277.3.5Clinicalsampledetection........................................................................1287.4Discussion..........................................................................................................128Chapter8-AninvestigationofanthelminticresistanceinwormsofgoatsalongtheHanRiverinHubeiProvince..................................................................................................1328.1Introduction........................................................................................................1328.2Materialsandmethods.......................................................................................1338.2.1Studyarea...............................................................................................1338.2.2Animalgrouping.....................................................................................1338.2.3Faecaleggcountreductiontest(FECRT)...............................................1348.2.4Post-mortemexamination.......................................................................1358.2.5Dewormingassays..................................................................................1358.3Results...............................................................................................................1358.4Discussion..........................................................................................................138Chapter9-DetectionofanthelminticresistanceingastrointestinalnematodesofDabieshanBlackGoatsinLuotian,HubeiProvince.......................................................1419.1Introduction........................................................................................................1419.2Materialsandmethods.......................................................................................1429.2.1Experimentaldesign...............................................................................1429.2.2Faecaleggcountreductiontest...............................................................1439.2.3Statisticalanalysis...................................................................................1439.2.4Dewormingassays..................................................................................1439.2.5Post-mortemexamination.......................................................................1449.3Results...............................................................................................................1449.4Discussion..........................................................................................................145Chapter10-Generaldiscussion......................................................................................148References........................................................................................................................154V HuazhongAgriculturalUniversityPhDDissertation2018Acknowledgements..........................................................................................................211Curriculumvitae..............................................................................................................213VI MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceAbstractTheobjectiveofthisthesiswastostudyaspectsofthemolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinHubeiProvince,China.Thethesisincludesaliteraturereview,eightchaptersdescribingtheoutcomesachievedandageneraldiscussion.Theliteraturereview(Chapter1)revealedsomeknowledgegapsthatneededtobeaddressed.Thesegapswereintheareasofepidemiologicalinvestigationsofgoatparasites,molecularidentificationanddiagnosisofgoatparasites,andanthelminticresistanceinnematodesofgoats.Theaimswereformulatedbasedonthetheseknowledgegapsidentifiedintheliteraturereview,namely(i)tosurveyparasitichelminthsofgoatsinHubeiProvincetounderstandandestimatetheirprevalence,(ii)tocharacterisethemitochondrialgenomesofimportantparasitestoprovideasourceofgeneticmarkersformolecularepidemiological,populationgeneticandphylogeneticstudies,(iii)toestablishaloop-mediatedisothermalamplificationmethodforthespecificdiagnosisofHaemonchuscontortusinfectionand(iv)toundertakesurveysofanthelminticresistanceingastrointestinalnematodesofgoatsasabasisforfutureworkinthisarea.TheepidemiologicalinvestigationsofgoathelminthsinZhanggang(Chapter2)andLuotian(Chapter3)usingmorphologicaltechniquesaswellasPCR-basedDNAsequencingindicatedthattheprevalenceofhelminthswashighthroughouttheyear,particularlyforgastrointestinalnematodes;ParamphistomumandHaemonchuscontortuswereprevalentinalmostallofthefarmsinvestigated.Mitochondrialgenomeswerecharacterisedforsomeoftheparasitesidentified,includingFischoederiuselongatus(Chapter4),Gastrothylaxcrumenifer(Chapter5)andHomalogasterpaloniae(Chapter6)inordertoprovidegeneticmakersforfuturemolecularepidemiological,populationgeneticandphylogeneticstudies.ThesestudiesrevealedthatallthreemitochondrialgenomessharedthesamegenearrangementandindicatedthatF.elongatus,G.crumeniferandH.paloniaehadarelativelycloserelationship(Chapters4to6).Aloop-mediatedisothermalamplification(LAMP)method,targetingthesecondinternaltranscribedspacer(ITS-2)ofH.contortus,wasdevelopedtospecificallydetectH.contortusinfectioningoats.Thismethodprovedtobeasensitive,specificandtime-savingapproachforspecificdetectionanddiagnosis(Chapter7).i HuazhongAgriculturalUniversityPhDDissertation2018Duringaninvestigationofgoathelminths,over-usageofanthelminticswascommon,especiallyforivermectin.Toinvestigateanthelminticresistance,studieswereconductedinZhanggangandLuotianusingfaecaleggcountreductiontesting(FECRT)andpostmortemexamination(Chapters8and9).Theresultsshowedtheexistenceofbenzimidazole,levamisoleandivermectinresistancesinZhanggang(Chapter8)andLuotian(Chapter9).Inconclusion,thisthesishascontributedtotheknowledgeoftheepidemiologyofgoatparasitesandhasmolecularlycharacterised,forthefirsttime,importantparasitictrematodesanddetectedanthelminticresistanceinhelminthsofgoatsinHubeiProvince(Chapter10).ThefindingsofthisthesishaveimplicationsforfuturestudiesandforthecontrolofgoatparasitesinHubeiProvince.Althoughthefocusofthethesiswaspredominantlyonparasitichelminthsofgoats,theoutcomesachievedalsohaveimplicationsforstudyingonthehelminthsofotherlivestockanimalsinChina.Keywords:Goat;helminth;gastrointestinalnematodes;epidemiology;mitochondrialgenome;loop-mediatedisothermalamplification;anthelminticresistanceii MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince中文摘要本论文对湖北省山羊主要寄生蠕虫开展分子流行病学调查方面的研究。论文主要包括文献综述，描述已取得成果的八个章节以及讨论。文献综述发现了一些需要解决的问题（第一章），这些问题主要包括山羊寄生虫分子流行病学调查、山羊寄生虫的分子鉴定与诊断以及山羊线虫的抗药性。本研究的目的是针对这些需要解决的问题开展相关方面的研究，包括：（1）通过对湖北省山羊寄生蠕虫的调查明确其流行情况；（2）对调查中发现的重要前后盘吸虫开展线粒体基因组的研究，为今后的分子流行病学、种群遗传结构以及遗传进化分析的研究提供更多的分子标记；（3）针对优势虫种捻转血矛线虫建立环介导等温扩增检测方法用于捻转血矛线虫病的特异性诊断；（4）同时，对湖北省山羊消化道线虫的抗药性进行调查，为该地区山羊寄生蠕虫病的防控提供参考。结合形态学检测技术及基于PCR的DNA测序方法对张港镇（第二章）及罗田县（第三章）放牧山羊寄生蠕虫感染情况进行调查，结果表明山羊寄生蠕虫，尤其是消化道线虫，全年都维持在较高的感染水平,线虫在2月份、5月份、9月份和12月份会出现感染的小高峰；吸虫感染主要出现在3-8月份，这可能与中间宿主螺蛳的活动相关；绦虫感染没有明显的季节动态。山羊寄生蠕虫主要包括捻转血矛线虫、蛇形毛圆线虫、奥斯特线虫、鞭虫、粗纹食道口线虫、前后盘吸虫、矛形双腔吸虫、肝片吸虫、胰阔盘吸虫、细颈囊尾蚴和扩展莫尼茨绦虫，优势虫种为捻转血矛线虫、前后盘吸虫及细颈囊尾蚴。针对重要前后盘吸虫长菲策吸虫（第四章）、荷包腹带吸虫（第五章）及野牛平腹吸虫（第六章）开展线粒体基因组的测序与分析，为后续的分子流行病学、种群遗传结构以及遗传进化分析的研究提供有用的分子标记。结果表明长菲策吸虫、荷包腹袋吸虫及野牛平腹吸虫线粒体基因组具有相同的基因排序，并且，基于线粒体全基因组的遗传进化分析表明三种前后盘吸虫与鹿前后盘吸虫的亲缘关系最近，这与它们在分类学上的关系相一致（第四至六章）。针对优势虫种捻转血矛线虫核糖体第二内转录间隔（ITS-2）建立了环介导等温扩增检测方法，并将该方法用于生产实践中捻转血矛线虫病的诊断，结果表明该方法具有操作简便、灵敏度高、特异性强及省时的优点（第七章）。在山羊寄生蠕虫调查过程中，发现驱虫药的超剂量使用在山羊驱虫过程中十分普遍，尤其是伊维菌素。为了验证是否存在抗药性，结合粪便虫卵计数减少实验（FECRT）及死后剖检在湖北省张港镇及罗田县放牧山羊中开展抗药性的调查，结果首次证明张港镇（第八章）和罗田县（第九章）山羊消化道线虫存在苯并咪唑、左旋咪唑及伊维菌素抗药性。iii HuazhongAgriculturalUniversityPhDDissertation2018本研究对于湖北地区山羊寄生蠕虫流行病学调查、重要吸虫分子生物学特性及抗药性检测等方面的研究具有重要的意义。本研究的结果为湖北省山羊寄生蠕虫的后续研究及其防控提供参考（第十章）。虽然本研究主要关注山羊优势寄生蠕虫，但本研究的结果对于我国其它家畜寄生蠕虫的系统研究具有借鉴意义。关键词：山羊；蠕虫；消化道线虫；流行病学；线粒体基因组；环介导等温扩增；抗药性iv MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceChapter1-Literaturereview1.1IntroductionThedomesticatedgoat(Capraaegagrushircus)originatesfromthewildgoatofthesouthwestAsiaandeasternEurope(ZederandHesse,2000).Goatsarerelativelycloselyrelatedtosheep;bothbelongtothesubfamilyCaprinae(Zhenetal.,1988).However,thesespeciesdifferintheirkaryotype,with60chromosomesinthegoatand54insheepaswellasintheirmorphologyandbiology(Zhenetal.,1988).Goatsareamongsttheoldestdomesticatedanimals,andhavebeenusedfortheirmilk,meatandskinsforthousandsofyears(Lindaetal.,2004).Therearemorethan300distinctbreedsofgoatworldwide(Hirst,2015).In2013,thereweremorethan1billiongoatsworldwide,accordingtotheFoodandAgricultureOrganization(FAO)oftheUnitedNations(http://www.fao.org/statistics/en/),andwith>140millionpresentinChina,accordingtotheNationalBureauofStatisticsofChina(http://data.stats.gov.cn/index).Inrecentyears,thegoatindustryinChinahasdevelopedandexpandedconsiderablyintermsofnumbersandbreeds.AccordingtotheNationalBureauofStatisticsofChina(http://data.stats.gov.cn/index.htm),thenumberofgoatsincreasedfrom16.13millionheadin1949to148.93millionheadin2015(66years),and,accordingtotheNationalInfrastructureofDomesticAnimalResources(http://www.cdad-is.org.cn/home)(Table1-1),thereareatleast71breedsinthiscountry(Table1-2).Althoughthenumberofgoatshasincreasedgreatly,thegoatindustryinChinaisstilllesswelldevelopedthanindevelopedcountriesintermsofproductionefficiency,managementandhusbandry(Zhaoetal.,2004;Nietal.,2007).Meanwhile,withthedevelopmentofthegoatindustry,thedamageandproductionlossescausedbyinfectiousdiseases,includingthoseinducedbyparasites,arebecomingveryapparent(Liu,2007;LiuandWei,2011;Xiaoetal.,2011;Gu,2016;Yu,2016).Accordingtosomepreviousstatistics,2169differentparasiteshavebeenrecordedin17animalsincludinggoat,sheep,horse,donkey,mule,cattle,waterbuffalo,yak,dzo,camel,swine,dog,cat,rabbit,chicken,duckandgoosehavebeenrecordedinChina(ShenandHuang,2004;Shen,2005),butacloseexaminationofthesestudiessuggeststhattherearesomeinaccuraciesintheserecords.AsforparasitesofgoatsandsheepinChina,ShenandHuang(2004)reportedthattherewere722speciesofparasites,ofwhich1 HuazhongAgriculturalUniversityPhDDissertation201845,250and427wereprotozoa,helminthsandarthropods.Basedonmorerecentstudies,diseasescausedbyparasitesareamongstthethreemostimportantanimalhealthproblemsinbreedingofgoats,leadingtosubstantialeconomiclosses(Li,2006;LiuandWei,2011;Xiaoetal.,2011;Gaoetal.,2016;Gu,2016;Yu,2016).Sincenoeffectivevaccinesareavailable,thecontrolofparasiticnematodeshaspredominantlyreliedontheusageofanthelmintics(Li,2006;Li,2011).Benzimidazoles,imidazothiazoles,macrocycliclactones,amino-acetonitrilederivativesandcyclooctadepsipeptidesarethemaingroupsofanthelminthics(Roeberetal.,2013a).However,theexcessiveanduncontrolleduseofanthelminticshasledtoananthelminticresistanceprobleminChinainrecentyears(LiuandWang,2006;Caietal.,2007;PuandYue,2009;Zhaoetal.,2010),andappearstobeexpanding.Nevertheless,thepreciseextentofsuchresistanceisunknown.KnowingandunderstandingthedistributionandextentofanthelminticresistanceinparasiticnematodesofgoatsinChinawillbecriticaltoimplementingfutureparasitecontrolprogramsinthiscountry.Table1-1:ProductionofgoatsandsheepinChinafrom1949to2015NumberofgoatsintheendofNumberofsheepintheendofYearyear(millionheads)year(millionheads)194916.1326.22195949.7661.881969N/AN/A197980.57102.57198998.13113.511999148.16131.102000149.46130.032001145.62130.632002148.41134.002003149.68143.402004151.96152.312005146.59151.342006137.68146.022007143.37142.282008152.29128.562009150.50134.022010142.04138.842011142.74139.622012141.36143.682013140.35150.202014144.66158.492015148.93162.06Note:DatacomefromtheNationalBureauofStatisticsofChina(http://data.stats.gov.cn/easyquery.htm?cn=C01).N/A:notavailable.2 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable1-2:GermplasmresourcesofgoatsinChinain2016NameSourceareaOriginNameSourceareaOriginTibetanCashmeregoatTibetNativeLingcangLongHairGoatYunnanNativeXinjianggoatXinjiangNativeLonglingGoatYunnanNativeNeimonggolCasheregoatInnerMongoliaNativeMaguanNohornGoatYunnanNativeHexiCashmeregoatGangsuNativeYunlingGoatYunnanNativeLiaoningCashmereLiaoningNativeZhaotongGaotYunnanNativeTaihanggoatShanxi,Hebei,HenanNativeZiwulingblackgoatShanxi,GansuNativeZhongweigoatNingxiaNativeChaidamugoatQinghaiNativeJinjingGreygoatShandongNativeVjimqinWhitegoatInnerMongoliaNativeHuanghuaigoatHenan,AnhuiNativeYaoshanWhitegoatHenanNativeShannanWhitegoatShanxiNativeMachengBlackgoatHubeiNativeMatougoatHunan,HubeiNativeYudongBlackgoatChongqingNativeYichangWightgoatHubeiNativeDazuBlackgoatChongqingNativeChengduMaGoatSichuanNativeYouzhouBlackgoatChongqingNativeJianchangBlackgoatSichuanNativeBeichuanWhitegoatSichuanNativeBanjiaogoatSichuanNativeChuannanBlackgoatSichuanNativeGuizhongWightgoatGuizhouNativeChuanzhongBlackgoatSichuanNativeFuqinggoatFujianNativeMeigugoatSichuanNativeLonglingoatGuangxiNativeGuizhouBlackgoatGuizhouNativeLeizhougoatGuangdongNativeQianbeiBrowngoatGuizhouNativeYangtseRiverDeltaWhitegoatJiangsu,Zhejiang,ShanghaiNativeLonglingYellowgoatYunnanNativeChengdeNohorngoatHebeiNativeLuopingYellowgoatYunnanNativeLuliangBlackGoatShanxiNativeMileRedBonegoatYunnanNativeDaiyungoatFujianNativeNinglangBlackHeadgoatYunnanNativeGanxiGoatJiangxiNativeMingdonggoatFujianNativeGuangfengGoatYunnanNativeLaiwuBlackgoatShandongNativeYimengBlackGoatShandongNativeChaidamuCashmeregoatQinghaiCultivatedLubeiwightgoatShandongNativeWendengDairygoatShandongCultivatedFuniuwightgoatHenanNativeHanshanWhiteCashmeregoatInnerMongoliaCultivatedXiangdongblackgoatHunanNativeGuanzhongDairygoatShanxiCultivatedDuangoatGuangxiNativeLaoshanDariyGoatShandongCultivatedBaiyublackgoatSichuanNativeNanjiangYellowSichuanCultivatedYaandairygoatSichuanNativeShanbeiwhitecashmereShanxiCultivatedGulinMagoatSichuanNativeSannenGoatSwitzerlandIntroducedChuandongWightgoatSichuanNativeAngoragoatAfricaIntroducedFengqingNohorngoatYunnanNativeBoerGoatSouthAfricaIntroducedGuishangoatYunnanNativeNote:DatacomefromNationalInfrastructureofDomesticAnimalResources(http://cdad-is.org.cn).3 HuazhongAgriculturalUniversityPhDDissertation2018Thepurposeofthischapteristo:(i)considerthecurrentexpansionofthegoatindustryinChinaandtheimportanceofparasiticdiseasesofgoats;(ii)reviewthecurrentknowledgeofparasitichelminthsofgoats,theirbiologyandepidemiologyandthediseasesthattheycause;(iii)reviewthemethodsusedforthediagnosisofparasitichelminthinfectionsanddiseasesofgoats;(iv)summarizethecurrentanthelminticsusedforthetreatmentofhelminthsofgoatsaswellasstrategiesusedtocontroltheseparasites;(v)establishwhatisknownaboutdrugresistanceinparasitichelminthsofgoatsinChina;and,(vi)identifythemajorgapsinknowledgeintheseareasinChina,inordertoformulatetheaimsofthisthesis.1.2ThedevelopinggoatindustryinChinaandtheimportanceofparasitesWithincreasinglivingstandardsinChinaandanurgentneedformoremilk,meatandleather,therehasbeenamassiveexpansioninthelivestockandpoultryindustries,includinggoatfarming(Luoetal.,2005;Zhang,2013;Hang,2014;XuandYu,2014;Zhouetal.,2014a;Li,2015;Ling,2015;Tuerxun,2015;Zhang,2015).TheChinesecentralgovernmentisgraduallyincreasingitssupportforthegoatindustryaswellaslocalgovernment(Nietal.,2007;Zhangetal.,2007;HanandLin,2012;Liuetal.,2012;Gu,2016),suchthatmanymorefarmersandinvestorsarenowbreedinggoats.Withthedevelopmentof66years,thenumberofgoatsincreasedfrom16.13millionheadin1949to148.93millionheadin2015(http://data.stats.gov.cn/index.htm),andthenumberofgoatsisstillgrowingyearbyyear.In2016,the71goatbreedsinChinaincluded61localbreeds,7artificiallycultivatedbreedsand3introducedbreeds(http://www.cdad-is.org.cn)(Table1-2).Attheendof2013,Henanprovincehadmostgoats(17.53million),followedbyShandong(16million),InnerMongolia(15.12million)andSichuan(14.61million)(Table1-3).However,indevelopedareasofChinawiththelargesthumanpopulationsoccur,e.g.,ShanghaiandGuangdong,thetotalnumberofgoatswaslessthan0.5million(http://data.stats.gov.cn/easyquery.htm?cn=C01)(Table1-3).Clearly,asustainablesupplyofgoat-derivedfoodsandcommoditiesarehighlydependentonimportfromregionalandremoteprovincesofChinaorfromabroad.4 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable1-3:DistributionofgoatandsheepinChinaintheendof2013ProvinceNumberofgoats(million)Numberofsheep(million)Tianjin0.050.40Beijing0.160.44Shanghai0.250.01Guangdong0.390Zhejiang0.420.68Jilin0.553.41Jiangxi0.560Hainan0.680Ningxia1.034.67Fujian1.160Chongqing1.850Qinghai1.9212.68Guangxi2.020Heilongjiang2.475.71Guizhou2.850.15Shanxi3.874.91Jiangsu3.940.09Gansu3.9914.27Liaoning4.013.34Hebei4.5110.04Hubei4.630Hunan5.130Xinjiang5.2631.37Shaanxi5.261.12Tibet5.639.95Anhui6.040.01Yunnan8.460.84Sichuan14.612.28InnerMongolia15.1237.27Shandong16.005.58Henan17.530.78Note:DatacomefromtheNationalBureauofStatisticsofChinaTheexpandinggoatindustry(breedingandproduction)arefacedwithmajorchallengesincludinginfectiousandnon-infectiousdiseases,ofwhichparasiticdiseasesareofmajorconcern(Li,2006;LiuandWei,2011;Gu,2016).Althoughsymptomaticinfectionscausedbyparasitescancausemajorsufferinganddeathingoats,manyinfectionsaresubclinical,andthushaveapersistentandchroniceconomicimpact(LiuandWei,2011;Xiaoetal.,2011;Gu,2016).AccordingtothesalesfrompharmaceuticalcompaniesinChina,theannualcostassociatedwithparasiticdiseasesofsheepandgoatsinChinaisproposedtobehundredsofmillionsofChinesedollars(RMB).Inaddition,thereisconsiderabletradeamongregionsandprovinces,andmanyfarmerstendtoimportgoatsfromareaswherehighqualitygoatsarebred.Thissituationcanleadtothe5 HuazhongAgriculturalUniversityPhDDissertation2018importationofparasitisedgoats,as,often,noprecautionsaretakentodewormanimalpriortotransportationtoorintroductionontofarms,potentiallyleadingtotheimportationofnewand/oranthelminticresistantpopulationsofparasites(AbbottandTaylor,2012;Wu,2015).1.3KeyparasitichelminthsofgoatsandtheirbiologyParasitichelminthsaremulticellulareukaryoticinvertebratesintube-likeorflattenedshapeexhibitingbilateralsymmetry,andmainlycontainthreelargegroups:nematodes,trematodes,andcestodes(e.g.,Li,2006;Li,2011;Tayloretal.,2016).Generally,thelifecyclefornematodesisdirect,butindirectfortrematodesorcestodeswhichneedtheexistenceofoneortwointermediatehosts(Fig.1-1).Fornematodes,sexuallydimorphicadultsmainlyexistinthegastrointestine,andonefertilizedfemalecanproduceaslargeasseveralthousandofeggsperday(Roeberetal.,2013a).Usually,eggsareexcretedtotheenvironmentwithfaecesanddevelopintoinfectiveL3within7to15daysundersuitablecondition,thenL3canenterintothebodyofanimalsbyoralinfection,anddevelopintoadultstocompletethelifecycleintheanimals(e.g.,Li,2006;Li,2011;Tayloretal.,2016).However,facingunfavorablecondition,theL3ofHaemonchus(BlitzandGibbs,1972a,b),Ostertagia(Lützelschwabetal.,2005)andTeladorsagia(Smith,2007)candevelopintoarrestedstageinthegoatstoshieldthemfromadverseenvironment.Whentheenvironmentisfavorable,arrestedlarvaecanresumedevelopmentintoadultsandcontributetothespringriseofnextyear(BlitzandGibbs,1971;Zhou,2010);fortrematodes,withsomeexception(e.g.,Schistosoma),mostspeciesarehermaphroditic.Generally,eggsdevelopintomiracidiumsintheenvironmentundersuitablecondition,theninvadetheintermediatehostanddevelopintocercariaeinthebodyofintermediatehost.Subsequently,cercariaeescapefromtheintermediatehost(e.g.,snails)andinfectfinalhost(e.g.,Li,2006;Li,2011;Tayloretal.,2016).Forsomespecies,suchasFasciolaspp.,cercariaewilldevelopintometacercariaeandattachonthesurfaceofwaterplants,finalhostsareinfectedbyintakingthewaterplantspollutedbymetacercariae;forcestodes,individualadultwormconsistsofmanysegments,andcanbeaslongasseveralmetersinlength.Usually,onematuresegmentcontainsbothmaleandfemaleorgans,andcanproducelargenumbersofeggs.Similarwiththedevelopmentoftrematodes,thedevelopmentandspreadofcestodesalsoneedtheexistenceofintermediatehosts(e.g.,mites)(Li,2006;Tayloretal.,2016).6 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceThroughmanyyears’surveysofpreviousscholars,parasitespeciesandtheirdistributionsofgoatsarepreliminarilyknowninChina(e.g.,Chen,1985;Wu,2001;ShenandHuang,2004;Shen,2005;Li,2006;Li,2011).Ofthenumerousgeneraandspeciesofparasitichelminthsthatinfectgoats,thecommonhelminthsofgoatsknowntooccurinChinaarelistedinTable1-4(fornematodes),Table1-5(fortrematodes)andTable1-6(forcestodes).Amongthesespecies,thedominantnematodesforgoatsinChinaincludeHaemonchuscontortus,Ostertagiaspp.,Teladorsagiacircumcincta,Trichostrongylusspecies,Cooperiaspecies,Bunostomumtrigonocephalum,OesophagostomumspeciesandTrichurisspecies;themajortrematodesincludeFasciolahepaticaandParamphistomumspecies;andthemajorcestodesincludeTaeniahydatigenaandMonieziaspecies.1.4Thepathogenesisofdiseasesandclinicalmanifestationofparasitichelminthsinfections/diseasesAlthoughparasitichelminthsofgoatsarecommonandofgreatimportancetothebreedingindustry,damagesinproductivepracticearemainlycausedbyoneormorehighlypathogenicspecies(e.g.,Huangetal.,1999;Li,2006;Zhao,2016).Thepathogenesisofdiseasescausedbyhelminthsofgoatsismainlyinfluencedbyparasitespecies,wormburden,immunologicalstate,environmentalproblem,drugresistanceandmanagement(e.g.,Kassaietal.,1999;Li,2006;Li,2011,Abbottetal.,2012;Tayloretal.,2016).Usually,goatsinfectedwithparasitichelminthswillshowaseriesofclinicalmanifestationincludinganemia,emaciated,diarrheaorevendeath(e.g.,Li,2006;Li,2011;Tayloretal.,2016).Andtwogroupsofanimalsaremorelikelytoacquirehighwormburdens:(i)animalswithoutimmunityorwithlowimmunity;(ii)animalsexposedtotheenvironmentwithhighriskstocontactinfectivelarvae(Zajac,2006).KeyhelminthspeciesofveterinaryimportanceofgoatsinChinaandtheirpathogenesisandclinicalmanifestationareasfollow.1.4.1HaemonchuscontortusHaemonchuscontortusisoneofthemostcommonstrongylenematodesingoatsinChina,individualfemalesatpregnantstagecanproducemorethan5000eggsperday,whichincreasedthelarvaeburdensheavilyinashorttimeandthreatedthehealthofanimalsseriously(e.g.,Li,2006;Tayloretal.,2016).ThemainpathogenesisofH.7 HuazhongAgriculturalUniversityPhDDissertation2018contortusiscausedbybloodsuckingofL4andadults,andsymptomsusuallyincludehaemorrhagicanaemia,edema,decreasedfoodintake,weakness,weightlossorevendeath(e.g.,Li,2006;Hu,2008;Sunetal.,2009).Everyadultcanintakeabout0.05mLofhostbloodperdaybyingestionandseepagefromthelesions,thusagoatinfectedwith5000H.contortusadultsmayloseabout250mLperday.Inacutehaemonchosis,anaemiabecomesobviousabouttwoweeksafterinfectionandischaracterisedbysignificantfallinthepackedredcellvolume(PCV).However,anaemiaisnotobviousduringchronicinfection(e.g.,Li,2006;Tayloretal.,2016).DiseasescausedbyH.contortushavebeenwidelyreportedinmanyprovincesinChina,e.g.,Guangxi(Huangetal.,2010),Hebei(Wang,2008),Hunan(Wangetal.,2008),Nanjing(Sunetal.,2016a),Shanxi(Quan,2005;WangandLi,2015),Sichuan(Lietal.,2007),Tianjin(Maetal.,2004)andYunnan(FengandSun,2006),andleadtonumerousdeathsinflockswithouteffectivedewormingandconsiderableeconomiclossestothebreeding(Caoetal.,1995;Chenetal.,2000;Yang,2005).Forsomeregions,theinfectionrateofH.contortusreachedto100%(e.g.,Caoetal.,1995;Chenetal.,2000a;Quan,2005;Yang,2005).Quan(2005)reportedonecaseofH.contortusinfectioninagoatflockofShaanxiProvince,thefarmerimported98lambsfromthemarketandotherfarmersfromApriltoMayin2003withoutdeworming.FromJuly,thelambsstartedtoshowaseriousofsymptomsincludinganaemia,edema,decreasedfoodintake,weaknessandweightloss,and31of98lambsdiedoneafteranother.Bysaturatedsodiumchlorideflotationandpost-mortemexamination,theinfectionrateofH.controtuswas100%,andlargenumberofH.contortusadultswerefoundintheabomasalandanteriorsegmentofsmallintestineofdiedlambs.WeiandDu(2011)reported43.88%(294/671)ofgoatsinthreefarmsinAnhuiProvinceshowedobvioussymptoms,suchasanaemia,edemaandweakness,and92goatsweredied.MassiveofH.contortuswerefoundintheabomasalofgoatsbypost-mortemexamination.8 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceFig.1-1:Lifecyclerepresentingnematode(A),cestode(B)andtrematode(C).Fornematode(useStrongylidasanexample),eggsdevelopintofirst,secondandthirdstagelarvae(L1,L2andL3,respectively)withinafewdaysunderfavorableenvironment,goatsinfectnematodesbyintakingfoodpollutedbyinfectiveL3.Adultsareparasiticstagesintheintestinaltract(adaptedbyDemeler,2005);forcestode(useMonieziaasanexample),eggsareintakenbyintermediatehost,andthendevelopedintometacestodeintheintermediatehost,goatsinfectcestodebyintakinginfectedintermediatehost,thenmetacestodedevelopsintoadultsintheintestinaltract;fortrematode(useFasciolahepaticaasanexample),eggsdevelopintomiracidiumwithinafewdays,thenmiracidiumcaninfectintermediatehostanddevelopintometacercariae,andmetacercariaeeacapesintotheenvironmentfromtheintermediatehostandinfectsgoats.Afterinfectinganimals,metacercariaewillimmigrateanddevelopintoadultsintheintestinaltract(adaptedby“www.county-vets.co.uk”).9 HuazhongAgriculturalUniversityPhDDissertation2018Table1-4:NematodesrecordedingoatsinChina.FamilySpeciesLocationFirstreportActionCommentReferenceTrichostrongylidaeHaemonchuscontortusAbomasum,smallFirstreportedinSichuanprovince,China(1956)BloodsuckingThemostcommonChen(1956)intestineparasiteofgoatH.similisAbomasumFirstreportedinShaanxiprovince,China(1983)BloodsuckingZhang(1983)MecistocirrusdigitatusAbomasum,smallReportedinShanghai,Chinainthereviewofparasites(Wuetal.,1965a);BloodsuckingWuetal.intestinehowever,originalliteratureisnotavailablefromthereview.(1965a)TeladorsagiaAbomasum,smallFirstreportedinGansuprovince,China(1963)MucosaldamageYang(1963)circumcinctaintestineT.davtianiAbomasumFirstreportedinShaanxiprovince,China(1983)MucosaldamageLifecycleunknownZhang(1983)MarshallagiamongolicaAbomasumReportedinZhejiang,Chinainthereviewofparasites(Wuetal.,1965a);MucosaldamageWuetal.however,originalliteratureisnotavailablefromthereview.(1965a)M.tarimanusAbomasumFirstreportedwithaprevalenceof6.45%(2/31)inTibet,China(1994)MucosaldamageLifecycleunknownZhangetal.(1994)M.marshalliAbomasumFirstreportedinShaanxiprovince,China(1983)MucosaldamageLifecycleunknownZhang(1983)M.hsuiAbomasumFirstreportedwithaprevalenceof16.13%(5/31)inTibet,China(1994)MucosaldamageLifecycleunknownZhangetal.(1994)M.lasaensisAbomasumFirstreportedwithaprevalenceof54.83%(17/31)inTibet,China(1994)MucosaldamageLifecycleunknownZhangetal.(1994)M.brevicaudaAbomasumReportedinTibet,Chinainthereviewofparasites(Liaoetal.,2003a);MucosaldamageLifecycleunknownLiaoetal.however,originalliteratureisnotavailablefromthereview.(2003a)OstertagiaostertagiAbomasum,smallFirstreportedinGansuprovince,China(1963)MucosaldamageYang(1963)intestineO.buriaticaAbomasumFirstreportedinShaanxiprovince,China(1983)MucosaldamageLifecycleunknownZhang(1983)O.orloffiAbomasumFirstreportedinShaanxiprovince,China(1983)MucosaldamageLifecycleunknownZhang(1983)O.trifurcataAbomasum,smallFirstreportedinGansuprovince,China(1963)MucosaldamageLifecycleunknownYang(1963)intestineO.skrjabiniAbomasumReportedinShandong,Chinainthereviewofparasites(Wuetal.,1965b);MucosaldamageLifecycleunknownWuetal.however,originalliteratureisnotavailablefromthereview.(1965b)O.wuhingensisAbomasumReportedinShandongandZhejiang,Chinainthereviewofparasites(WuMucosaldamageLifecycleunknownWuetal.etal.,1965a);however,originalliteratureisnotavailablefromthereview.(1965a)O.heterospiculagiaAbomasumFirstreportedinShaanxiprovince,China(1983)MucosaldamageLifecycleunknownZhang(1983)O.dahuricaAbomasum,smallFirstreportedinShaanxiprovince,China(1983)MucosaldamageLifecycleunknownZhang(1983)intestineO.occidentalisAbomasum,smallFirstreportedinGuizhouprovince,China(1995)MucosaldamageLifecycleunknownDuetal.(1995)intestineO.xizangensisAbomasumFirstreportedwithaprevalenceof70.96%(22/31)inTibet,China(1994)MucosaldamageLifecycleunknownZhangetal.(1994)O.sinensisAbomasumFirstreportedwithaprevalenceof22.58%(7/31)inTibet,China(1994)MucosaldamageLifecycleunknownZhangetal.(1994)10 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceO.niangingtangulaensisAbomasumFirstreportedwithaprevalenceof6.45%(2/31)inTibet,China(1994)MucosaldamageLifecycleunknownZhangetal.(1994)TrichostrongylusaxeiAbomasum,smallReportedinShandongandZhejiang,Chinainthereviewofparasites(WuMucosaldamageWuetal.intestineetal.,1965a);however,originalliteratureisnotavailablefromthereview.(1965a)T.colubriformisAbomasum,smallFirstreportedinJiangsu,China(1963)MucosaldamageWang(1963)intestine,pancreasT.probolurusAbomasum,smallFirstreportedinGansuprovince,China(1963)MucosaldamageYang(1963)intestine,pancreasT.tenuisCecumReportedinSichuan,Chinainthereviewofparasites(Liaoetal.,2003a);MucosaldamageLifecycleunknownLiaoetal.however,originalliteratureisnotavailablefromthereview.(2003a)CooperiaoncophoraSmallintestine,Describedinthebookofnematodes(Wu,2001);however,originalMucosaldamageLifecycleunknownWu(2001)pancreasliteratureisnotavailablefromthebookC.zurnabadaSmallintestineDescribedinthebookofnematodes(Wu,2001);however,originalMucosaldamageLifecycleunknownWu(2001)literatureisnotavailablefromthebookC.pectinataSmallintestine,ReportedinYunnan,Chinainthereviewofparasites(Wuetal.,1965b);MucosaldamageWuetal.pancreashowever,originalliteratureisnotavailablefromthereview.(1965b)C.punctataAbomasum,smallFirstreportedinShaanxiprovince,China(1983)MucosaldamageZhang(1983)intestineC.erschoviAbomasum,smallFirstreportedinShaanxiprovince,China(1983)MucosaldamageLifecycleunknownZhang(1983)intestine,pancreasC.laterouniformisSmallintestineFirstreportedinShaanxiprovince,China(1983)MucosaldamageLifecycleunknownZhang(1983)NematodirusabnormalisAbomasum,smallFirstreportedinShaanxiprovince,China(1983)MucosaldamageLifecycleunknownZhang(1983)intestineN.archariAbomasum,smallDescribedinthebookofnematodesofgoat(Wu,2001);however,originalMucosaldamageLifecycleunknownWu(2001)intestineliteratureisnotavailablefromthebookN.davtianiAbomasum,smallDescribedinthebookofnematodesofgoat(Wu,2001);however,originalMucosaldamageLifecycleunknownWu(2001)intestineliteratureisnotavailablefromthebookN.filicollisSmallintestineReportedinShanghai,JiangsuandYunnan,ChinaintworeviewofMucosaldamageLifecycleunknownWuetal.parasites(Wuetal.,1965a,b);however,originalliteratureisnotavailable(1965a,b)frombothofthereviews.N.oiratianusAbomasum,smallReportedinKunming,Chinainthereviewofparasites(Wuetal.,1965b);MucosaldamageLifecycleunknownWuetal.intestinehowever,originalliteratureisnotavailablefromthereview.(1965b)N.spathigerSmallintestineFirstreportedinShaanxiprovince,China(1983)MucosaldamageZhang(1983)NematodirellaSmallintestineFirstreportedwithaprevalenceof9.68%(3/31)inTibet,China(1994)MucosaldamageLifecycleunknownZhangetal.longispiculata(1994)N.gazelliSmallintestineFirstreportedwithaprevalenceof3.23%(1/31)inTibet,China(1994)MucosaldamageLifecycleunknownZhangetal.(1994)HyostrongylusrubidusStomachFirstreportedwithaprevalenceof5%(1/20)inSichuanprovince,ChinaMucosaldamageMorphologicalJiangetal.(1986)descriptionsavailable(1986a)11 HuazhongAgriculturalUniversityPhDDissertation2018AncylostomatidaeBunostomumSmallintestineFirstreportedinGansuandJiangsuprovince,China(1963)BloodsuckingWang(1963)trigonocephalumYang(1963)B.phlebotomumSmallintestineFirstreportedwithaprevalenceof1.38%(10/727)inGuizhou,ChinaBloodsuckingDuetal.(1995)(1995)StrongyloididaeStrongyloidespapillosusSmallintestineFirstreportedinGansuprovince,China(1963)MucosaldamageYang(1963)ChabertiidaeOesophagostomumLargeintestine,FirstreportedinSichuanprovince,China(1956)Mucosaldamage,LifecycleunknownChen(1956)columbianummainlyincolonnodulesO.venulosumLargeintestine,Firstreportedwithaprevalenceof18.06%(131/727)inGuizhou,ChinaMucosaldamage,LifecycleunknownDuetal.(1995)mainlyincolon(1995)nodulesO.asperumLargeintestine,FirstreportedinJiangsu,China(1963)Mucosaldamage,Wang(1963)mainlyincolonnodulesO.radiatumLargeintestine,ReportedinShandongandJiangsu,Chinainthereviewofparasites(WuMucosaldamage,LifecycleunknownWuetal.mainlyincolonetal.,1965a);however,originalliteratureisnotavailablefromthereview.nodules(1965a)O.kansuensisLargeintestine,FirstreportedinHebei,China(1984)Mucosaldamage,YangandmainlyincolonnodulesWang(1984)O.hubeinsisLargeintestineFirstreportedwithaprevalenceof19.2%(28/144)inSichuan,ChinaMucosaldamage,LifecycleunknownHeandLuo(1995)nodules(1993)ChabertiaovinaCecum,colonFirstreportedinHebei,China(1984)MucosaldamageYangandWang(1984)C.erschowiCecum,colonFirstreportedwithaprevalenceof0.96%(7/727)inGuizhou,ChinaMucosaldamageDuetal.(1995)(1995)TrichuridaeTrichurisglobulosaLargeintestine,ReportedinJiangsuandZhejiang,Chinainthereviewofparasites(WuetHemorrhagicenteritis,Wuetal.mainlyincecumal.,1965a);however,originalliteratureisnotavailablefromthereview.edema,ulcers(1965a)T.ovisLargeintestine,FirstreportedinJiangsu,China(1963)Hemorrhagicenteritis,Wang(1963)mainlyincecumedema,ulcersT.skrjabiniLargeintestine,FirstreportedinShaanxiprovince,China(1983)Hemorrhagicenteritis,Zhang(1983)mainlyincecumedema,ulcersDictyocaulidaeDictyocaulusfilariaTrachea,bronchusFirstreportedinSichuanprovince,China(1956)BronchitispneumoniaChen(1956)D.viviparusTrachea,bronchusFirstreportedinGansuprovince,China(1963)BronchitispneumoniaYang(1963)D.eckertiTrachea,bronchusReportedinChongqingandSichuan,Chinainthereviewofparasites(WuBronchitispneumoniaLifecycleunknownWuetal.etal.,1965b);however,originalliteratureisnotavailablefromthereview.(1965b)GongylonematidaeGongylonemapulchrumEsophagusReportedinShandong,Chinainthereviewofparasites(Wuetal.,1965a);MucosaldamageWuetal.however,originalliteratureisnotavailablefromthereview.(1965a)ProtostrongylinaeProtostrongylusTrachea,bronchusFirstreportedinShaanxiprovince,China(1983)BronchitispneumoniaZhang(1983)hobmaieriP.raillietiTrachea,bronchusFirstreportedwithaprevalenceof4.76%(2/42)inSichuan,China(1995)BronchitispneumoniaLifecycleunknownYang(1997)12 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceP.rufescensTrachea,bronchusReportedinSichuan,Chinainthereviewofparasites(Liaoetal.,2003a);BronchitispneumoniaLiaoetal.however,originalliteratureisnotavailablefromthereview.(2003a)SpiculocauluskwongiTrachea,bronchusFirstreportedwithaprevalenceof27.8%(17/62)inGansuprovince,BronchitispneumoniaLifecycleunknownHui(2000)China(1986)Note:Partof“Action”wasreferencedtoWu(2001)andLi(2011).13 HuazhongAgriculturalUniversityPhDDissertation2018Table1-5:TrematodesrecordedingoatsinChina.FamilySpeciesLocationFirstreportActionCommentReferenceFasciolidaeFasciolahepaticaBileductFirstreportedinJiangsu(1963)andGansu(1963),ChinaDestructionofWang(1963)tissue,fibrosisYang(1963)F.giganticaBileductFirstreportedinJiangsu(1963)andGansu(1963),ChinaDestructionofWang(1963)tissue,fibrosisYang(1963)SchistosomatidaeSchistosomajaponicumMesentericDescribedinthebookoftrematode(Chen,1985);however,originalMechanicalChen(1985)vein,hepaticliteratureisnotavailablefromthebookinjury,portalveinimmunopathologicaleffectsOrientobilharziaMesentericReportedinKunming,Chinainthereviewofparasites(Wuetal.,1965a);MechanicalRecognizedasaWuetal.(1965b)turkestanicumtvein,hepatichowever,originalliteratureisnotavailablefromthereview.injury,memberofWangetal(2011b)portalveinimmunopathologSchistosomabasedonicaleffectsmitochondrialgenome(Wangetal.,2011b).DicrocoeliidaeDicrocoeliumLiver,bileFirstreportedinGansuprovince,China(1963)DestructionofYang(1963)dendriticumduct,galltissue,fibrosisbladderD.chinensisLiver,bileFirstreportedinGansuprovince,China(1963)DestructionofYang(1963)duct,galltissue,fibrosisbladderEurytremacoelomaticuPancreaticFirstreportedinShaanxiprovince,China(1983)DestructionofZhang(1983)duct,seldomtissue,fibrosisinbileductandduodenumE.pancreaticumPancreaticFirstreportedinGansu(1963)andJiangsu(1963)province,ChinaDestructionofWang(1963)duct,seldomtissue,fibrosisYang(1963)inbileductandduodenumE.cladorchisPancreasFirstreportedwithaprevalenceof11.55%(84/727)inGuizhou,ChinaDestructionofLifecycleunknownDuetal.(1995)(1995)tissue,fibrosisE.fukienensisPancreas,Firstreportedwithaprevalenceof0.14%(1/727)inGuizhou,ChinaDestructionofLifecycleunknownDuetal.(1995)pancreaticduct(1995)tissue,fibrosisE.sphaeriorchisPancreaticReportedinSichuan,Chinainthereviewofparasites(Liaoetal.,2003b);DestructionofLifecycleunknownLiaoetal.(2003b)ducthowever,originalliteratureisnotavailablefromthereview.tissue,fibrosisPlatynosomumLiver,bileFirstreportedwithaprevalenceof8%(58/727)inGuizhou,China(1995)DestructionofLifecycleunknownDuetal.(1995)capranumducttissue,fibrosisParamphistomidaeParamphistomumcerviRumenFirstreportedinSichuanprovince,China(1956)MucosaldamageChen(1956)P.gotoiRumenFirstreportedwithaprevalenceof3.58%(26/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)P.ichikawaiRumenFirstreportedwithaprevalenceof0.56%(4/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)P.microbothriumRumenFirstreportedwithaprevalenceof0.41%(3/727)inGuizhou,ChinaMucosaldamageDuetal.(1995)(1995)P.bothriophoronRumenFirstreportedwithaprevalenceof0.14%(1/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)14 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceP.procaprumRumenFirstreportedwithaprevalenceof0.14%(1/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)P.gracileRumenFirstreportedinGuangxi,China(1989)MucosaldamageLifecycleunknownYangetal(1989)CalicophoronRumenFirstreportedwithaprevalenceof0.97%(7/727)inGuizhou,ChinaMucosaldamageDuetal.(1995)calicophorum(1995)C.ijimaiRumenFirstreportedwithaprevalenceof0.84%(6/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)C.fusumRumenFirstreportedinGuangxi,China(1989)MucosaldamageLifecycleunknownYangetal(1989)C.ovillumRumenFirstreportedinGuangxi,China(1989)MucosaldamageLifecycleunknownYangetal(1989)C.skrjabiniRumenFirstreportedinGuangxi,China(1989)MucosaldamageLifecycleunknownYangetal(1989)C.wuchengensisRumenReportedinGuizhouandYunnan,Chinainthereviewofparasites(LiaoMucosaldamageLifecycleunknownLiaoetal.(2003b)etal.,2003b);however,originalliteratureisnotavailablefromthereview.CeylonocotyleRumenFirstreportedwithaprevalenceof0.69%(5/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)streptocoelium(1995)C.scoliocoeliumRumenFirstreportedwithaprevalenceof3.58%(26/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)C.cheiniRumenFirstreportedwithaprevalenceof3.99%(29/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)C.dicranoceliumRumenFirstreportedwithaprevalenceof4.68%(34/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)C.sinuocoeliumRumenFirstreportedwithaprevalenceof1.93%(14/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)C.longicoeliumRumenReportedinSichuan,Chongqing,GuizhouandYunnan,ChinaintheMucosaldamageLifecycleunknownLiaoetal.(2003b)reviewofparasites(Liaoetal.,2003b);however,originalliteratureisnotavailablefromthereview.C.parastreptocoeliumRumenReportedinSichuan,Chongqing,GuizhouandYunnan,ChinaintheMucosaldamageLifecycleunknownLiaoetal.(2003b)reviewofparasites(Liaoetal.,2003b);however,originalliteratureisnotavailablefromthereview.CotylophoronRumenFirstreportedwithaprevalenceof5.64%(41/727)inGuizhou,ChinaMucosaldamageDuetal.(1995)cotylophorum(1995)C.indicumRumenFirstreportedwithaprevalenceof2.61%(19/727)inGuizhou,ChinaMucosaldamageDuetal.(1995)(1995)C.shangkiangensisRumenFirstreportedwithaprevalenceof1.1%(8/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)C.sinuointestinumRumenFirstreportedwithaprevalenceof0.41%(3/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)C.kwantungensisRumenFirstreportedwithaprevalenceof1.93%(14/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)GigantocotyleAbomasumFirstreportedinZhejiangprovince,China(1988)MucosaldamageLifecycleunknownZhangetal.(1988)nanhuenseMorphologicaldescriptionsavailableG.formosanumAbomasumFirstreportedinZhejiangprovince,China(1988)MucosaldamageLifecycleunknownZhangetal.(1988)MorphologicaldescriptionsavailableMacropharvnxAbomasumFirstreportedinHenanprovince,China(2011)MucosaldamageLifecycleunknownNingetal.(2011)chinensis15 HuazhongAgriculturalUniversityPhDDissertation2018GastrodiscidaeHomalogasterpaloniaeCecum,colonMucosaldamageWuetal.(1965b)ReportedinYunnan,ChongqingandSichuan,Chinainthereviewofparasites(Wuetal.,1965b);but,originalliteratureisnotavailablefromthereview.GastrothylacidaeGastrothylaxcrumeniferRumenFirstreportedinJiangsu,China(1963)MucosaldamageWang(1963)G.glandiformisRumenFirstreportedwithaprevalenceof0.69%(5/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)G.globoformisRumenReportedinGuangxi,Chinainthereviewofparasites(ZhangandHuang,MucosaldamageLifecycleunknownZhangandHuang(2001)2001);however,originalliteratureisnotavailablefromthereview.CarmyeriussynethesRumenReportedinAnhui,Chinainthereviewofparasites(Lu,2004);however,MucosaldamageLifecycleunknownLu(2004)originalliteratureisnotavailablefromthereview.FischoederiuselongatusRumenFirstreportedwithaprevalenceof4.68%(34/727)inGuizhou,ChinaMucosaldamageLifecycleunknownDuetal.(1995)(1995)F.ovatusRumenFirstreportedinGuangxi,China(1989)MucosaldamageLifecycleunknownYangetal(1989)F.siamensisRumenFirstreportedinGuangxi,China(1989)MucosaldamageLifecycleunknownYangetal(1989)F.japonicusRumenReviewSichuanChongqingGuizhouYunnanMucosaldamageLifecycleunknownLiaoetal.(2003b)F.fischoederiRumenReviewSichuanChongqingGuizhouMucosaldamageLifecycleunknownLiaoetal.(2003b)F.compressusRumenReportedinAnhui,Chinainthereviewofparasites(Lu,2004);however,MucosaldamageLifecycleunknownLu(2004)originalliteratureisnotavailablefromthereview.F.ceylonensisRumenFirstreportedwithaprevalenceof3.23%(1/31)inChongqing,ChinaMucosaldamageLifecycleunknownWuetal.(2007)(1995)NotocotylidaeOgmocotylesikaeSmallintestineFirstreportedinGansuprovince,China(1963);HemorrhagicYang(1963)MorphologicaldescriptionavailableinWei(1965).enteritisWei(1965)O.indicaSmallintestineFirstreportedinGansuprovince,China(1965);HemorrhagicWei(1965)MorphologicaldescriptionavailableinWei(1965).enteritisO.pygargiSmallintestineReviewwithaprevalenceof11.11%(1/9)inKunming,China(Wuetal.,HemorrhagicWuetal(1965b)1965b);however,originalliteratureisnotavailablefromthereview.enteritisBrachylaemidaeSkrjabinotremaOrloffSmallintestineReportedinSichuanandTibet,Chinainthereviewofparasites(LiaoetHemorrhagicLiaoetal.(2003b)al.,2003b);however,originalliteratureisnotavailablefromthereview.enteritisNote:Partof“Action”wasreferencedtoChen(1985)andLi(2011).16 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable1-6:CestodesrecordedingoatsinChina.FamilySpeciesLocationFirstreportActionCommentReferenceAnoplocephalidaeMonieziaexpansaSmallintestineReportedinShandong,ShanghaiandZhejiang,ChinainthereviewofMucosaldamage,Wuetal.(1965a)parasites(Wuetal.,1965a);however,originalliteratureisnotavailablemechanicaldamagefromthereview.M.benedeniSmallintestineReportedinYunnanandGuizhou,Chinainthereviewofparasites(WuetMucosaldamage,al.,1965b);however,originalliteratureisnotavailablefromthereview.mechanicaldamageWuetal.(1965b)AvitellinaSmallintestineFirstreportedinShaanxiprovince,China(1983)Mucosaldamage,Zhang(1983)centripunctatamechanicaldamageHelictometragiardiaSmallintestineFirstreportedinHebeiprovince,China(1984)Mucosaldamage,YangandWang(1984)mechanicaldamageTaeniidaeEchinococcusLiver,lungFirstreportedinSichuanprovince,China(1956)MechanicalNoeffectiveChen(1956)granulosusdamage,methodsfornutrientdiagnosisandconsumptiontreatment.TaeniahydatigenaLiver,FirstreportedinSichuanprovince,China(1956)MechanicalNoeffectiveChen(1956)mesenterydamage,methodsfornutrientdiagnosisandconsumptiontreatment.CoenuruscerebralisBrainFirstreportedinSichuanprovince,China(1956)MechanicalChen(1956)damage,nutrientconsumptionNote:Partof“Action”wasreferencedtoLi(2011).17 HuazhongAgriculturalUniversityPhDDissertation20181.4.2OstertagiaspeciesOstertagiaspeciesarecommonspeciesinhabitingintheabomasumandsmallintestineofsmallruminants.AccordingtotheChinaAnimalScientificDatabase,asmanyas15OstertagiaspecieshavebeenreportedingoatsinChina,namelyOstertagiaostertagi,Ostertagiasinensis,Ostertagianianqingtangulaensis,Ostertagiaxizangensis,Ostertagiaskrjabini,Ostertagiaheterospiculagia,Ostertagianingshaanensis,Ostertagiagansuensis,Ostertagiatrifurcate,Ostertagialanceata,Ostertagiawuhingensis,Ostertagiaburiatica,Ostertagiaorloffi,OstertagiadahuricaandGrosspiculagiaoccidentalis(Wu,2001).Amongthesespecies,Ostertagiatrifurcateisacommonspecies(Li,2006;Li,2011).ThelifecycleofOstertagiaissimilarwithHaemonchuscontortus,usually,eggscanbefoundinthefaecalsamplesofanimalsinfectedwithOstertagiafor15-17days,andfewwormscanbefoundintheanimals60daysafterinfections(Li,2006).Ostertagiaspeciescantoleratecoldweather,themainpathogeniceffectiscausedbylarvalsthatcanenterintoanydepthofmucousmembrane,andcausenodulesandinflammatoryreaction.Underheavyburden,animalscanshowaseriousofsymptomsincludingemaciated,anaemia,weakandintermittentconstipation,orevendeath(Li,2011;Tayloretal.,2016).OstertagiaspeciesinfectioniscommoningoatsinChina(e.g.,Ma,1982;Lietal.,2006;Wangetal.,2014).In2016,Ba(2016)reportedtheinfectionrateofOstertagiaspeciesreachedto83%bypost-mortemexaminationinsheepandgoatsofGongheCounty,Qinghai.1.4.3TeladorsagiacircumcinctaTeladorsagiacircumcinctaisaparasiticnematodeinhabitingintheabomasumofgoats.ThelifecycleofT.circumcinctaisalsosimilarwithH.contortus,however,femalesofT.circumcinctacanonlyproduceabout100-200eggsperday,whichissignificantlylessthanthatofH.contortus(Tayloretal.,2016).Thelarvalcandevelopintoadult15dayspost-infection,andmostadultwormswilldisappearwithin60days(Li,2011).Thebrownishwormsdonotfeedonblood,andthemainpathogeniceffectsarecausedbyitslarvalstages(Tayloretal.,2016).Larvaldevelopmentingastricglandscausenoduleformationinabomasalmucosaandsignificantdamagetotheparietalcells,thenhypochloricacidproductionwilldecrease(Levine,1968).Consequently,disorderedpHleadtotheincreasingofplasmapepsinogenlevelsanddecreasingofproteindigestion18 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince(Tayloretal.,2016).Ingeneral,moderateorsubclinicalinfectionsleadtodiarrhoea,growthinhibition,bodyweightlossandreducedwoolproduction(Zajac,2006).AlthoughT.circumcinctaisacommonspeciesinChina,damagecausedbythisspeciesislimitedingoats(Li,2011).1.4.4TrichostrongyluscolubriformisTrichostrongylusspeciesisanimportantgroupofgastrointestinalhelminthsofgoats,andalsozoonotic.InChina,TrichostrongyluscolubriformisisthecommonestTrichostrongylusspeciesingoatsandsheep,butseldomlyfoundinpigsandhumans(Li,2006;Li,2011).Usually,Trichostrongyluscolubriformisissmallercomparedwithotherfamilies(lessthan10mminlength)(Li,2011).ThelifecycleofTrichostrongyluscolubriformisissimilarwithH.contortus,larvalscanenterintotheglandsofmucosalepithelialcellsandmembranaepropriatoformpassageway,andthencausehemorrhage,edemaandhypoproteinemiabyflowingofserumalbuminintomesocaval(Li,2011).Goatsinfectedwiththisspeciescanshowclinicalsignsincludingbodyweightloss,diarrheaanddeathunderheavyburden.Meanwhile,highdeathcanhappenundertheinfectionoflargenumberofwormswithinashortperiods.Underlightburden,thesignsarenotobvious.Thisparasitesurvivesonthesurfaceofabomasumandsmallintestine,acuteinfectioncancausemucosalswellingofsmallintestine,andchronicinfectionleadtomarasmus,anemia,hepaticsteatosis,mucosalhyperplasia,inflammationandulcer(Li,2011).Theliberationofyoungadultsisrelatedwithextensivedamagetoduodenalmucosa,andcauseshaemorrhage,proteinloss,hypoalbuminaemiaandhypoproteinaemia(Li,2006;Li,2011).In1987,morethantenthousandT.colubriformiswererecoveredfromadeadgoatinaflockinSheyang,JiangsuProvince,andoutbreakofT.colubriformiscausedseriousdiarrheaandleadedto45deathsamongthewholeflock(Xuetal,1990).1.4.5CooperiaspeciesAlthoughCooperiaspeciesarelargelyconsideredasmildpathogens,thereareafewspeciesthatareofgreaterveterinaryimportance,namelyC.punctataandC.pectinata(Shen,2005).Thesespeciesareparasitesofsmallruminantsandmostspeciesofthisgenuspreferwarmerclimates.Ingeneral,Cooperiaspeciesarespecies-specific,andthethirdstagelarvacandevelopintoadultswithin15dayspostinfection(Li,2006).The19 HuazhongAgriculturalUniversityPhDDissertation2018mainpathogenesisofCooperiaspeciesiscausedbyintestinalmucosalinjurycausedbytheparasitismofworms,anteriorsmallintestinalmucosawillshowanumberofblutenechloaide,andposteriorpartswillshowmildcatarrhalexudateatnecropsy(Li,2006).Animalsinfectedwiththisspecieswillshowdiarrhea,anorexiaandprogressiveemaciation.Cooperiaspecieshavebeenreportedtobeprevalent(withtheinfectionraterangesfrom50%to80%)ingoatsinXinjiang(Suwaetal.,2012),andShanxi(Wangetal.,2011a).1.4.6BunostomumtrigonocephalumBunostomumtrigonocephalumisworldwide,andusuallyendemic(Lane,1917;Wuetal.,1965).Goatsorothersmallruminentsareinfectedbyskincontactwithlarvaorintakingbypollutedfood,larvaemigratealongtracheaandbronchiole,thentotheintestineanddevelopintoadultworms.Younganimalsareeasytobeinfected,larvaecaninvadeanddestroytheskinofhosts,thenleadtosecondaryinfectionofbacteria(Li,2006;Li,2011;Tayloretal.,2016).Whenmigratingtolung,larvaecancausehemorrhageandeffectbreathingfunction,andtheycanalsocausebronchitisorbronchiectasiaunderheavyburden.Adultsintheintestinesdestroytheintestinalmucosaofhostsandcausecapillaryhemorrhage.Meanwhile,adultsfeedonbloodfromhosts,andcanleadtoanaemiaandemaciated(Li,2011;Tayloretal.,2016).Wangetal(2004)reportedover50%ofgoatsshowedemaciated,anaemia,diarrhea,abortionorevendeathinaflockinShanxiprovince,andprovedtobecausedbytheinfectionofB.trigonocephalumbypost-mortemexamination.In2014,therewasareportaboutB.trigonocephaluminfectioninaflockinHenanprovince,andabout20%ofthegoatsshowedanaemia,emaciatedanddiarrhea,and5%ofgoatsweredeadafterafewdays(YuandXu,2014).1.4.7OesophagostomumcolumbianumOesophagostomumcolumbianumisoneofthecommonestspeciesingoats.Thelarvaecanformnodulesinthesurfaceoflargeintestine,whichisalsocalledthe“noduleworm”(Linetal.,2010;Li,2014).Usually,larvaissusceptibletotemperature,andwilldiesoonwhenthetemperatureis>35°C.Themainpathogenesisiscausedbylarvae,larvaecanenterintotheintestinalwallandleadtoinflammation,andthentoformnodulesduetoimmunereactionofhost,thedisruptionofnodulesinserosacancauseperitonitis.Adultsinhabitinthelargeintestines,cansecretetoxinandcauseinflammation.20 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceO.columbianuminfectionwillshowpersistentdiarrhea,diarrheaalwayshappensixdaysafterinfection.Inchronicinfection,constipationanddiarrheaoccuralternately,andanimalsmaydiebecauseofbodyfluidimbalance(Li,2011).Zongetal(1995)investigatedtheO.columbianuminfectionofsheepinInnerMongolia,theresultindicatedO.columbianumwasfoundinallthe17investigatedsheepwiththewormburdenrangedfrom47to455.1.4.8OesophagostomumasperumOesophagostomumasperumisalsooneofthecommonestspeciesingoats.ThepathogenesisandclinicalsignsofOesophagostomumasperumissimiarwiththatofO.columbianum(Li,2011).In2009,YangreportedthegastrointestinalparasitesofgoatsataslaughterhouseinKunming,andfound27of31goatsinfectedwithO.asperum(Yangetal.,2009).1.4.9TrichurisspeciesTrichurisspeciesinhabitinthelargeintestine,especiallyforcecum(Li,2011;Tayloretal.,2016).Unlikeothernematodes,thebodyofTrichurisspeciesconsistsoffilamentousheadandintumescenttail.TrichrisovisandTrichrisglobulosaarecommonspeciesingoatsinChina(Li,2011).Adultwormscanstickintotheintestinalwallusinglongandthinhead,causingmechanicaldamageandsecretetoxins,then,cecumandcoloncanshowseriouscatarrhalinflammationandhemorrhagicinflammation,andyounganimalsaresusceptibletothesespecies.TrichurisspeciesarealsocommonlyreportedingoatsinChina(e.g.,Xiaoetal.,2013;Chenetal.,2015;Huang,2015).Chenetal(2015)reportedtheinfectionofTrichurisspeciesreached50%intheinvestigatedgoatfarminChongqing.1.4.10ParamphistomumcerviParamphistomumcervi,thetypespeciesofgenusParamphistomum,isaparasiticflatwormbelongingtotheclasstrematoda.Itisatinyflukemostlyparasitinginlivestock,aswellassomewildmammals(Li,2011).Uniquely,unlikemostparasites,theadultwormsarerelativelyharmless,butitisthedevelopingjuvenilesthatcauseseriousdiseasecalledparamphistomiasis,themigrationofthedevelopingjuvenilescancauseacuteandseriousclinicalsymptomsofgastrointestinalmucosaandinternalorgans.Thelifecycleof21 HuazhongAgriculturalUniversityPhDDissertation2018P.cerviisindirect,andneedtheexistenceoffreshsnailsservedasintermediatehost.Itssymptomsinsmallruminantsincludeprofusediarrhoea,anaemia,lethargy,andoftenresultindeathunderheavyburdenifuntreated(Horak,1971;Olsen,1974).Zhang(2014)reportedonecaseofP.cerviinfection,fiveof40goatsdiedafterpersistentdiarrheaandmarasmusinaflockofFujianprovince.Xiaoetal(2015)reported280goatsofaflockinGuangdongprovinceshowedmarasmus,anorexiaandanaemia,andmassiveP.cerviadultswerefoundinfivedeadgoats.1.4.11FischoederiuselongatusFischoederiuselongatusisakindofParamphistomuminhabitinginthesurfaceofrumeningoats,itisdeepred,cylindricalinshape.Thelifecycle,pathogenesisandclinicalmanifestationofdiseasescausedbyF.elongatusaresimilarwithP.cervi(Li,2006;Li,2011;Tayloretal.,2016).In1995,LininvestigatedhelminthsofgoatsinSanming,FujianProvincefromJune,1987toOct,1990,andfound52.31%ofgoatswereinfectedwithF.elongatus(Lin,1995).1.4.12GastrothylaxcrumeniferGastrothylaxcrumeniferisalsoacommonParamphistomuminhabitingintherumenofsmallruminantsincludinggoats.ThepathogenesisandclinicalmanifestationofGastrothylaxcrumeniferislikelytootherParamphistomum,suchasParamphistomumcerviandFischoederiuselongatus(Li,2011).1.4.13HomalogasterpaloniaeHomalogasterpaloniaeisalsoanimportantParamphistomuminsmallruminants.However,unlikeotherParamphistomum,Homalogasterpaloniaeisleaf-like,andinhabitesinthececumofgoats.Thelifecycle,pathogenesisandclinicalmanifestationofdiseasescausedbyH.paloniaearesimilarwithP.cerviandF.elongatus(Li,2006;Li,2011;Tayloretal.,2016).Lin(1995)foundtheprevalentofH.paloniaeingoatsinSanming,FujianProvincewas10.77%.22 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince1.4.14FasciolahepaticaFasciolahepaticaadultsusuallyparasiteinthehepaticductandportalveininsmallruminants.ThedevelopmentofFasciolahepaticaisalsoindirect,freshwatersnailsserveasintermediatehost,andmammalsarefinalhost.PathogenesisofFasciolahepaticavariesaccordingtothenumberofingestedmetacercariae,thephaseofparasiticdevelopmentintheliverandthespeciesofhostinvolved.Essentially,thepathogenesisistwo-fold,thefirstphaseoccursduringmigrationintheliverparenchymaandisassociatedwithliverdamageandhaemorrhage;thesecondoccurswhentheparasiteisinthebileducts,andresultsfromthehaematophagicactivityoftheadultflukesandfromdamagetothebiliarymucosabytheircuticularspines(Xuetal.,2000;Li,2006;Tayloretal.,2016).YounganimalsaremoresusceptibletoF.hepaticacomparedwithadults.Massiveinfectioninyounganimalscancauseacuteinfection,andleadtoelevatedbodytemperature,mentalfatigue,accidentaldiarrhea,susceptibletopalpationinliverarea,anemiaorevendeathwithinafewdays,andsomebecomechronicinfection.Chronicinfectionismorecommon,andanimalscanshowsymptomsincludingemaciation,coarsehair,palemucousmembranesandanorexia(Wu,2001;Li,2011;Tayloretal.,2016).Lu(2011)reportedonecaseofF.hepaticainfectioninagoatflockinYunnanprovince,47of146goatsshowedobvioussymptoms,and16goatsdiedafewdayslater.Yang(2014)alsoreportedF.hepaticainfectioninagoatflockinFujian,andmostofthe156goatsshowedmarasmus,anemia,astrictionanddiarrhea,11kidsdiedwithinonemonth.1.4.15FasciolagiganticaFasciolagiganticaissimilarwithFasciolahepaticainmorphology,themaindifferenceisthatthebodyofF.giganticaismoretransparent,theconicalanteriorendisveryshort,shouldersarebarelyperceptible(Li,2011;Tayloretal.,2016).ThepathogenesisandclinicalmanifestationofF.giganticaaresimilarwiththatofF.hepatica(Li,2011;Tayloretal.,2016).In2015,Chen(2015)reportedonecaseofmixedinfectionofF.giganticaandF.hepaticainagoatfarmwith197goatsinYunnanProvince,andledto10deathswithinafewdays,massofF.giganticaandF.hepaticawerefoundbypost-mortemexamination.23 HuazhongAgriculturalUniversityPhDDissertation20181.4.16DicrocoeliumdendriticumDicrocoeliumdendriticumisacommonspeciesintheliver,bileductandgallbladderofgoats.ThelifecycleofD.dendriticumneedstwointermediatehost(snailsandants)tofinishthedevelopmentandtransmission(e.g.,Li,2011;Tayloretal.,2016).AlthoughD.dendriticumarecommonlyfoundinthebileducts,theliversarerelativelynormal.Adultsintheliver,bileductandgallbladderofgoatscancausemechanicaldamageandtoxicityduetothehaematophagicactivityofadults,thenaffectthefunctionofdigestivesystem.Condemnationofliversatslaughtermaycausesevereeconomiclossesamongcattleherdsandsheepflocks.Thesymptomsarenotobviousundermildinfection,andsevereinfectioncanleadtodigestivedisorder,alternationofdiarrheaandconstipation,andemaciation(e.g.,YangandWang,1984;Li,2006;Li,2011;Tayloretal.,2016).1.4.17EurytremapancreaticumEurytremapancreaticumisacommonspeciesinhabitinginthepancreasofgoats(Li,2006;Li,2011).ThelifecycleofE.pancreaticumisalsoindirect,needsnails(servesasfirstintermediatehost)andConocephalusmaculatus(servesassecondintermediatehost)toaccomplishthedevelopmentandtransmission(Li,2011).Lowtomoderateinfectionscauselittleeffectonthehost,ageneralweightlossmayoccurinheavyinfections.Duetomechanicaldamageandtoxiceffect,pancreiaticductoccurshronichyperplasticinflammation,andtheductwallwillbecomethickerorevenbeblockedunderheavyburden.SymptomsforE.pancreaticuminfectionincludeemaciation,anemia,edema,thinstoolorevendeath(Sun,1984;Li,2006;Li,2011;Tayloretal.,2015).E.pancreaticumiscommonlyfoundingoatsinChina(e.g.,Sun,1984,Li,2011),Dingetal(2014)reported29%(84/290)ofgoatsinfectedwithE.pancreaticuminoneflockinChongqing,and27goatsunderheavyburdendiedaheadofdiagnosis.1.4.18TaeniahydatigenaTaeniahydatigenaisazoonoticparasitewithworldwidedistribution,andiscommonasalarvalormetacestodestage(cysticercus)ingoats(Li,2006;Tayloretal.,2016).ThemetacestodestageofTaeniahydatigenaparasitizesintheserosalsurfaces(usuallyliver),omentum,mesenterium,uterusandotherorgansofgoats,sheepandswine,andalsohumanbeings.Intermediatehostsareinfectedbyingestinginfectiveeggsfrom24 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincetheenvironment.Eggswillreleaseoncosphereinthegastrointestinaltract,invadetheintestinalvascularwall,thenflowthroughliverandinhabitonthesurfaceofliver,andenterabdomenforfurtherdevelopment.ThemainpathogeniceffectofTaeniahydatigenaiscausedbymechanicalcompression,toxinactionandsupersensitivity.Younganimalswillshowemaciatedandjaundice,however,adultanimalsdonotshowobvioussymptoms(Li,2006;Tayloretal.,2016).Forthisspecies,noeffectivediagnosticmethodsandtreatmentareavailable(Liu,2000;Bai,2006;Li,2006;Li,2011).It’scommontofindT.hydatigenainfectioningoatsinChina,especiallyforflockswithdogs,sincefarmersliketofeedtheirdogswithdiseasedorganspollutedbyTaeniahydatigenaorotherpathogens,andthiswillleadtothespreadandprevalenceofT.hydatigenainthewholeflock,aswellastreatthehealthofhumans(e.g.,Lang,2013;Ningetal.,2015;Wangetal.,2016).Wen(2017)reportedonecaseofTaeniahydatigenacystsinfectioninblackgoatsinChongqingCitywhichledtothemorbidityof13lambs,andsixofthemdied.1.4.19MonieziaexpansaMonieziaexpansaisalargetapeworminhabitinginthesmallintestinesofruminantssuchassheep,goats,cattleandwaterbuffalo,andyounganimalsaremoresensitivetothisparasite(JingandZhao,2009;Guietal.,2011).ThedevelopmentandspreadofM.expansaneedtheexistenceofOribatei(intermediatehost).AnimalscanbeinfectedwithM.expansabyintakingfoodorwaterpollutedbycysticercoid(Li,2006;Li,2011).PathogenicmechanismsofM.expansacontainmechanicaldamage,nutrientconsumptionandtoxiceffectofadultsinhabitingintheintestinaltract.Infectedanimalswillshowanorexia,diarrhea,emaciated,anemia,inhibitiontogrowthanddevelopmentorevendeath(Yang,2006;Li,2011;Wangetal.,2014).In2005,thirty-four(lambs)of130goatsofaflockinFujianprovinceshowedanorexia,lethargyandweightloss,andthreekidsdiedafterafewdays.M.expansaadultswerefoundinallthethreelambsafterpost-mortemexamination(Yang,2006).Li(2006)reported40%ofkidswereinfectedwithM.expansainaflockinShangdongprovince.WangandLin(2014)reported32(lambs)of75goatswereinfectedwithM.expansa,andshowedobvioussymptoms.25 HuazhongAgriculturalUniversityPhDDissertation20181.5Diagnosisofparasitichelminthsinfections/diseasesofgoatsDifferentspeciesofparasitichelminthsaregreatlyvariableintheirdistribution,epidemiology,pathogenicity,susceptibilitytowardsanthelminticsordamagetothebreedingindustry(ShenandHuang,2004;Aguayo-Ortizetal.,2013).Therefore,theaccuratediagnosisofinfectionsandunderstandingofdominantspeciesareofmajorimportance(Li,2006;Demeleretal.,2012;Roeberetal.,2013a).Knowledgeabouthelminthsingoatscanprovidesupportforparasitichelminthscontrolstrategies,andisalsousefulforstudiesonbiology,epidemiologyanddrugresistance.1.5.1DiagnosticmethodsbasedonclinicalsymptomsofanimalsAnimalsinfectedwithparasitichelminthswillshowavarietyofsymptoms,suchasanemia,diarrhea,weakness,lossofbodyweightorevendeath(Li,2006;Li,2011;Tayloretal.,2016).Thedegreeofclinicalsymptomsareinfluencedbyspecies,burdenofworms,nutritionconditionandimmunelevelofanimals(Li,2006;Li,2011).Forthediagnosisofparasitichelminths,someapproachesbasedonclinicalsymptomshavebeendevelopedandcanreflectinfectionintensityofworms.ThesemethodscontainFAMACHAscore(FAffaMAlanCHArt;parasiticanaemiaindex)andBCS(bodyconditionscore),whichcanreflectthehealthconditiontosomeextentandprovidereferencesforselectivedeworming(e.g.,Van-WykandBath,2002;Sotomaioretal.,2012;Torres,2014;Maiaetal.,2015).TheFAMACHAmethodwasfirstlydevelopedforselectivetreatmentagainstthebackdropofmajoranthelminticresistanceinSouthAfrica(Bathetal.,1996),then,themethodhasbeenappliedinotherplacesaroundtheworld(Besier,2008).Inbriefly,theFAMACHAisbasedonassessinganaemiaofanimalsviaacolourguidedchart:ananimalwithapink-redmucousmembranecolour(withascoreof1to3)doesn’tneedtreatment,whileananimalwithwhitemucousmembrane(withascoreof4to5)requireemergencytreatmentforhaemonchosis.Thismethodistime-saving,suitableinclinicalapplication,andcanreducethecostfortreatment.However,itneedsexperiencedveterinarians,andnotaccuratetosomeextentsinceotherdiseasesmaycausesimilarsymptoms.Torresetal(2014)appliedatargetedselectivetreatmentagainstgastrointestinalnematodesingoatscombingdatafromFAMACHAscore,BCS(bodyconditionscore)andEPG(eggspergram),onlythegoatsunderheavyinfectionofgastrointestinal26 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincenematodesafterassessmentneedtobedewormed,thus,thiscanreducetheuseofdruganddelaythedevelopmentofdrugresistance.Ifusedreasonably,thecostfortreatmentwillbedecreasedandanthelminticresistancewillbedelayed.However,thesemethodsaremainlyappliedinafarmlevelandneedthemanagementhistoryofrecentyearsandexperiencedveterinarians,andlackspecificitysinceclinicalsymptomsmaybevariableandrelatetootherunrelateddiseasesorconditionofthehost(Van-WykandBath,2002;Tayloretal.,2016).1.5.2FaecaleggcountsFaecaleggcountsbasedontheeggsoffaecesisthecommonestmethodforthediagnosisofhelminthsinfection.Thetechniqueiseasytoperform,inexpensiveanddoesn’tneedspecificequipments,whichmakesitbecomearoutinediagnosticmethodforhelminthsinfection.Asausefulmethod,faecaleggcounts(FECs)hasbeenwidelyusedintheinvestigationofhelminths(e.g.,McKenna,1987;McKennaandSimpson,1987;Lietal.,2012;Dong,2014),assessingtheeffectivenessofdeworming(e.g.,Colesetal.,1992;CaiandBai,2009;Zhaoetal.,2010;Barrereetal.,2013a,b),andprovidingguidesforhelminthscontrol(e.g.,LiuandWang,2006;Zhaoetal.,2010).Themethodisbasedonthatwheneggsaresuspendedinasaturatedsolutionwithhighergravity(e.g.,saturatedsaltwaterorsodiumnitrate)thaneggs,theeggswillfloatandconcentratetothesurfacewithinseveralminites,theneggsinanaliquotofthesuspensionareappliedintoexaminationunderalightmicroscopetochecktheexistenceofeggsorassesseggsperonegram(EPG)(Li,2006;Tayloretal.,2016).Basedontheaboveprinciples,somemethodsaboutFECshavebeendeveloped,e.g.,thedirectcentrifugalflotationmethod(Lane,1922),theMcMastermethod(GordonandWhitlock,1939)andtheWisconsinflotationmethod(CoxandTodd,1962).Forthesemethods,theMcMastermethodisthemostwidelyusedandmodifiedinpractice(RobertsandO’Sullivan,1950;Levineetal.,1960;Raynaud,1970;NichollsandObendorf,1994).Modificationsoffaecaleggcountsusuallybaseonflotationsolutions,sampledilutionsandcountingprocedures,whichcanleadtodifferentsensitivities,andmakeithardtocompareamongdifferentmethods.Meanwhile,theFECsresultsaresignificantlyinfluencedbyfecundityofworms(McKenna,1981;Thienpontetal.,1986;Villanuaetal.,2006),watercontentoffaeces(Thienpontetal.,1986;McKenna,2002)andstorageoffaeces(Nielsenetal.,2010).Toovercometheseproblems,aseriousofmethodshave27 HuazhongAgriculturalUniversityPhDDissertation2018beentested,includingthediagnostictest-kit(www.techiongroup.co.nnz),FLOTAC(Cringoli,2006;Kochanowskietal.,2014)andMini-FLOTAC(Limaetal.,2015;Coulibalyetal.,2016)eggcountingmethod.EspeciallyforFLOTACandMini-FLOTAC,theyarenovelmultivalentFECtechniqueswithhighsensitivity(e.g.,Cringoli,2006;Kochanowskietal.,2014;Limaetal.,2015;Coulibalyetal.,2016),andhavethepotentialitytobealternativemethodstothetraditionalflotationmethods,butlargescalesamples,moreparasiticspeciesandhostsneedtobetestedtoprovethesemethodsarevalidatedfordifferenthostsandspecies.1.5.3LarvalcultureLarvalcultureisamethodappliedfordiagnosinggastrointestinalnematodeinfection.Generally,thismethodisusedtoidentifythefirstorthirdstagelarvaeofstrongylenematodesrecoveredfromeggspresentinfaeces(Li,2006;Tayloretal.,2016).ThecommonestmethodistoharvestinfectiveL3fromfaecesformorphologicalanalysis.Inbriefly,eggsinfaeceswillhatchanddevelopintotheinfectivethirdstagelarval(L3)after7to15daysundersuitablehumidityandtemperature(Van-Wyketal.,2004;Li,2006;Tayloretal.,2016).Subsequently,thelarvaecanberecoveredbyBaermanntechniqueandusedforidentificationduetothestrongmotilityofL3(Li,2006;Tayloretal.,2016).Untilnow,manyprotocolshavebeenreportedforspeciesidentification(e.g.,HansenandPerry,1994;Van-Wyketal.,2004;Tayloretal.,2016).ThemorphologicalcharactersusedtoidentifyL3includeanteriorpart(headshapeandtheinternalstructureoftheanteriorend),middlepart(thenumberandshapeofgutcells)andposteriorpart(thelengthofthesheathtailandthepresenceorabsenceoflobesonthelarvaltail)oflarvae(Fig.1-2).However,it’snotablethatstudiesonthedevelopmentofgastrointestinalnematodesinfectingruminantsindicatedthatthetemperature,relativehumidityrequiredforeachspeciesweredifferent(Beveridgeetal.,1989;O’Connoretal.,2006),therefore,LCsarenotreliableinestimatingthecontributionofindividualspeciesatthesameculturecondition.Forexample,cultureconditionat27°Cfor7daysissuitableformostspecies,butnotforOstertagiaspecies,sincethisspeciescandevelopbetterundersomewhatlowertemperature(Whitlock,1956).Berrieetal(1988)reportedsimilarobservationforHaemonchusplacei,OesophagostomumraddiatumandCooperiapectinata.Meanwhile,28 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincethecomposition,pH,moistureandoxygenofculturemediumalsosignificantlyinfluencethedevelopmentoflarvae(HubertandKerboeuf,1984),thusastandardizedmediumwasnecessaryforlarvalculture(HubertandKerboeuf,1984).Toimprovethelarvalculturemethods,HubertandKerboeuf(1984)improvedamodifiedmethodforlarvalcultureusingan‘on-agar’approachtoprovidestandardizedconditions,thismethodcangethighhatchabilitycomparedwithtraditionalmethods.Exceptfortheabove-mentionedproblems,identificationofthethirdstagelarvaetogeneand/orspecieslevelchallengesduotosubstantialoverlapsamongthebodylengthofdifferentspecies,indistinguishablecharacteristics(McMurtryetal.,2000;Bottetal.,2009).Van-Wyketal(2004)developedamethodfordistinguishingL3amongChabertia,Haemonchus,Oesophagostomum,TeladorsagiaandTrichostrongylus.But,thismethodcannotacquireaccuratecompositionofdifferentspecies,andadditionalfeaturesareneededforfurtheridentification(LancasterandHong,1987).Meanwhile,thefeaturesforL3identificationaresometimessubtleandsubjective.Althoughsomeothermethodshavealsobeentestedforlarvalidentification(e.g.,Gordon,1933;Lichtenfelsetal.,1997;McMurtryetal.,2000),thesemethodsaretime-consuming,inaccurate,laboriousandalsoneedexperiencedperformers.Fig.1-2:Proceduresforidentifyingthethirdstagelarvals(L3)ingoats(adaptedtoTheRVC/FAOGuidetoVeterinaryDiagnosticParasitology“http://www.rvc.ac.uk/review/parasitology/RuminantL3/_ID_Overview.htm”)29 HuazhongAgriculturalUniversityPhDDissertation20181.5.4ImmunologicalmethodsAnumberofimmunologicalmethodsbasedonthedetectionofaspecificimmuneresponsebetweenantigenandantibody,andhavebeenappliedinthetestsfornematodescontainingH.contortus(e.g.,Schalligetal.,1995;Kooymanetal.,1997;Gómezmuñozetal.,2000;Lietal.,2007;Prasadetal.,2008),T.circumcincta(Huntleyetal.,2001;Molinaetal.,2009),Trichostrongylus(Shawetal.,1998;Bendixsenetal.,2004)andOesophagostomum(Jasetal.,2010);trematodescontainingDicrocoeliumdendriticum(OtrantoandTraversa,2002),FasciolahepaticaandFasciolagigantica(e.g.,Yadavetal.,2005;Phirietal.,2006),P.cervi(Anuracpreedaetal.,2008)andSchistosomaspp.(Santosetal.,2000).Althoughtheabove-mentionedimmunologicalmethodsshowcapabilitytodetecthelminthsinfectiontosomeextent,forexample,thesensitivityandspecificityfortheELISAdetectionofHaemonchuscontortusexcretory/secretory(ES)andcrudesomaticantigens(CSA)canreachto87.2%and97.7%,82.7%and82.7%insmallsampledetection,respectively(Schalligetal.,1995),theirapplicationsonclinicaldiagnosisarestillunavailable,andneedtobetestedonlargesamplestoassessthevalueofthesemethodsindifferentregions,hostsaswellasmatrix.Accordingtothedifferentdetectioncomponents(antigenorantibody),theimmunologicaldiagnosticmethodscanberecognizedasdirectandindirectmethods.Directmethodsarebasedontheparasiteantigens,thusprovidedirectevidenceforparasiteinfections.However,somespeciescansharesimilarantigens(CohenandSadun,1976),andantigensfromlarvalstageandadultstagemayvaryinthediagnosisofinfection(McLarenetal.,1978).Althoughlimitationsareexisting,Johnsonetal(1996)describedanimmunologicalmethodinamurinemodelsystemusingHeligmosomoidespolygyrus.Andthediagnosticmethodshowedpromisingresultsunderexperiments,however,thereactivitycanbeinterferedbyfaecalcontentsandcross-reaction,andantigensinfaecesmaybelost(Johnsonetal.,1996).Indirectmethodsarebasedontheexistenceofanti-parasiteantibodiesorcell-mediatedimmuneresponses.Berghenetal(1993)reportedamethodforthedetectionofanti-Ostertagiaantibodiesinserumsamplesfromcattle,however,serumantibodylevelscanbeinfluencedbyaseriousoffactors,includingimmunestateofanimalsandparasiteburdens;atthesametime,immunologicalmethodsarehardtodistinguishpreviousinfectionsfromtherecentones,andalsolackofsensitivityandspecificityinclinicaldetection(Lichtenfelsetal.,1997;Demeleretal.,2012).30 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince1.5.5Post-mortemdiagnosisPost-mortemdiagnosisisacommonmethodinvivoforthediagnosisofparasitesinfectionandroutinelyemployedinparasitologyworldwide.Thismethodisaccurateandcomprehensivesincemostoftheorgansandtissuesareunderexaminationforparasitesrecoveredpartsbypartsfrompostmortemanimals(e.g.,HansenandPerry,1994;Li,2006;Wang,2013;Tayloretal.,2016),then,identifyingthecleanwormsunderalightmicroscopebaseonmorphologicalfeaturesandparasiticsiteafterdyeingifneeded(eg.HansenandPerry,1994;Li,2006;Li,2011;Tayloretal.,2016).ThismethodisoneofthecommonestmethodfortheinvestigationofparasitesingoatsinChina,andhasbeenwidelyusedinmanyprovinces,suchasAnhui(e.g.,LiandLiu,2004;Lietal.,2012;Guetal.,2014),Gansu(e.g.,Hui,2000;Jia,2005;Jiaetal.,2009;Hui,2014),Guizhou(e.g.,Duetal.,1995;Ruanetal.,1995;Miaoetal.,1997;Luetal.,2015;Songetal.,2015),Henan(e.g.,Zhuetal.,1982;Li,2009;Lietal.,2012;FanandWang,2014),Shaanxi(e.g.,Guoetal.,2003;Fengetal.,2008;Fengetal.,2012;Weietal.,2013)andShandong(e.g.,Zhouetal.,2006;WangandCao,2008).Anumberofdifferenttechniqueshavebeendevelopedandappliedinclinicaldetection(e.g.,RobertsonandElliott,1966;EyskerandKooyman,1993).Thedifferencesamongthesemethodscontain:usingsieveswithdifferentmeshsizestoremoveplantdebrisorotherlargeimpurity;countingwormsfromdigestaand/orwashes;incubatingtheorgansinwaterorsalineforrecoveryofimmatureparasitesornot;examingpartialortotalgastrointestine(e.g.,Gabaetal.,2006;McKenna,2008).However,thismethodistime-consumingsincenumerousworkneedtobedone,andcan’tbeusedtodetectparasitesinfectionwhenanimalsarealive(Li,2006;Tayloretal.,2016).1.5.6MolecularmethodsObviously,traditionalmethodsbasedonmorphologicalcharacteristicsandimmunologyhavelimitationsonsensitivityandspecificity(e.g.,Bottetal.,2009;Demeleretal.,2012).Withthedevelopmentoftechniquesandbioinformatics,DNA-basedamplificationinvitromethodshavebeenwidelyusedinspecificidentificationandcharacterisationofbacterium,virusandparasites(e.g.,Gasseretal.,2008;Demeleretal.,2012).ToapplyDNA-basedmethodsfortheidentificationandcharacterisationofparasitichelminthsingoats,someaspectsthatinfluencetheresultsneedtobefocused.Firstly,31 HuazhongAgriculturalUniversityPhDDissertation2018PCRinhibitors(suchasBilesalts,collagenandhaeme)arecommonlyfoundinfaecalsamplesandsignificantlydecreasethePCRefficience(Wilson,1997).Thus,PCRinhibitorsinsamplesshouldbeeliminatedaheadofPCRprocessing,usually,acommercialkitcaneffectivelyeliminatePCRinhibitors(Roeberetal.,2013b).Secondly,it’stoselectspecificmolecularmarkers.Thefirstinternaltranscribedspacer(ITS1)andthesecondinternaltranscribedspacer(ITS2)ofribosomehavebeenprovedtobereliablemolecularmarkersforspeciesidentification,andcommonlyusedinpractice(e.g.,Gasseretal.,1993;Aietal.,2007;Linetal.,2007;Lotfyetal.,2010;Nguyenetal.,2012).Mitochondrialmarkers,suchascytochromecoxidasesubunit1(cox1),havealsobeenused,andprovedtobemoresuitablethanribosomalmarkersinidentifyingsomedistantlyrelatedtrematodespecies(Jiaetal.,2011a).1.5.6.1PCRandsequencingConsideringallthemoleculartechniquesappliedinmolecularbiosciences,thePolymeraseChainReaction(PCR)maybethemostwidelyusedtechnique.PCRisanefficientamplificationtechniqueofspecificnucleicacidsinvitro,it’ssensitive,specificandcanobtainmillionsofcopiesofaspecifictargetDNAsequence(Welch,2012;Tayloretal.,2016).In1985,PCRwasfirstlyappliedintheamplificationoftheβ-globingeneforthediagnosisofsicklecellanaemia(Saikietal.,1985).Fromthenon,PCRtechniquesareimprovedgreatlyduetothedevelopmentofscienceandtechnology(Erlichetal.,1991).InbasicPCR(Fig.1-3),thesystemusuallycontainstemplateDNA,forwardandreverseprimersflankingthetarget,DNApolymerase,deoxynucleotidetriphosphates,suitablebuffer.Asforthecondition,itmainlycontains30-40cyclesofthreebasicstepsincludingdenaturation,annealingandextension(Welch,2012;Tayloretal.,2016).Combinedwiththesequencing,PCRcanbeemployedinthestudyofspeciesidentification,molecularepidemiology,populationdiversityandphylogeneticanalysis.Forexamples,Gasseretal(1993)appliedaPCR-basedsequencemethodtargetingtheITS-2regiontorapidlyidentifyparasiticnematodes;Chiltonetal(2006)usedPCR-basedsequencemethodstargetingthe18Sand28SrDNAsequencetoexploretheevolutionaryoriginsofnemaodeswithintheorderStrongylida;Aietal(2011)appliedPCR-basedsequencemethodstargetingthecox1,nad4andnad5ofmitochondrialDNAtocomparethegeneticdiversityofFasciolaspp.isolatesfromdifferenthostsandregions;Yinetal(2013)usedPCR-basedsequencemethodstargetingtheITS-2andnad4regionsto32 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceexploregeneticvariabilitywithinandamongHaemonchuscontortusfromgoatsandsheepinChina;Liuetal(2014a)appliedthePCR-basedsequencemethodtoobtainandanalysisthecompletemitochondrialgenomeamongFasciolahepatica,F.giganticaandFasciolaspp.Fig.1-3:SchematicdiagramofPCR.EachcycleofPCR(heating-denaturing,cooling-annealingandextending)doublestheamountoftargetDNA,increasingtheconcentrationoftargetDNAexponentially(adaptedbyTayloretal.,2016).Asusefulmolecularmarkers,thefirsttranscriptioninterval(ITS-1)and/orsecondtranscriptioninterval(ITS-2)ofribosomalDNAhavebeenwidelyusedinaccuratediagnosis(Roeberetal.,2013a).PCR-basedmolecularmethodstargetingthefirsttranscriptioninterval(ITS-1)and/orsecondtranscriptioninterval(ITS-2)ofribosomalDNA,suchasPCR(e.g.,Gasseretal.,1993;Aietal.,2007;Linetal.,2007;Lotfyetal.,2010;Nguyenetal.,2012),species-specificPCR(e.g.,Bottetal.,2009),real-timePCR(e.g.,Bottetal.,2009;Roeberetal.,2015),multiplexPCR(e.g.,Zarlengaetal.,2001;Bissetetal.,2014)andmultiplexed-tandemPCR(Roeberetal.,2012)havebeenusedfor33 HuazhongAgriculturalUniversityPhDDissertation2018speciesidentification,andprovedtobespecific,sensitive,accurateandtime-saving(Roeberetal.,2013a;Bissetetal.,2014).ExceptforribosomalDNA,mitochondrialDNAhasalsobeenusedasmolecularmarkersforthestudyofspeciesidentification(e.g.,Wangetal.,2011b),aswellasgeneticdiversity(e.g.,Bowlesetal.,1992;Aietal.,2011;Yinetal.,2013)andphylogeneticanalysis(e.g.,Littlewoodetal.,2006;Shekhovtsovetal.,2010;Liuetal.,2013).Althoughribosomalgenome(ITS-1andITS-2)havebeenprovedtobereliablemolecularmarkers,applicationofmitochondrialgenomeinsteadof18sandITSasmolecularmarkersinidentifyingsynonymsmaybemoresuitable(Jiaetal.,2011a,b,c).1.5.6.2IsothermalamplificationtechniquesComparedwiththePCRtechniques,whichrequirecyclesofdenaturation,annealingandextension,isothermalamplificationofnucleicacidscanbeaccomplishedunderconstanttemperature(e.g.,Gaoetal.,2016).Fromtheearlyof1990s,anumberofisothermalamplificationtechniqueshavebeendevelopedandusedfordetectingtargetsofcells,proteins,smallmolecules,ions,DNAandRNA(Zhaoetal.,2015),includingnucleicacidsequence-basedamplification(NASBA),exponentialstranddisplacementamplification(E-SDA),hyperbranchedrollingcircleamplification(HRCA),primer-generationrollingcircleamplification(PG-RCA),loop-mediatedisothermalamplification(LAMP),helicase-dependentamplification(HDA),recombinasepolymeraseamplification(RPA),exponentialamplificationreaction(EXPAR),andwholegenomeamplification(WGA)(Table1-7).Loop-mediatedisothermalamplification(LAMP)isacommonlyusedisothermalamplificationtechniquefirstlyreportedbyNotomietal(2000).Briefly,LAMPuseshightemperaturetoseparateadoublestrandDNApriortoamplification,andcanachieveexcellentspecificityusingtwopairsofspecificprimerswithinonereaction,thefourprimerscanrecognizesixregionsofthetargetduringamplification.DetailsabouttheprocedureofLAMPmethodwerepresentedinFig.1-4.Inbrief,LAMPcontainstwosteps,thestartingstructure-producingstepandthecyclingamplificationstep,andcanbeaccomplishedwithinonehour(Tomitaetal.,2008).ForthedetectionofLAMPproduct,itwillshowladderpatternbyagarosegelelectrophoresisorchangecolourbyaddingdyes,suchasSYBRGreenIandhydroxynaphtholblue.Forrecentyears,LAMPhasbeenusedinthedetectionofhelminthsincludingFasciolahepaticaandFasciolagigantica34 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince(Aietal.,2010),Haemonhuscontortus(Melvilleetal.,2014),Necatoramericanus(Mugambietal.,2015),Schistosomajaponicum(Xuetal.,2010),Strongyloidesstercoralis(Wattsetal.,2014)andTaeniatapeworms(Sakoetal.,2013)basedonitsadvantagesincludinghighsensitivityandspecificity,cost-savingandpotentialapplicationinclinicaldiagnosis.ExceptforLAMP,recombinasepolymeraseamplification(RPA)isalsoapowerfulisothermaltechniquethatappliestherecombinasetoseparateadoublestrandDNAtarget,thenthesingle-strandedDNA-bindingproteinwillbindtoonesinglestrand,theprimerwillbindtotheotherstrandtocompletetheamplificationwiththehelpofDNApolymerasewhichcanrecognizethe3’-endsoftheprimer(Piepenburgetal.,2006).Theextensionoftwoopposingprimerscangenerateonefullcopyoftheamplicontogetherwiththeoriginaltemplate.Thecyclicrepetitionofthisstepleadstoexponentialamplificationofthetargetsequence(Fig.1-5).Usually,millionsoftargetcopiesaccumulatecanbeaccomplishedat37-42°Cwithin40min(Zhaoetal.,2015).AsfordetectingtheRPAproduct,auniqueFRET-basedfluorescentprobeandalateral-flowstripsensingsystemareappliedinthereaction.RPAhasbeenusedinthedetectionofSchistosomahaematobium(Rosseretal.,2015)andSchistosomajaponicum(Sunetal.,2016b),andprovedtobetime-saving,highspecificityandsensitivy,andalsohavepotentialabilityinclinicaldiagnosis.1.5.6.3CharacterisationofparasitichelminthsusingmitochondrialgenomeMitochondriaisanimportantsubcellularorganellesinwhichimportantbiochemicalfunctionsrelatedwithenergetakeplacewithincellsofmosteukaryotes.Withintheorganelleismitochondrial(mt)genome,whichisindependentofnucleargenome,butalsohascloserelationshipwithnucleargenome.Comparedwithnucleargenome,mitochondrialgenomehassomespecificfeatures:1)itismaternalinherited;2)ithasrelativelystablegenes;3)thegenearrangementandusageofgeneticcodearerelativelystationary;4)geneevolutionrateofmitochondrialgenomeisfasterthanthatofnucleargenome(Jiaetal.,2011a,b,c).Duetotheabove-mentionedfeatures,mitochondrialgenomehasbeenappliedinthestudyofmolecularepidemiology,populationgeneticsandsystematics(e.g.,Leetal.,2000;Huetal.,2006;Jiaetal.,2011a,b,c;Wangetal.,2011b;Yanetal.,2013).Ingenral,mitochondrialgenomesareusuallysmall(13-20kb),compactandcirculargenomes,andcontainabout12or13proteingenes(withorwithout35 HuazhongAgriculturalUniversityPhDDissertation2018ATPsynthaseprotein8)forenzymesinoxidativephosphorylation,tworibosomalRNAgenesforconstructingRNAcomponentsofribosomeand22thansferRNAfortransferringRNAduringthetranslationofmitochondrialproteins(Boore,1999).Duetoahighmutationrate,mitochondrialgenomehasbeenappliedonthestudiesofmolecularepidemiology,populationgeneticsandphylogeneticanalysis(e.g.,Andersonetal.,1998;GasserandNewton,2000;Huetal.,2004;Jiaetal.,2011a,b,c).Usefulmolecularmarkerswithinmtgenome,suchascox1andnad4,havebeenappliedintheidentification,differentiationanddeterminationofcloselyrelatedspecies(e.g.,Andersometal.,1998;GasserandNewton,2000;Aietal.,2011).Recently,studiesonthemolecularepidemiology,populationgeneticsandphylogeneticanalysisofgoathelminthsbasedonpartialorcompletemtgenomehavebeenperformed.Forexample,Aietal(2011)investigatedgeneticdiversityofFasciolaspp.isolatesfromcattle,sheepandgoatindifferentcountriesbasedoncox1,nad4andnad5,andphylogeneticanalysisbasedonthecombinedsequenceofcox1,nad4andnad5revealedFasciolaspp.isolatesfromChinaandNigerweregroupedindifferentsub-clusters;Yinetal(2013)amplifiednad4geneof152HaemonchuscontortusfromsheepandgoatsinsevenregionsinChinatoanalysisgeneticvariabilitywithinandamongsevenisolates,andtheresultsindicatedthathighvariationswithinthesevenpopolations,andlowgeneticdifferentictionandhighgeneflowwerefoundamongdifferentH.contortuspopulationsinChina;Yanetal(2013)sequencedthecompletemitochondrialgenomeofParamphistomumcervi,andcomparativelyanalysedwithothertrematodes,thephylogeneticanalysisresultsofconcatenatedsequencesofthe12protein-codinggenesofP.cerviandothertrematodesindicatedP.cerviwasplacedwithinthePlagiorchiida,whichwerenotthesameaspreviousstudies.Lietal(2016)analysedandcomparedthesequencevariationwithinOesophagostomumasperumandamongOesophagostomumspeciesofgoatsinHunanProvinceusingpartialcox1andnad1genesofmitochondrialgenome,theresultsindicatedintra-specificsequencevariationwithinO.asperumwere0-1.6%and0-1.9%forcox1andnad1,respectively,andinter-specificsequencedifferencesamongOesophagostomumspecieswere11.1-12.5%and13.3-17.7%forcox1andnad1,respectively.Later,phylogeneticanalysisbasedonthecombinedsequencesofcox1andnad1revealedthatdistinctgroupswerehighstatisticalsupported.36 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable1-7:Characteristicsofisothermalamplificationtechniques.TechniquesNo.ofenzymesNo.ofPrimersTemperature(°C)Time(h)TargetEfficiencyReferenceNASBA2or3:reverse2~411.5-2RNAorDNA106-109Compton(1991)transcriptaseandRNAGuatellietal.(1990)polymerase(RNaseH)E-SDA2:DNApolymerase2or6372DNA107Walkeretal.(1992a)andNEaseWalkeretal.(1992b)Shietal.(2014)HRCA2:ligaseandDNA2601.5DNAorRNA109Lizardietal.(1998)polymeraseDeanetal.(2001)CaoandZhang(2012)PG-RCA2:DNApolymerase0601-3DNAorRNA~60copiesofMurakamietal.(2009)andNEasegonomicDNAMurakamietal.(2012)Zengetal.(2013)LAMP1:DNApolymerase460-65<1DNA109Notomietal.(2000)Tomitaetal.(2008)HDA2:DNApolymerase237-650.5-2DNA107Vincentetal.(2004)andhelicaseGaoetal.(2013)RPA2:DNApolymerase237-420.5-1.5DNA10copiesofPiepenburgetal.(2006)andrecombinasegonomicDNAHeetal.(2012)EXPAR2:DNApolymerase0~60<0.5shortDNAorRNA106-108VanNessetal.(2003)andNEaseXuetal.(2015)Zhouetal.(2014b)Jiaetal.(2010)MDA1:DNApolymeraserandomprimers30-37>8DNA106Deanetal.(2002)Henkeetal.(1997)pWGA2:T7gp4primaseand0370.5-2DNA103-108Lietal.(2008)DNApolymeraseNote:ThetablewasadaptedbyZhaoetal(2015)37 HuazhongAgriculturalUniversityPhDDissertation2018Fig.1-4:Principleofloop-mediatedisothermalamplification(LAMP).(a)PrimerdesignoftheLAMPreaction.Foreaseofexplanation,sixdistinctregionsaredesignatedonthetargetDNA,labeledF3,F2,F1,B1c,B2c,andB3fromthe5’end.(b)Startingstructureproducingstep.(c)Cyclingamplificationstep(adaptedbyTomitaetal.,2008).38 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceFig.1-5:Principleofrecombinasepolymeraseamplification(RPA).Combination(ingray)ofrecombinaseandprimerscantargetDNAforhomologoussequences(inredorblue)andopenthedoublestrandDNA.Then,thedisplacedstrandisboundbythesinglestrandDNA-bindingprotein(ingreen)tokeepsinglestrand,primersareextendedbytheexistenceofpolymerase(inblue).Thenetresultoftwoopposingextensioneventsistogenerateonecompletecopyoftheamplicon.(adaptedbyPiepenburgetal.,2006).39 HuazhongAgriculturalUniversityPhDDissertation2018Currently,over6130mtgenomesareavailableintheEuropeanBioinformaticsInstitute(EBI)resource(http://www.ebi.ac.uk/genomes/organelle.html).Theavailabilityofmtgenomedatabasecanprovidesourcesofmarkersforstudiesonthemolecularepidemiology,populationgeneticsandsystematics(e.g.,Andersonetal.,1998;GasserandNewton,2000;Huetal.,2004;Jiaetal.,2011a,b,c).Usually,thecompletemitochondrialgenomesequencecanbeobtainedbycombinationsofseveralconventionalPCRandlong-PCRspanningtheentiregenome(e.g.,Leetal.,2001a;Yanetal.,2013;Zhaoetal.,2013).Huetal(2002)appliedtwolong-PCRtoobtainthecompletemitochondrialgenomeofparasiticnematodes,andprovedtobeeffectiveforamplifyingthecompletemitochondrialgenomeof14differentspeciesofparasiticnematodesincludingHemonchuscontortus,BunostomumphlebotomumandDictyocaulusviviparus.However,limitedmtgenomeofhelminthsinsmallruminantswerereportedandcharacterizeduntilnow,suchasHaemonchuscontortus(Jexetal.,2008),Trichostrongylusaxei(Jexetal.,2010),Cooperiaoncophora(VanderVeeranddeVries,2004),Oesophagostomumasperum(Zhaoetal.,2013),Teladorsagiacircumcincta(Jexetal.,2010),Paramphistomumcervi(Yanetal.,2013),Fasciolahepatica(Leetal.,2001a),Dicrocoeliumdendriticum(Liuetal.,2014b)andEurytremapancreaticum(Changetal.,2016).Moremtgenomeinformationofhelminthsofsmallruminantareneededtoenrichthedatabaseandcharacterizespecies,andprovideusefulinformationforfurtherstudiesonspeciesidentification,geneticdiverdityandphylogeneticanalysis.Especiallyfortrematodespecies,traditionalmethodsaredifficulttoidentifysynonymswithsimilarmorphologybutdifferentingenetics(e.g.,Lietal.,2000;Littlewoodetal.,2006;Jiaetal.,2011a,b,c).FortheidentificationofgoathelminthsinChina,becauseofrestrictionsoftechniques,traditionalmorphologicalmethodsand/orpost-mortemidentificationwerecommonlyusedforthestudyofepidemiology,whilemolecularmethodswereseldomlyapplied.Comparedwithmolecularmethods,traditionalmethodsaretime-consumingandinaccurateduetosharingsimilarmorphologicalcharacteristicsamongspecies,andalsoneedexperiencedpersons(Bottetal.,2009).Therefore,molecularmethodsbasedonPCRorisothermalamplificationtechniquesarenecessaryforthefurtherstudyofepidemiologytoincreaseaccuracyandsavetime(e.g.,Gasseretal.,2008;Demeleretal.,2012;Zhaoetal.,2015).40 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince1.6Epidemiologyofparasitichelminthsinfections/diseasesofgoatsWiththerapiddevelopmentofbreedingindustryofgoats(Li,2015;Ling,2015;Tuerxun,2015;Zhang,2015),problemslinkedwithparasiticdiseaseshaveledtosignificanteconomiclossestothebreedingindustry(LiuandWei,2011;Xiaoetal.,2011;Gu,2016).However,thepathogenicitymayvarygreatlyamongparasitichelminths,somespeciesarehighlypathogenicandcanleadtoseriousdamage,suchasHaemonchuscontortus,FasciolahepaticaandCoenuruscerebralis(Huangetal.,1999;Li,2006;Zhao,2016),whilesomespeciesarelowpathogenic,suchasChabertiaovinaandParamphistomumcervi(HansenandPerry,1994;Li,2006),andcancauselimiteddamage.Thus,investigationsofparasitichelminthsspeciesingoatsareessentialforpreventingandcontrollingparasites,aswellasforthestudyofdrugresistance.TounderstandthestatusofparasitesinfectionofeconomicanimalsinChina,ShenandHuang(2004)reviewedinvestigationsaboutparasitespeciesof17livestockandpoultry.Fromtheirreport,it’snotablethatthespecies,prevalenceofhelminthsindifferentareasvarygreatlybecauseofthediversityofgeography,climate,managementandsoon(Shen,2005).Asfortheinvestigationofhelminthsinfectionofgoats,Chen(1956)firstlyreportedtheinfectionofFasciola,Paramphistomumcervi,Echinococcusgranulosus,Taeniahydatigena,Coenuruscerebralis,Moniezia,OesophagostomumcolumbianumandHaemonchuscontortusofgoatsinafarmofSichuanProvince.Fromthenon,goathelminthswerewidelyreportedin28provincesbelongingtosevengeographicaldivisions,namelyNortheastChina,NorthChina,NorthwestChina,SouthernChina,SouthwestofChina,CentralChinaandEastChina(Table1-8),includingAnhui(e.g.,LiandLiu,2004;Lietal.,2012;Guetal.,2014),Beijing(e.g.,Caietal.,2011;Guanetal.,2012),Chongqing(e.g.,Lietal.,2001;Tangetal.,2003),Fujian(e.g.,Linetal.,1996;Fang,1998),Gansu(e.g.,Hui,2000;Jia,2005;Jiaetal.,2009;Hui,2014),Guangdong(Lietal.,1999),Guangxi(Taoetal.,2010),Guizhou(e.g.,Duetal.,1995;Ruanetal.,1995;Miaoetal.,1997;Luetal.,2015;Songetal.,2015),Hebei(e.g.,YangandWang,1984;Dong,2014),Heilongjiang(e.g.,Quan,1981;Wangetal.,2005),Henan(e.g.,Zhuetal.,1982;Li,2009;Lietal.,2012;FangandWang,2014),Hubei(e.g.,Caoetal.,1995),Hunan(e.g.,Liuetal.,2006;Liangetal.,2007;Liu,2014),InnerMongolia(e.g.,Zhaoetal.,1981;Guo,1985;Duanmugesurongetal.,1995;Yangetal.,2006;Yongetal.,2009;),Jiangsu(e.g.,PengandKuang,1994;Pengetal.,1994;Chenetal.,2000a,b),41 HuazhongAgriculturalUniversityPhDDissertation2018Jiangxi(e.g.,Wei,1984;Jianetal.,1993),Jilin(Yuetal.,2011),Liaoning(Liuetal.,2011),Qinghai(e.g.,Ma,1982;Lietal.,2006;Wangetal.,2014),Shaanxi(e.g.,Guoetal.,2003;Fengetal.,2008;Fengetal.,2012;Weietal.,2013),Shandong(e.g.,Zhouetal.,2006;WangandCao,2008),Shanghai(Heetal.,1999a),Shanxi(Zhuetal.,2010),Sichuan(e.g.,Yang,1988;Xuetal.,2014;Zhangetal.,2014;JiangandHao,2015),Tibet(e.g.,Hanetal.,1984;Nimaciren,1998;Labacidan,2014),Xinjiang(Mamuerhanetal.,1988),Yunnan(e.g.,Heetal.,1999d;Jiangetal.,2000;Lietal.,2002;Tianetal.,2008)andZhejiang(Jinetal.,2011).Ingeneral,helminthsingoatsarecommonandserious,especiallyforgastrointestinalnematodes(Table1-8).Insomeareas,theinfectionrateofgastrointestinalnematodescanreachto100%(e.g.,Caoetal.,1995;Chenetal.,2000a;Yang,2005).Accordingtothesesurveys,predominantspeciesofhelminthsofgoatsinChinacontainH.contortus,T.circumcincta,Trichostrongylusspecies,Oesophagostomumspecies,Cooperiaspecies,B.trigonocephalum,Paramphistomumspecies,Fasciolahepatica,Taeniahydatigena(larvae)andMonieziaspecies.Althoughstrongyle,ParamphistomumsppandFasciolaspparecommoninallthereportedprovinces,thenumber,speciesandinfectionrateofhelminthspeciesdiffergreatlyduetothedifferencesinclimate,environment,animalvarietiesandanimalhusbandrypracticesinfluencingonthesurvivalanddevelopmentofparasites(e.g.,Shen,2005;Abbottetal.,2012;Tayloretal.,2016).KnowledgeoftheepidemiologyofhelminthsofgoatsinChinacanprovideusefulinformationforstudiesonbiology,ecologyandpopulationgeneticsofhelminthsingoats.Besides,seasonaldynamicscanhelpusunderstandthechangeregularityofparasiteinfectionandwhenthehighestandlowestinfectionoccur.Withtheseasonaldynamicsdata,wecanunderstandthedevelopmentofprominentspeciesandchoosethebesttimefordewormingtoreducethedamages.ChenandLin(1982)reportedtheseasonaldynamicsofHaemonchuscontortusinXiamen,FujianProvince,andindicatedtwoinfectionpeaksinJuneandAugust.Heetal(1999a)reportedtheseasonaldynamicsofHaemonchuscontortusandMonieziaofwhitegoatsinChongming,Shanghai,theresultsindicatedtherewerethreeinfectionpeaksinMay,AugustandNovemberforHaemonchuscontortus,andtwoinfectionpeaksinJuneandAugustforMoniezia.Combiningwiththeepidemiologyandseasonaldynamicsdata,suitabletimefordewormingtargetedondominantspeciesbyepidemiologicalinvestigationscanbedeterminedaheadoftheinfectionpeaktoeffectivelycontrolparasitesintoalowlevelandminimizethedamage(WangandWang,1993;Wangetal.,2015).42 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceHowever,duetotherestrictionsoftechnologies,traditionalmicroscopicexaminationsofeggsand/orpostmortemexaminationwerecommonlyappliedinalltheinvestigations,atthesametime,onlyasmallamountofanimalswereusedforexaminationforsomeinvestigations,therefore,investigationofparasitespeciesinanimalsmaybenotsufficientandneedfurtherverificationstosomeextent,especiallyforStrongylidawithsimilarmorphology(Chilton,2004;Chiltonetal.,2006;Bottetal.,2009).HubeiProvincelocatesinthecentralofChina,plentyofvegetationresourcesandwaterresourcesmakeitsuitableforlivestockbreeding,inculdinggoatbreeding.AlthoughtheLivestockSchistosomiasisControlStationofHubeiProvinceetal(1981)andCaoetal(1995)investigatedandreportedtheinfectionconditionofhelminthsofgoatsinHubeiProvince,thecurrentstateofgoathelminthsinHubeiProvinceisunkownsincethebreedingenvironmentandscaleforgoatschangedgreatlyinrecentyearscomparedwithbefore(e.g.,Lietal.,2009;Li,2015;Ling,2015).Thus,thereisanurgentneedtoinvestigateandunderstandthecurrentepidemiologicalconditionofgoathelminthsinHubeitoprovideusefulsuggestionsforeffectivepreventionandcontrollingofgoathelminths.1.7CurrenttreatmentandcontrolstrategiesinChinaSincehelminthisprevalentingoatsinallthereportedprovinces,stratigesneedtobetakentopreventingandcontrollinghelminthstopromotethehealthydevelopmentofgoatbreedingindusry.Duetodifferentbreedingscaleandlevelofexperience,thetreatmentandcontrolstrategiesdiffergreatlyamongfarmers.Currently,althoughvaccinesagainstHaemonchuscontortushavemadeprogressinrecentyears(e.g.,Yanetal.,2006,2007;Sunetal.,2007;Zhouetal.,2010),thereisstillnoeffectivevaccinesforgoatsinChina,andthestrategiesforpreventionandcontrollingofgoathelminthsmainlybaseontheapplicationofanthelminticsandimprovementofmanagement.1.7.1VaccineRecently,recombinantsubunitvaccineandDNAvaccinehavebeentestedforthestudiesonthevaccinesagainstHaemonchuscontortusofgoatsinChina,andachievedsomeprogress.43 HuazhongAgriculturalUniversityPhDDissertation2018Table1-8:InvestigationsofparasitichelminthsingoatsinChina.GeographicalLandformClimateInvestigationofparasitichelminthsingoatsandsheepdivisionsProvinceMethodsSpeciesReferencesNortheastBasin;MonsoonclimateofHeilongjiangNecropsyHaemonchuscontortus,Trichostrongyluscolubriformis,NematodirusWangetal.(2005)ChinaPlainmediumlatitudes;Oiratianus,Bunostomumtrigonocephalum,OesophagostomumTemperatecontinentalcolumbianum,Strongyloidespapillosus,Dictyocaulusfilarial,TrichurisclimatelaniParamphistomumcervi,Fasciolahepatica,SchistosomaturkestanicaMonieziaexpansa,Monieziabenedeni,CoenurusandTaeniahydatigenaJilinFlotationmethodsHaemonchuscontortus,Trichostrongyluscolubriformis,MecistocirrusLiuetal.(1999)andnecropsydigitatus,Ostertagiawuhingensis,Ostertagiaskrjabini,OesophagostomumYuetal.,2011columbianum,Oesophagostomumradiatum,Skrjabinemaovis,Trichurislam,Trichurisglobulosa,Bunostomumtrigonocephalum,Setarialabito-papillosa,Setariadigitata,Fasciolahepatica,Paramphistomumcervi,Calicophoronerschowi,Monieziaexpansa,MonieziabenedeniLiaoningNecropsyHaemonchuscontortus,Oesophagostomumcolumbianum,Xu(2001)Oesophagostomumasperum,Bunostomumtrigonocephalum,NematodirusLiuetal.(2011)filicollis,Trichostrongyluscolubriformis,Strongyloidespapillosus,Gongylonemapulchrum,Dictyocaulusfilarial,Chabertiaerschowi,Trichurisglobulosa,Fasciolahepatica,Eurytremapancficum,Eurytremacoelomaicum,Dicrocoeliumdendfiticum,Paramphistomumcervi,Schistosomaturkestanica,Monieziaexpansa,Monieziabenedeni,Helictometragiardia,Avitellinacentripunctata,Coenuruscerebralis,Taeniahydatigena,EchinococcusNorthChinaPlateau;MonsoonclimateofBeijingFlotationmethodsHaemonchuscontortus,Trichostrongylus,Ostertagia,Cooperia,Caietal.(2011)PlainmediumlatitudesandLarvalcultureOesophagostomum,Marshallagia,Nematodirus,TrichurisandGuanetal.(2012)StrongyloidespapillosusHebeiFlotationmethodsHaemonchuscontortus,Trichostrongylusaxei,Ostertagiawuhingensis,YangandWangandnecropsyTeladorsagiacircumcincta,Nematodirus,Bunostomumtrigonocephalum,(1984)Trichurisovis,Chabertiaovis,Oesophagostomumcolumbianum,Liuetal.(2002)O.asperum,O.kansuensis,Cooperiaerschowi,Dictyocaulusfilaria,Dong(2014)Muelleriuscapillaris,Gongylonemapulchrum,Strongyloidespapillosus,Fasciolahepatica,Eurytremapancficum,Dicrocoeliumdendfiticum,Paramphistomumcervi,Monieziaexpansa,M.benedeni,Helictometragiardia,Taeniahydatigena,Echinococcus44 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceShanxiFlotationmethodsHaemonchuscontortus,Trichostrongylus,Nematodirus,Ostertagia,Langetal.(2005)Trichuris,Dictyocaulus,Bunostomum,Oesophagostomum,Ascaris,Zhuetal.(2010)Chabertia,Gongylonemapulchrum,Paramphistomum,Fasciolahepatica,Dicrocoeliumdendfiticum,Moniezia,TaeniahydatigenaNorthwestPlateau;MonsoonclimateofShaanxiFlotationmethodsHaemonchuscontortus,H.similis,H.bedfordi,H.longistipes,NematodirusZhang(1983)ChinaBasinmediumlatitudes;andnecropsyabnormalis,N.filicollis,N.hsuii,N.oiratianus,N.helvetianus,N.Guoetal.(2003)Temperatecontinentalspathiger,Protostrongylushobmaieri,Dictyocaulusfilarial,D.eckerti,Songetal.(2007)climateBunostomumtrigonocephalum,Oesophagostomumcolumbianum,Fengetal.(2008)Parabronemaskrjabin,Gongylonemapulchrum,Strongyloidespapillosus,Fengetal.(2012)Skrjabinemaovis,Chabertiaovina,Trichostrongyluscolubriformis,T.axei,Mengetal.(2012)Trichostrongylusprobolurus,Teladorsagiacircumcincta,OstertagiaWeietal.(2013)wuhingensis,Ostertagiatrifurcate,O.dahurica,Ostertagiaostertagi,O.buriatica,O.orloffi,O.heterospiculagia,O.skrjabini,Marshallagiamarshalli,Cooperiaerschovi,C.laterouniformis,C.pectinata,Spicutopteragiaschulzi,Setarialobiato-papillose,T.concolor,T.gazellae,T.lani,T.longispiculus,T.ovis,T.skrjabini,Capillariabovis,C.bilobata,Ascaropsstrongylina,Fasciolahepatica,Dicrocoeliumdendriticum,D.orientalis,Eurytremapancficum,E.coelomaticum,E.ovis,E.minutum,,Ceylonocotylescoliocoelium,Ceylonocotylestreptocoelium,Cotylophoroncotylophorum,C.indicum,Fischoederiuselongatus,F.ceylonensis,Homalogasterpaloniae,Ogmocotylesikae,Ogmocotyleindica,Schistosomaturkestanica,Orientobilharziabomfordi,Monieziaexpansa,Monieziabenedeni,Avitellinacentripunctata,Helictometragiardia,Taeniahydatigena,Coenuruscerebrais,Coenurusskrjsbini,Echinococuscysticus,GansuNecropsyHaemonchuscontortus,H.similis,Mecistocirrusdigitatus,OstertagiaWeietal.(1963)trifurcate,O.ostertagi,O.erschowi,Teladorsagiacircumcincta,Yangetal.(1963)Trichostrongylusprobolurus,T.colubriformis,BunostomumYuanandLi(1990)trigonocephalum,Trichurisovina,Oesophagostomumcolumbianum,Hui(2000)O.asperum,O.kansuensis,Skrjabinemaovis,Protostrongyluskochi,Jia(2005)Strongyloidespapillosus,Gongylonemapulchrum,Chabertiaovina,Jiaetal.(2009)C.erschowi,Marshallagiamarshalli,M.mongolica,Nematodirusfilicollis,Xuetal.(2010)N.oiratianus,Protostrongyluscamereni,P.hobmaieri,P.kochi,BicauHui(2014)schulzi,Dictyocaulusfilarial,Spiculocauluskwongi,Fasciolahepatica,Li(2014)Dicrocoeliumdendriticum,D.orientalis,Eurytremapancreaticum,Ogmocotylesikae,Monieziaexpansa,Monieziabenedeni,Avitellinacentripunctata,Helictometragiardia,Taeniahydatigena,Echinococcus,Coenuruscerebralis45 HuazhongAgriculturalUniversityPhDDissertation2018QinghaiFlotationmethodsHaemonchuscontortus,Trichostrongylus,Marshallagia,Ostertagia,Ma(1982)andnecropsyBunostomumtrigonocephalum,Oesophagostomum,Nematodirus,ChenandLuoDicrocoeliumdendriticum,Fasciolahepatica,Avitellinacentripunctata,(1985)Taeniahydatigena,Monieziabenedeni,CysticercuscerebralisLietal.(2006)Zhuetal.(2013)InnerMongoliaFlotationmethodsHaemonchus,Ostertagia,Nematodirus,Trichostrongylus,Jinetal.(1986)andnecropsyOesophagostomum,Bunostomum,Trichuris,Fasciola,Moniezia,TaeniaZhuetal.(2013)hydatigenaXinjiangNecropsyChabertiaovina,C.erschowi,Trichostrongyluscolubriformis,Mamuerhanetal.Teladorsagiacircumcincta,Ostertagiatrifurcate,O.dahurica,(1988)O.occidentalis,Marshallagiamongolica,Marshallagiabrevicauda,Skrjabinemaovis,Trichurisglobulosa,Dictyocaulusfilarial,D.viviparousSouthernHillySubtropicalmonsoonGuangxiNecropsyHaemonchuscontortus,H.longistipes,Capillariabovis,TrichurisZhangandHuangChinaclimate;globulosa,T.lani,T.ovis,Bunostomumtrigonocephalum,Chabertia(1998,2001a,b,c)Tropicalmonsoonerschowi,Oesophagostomumasperum,O.columbianum,O.hupensis,Taoetal.(2010)climateO.venulosum,Dictyocaulusfilarial,Marshallagiamongolica,OstertagiaQuanetal.(2014)trifurcata,O.wuhingensis,O.wuia,Trichostrongylusaxei,T.colubriformis,Skrjabinemaovis,Dicrocoeliumdendriticum,Eurytremacladorchis,E.coelomaticum,E.pancreaticum,Fasciolagigantica,F.hepatica,Homalogasterpaloniae,Fischoederiuselongatus,F.ovatus,F.siamensis,Gastrothylaxcrumenifer,G.glandiformis,G.globoformis,Ogmocotyleindica,Calicophoronfusum,C.ovillum,C.skrjabini,C.zhejiangensis,Ceylonocotylecheni,C.sinuocoelium,Cotylophoroncotylophorum,C.indicum,C.kwantungensis,C.orthocoelium,C.sinuointestinum,GuangxiNecropsyParamphitomumcervi,P.gotoi,P.gracile,P.microbothrium,P.procaprum,Schistosomajaponicum,Ogmocotylesikae,Monieziaexpansa,Avitellinacentripunctata,Echinococuscysticus,Coenuruscerebrais,Coenurusskrjsbini,TaeniahydatigenaGuangdongFlotationmethodsHaemonchuscontortus,Trichostrongylus,Ostertagia,CooperiaandLietal.(1999)andnecropsyMarshallagiaSouthwestofMountain;SubtropicalmonsoonSichuanFlotationmethodsHaemonchuscontortus,H.similis,Ascarisovis,Chabertiaovina,Chen(1956)ChinaPlateau;climate;andnecropsyC.erschowi,Bunostomumtrigonocephalum,B.phlebotomum,Wuetal.(1965b)BasinAlpineclimateTrichostrongyluscolubriformis,T.axei,Teladorsagiacircumcincta,Yuetal.(1985)Ostertagiawuhingensis,O.ostertagi,O.trifurcata,O.skrjabin,O.orloffi,Jiangetal.Hyostrongylusrubidus,Marshallagiamongolica,M.marshalli,Cooperia(1986a,1986b,1998)laterouniformis,C.erschovi,Nematodirusfilicollis,N.oiratianus,Yang(1988)46 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceNematodirellalongispiculata,Mecistocirrusdigitatus,OesophagostomumHeandLuo(2000)radiatum,O.asperum,O.kansuensis,O.columbianum,O.venulosum,Liaoetal.(2002,Dictyocaulusviviparous,D.filarial,D.eckerti,Varestrongylus2003a,b,2006)pneumonieus,Protostrongylushobmaieri,P.raillieti,P.kocbi,P.davtiani,Zhangetal.(2014)P.rufescens,Cystocaulusocreatus,Skrjabinemaovis,Gongylonemapulchrum,Thelaziarhodesii,Setarialabiato-papillosa,S.digitata,Strongyloidespapillosus,Trichurislongispiculus,T.ovis,T.globulosa,T.concolor,T.lani,T.skrjabini,T.gazellae,Capillariabilobata,C.bovis,Schistosomajaponicum,S.turkestanicum,O.cheni,O.bomfordi,Fasciolahepatica,F.gigantica,Eurytremapancreaticum,E.coelomaticum,E.cladorchis,E.sphaeriorchis,Dicrocoeliumdendriticum,D.orientalis,D.chinensis,Platynosomumcapranum,P.ovis,Paramphistomumcervi,SichuanFlotationmethodsP.gotoi,P.ichikawai,P.microbothrium,P.gracile,Cotylophoronandnecropsycotylophorum,C.indicum,C.shangkiangensis,C.sinuointestinum,Calicophoroncalicophorum,C.skrjabini,C.fusum,Ceylonocotylestreptocoelium,C.dicranocelium,C.longicoelium,C.parastreptocoelium,C.cheini,C.sinuocoelium,C.scoliocoelium,Fischoederiussiamensis,Fischoederiuselongatus,F.japonicas,F.ovatus,F.sichuanensis,F.fischoederi,Gastrothylaxcrumenifer,G.glandiformis,Homalogasterpaloniae,Skrjabinotremaovis,Ogmocotyleindica,O.pygargi,Monieziaexpansa,M.benedeni,Avitellinacentripunctata,A.minuta,Stilesiaglobipunctata,Helictometragiardia,Echinococcusveterinarum,Coenuruscerebralis,C.skrjabini,TaeniahydatigenaYunnanFlotationmethodsHaemonchuscontortus,H.similis,Ascarisovis,Chabertiaovina,Wuetal.(1965b)andnecropsyC.erschowi,Bunostomumtrigonocephalum,B.phlebotomum,Heetal.(1999d)Trichostrongyluscolubriformis,T.axei,T.robelurus,TeladorsagiaJiangetal.(2000)circumcincta,Ostertagiawuhingensis,O.ostertagi,O.trifurcate,Lietal.(1998,O.skrjabin,O.xizangensis,O.shortodoraslrsy,Hyostrongylusrubidus,2002)Marshallagiamongolica,Cooperialaterouniformis,C.erschovi,Liaoetal.(2002,Nematodirusfilicollis,N.oiratianus,N.hsui,Mecistocirrusdigitatus,2003a,b,2006)Oesophagostomumradiatum,O.asperum,O.kansuensis,O.columbianum,Yangetal.(2007)O.venulosum,O.hubeinsis,Dictyocaulusviviparous,D.filarial,D.eckerti,Tianetal.(2008)Protostrongylushobmaieri,P.kocbi,Bicaulusschulzi,B.xinanensis,Skrjabinemaovis,Gongylonemapulchrum,Thelaziarhodesii,Setarialabiato-papillosa,S.digitata,Strongyloidespapillosus,Trichurislongispiculus,T.ovis,T.globulosa,T.lani,T.skrjabini,T.gazellae,Parabronemaskrjabini,Schistosomajaponicum,S.turkestanicum,O.cheni,47 HuazhongAgriculturalUniversityPhDDissertation2018YunnanFlotationmethodsFasciolahepatica,F.gigantica,Eurytremapancreaticum,andnecropsyE.coelomaticum,E.cladorchis,Dicrocoeliumdendriticum,Platynosomumcapranum,Paramphistomumcervi,P.gotoi,P.ichikawai,P.microbothrium,P.gracile,Cotylophoroncotylophorum,C.indicum,C.shangkiangensis,C.sinuointestinum,Calicophoroncalicophorum,C.skrjabini,C.ijimai,C.wuchengensis,C.fusum,Ceylonocotylestreptocoelium,C.dicranocelium,C.longicoelium,C.parastreptocoelium,C.cheini,C.sinuocoelium,C.scoliocoelium,Fischoederiuselongatus,F.japonicas,F.ovatus,Fischoederiussiamensis,Gastrothylaxcrumenifer,G.glandiformis,Homalogasterpaloniae,Ogmocotyleindica,O.pygargi,Monieziaexpansa,M.benedeni,M.alba,Avitellinacentripunctata,A.minuta,Helictometragiardia,Echinococcusveterinarum,Coenuruscerebralis,TaeniahydatigenaGuizhouFlotationmethodsHaemonchuscontortus,H.similis,H.longistipes,H.lunatus,ChabertiaWuetal.(1965b)andnecropsyovina,C.erschowi,Bunostomumtrigonocephalum,B.phlebotomum,Duetal.(1995)Trichostrongyluscolubriformis,T.axei,T.robelurus,T.capricola,Luo(1995)T.skrjabini,T.longispicularis,T.pietersei,Teladorsagiacircumcincta,Ruanetal.(1995a,O.ostertagi,O.trifurcate,O.skrjabin,O.orloffi,O.oceidentalis,b)O.argunica,Hyostrongylusrubidus,Cooperialaterouniformis,Fanetal.(1996)N.spathiger,Mecistocirrusdigitatus,Oesophagostomumradiatum,Yang(1996)O.asperum,O.kansuensis,O.columbianum,O.venulosum,DictyocaulusMiaoetal.(1997)viviparous,D.filarial,Protostrongylushobmaieri,P.raillieti,P.davtiani,Panetal(1997)Bicaulusschulzi,Spiculocauluskwongi,Skrjabinemaovis,ThelaziaTanetal.(1999)rhodesii,Setarialabiato-papillosa,S.digitata,Trichurislongispiculus,Qianetal.(2000)T.globulosa,T.concolor,T.lani,T.skrjabini,Capillariabilobata,Wangetal(2000)S.turkestanicum,O.cheni,O.bomfordi,Fasciolahepatica,F.gigantica,Zhaoetal.(2000)Eurytremapancreaticum,E.coelomaticum,E.cladorchis,E.fukienensis,Dongetal.(2002)Dicrocoeliumdendriticum,Paramphistomumcervi,P.gotoi,P.ichikawai,Longetal.(2002)P.microbothrium,P.gracile,P.procaprum,P.orthocoelium,Liaoetal.(2002,Cotylophoroncotylophorum,C.indicum,C.shangkiangensis,2003a,b,2006)C.sinuointestinum,C.kwantungensis,Calicophoroncalicophorum,Pengetal.(2004)C.ijimai,C.wuchengensis,Ceylonocotylestreptocoelium,Yuanetal.(2004)C.dicranocelium,C.longicoelium,C.parastreptocoelium,C.cheini,Xiaoetal.(2012)C.sinuocoelium,C.scoliocoelium,Fischoederiuselongatus,F.japonicas,Fanetal.(2013)F.ovatus,F.fischoederi,Gastrothylaxcrumenifer,G.glandiformis,Weietal.(2013)Homalogasterpaloniae,Ogmocotyleindica,O.pygargi,Monieziaexpansa,Mengetal.(2014)M.benedeni,M.alba,Avitellinacentripunctata,A.minuta,HelictometraSongetal.(2015)giardia,Echinococcusveterinarum,Coenuruscerebralis,TaeniaLuetal.(2015)hydatigena48 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTibetFlotationmethodsHaemonchuscontortus,H.similis,H.longistipes,Chabertiaovina,Wuetal.(1965b)andnecropsyC.erschowi,Bunostomumtrigonocephalum,B.phlebotomumNimaciren(1998)Trichostrongyluscolubriformis,T.axei,Teladorsagiacircumcincta,Liaoetal.(2002,O.ostertagi,O.trifurcate,O.skrjabin,O.orloffi,O.oceidentalis,2003a,b,2006)O.xizangensis,O.sinensis,O.niangingtangulaensis,O.volgaensis,Liuetal.(2016)Marshallagiamongolica,M.hsui,M.lasaensis,M.marshalli,M.orientalis,M.tarimanus,M.brevicauda,Cooperialaterouniformis,Nematodirusfilicollis,N.spathiger,N.oiratianus,N.hsui,N.abnorm,Nematodirellalongispiculata,N.gazelle,Mecistocirrusdigitatus,Oesophagostomumradiatum,O.asperum,O.kansuensis,O.columbianum,O.venulosum,Dictyocaulusviviparous,D.filarial,Protostrongylushobmaieri,P.raillieti,P.kocbi,Bicaulusschulzi,Spiculocauluskwongi,Cystocaulusnigrescens,C.nigrescens,Muelleriuscapillaris,Skrjabinemaovis,Gongylonemapulchrum,Thelaziarhodesii,Trichurislongispiculus,T.ovis,T.globulosa,T.concolor,T.lani,T.skrjabini,T.gazellae,S.turkestanicum,O.cheni,O.bomfordi,Fasciolahepatica,F.gigantica,Dicrocoeliumdendriticum,D.orientalis,D.chinensis,Paramphistomumcervi,P.procaprum,Skrjabinotremaovis,Ogmocotyleindica,Monieziaexpansa,M.benedeni,M.alba,Avitellinacentripunctata,Helictometragiardia,Echinococcusveterinarum,Coenuruscerebralis,C.skrjabini,TaeniahydatigenaChongqingFlotationmethodsHaemonchuscontortus,Chabertiaovina,C.erschowi,BunostomumLietal.(2001)andnecropsytrigonocephalum,Hyostrongylusrubidus,C.erschovi,OesophagostomumTangetal.(2003a)radiatum,O.asperum,O.kansuensis,Dictyocaulusviviparous,D.filarial,Tangetal.(2003b)D.eckerti,Varestrongyluspneumonieus,Gongylonemapulchrum,ThelaziaLiaoetal.(2002,rhodesii,Setarialabiato-papillosa,S.digitata,Trichurislongispiculus,2003a,b,2006)T.ovis,T.globulosa,T.concolor,T.lani,Capillariabilobata,C.bovis,Yang(2005)Schistosomajaponicum,O.cheni,O.bomfordi,Fasciolahepatica,Wuetal.(2007)F.gigantica,Eurytremapancreaticum,E.coelomaticum,E.cladorchis,Zhuetal.(2013)Dicrocoeliumdendriticum,D.orientalis,D.chinensis,PlatynosomumMengetal.(2014)capranum,P.ovis,Paramphistomumcervi,P.gotoi,P.ichikawai,Chenetal.(2015)P.microbothrium,P.gracile,Cotylophoroncotylophorum,C.indicum,C.shangkiangensis,C.sinuointestinum,Calicophoroncalicophorum,Ceylonocotylestreptocoelium,C.dicranocelium,C.longicoelium,C.parastreptocoelium,C.cheini,C.sinuocoelium,C.scoliocoelium,Fischoederiuselongatus,F.japonicas,F.ovatus,F.sichuanensis,F.fischoederi,Gastrothylaxcrumenifer,G.glandiformis,Homalogasterpaloniae,Ogmocotyleindica,Monieziaexpansa,M.benedeni,Avitellinacentripunctata,Taeniahydatigena49 HuazhongAgriculturalUniversityPhDDissertation2018CentralChinaPlain;TemperatemonsoonHenanFlotationmethodsHaemonchuscontortus,H.similis,Ascarisovis,Strongyloidespapillosus,Wangetal.(2005)Hillyclimate;andnecropsySkrjabinemaovis,Chabertiaerschowi,C.ovina,OesophagostomumNingetal.(2011)Subtropicalmonsooncolumbianum,O.venulosum,O.asperum,O.radiatum,O.kansuensis,Zhaoetal.(2011)humidclimateBunostomumtrigonocephalum,Trichostrongylusaxei,T.colubriformis,Lietal.(2012)Cooperiaoncophora,C.lanchowensis,Marshallagiamarshalli,Zhuetal.(2013)M.mongolica,Mecistocirrusdigitatus,Nematodirellalongispiculata,FangandWangNematodirusabnormalis,N.filicollis,N.spathiger,N.longispiculata,(2014)N.oiratianus,Teladorsagiacircumcincta,Ostertagiaerschowi,Caoetal.(2015)O.heterospiculagia,Ostertagiaostertagi,O.trifurcate,O.wuhingensis,Suietal.(2015)Metastrongylidaeapri,Dictyocaulusfilarial,D.eckerti,Protostrongylushobmaieri,Muelleriuscapillaris,M.minutissimus,Gongylonemapulchrum,Parabronemaskrjabin,Trichurisglobulosa,T.concolor,T.gazellae,T.lani,T.longispiculus,T.ovis,Fasciolahepatica,F.gigantica,Paramphistomumcervi,P.gotoi,Gigantocotyleformosanum,Cotylophoroncotylophorum,C.indicum,Ceylonocotyledicranocoelium,C.parastreptocoelium,C.scoliocoelium,C.streptocoelium,Macropharvnxchinensis,Homalogasterpaloniae,Gastrothylaxcrumenifer,G.glandiformis,Fischoederiuselongatus,F.japonicas,F.ovatus,Eurytremapancreaticum,E.coelomaticum,E.cladorchis,Dicrocoeliumdendriticum,Monieziaexpansa,M.benedeni,Helictometragiardia,Coenurusskrjabini,TaeniahydatigenaHubeiFlotationmethodsFasciolahepatica,F.gigantica,Eurytremapancreaticum,LSCSHPetal.andNecropsyParamphistomumcervi,Homalogasterpaloniae,Dictyocaulusfilarial,(1981)Haemonchuscontortus,Ostertagiaskrjabini,O.wuhingensis,OstertagiaCaoetal.(1995)ostertagi,Cooperialaterouniformis,Cooperiapectinata,TrichostrongylusChengetal.(2014)colubriformis,T.axei,Capillaria,Nematodirus,Bunostomumtrigonocephalum,Strongyloidespapillosus,Oesophagostomumasperum,O.hubeinsis,Oesophagostomumcolumbianum,Monieziaexpansa,Taeniahydatigena,CoenurusHunanFlotationmethodsHaemonchuscontortus,H.placei,H.similis,Trichostrongylusaxei,Fuetal.(1999)andnecropsyT.colubriformis,Trichostrongylusprobolurus,Teladorsagiacircumcincta,WangandHeBunostomumtrigonocephalum,Ostertagia,Oesophagostomum(2005)columbianum,O.kansuensis,Ostertagiatrifurcate,Cooperiaerschovi,Liuetal.(2006)C.laterouniformls,Nematodirusspathiger,Marshallagia,TrichurisLiangetal.(2007)globulosa,T.concolor,T.gazellae,T.lani,T.longispiculus,T.ovis,Heetal.(2008)Ancylostoma,Chabertiaovina,C.erschowi,OesophagostomumTangetal.(2008)columbianum,O.asperum,Mulleriuscapillaris,Setariadigitata,Daietal.(2009)Protostrongyluskochi,Strongyloidespapillosus,Dictyocaulusfilarial,Lietal.(2013)Paramphistomum,Fasciolahepatica,Dicrocoelium,HomalogasterChengetal.(2014)paloniae,Eurytremapancreaticum,E.coelomaticum,FischoederiusLiu(2014)Maetal.(2014)50 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceHunanFlotationmethodselongatus,Gastrothylaxcrumenifer,Ogmocotyle,Monieziaexpansa,Zhouetal.(2015)andnecropsyM.benedeni,Avitellinacentripunctata,Taeniahydatigena,CysticercusHouetal.(2016)cerebralis,CoenurusEastChinaPlain;MonsoonclimateofShangdongFlotationmethodsHaemonchuscontortus,Bunostomum,Oesophagostomum,Strongyloides,Cheng(1989)Hillymediumlatitudes;Fasciola,MonieziaLietal.(2006)SubtropicalmonsoonZhouetal.(2006)climateWangandCao(2008)JiangsuFlotationmethodsHaemonchuscontortus,Trichostrongyluscolubriformis,OesophagostomumWang(1963)andnecropsyasperum,O.venulosum,O.columbianum,Trichurisglobulosa,T.ovis,ZhangandTianStrongyloidespapillosus,Ostertagia,Nematodirus,Bunostomum(1986)trigonocephalum,Capillariabovis,Paramphistomumcervi,HomalogasterPengandKuangpaloniae,Fasciolahepatica,F.gigantica,Monieziaexpansa,Avitellina,(1994)Helictometra,Taeniahydatigena,CoenuruscerebralisPengetal.(1994)Chenetal.(2000a)Chenetal.(2000b)Guietal.(2010)AnhuiFlotationmethodsHaemonchuscontortus,H.similis,Teladorsagiacircumcincta,Taoetal.(2001)andNecropsyTrichostrongyluscolubriformis,Trichuris,Cooperia,OstertagiaLiandLiu(2004)wuhingensis,Chabertiaovina,Bunostomumtrigonocephalum,Lu(2004a,2004b)Oesophagostomumasperum,O.columbianum,Strongyloidespapillosus,Yangetal.(2008)Marshallagia,Nematodirus,Skrjabinemaovis,Dictyocaulusfilaria,Jinetal.(2011)Protostrongylushobmaieri,P.kochi,Spiculocauluskwongi,Lietal.(2012)Paramphistomumcervi,P.gotoi,Cotylophoroncotylophorum,C.indicum,Zhuetal.(2013)Calicophoronovillum,Ceylonocotyledicranocoelium,ChenocoeliumGuetal.(2014)kingsiensis,Homalogasterpaloniae,Carmyeriussynethes,Gastrothylaxcrumenifer,Fischoederiuselongatus,F.compressus,F.ovatus,Eurytremapancreaticum,E.coelomaticum,E.cladorchis,Dicrocoeliumdendriticum,Fasciolahepatica,F.gigantica,Schistosomajaponicum,Monieziaexpansa,Monieziabenedeni,Helictometragiardia,TaeniahydatigenaShanghaiFlotationmethodsHaemonchuscontortus,Oesophagostomumasperum,O.columbianum,Heetal.(1999a)andnecropsyO.venulosum,Trichurisovis,Spiculocauluskwongi,Nematodirus,ShenandChangSchistosomajaponicum,Paramphistomumcervi,Eurytremapancreaticum,(1999)Monieziaexpansa,MonieziabenedeniandTaeniahydatigenaShenetal.(2001)ZhejiangNecropsyHaemonchuscontortus,Trichuris,Oesophagostomum,Marshallagia,Zhang(1984)Nematodirus,Trichostrongylus,Bunostomum,Paramphistomum,Zhangetal.(1988)Giantocotylenanhuense,Fasciolahepatica,Avitellinacentripunctata,Jinetal.(2011)Monieziaexpansa,MonieziabenedeniandCoenurus51 HuazhongAgriculturalUniversityPhDDissertation2018JiangxiFlotationmethodsHaemonchuscontortus,Ostergagia,Bunostomum,Oesophagostomum,Jianetal.(1993)andnecropsyMarshallagia,Chabertia,Strongyloidespapillosus,Trichurid,Dictyocaulus,Fasciolahepatica,F.gigantica,Paramphistomum,EurytremapancreaticumandMonieziaFujianFlotationmethodsHaemonchuscontortus,Oesophagostomumcolumbianum,O.asperum,LinandLin(1996)andnecropsyTrichostrongylus,Ostertagia,Chabertia,Strongyloidespapillosus,Fang(1998)Paramphistomum,Fasciolahepatica,Dicrocoelium,Monieziaexpansa,Monieziabenedeni,Eurytremapancreaticum,andTaeniahydatigena52 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceForrecombinantsubunitvaccine,YanandLi(2005)expressedthreeaminopeptidaseH11genes(H11-1,H11-2andH11-3)togetherinPichiapastoris,however,protectiontestwasnotavailable;then,YanandLi(2006)expressedtheaminopeptidaseH11forms(H11-1andH11-2)inEscherichiacoli,andfoundtheaminopeptidaseenzymeactivityofH11-1issignificantlylowerthanthatofH11-2;subsequently,Yanetal(2007)immunedthegoatswithrecombinantofH11-1,H11-2andH11-1plusH11-2expressedinE.coli,andtheresultindicatedH11-1plusH11-2immunedgroupprovidedpartialprotectionwith29%reductiononfaecaleggcountand18%reductiononwormburdenofadults;later,CaenorhabditiseleganswasusedtoexpressH11(Zhouetal.,2010),Zhouetal(2014c)appliedtheC.eleganssystemstoexpressafragmentofH11namedTrans-HPS,andthegroupimmunedwiththerecombinedTrans-HPSindicated38%reductiononfaecaleggcountand25%reductiononwormburdenofadultsbyimmunedexperimentingoats.ExceptforH11gene,galectinisolatedfromH.contortusadultswasalsotested,Sunetal(2007)expressedthegalectininE.coli,therecombinedgalectinimmunedgroupshowed48%reductiononfaecaleggcountand46%reductiononwormburdenofadultsbyimmunedexperimentingoats.DNAvaccinationisamethodbyimmuningtheanimalswithtransgenicDNAwhichcanbeexpressedinimmunedanimals.Comparedwithconventionalvaccines,DNAvaccinescaninducelong-periodimmunereseponseinanimals(Gurunathanetal.,2000).Recently,aminopeptidaseH11(Zhaoetal.,2012),glutathioneperoxidaseHC29(Sunetal.,2011),glyceraldehyde-3-phosphatedehydrogenase(GAPDH)(Hanetal.,2012),gadisorganizedmusclefamilymember1(Dim-1)(Yanetal.,2013)andactin(Yanetal.,2014)havebeenappliedinDNAvaccineforgoatsagainstH.contortusinfectionwith34-57%reductiononfaecaleggcountand33-47%reductiononwormburdenofadults.AlthoughthereisstillnoavailablevaccineswidelyusedagainstHaemonchuscontortusinChina,theprospectforvaccinestoeffectivelyprotectanimalsandsignificantlyreducetheusageofanthelminticsisobvioussinceacommercializedvaccine(BarbervaxR)availableinAustraliacansignificantlyreduceFECsinMerinosheep(Nisbetetal.,2016),andhavebeentestedinAustralia,Brazil,Merida,SouthAfrica,SwitzerlandandTanzania(www.moredun.org.uk).53 HuazhongAgriculturalUniversityPhDDissertation20181.7.2AnthelminticsDuetotheabsentofeffectivevaccines,themainwayforthepreventionandcontrollingofparasitichelminthsingoatsstillbasesontheusageofchemicalanhelmintics(Li,2006;Tayloretal.,2016).Benzimidazoles,imidazothiazoles,macrocycliclactones,amino-acetonitrilederivativesandcyclooctadepsipeptidesarethefivemainclassesofanthelminticdrugsingoats(Roeberetal.,2013a).Exceptforamino-acetonitrilederivativesandcyclooctadepsipeptides,theremainingthreeclasseshavebeenwidelyusedfordeworminginChina.Amongtheseanthelmintics,benzimidazoleswasthefirstlyusedanthelminticdruginChina,becauseoflong-termandunreasonableusage,benzimidazolesresistanceforgastrointestinalnematodeshavebeenreportedinsomeprovinces(Guetal.,1999;Heetal.,1999b;Heetal.,1999c;Caietal.,2007;Zhangetal.,2016).Recently,ivermectin(Macrocycliclactones)iswidelyused,however,insomeareas,dewormingswerenotidealunderorovertherecommendeddosage(e.g.,Cai,2007;Chen,2015;Fengetal.,2016).Usually,farmerswilldeworminAprilbeforethespringtideofparasitesandOctoberbeforethewinter,alsosomefarmerswilldewormwhentheysuspectthattheirgoatsmaybethreatenbyparasiticdiseases.Duetotheabsentofexperiencedtechniciansinthebreeding,preventionandtreatmentofparasitic,infectiousandgeneraldiseasesarealwaysbasedonpersonalcommunicationwithotherexperiencedfarmersand/orlocalveterinarianfromanimalhusbandryandveterinarystation.Atthesametime,farmersalwayschoosedrugsbasedonpersonalexperience,communicationswithotherexperiencedfarmers,aswellasconsideringthecost.Forsomefarmers,themoredrugtheyuse,thebetterresultstheycanget(personalcommunicationwithZhangSS,ZhangYHandZhouQZin2016).1.7.3ManagementInChina,managementofgoatbreedingmainlybaseson“homemodel”,farmersusuallybuildsheepfoldsforlessthan200goatsclosetotheirhousesforconvenientmanagement.Usually,farmerwillfeedtheirgoatsathillside,naturepasturesoralongriverswithplentyofgrassonsunnydays,andsupplytheirgoatswithhay,straworvegetablesbasedonlocalconditions,however,rotationalgrazingisalmostunavailable,andmostfarmerssharepastureswithothers,thusre-infectionarecommonandparasitesareeasytospreadamonggoatsfromdifferentfarmers.Duetolackofscientific54 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincemanagementandtechniques,theefficiencyofbreedingislimited,diseasescausedbyparasitesarecommoningoatsandcanleadtoconsiderableeconomiclossestothegoatbreedingindustry(e.g.,Chen,2016;Xie,2016),thusthe“homemodel”isrelativelybad-managedforcontrollinghelminths,goatbreedingalongtheHanRiverinZhanggang,HubeiProvinceisatypical“homemodel”.Withtheincreasingneedsforeffectivelybreedinggoats,somefarmersgettogethertoestablishcooperativesforsharingmanagementexperiencetoimproveefficiencyofbreedinggoats,atthesametime,therearealsoprofessionalcooperativesofforestryandanimalhusbandrythatcanprovidebreedsandtechnicalsupportsfortheircommunemembersinbreeding.Thus,this“cooperativemodel”isrelativelywell-managedcomparedwiththe“homemodel”,e.g.,goatbreedingalongtheDabieMountaininLuotian,HubeiProvinceisatypical“cooperativemodel”.However,the“homemodel”isstillthemajorapproachfordewormingingoatbreedingcurrently(Chen,2016;Xie,2016).Inconclusion,thedewormingstrategiesforgoatsareblindtosomeextentandlackofguidanceinsomeregionsinChina,what’smore,overrecommendeddosageindewormingcanacceleratethedevelopmentofdrugresistance.Thus,moresuggestionsareneededforfarmerstocontrolhelminthsbasedonparasitesinfection,climateandmanagementingoatseffectively.Besidestheapplicationofanthelmintics,grazingmanagement,nutritionalmanagement,biologicalcontrol,alternativeanthelminticcompounds,combinedmedicationandvaccinesarealsousefulandcanbeconsideredincontrolstrategies(Besieretal.,2016).1.8DrugresistanceinparasitichelminthsofgoatsinChinaBenzimidazoles,imidazothiazoles,macrocycliclactones,amino-acetonitrilederivativesandcyclooctadepsipeptidesweremainanthelminticsappliedinthetreatmentofnematodesinfectioninlivestock(Roeberetal.,2013a).Duetolong-termandwideuseofanthelmintics,drugresistanceisbecomingmoreandmoreprominent(e.g.,Kaplan,2004;Zhaoetal.,2010;KaplanandVidyashankar,2012).What’smore,unreasonalapplicationofanthelminticsacceleratethedevelopmentofdrugresistance(Kaplan,2004).Benzimidazoleisthefirstdrugappliedinthecontrollingofnematodesinruminants,andhavebeenusedforoverhalfacentury.Inrecentyears,diagnositicmethods,includingfaecaleggcountreductiontest,controlledtest,egghatchassay,larvaldevelopmenttest,larvalparalysisandmigrationtestsandPCR-basedmethod,wereappliedinthe55 HuazhongAgriculturalUniversityPhDDissertation2018investigationofdrugresistanceinpractice,andprovedtheexistenceofanthelminticresistanceinChina.Inthissection,themechanism,diagnosticmethodsaswellascurrentconditionforanthelminticresistancewerereviewedtoprovideusefulinformationforunderstandinganthelminticresistanceofgoatsinChina.1.8.1Mechanismofdrugresistance1.8.1.1MechanismofbenzimidazoleresistanceThiabendazolewasthefirstbenzimidazolesusedinpracticesince1960s(Brownetal.,1961).Fromthenon,manydrugsfrombenzimidazolesclasswithroadspectrum,highefficiencyandlowtoxicity,suchasalbendazole,fenbendazoleandoxfendazole,weresynthesisandappliedindeworming.Benzimidazolesbelongtocelltubulininhibitors,itcancombinewithtubulinofnematodestoinhibitpolymerizationofthetubulins,thenaffectcellularmetabolismincludingproteinassembly,mitosisandenergymetabolismsincetubulinsarebasicunitfororganelles,thenthewormswilldie(Kohler,2001).Themechanismofbenzimidazoleresistanceiswell-understood,previousstudiesindicatedthreesinglenucleotidepolymorphismoftypeIβ-tubulingenewererelatedwiththeemergenceofbenzimidazoleresistance,namelyatcodons167(TTCtoTAC;F167Y)(SilvestreandCabaret,2002),198(GAAtoGCA;E198A)(Ghisietal.,2007;Rufeneretal.,2009)and200(TTCtoTAC;F200Y)(Kwaetal.,1994;Kwaetal.,1995)oftypeIβ-tubulinprotein-codinggene.1.8.1.2MechanismofimidazothiazolesresistanceImidazothiazolesaffectthenicotinicacetylcholinereceptor(nAChR)bysimulatingtheactionofacetylcholine,thentheacetylcholinegatedionchannelcanbeopened,cationentersintothecellandleadtocontinuousmusclecontractionparalysisofworms,paralyticwormsareexpelledtotheoutsideofthehostbytheperistalsisofthegastrointestinaltract(Roeberetal.,2013c).Tillnow,themechanismofimidazothiazolesresistanceispoor-understood.Therearethreemainaspectsmayberelatedwiththeemergencyofimidazothiazolesresistance:thefirstaspectistheexpressionoftruncatednAChRsubunitgene.Fauvinetal(2010)comparativelyanalysedthetranscriptionalleveloflevamisoleresistantandsusceptibleHaemonchuscontortus,foundthecoexpressionofHco-acr-8geneandtruncatedmutant56 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceHco-acr-8bonlyexistedintheresistantstrain.Neveu(2010)foundthecoexpressionofHco-unc-63geneandtruncatedmutantHco-unc-63binlevamisoleresistantHaemonchuscontortus,TeladorsagiacircumcinctaandTrichostrongyluscolubriformis,theexistenceofHco-unc-63bcanblockthefunctionofnAChR,thuscauseresistantphenotype;thesecondaspectisthedecreaseintranscriptionlevelsofnAChRsubunitgene.Saraietal(2013)foundthedecreaseintranscriptionlevelsofHco-unc-63andfourhomologousgeneofHco-unc-29inlevamisoleresistantHaemonchuscontortus.Later,Saraietal(2014)foundthesignificantdecreaseintranscriptionlevelsofHco-acr-8a,Hco-unc-26,Hco-unc-29.2,Hco-unc-29.4,Hco-unc-63aandHco-unc-63b;thethirdaspectisthedecreaseintranscriptionlevelsofhelperproteingenes.Saraietal(2013;2014)foundthesignificantdecreaseintranscriptionlevelsofHco-ric-3.1,Hco-ric-3.2,Hco-unc-50andHco-unc-74thatresponsiblefortheassemblingofnAChR.Althoughsomemolecularmechanismsarefoundtoberelatedwithimidazothiazolesresistance,thereisstillnomolecularmechanismthatcanappliedinallresistantpopulations.1.8.1.3MechanismofmacrocycliclactonesresistanceMacrocycliclactonesisaclassofanthelminticswidelyusedintheeliminationofendoparasiteandectoparasites,includingivermectin,doramectinandmoxidectin.Thetargetofmacrocycliclactonesisglutamategatedchloridechannelreceptor(GluClR),thedrugirreversiblestimulustheGluClRtokeepopen,then,leadtotheinfluxofchloridionintoneuronandinhibittheactivityofneuronsandthecontractilityofmuscles,wormswillshowflaccidparalysisanddie(Cullyetal.,1994).OtherstudiesindicatedGABAClRandGlyClRwerealsothetargetofmacrocycliclactones,buttheyneededhigherconcentrationofdrugtobeactivatedcompairedwithGluClR,thusGluClRiscommonlyrecognizedasthemaintarget(Adelsbergeretal.,2000).Themechanismofmacrocycliclactonesresistanceiscomplicated,andstillpoorlyunderstood.Therearefivecommonopinionsonthisissue:thefirstisrelatedwiththegenemutationofGluClR(Blackhalletal.,1998b;Njueetal.,2004;Mccaveraetal.,2009);thesecondiscausedbythatthemutationofglycineresidueinthethirdtransmembraneregionofreceptor(M3-Gly)canreducethesensitivityofivermectin(LynaghandLynch,2010;LynaghandLynch,2012);thethirdiscausedbythechangesoftranscriptionallevelofGluClR(El-Abdellatietal.,2011;Williamsoetal.,2011;Martinez-Valladaresetal.,2012);thefouthiscausedbygenediversityofdrugefflux57 HuazhongAgriculturalUniversityPhDDissertation2018pumpPglycoprotein(Pgp)(Blackhalletal.,1998a;Sangsteretal.,1999);andthefifthistheincreasingoftranscriptionallevelofPgp(Dickeretal.,2011;Williamsoetal.,2011).1.8.1.4Mechanismofamino-acetonitrilederivativesresistanceMonepantelisanewanthelmintcsscreenedfrommorethan700amino-acetonitrilederivatives,andcaneffectivelycontrollingmultidrugresistantparasiticnematodes(Kaminskyetal.,2008a;Kaminskyetal.,2008b).ThetargetofmonepantelisnicotinicacetylcholinereceptorofmuscleornervecellwithMPTL-1subunit.Monepantelsensitivereceptorkeepssustainableopeningafterthecombinationofdrugandreceptor,cationflowsintocellsandleadstopersistentdepolarizationofmuscleorneuroncellmembrane(Kotzeetal.,2014).Kaminskyetal(2008a)foundmonepantelresistantstrainhadnAChRwithHco-des-2HsubunitmutationorHco-acr-23Hsubunitmutationbysuccessivegenerationsofdrugscreening,however,thesewerenotobservedinpractice.1.8.1.5MechanismofcyclooctadepsipeptidesresistanceForcyclooctadepsipeptides,itmayfunctionbybindingtoapresynapticlatrophilinreceptorinnematodes(Harderetal.,2003;Harderetal.,2005).However,theproposedmechanismofcyclooctadepsipeptidesresistanceisstillunknown.1.8.2Diagnosticmethodsfordetectingdrugresistance1.8.2.1FaecaleggcountreductiontestFaecaleggcountreductiontest(FECRT)isthecommonestdiagnosticmethodfordrugresitance,andisalsotherecommendedmethodfortheWorldAssociationfortheAdvancementofVeterinaryParasitology(WAAVP)(Woodetal.,1995).FECRTisappliedindetectionbyassessingtheeggburdenoffaecesbeforeandafterdeworming,it’snotablethattheresultcanbereliableonlywhenthenumberofresistantwormsisover25%ofthetotalworms.Forthismethod,whentheFECRT<95%andCI95%<90%,itindicatestheexistenceofdrugresistance.However,theFECRTisaffectedbyaspectsincludingspecies,host,druganddetectiontime.Meanwhile,themethodiscostlyandlackofaccuracyandsensitivity(Colesetal.,1992).58 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince1.8.2.2ControlledtestControlledtestisthemostreliablemethodforassessingresistance,butalsomostlabor-consumingandtime-consuming,andneedhighcostscompairedwithothermethods.Currently,themethodisseldomlyused.Inbriefly,theinfectiveeggswereusedtoinfectparasite-freeanimals,then0.5,1and2timesofrecommenddosagewereappliedindeworming,respectively.Drugresistanceisconfirmedifthewormreductionrateislessthan90%orover1000wormssurviveaftertreatment(Tayloretal.,2002).1.8.2.3EgghatchassayEgghatchassay(EHA)isamethodappliedinthedetectionofbenzimidazolesresistance.EHAissuitablefordetectiontheresistanceofwater-solubledrugs.Byinhibitingegghatch,EHAcanqualitativelyandquantificationallyassesstheeffectofdrugsonfirst-stageorthird-stagelaevae.Inbrief,infectiveeggsareincubatedindrugswithdifferentconcentration,then,egghatchrateofeachconcentrationiscalculatedafterapermanenttime,oneblankcontrolwithoutdrugisincluded.DoseeffectcurveismadetocalculateED50,apopulationisprovedtoberesistantwhenED50isover0.1μg/ml.Thedisadvantageisthattheresultcanbereliableonlywhentheratioofresistantwormsisover25%(Colesetal.,1992;Colesetal.,2006).1.8.2.4LarvaldevelopmenttestLarvaldevelopmenttest(LDT)isbasedonthedevelopmentofeggsintothird-stagelarvaeindifferentconcentrationofdrugtoassessdrugresistance.Itcanbedevidedintotwotypesbasedonthethetypesofmedium,oneisliquidmedium(LDT),andtheotherisagarmedium(MALDT).Colesetal(1988)firstlydetectedbezimidazolesresistancebyLDT,however,thismethodcannotbeappliedinthedetectionoflevamisoleandivermectinresistanceintheliquildmediumwithEscherichiacoli.Tayloretal(1990)improvedthemediumwithyeastextract,anddetectedtheresistanceoflevamisoleandivermectin.HubertandKerboeuf(1992)usedyeastextractandbacteriumstofeedlarvae,andcantheoreticallydetecttheresistanceofbenzimidazoles,imidazothiazolesandmacrocycliclactones.Amaranteetal(1997)foundapplicationofavermectinanaloguescouldhelptodetectthemacrocycliclactonesresistance.Colesetal(2006)developedMALDTmethod,andsuccessfullydetectedtheivermectinresistanceinpractice.Through59 HuazhongAgriculturalUniversityPhDDissertation2018continuoustests,LDTcanbeappliedinthedetectionofresistanceforalmostallthedrugsinpractice.1.8.2.5LarvalparalysisandmigrationtestMartinandJambre(1979)developedalarvalparalysistestforthedetectionoflevamisoleandmorantelresistanceinOstertagia.Duringthetest,theinfectivethird-stagelarvaearetreatedwithdifferentconcentrationofanthelminticsfor24hours,thencountthenumberofparalyticlarvaeindifferentconcentration,drawdoseeffectcurveandcomparewithreferencedstrain.Themethodmustchooselarvaeinsuitabledevelopmentalstage,orthelarvaecanrecoveryfromparalysisandtherepeatabilityreduces.SutherlandandLee(1990)detectedthiabendazoleresistanceundertheexistenceofacetylcholineinhibitors,theresultindicatedthesensitivelarvaewereeasiertoproduceparalysisthanresistantones.Later,BennettandPax(1986)developedmicroactivitytestertodetecttheactivitychangeoflarvaeandadultsaftertreatedwithanthelmintics.Gilletal(1991)appliedthetestertodetectivermectinresistanceofthird-stagelarvaeofHaemonchuscontortus,andfoundtherelevancebetweentheconcentrationofanthelminticsandactivityoflarvae.1.8.2.6PCR-basedmethodUntilnow,onlythemechanismofbenzimidazolesresistancewaswellunderstood,threesinglenucleotidepolymorphisms(designatedF167Y,E198AandF200Y)ofthetypeIβ-tubulingenecausesstructuralchanges,therefore,thedrugcan’tbindtothetargetsite(Lacey,1988;Winterrowdetal.,2003).Colesetal(2006)establishedanallelespecificPCR(AS-PCR)methodtodetecttheF200YofthetypeIβ-tubulingeneofHaemonchuscontortus,TrichostrongyluscolubriformisandTeladorsagiacircumcincta.Tiwarietal(2006)designedarestrictionfragmentlengthpolymorphism-polymerasechainreaction(RFLP-PCR)todetecttheF200YofthetypeIβ-tubulingeneofHaemonchuscontortus.WhenthecodonschangedfromTTCintoTAC,therewouldbearestrictionsiteforTaaI,itcanbeusedtodistinguishthesensitivetypeandresistanttype.Ghisietal(2007)foundtherewouldbearestrictionsiteforHpyCH4-Vwhenthe198thaminoacidofthetypeIβ-tubulingeneofHaemonchuscontortuschangedfromGlutamate(GAA)intoAlanine(GCA).Basedonthisreason,aPCR-RFLPbasedonE198AwasestablishedtodetectbenzimidazoleresistanceinHaemonchuscontortus.Von60 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceSamson-Himmelstjernaetal(2009)appliedpyrosequencingtoquantitativelydetectthebenzimidazoleresistanceofHaemonchuscontortuspopulations,andprovedtobereliableinfieldtestandhavehighersensitivitythanEHA.Niciuraetal(2012)developedanested-PCR(TetraPrimerARMS-PCR)todetecttheallelefrequencyofF200Y,andcandetecthomozygousresistanttype,heterozygousresistanttypeandsensitivetypewithinonereaction.DosSantosetal(2014)detectedthebenzimidazoleresistanceoffiveHaemonchuscontortuspopulationsusingFECRT,EHAandPCR-RFLP,theresultsindicatedthefrequencyofF200YandF167Ywerehigh,butnoE198Awasdetected.PCR-basedpyrosequencingisalsoausefulmethodfordetectingresistance,andcandirectlytesttheallelefrequencyofF167Y,E198AandF200Ywithinpopulationlevel.1.8.3CurrentdrugresistanceinChinaAnthelminticresistanceisaglobalproblem,forrecentyears,testsonanthelminticresistancehavebeenappliedinFujian,Guangxi,Hebei,Heilongjiang,Hubei,Hunan,InnerMongolia,Jiangsu,Liaoning,Ningxia,Shanghai,Shaanxi,Sichuan,XinjiangandYunnan(Table1-9).Untilnow,benzimidazolesresistancehavebeenreportedingastrointestinalnematodesofgoatsinFujian(Linetal.,2016),Heilongjiang(Heetal.,1999b),Hunan(Chengetal.,2015),Guangxi(Zhangetal.,2016),InnerMongolia(Zhangetal.,2016),Jiangsu(Guetal.,1999),Liaoning(Zhangetal.,2016),Ningxia(Caietal.,2007),Shaanxi(Fengetal.,2016),Shanghai(Heetal.,1999c),Xinjiang(Zhaoetal.,2010)andYunnan(Zhangetal.,2016)ofChina(Table1-9),especiallyforHaemonchuscontortus(Heetal.,1999c;Zhangetal.,2016).Meanwhile,Fengetal(2016)reportedivermectinresistanceofHaemonchuscontortusbyFECRTatagoatfarminShaanxiProvince,China;Chengetal(2015)reportedivermectinresistanceofgastrointestinalnematodesandlevamisoleresistanceofHaemonchuscontortusinHunanProvince,China.Morestudiesurgentlyneedtobeperformedtocomprehensivelyunderstandthecurrentstateofanthelminticresistanceingoats.61 HuazhongAgriculturalUniversityPhDDissertation2018Table1-9:AnthelminticresistancedetectedingastrointestinalnematodesofgoatsinChinaRegionAnthelminticsMethodResistancestatusResistantspeciesReferenceFujianAlbendazoleFECRTRHaemonchuscontortusLinetal(2016)IvermectinFECRTSLevamisoleFECRTSAlbendazoleplusFECRTSIvermectinGuangxiBenzimidazolesPCRandSequencingRHaemonchuscontortusZhangetal.(2016)HebeiBenzimidazolesPCRandSequencingSZhangetal.(2016)HeilongjiangAlbendazoleFECRTRGastrointestinalnematodesHeetal.(1999b)HeilongjiangBenzimidazolesPCRandSequencingSZhangetal.(2016)HubeiBenzimidazolesPCRandSequencingSZhangetal.(2016)HunanAlbendazoleEHTRGastrointestinalnematodesChengetal.(2015)IvermectinEHTRGastrointestinalnematodesLevamisoleEHTRHaemonchuscontortusInnerMongoliaAlbendazoleEHTSHaoetal.(2007b)InnerMongoliaBenzimidazolesPCRandSequencingRHaemonchuscontortusZhangetal.(2016)JiangsuAlbendazoleEHTRGastrointestinalnematodesGuetal.(1999)JiangsuAlbendazoleEHTSWangetal.(2000)LiaoningBenzimidazolesPCRandSequencingRHaemonchuscontortusZhangetal.(2016)NingxiaAlbendazoleFECRTRGastrointestinalnematodesCaietal.(2007)NingxiaBenzimidazolesEHA,PCR-SSCPRHaemonchuscontortusHaoetal.(2007a)62 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceShanghaiAlbendazoleEHTRHaemonchuscontortusHeetal.(1999c)ShanghaiLevamisoleEHTSXiaandWang(2010)ShaanxiAlbendazoleFECRTRHaemonchuscontortusFengetal.(2016)IvermectinFECRTRHaemonchuscontortusShaanxiBenzimidazolesPCRandSequencingSZhangetal.(2016)SichuanAlbendazoleEHTRGastrointestinalnematodesLuo(2005)XinjiangAlbendazoleEHTRGastrointestinalnematodesYueetal.(2006)XinjiangAlbendazoleEHTROstertagiaspp.PuandYue(2009)XinjiangAlbendazoleEHTRGastrointestinalnematodesZhan(2010)XinjiangAlbendazoleEHTRTrichostrongyloidnematodesZhaoetal.(2010)YunnanBenzimidazolesPCRandSequencingRHaemonchuscontortusZhangetal.(2016)Note:EHT-egghatchtest;FECRT-faecaleggcountreductiontest;PCR:polymerasechainreaction;SSCP:singlestrandconformationpolymorphism;R-resistant;S-susceptible63 HuazhongAgriculturalUniversityPhDDissertation2018HubeiProvincelocatesinthecentralofChina,plentyofwaterresourcesandvegetationresourcescanprovidesuitableconditionforlivestockbreedingindustry,includinggoatbreeding.BenzimidazolesandmacrocycliclactonesarecommonlyusedanthelminticsinthepreventionandcontrollingofgastrointestinalnematodesingoatsinHubeiProvince,especiallyforivermectinforrecentperiods.However,duetotheabsentofcomprehensiveinvestigations,thereisstillnoreportaboutanthelminticresistanceinHubeiProvince.Moreover,insomeareas,effectofanthelminticsindeworming,suchasivermectin,isnotgoodevenwithseveraltimesrecommenddosage,thus,drugresistancemayalsoexistinHubeiProvince,butitneedstobeconfirmedinfieldexperiments.1.9Conclusionsfromtheliteraturereview,andaimsofthethesisWiththemassiveexpansionofthegoatindustryinChina,problemslinkedtohelminthicdiseaseshavebecomeincreasinglyprominent.AlthoughformerscholarshavemadegreatcontributionsthroughepidemiologicalinvestigationsandthepreventionandcontrolofhelminthsinChina,therearesomeconsiderableknowledgegapsinthisarea.First,sinceonlytraditionaldiagnosticmethods,basedonmorphologicalfeaturesofeggs,larvaeoradults,wereused,resultsachievedarelikelytohavebeeninaccurateandinsufficientinsomeregard.Second,moleculardiagnosticmethodsbasedontheuseofnucleicacidshavebeendeveloped,molecularmethodshavenotbeenappliedtoparasitichelminthsofgoatsinChina.Third,anthelminticresistanceisbecomingmoreprominentwiththewidespreadandexcessiveuseofanthelmintics.Althoughresistancetobenzimidazoleshasbeenrecordedinsomegeographicalregions,acomprehensiveunderstandingresistancetootheravailableanthelminticsislacking.Last,sustainableparasitepreventionandcontrolprogrammesareabsent.Clearly,improveddiagnosticmethodsareneededtoinvestigatethedistributionandprevalenceofhelminthsingoatsinregionsofChinawherebreedingandproductionareintense,andthentosurveyanthelminticresistanceasabasisforsettingasustainableprogramtocontrolparasiticnematodesofgoats.Asthegoatindustryhasdevelopedsubstantiallyinrecentyears,thereisanincreasedneedtounderstandtheextentoftheparasiteproblemingoats,themethodsusedbyfarmerstocontrolgoatparasitesandtoestablishthedistributionandextentofdrugresistanceinparasitepopulationsofgoats.Thisknowledgeandunderstandingwillprovideastartingpointforeffectivecontrolandthechoiceeffectiveanthelminticsforstrategictreatment.Moreover,gainingimproved64 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceinsightsintosomeaspectsoftheepidemiologyofparasitesinHubeiandotherpartsofChinawithanexpandinggoatproductionwillassistinestablishingintegratedmethodsforthecontroloftheseparasites,withafocusonincreasedanimalhealthandeconomicbenefits.Thespecificaimsofthisthesisareto:StudytheepidemiologyofgoathelminthsalongtheHanRiverandDabieMountaininHubeiProvince(Chapters2and3);Geneticallycharacteriseimportantparasitesofsmallruminantsusingmitochondrialgenomesequencing,asaresourceforDNAmarkersforfuturesystematic,populationgeneticandepidemiologicalstudies(Chapters4to6);Establishaloop-mediatedisothermalamplification(LAMP)methodforthespecificdetectionanddiagnosisofHaemonchuscontortusingoats(Chapter7);SurveyanthelminticresistanceingastrointestinalnematodesofgoatsinpartsofHubeiProvince(Chapters8and9).Discussthefindings(Chapters2to9)inageneralcontext,toassesshowthethesishascontributedtotheareaofgoatparasitology,toiteratetheimplicationsoftheresearchresultsforthecontrolofcaprineparasitesinChinaandtoidentifyareasforfutureinvestigation(Chapter10).65 HuazhongAgriculturalUniversityPhDDissertation2018Chapter2-SurveyofparasitichelminthsofgoatsalongtheHanRiverinHubeiProvince,China2.1IntroductionParasitichelminthsarecommoningoatsandoftenleadtoconsiderableeconomiclossesduetoassociateddiseasesand/ordeath(PerryandRandolph,1999;VercruysseandClaerebout,2001).Moreover,somehelminthsofgoats,suchasFasciolahepaticaandSchistosomajaponicum,arezoonosesandalsoposeathreattohumanhealth(Mansoorlakoorajetal.,2011;Kheirandishetal.,2012).Todate,theprevalenceofhelminthsofgoatshasbeenreportedfrommanycountries,includingBrazil(Vieiraetal.,2014),China(Yang,1988;Guoetal.,2003;Yang,2005;Wangetal.,2006;Fengetal.,2008;Maetal.,2014),Denmark(Holmetal.,2014),Ethiopia(Sissayetal.,2007),India(Guptaetal.,1987;Khajuriaetal.,2013),Italy(Zanzanietal.,2014),Malaysia(Chandrawathanietal.,2009),Mongolia(Sharkhuu,2001),Netherlands(BorgsteedeandDercksen,1996),Norway(Domkeetal.,2013),Pakistan(Khanetal.,2010;Ayazetal.,2013),PapuaNewGuinea(Koinarietal.,2013)andSwitzerland(Murrietal.,2014),withgastrointestinalstrongylidnematodesbeingverycommon.However,prevalenceanddistributionofhelminthscanvaryconsiderablyamongdifferentcountriesasaconsequenceofdifferencesinenvironmentandmanagement(Maetal.,2014).HubeiProvince,withmorethanonethousandlakesandrivers,islocatedincentralChina.Theabundanceofwaterprovidesasuitablehabitatformanyparasites,particularlygastrointestinalworms,tothrive.AlthoughShenandHuang(2004)reportedthatmanyparasitesarefoundinlivestockinHubei,thereisnopublishedreportoftheprevalenceofhelminthsofgoatsinthelakeareaofthisprovince.Toaddressthisknowledgegap,IundertookthisfirstsurveyalongtheHanRiverinHubeitoprovideafoundationforfuturecontroleffortsofhelminthsofgoats.2.2Materialsandmethods2.2.1Studyarea,animalsandsamplecollectionThestudywasconductedinZhanggang,HubeiProvince,China,fromJanuarytoDecemberin2014.ZhanggangislocatedinthemiddleofHubeiProvince(112°33’E,66 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince30°22’N).TheHanRiverflowsthroughthisregionandprovideswatertorichpasturelandsinZhanggangforgrazinganimals.Zhangganghasasubtropicalmonsoonclimate.InZhanggang,goatsusuallyfeedongrassesandshrubsalongtheHanRiver,butfarmersalsosupplementwithpeanutstalksinwinter.Inthepresentstudy,741faecalsampleswerecollecteddirectlyfromtherectumofgoatsfromeightflocksalongtheHanRiver(6-12monthsofage)fromJanuarytoDecember2014.InformationaboutfaecalsampleswerepresentedinTable2-1.FreshfaecalsamplesweretransportedtotheLaboratoryofParasitology,HuazhongAgriculturalUniversity,undercooledandanaerobicconditions.2.2.2ConventionalfaecalexaminationAllthefaecalsampleswerestoredat4°Cunderanaerobicconditionsandexaminedwithinsevendays(Nielsenetal.,2010).Sampleswereexaminedusingstandardflotationandsedimentationtechniques(Wang,2013).Inbrief,twoaliquotsof2goffaeceswerehomogenisedin20mlofsaturatedsodiumnitrateandtapwater,respectively,separatelysievedthroughateastrainer(meshsize:250µm)andthentransferredtoseparatetubestoconcentrateeggsbyflotation(sodiumnitrate)andsedimentation(water),respectively.Helmintheggswereidentifiedtoasfaraspossibleusingmorphologicalcharacteristicsusingalightmicroscope(NikonECLIPSE80i)(Tayloretal.,2007).2.2.3MoleculardiagnosisSomesampleswereusedformolecularidentification.APCR-basedsequencingapproachwasalsoemployedforthespecificdetectionofselectedstrongylids.Inbrief,totalgenomicDNAwasisolateddirectlyfrom500mgoffaecesfromeachsampleusingakit(OMEGASoilDNAKit,USA),accordingtotheprotocol.ForHaemonchuscontortus,part(265bp)ofthesecondinternaltranscribedspacer(ITS-2)ofnuclearribosomalDNAwasPCR-amplifiedusingthespecies-specificprimerHAE(5’-CAAATGGCATTTGTCTTTTAG-3’)andaconservedprimerNC2(5’-TTAGTTTCTTTTCCTCCGCT-3’)(Bottetal.,2009).ForTrichostrongylusspecies,part(267-268bp)ofITS-2wasamplifiedusingagenus-specificprimerTRI(5’-TCGAATGGTCATTGTCAA-3’)andNC2(5’-TTAGTTTCTTTTCCTCCGCT-3’)(Bottetal.,2009).EachPCRwasconductedinavolumeof50µlcontainingGoTaqFlexibuffer(Promega,USA),3.0mMofMgCl2,200µMofeachdeoxynucleotidetriphosphate,25pmolofeachprimerand1UofGoTaq(Promega)DNApolymerase67 HuazhongAgriculturalUniversityPhDDissertation2018(Bottetal.,2009).Knowntest-positive,test-negativeandno-templatecontrolswereincludedineachsetofPCRs.Thecyclingprotocolinaconventionalthermalcycler(EastwinLifeSciences,Inc.,China)was:94°Cfor5min(initialdenaturation),followedby35cyclesof94°Cfor45sec(denaturation),50°Cfor45sec(annealing)and72°Cfor1min(extension),withafinalextensionof72°Cfor10min.Subsequently,thesizeandintensityofallampliconswereassessedbyelectrophoresis(7V/cm)in1.5%agarosegelsusingTBE(65mMTris-HCl,27mMboricacid,1mMEDTA,pH9;Bio-Rad,USA)asthebuffer.Followingelectrophoresis,gelswerestainedwithethidiumbromideandtheirsizeestimatedbycomparisontoPhi-X174-HaeIII(Promega,USA)markers.Aliquots(5µl)ofindividualampliconswerepurifiedusingmini-columns(WizardPCR-Preps,Promega),andthensubjectedtodirect,automatedsequencing(BigDyeTerminatorv.3.1chemistry,AppliedBiosystems,USA)inbothdirectionsusingthesameprimersasemployedforPCRamplification.2.3ResultsTheestimatedprevalenceofhelminthsofgoatsstudiedinZhanggangin2014isgiveninFig.2-1.Ofthe741goatsexamined,592(79.9%)excretedeggsofoneormorehelminths.TheprevalencewerehigherthanthoseinShaanxi(78.57%),butlowerthanthatfoundinHunan(86%),Chongqing(100%)andSichuan(99.5%)(Yang,1988;Guoetal.,2003;Yang,2005;Maetal.,2014).Ofthehelminthsdetected,strongylidswerecommon,particularlyH.contortus;thisfindingisinagreementwithsurveysinHunan(Maetal.,2014),Shanxi(Guoetal.,2003;Fengetal.,2008),Chongqing(Yang,2005),Sichuan(Yang,1988)andHeilongjiang(Wangetal.,2006),buttheprevalenceoftrematodesinHubeiwashigherthanthatofotherprovinces,exceptHeilongjiang(Wangetal.,2006).Thedifferencesbetweenourandinvestigationinotherprovincesmightrelatetodifferencesofsamplesexamined,climate,rainfall,managementandotherfactors(Maetal.,2014).Theprevalenceofnematodeswashighthroughouttheyear,withfourpeaksinFebruary,May,SeptemberandDecember.Theprevalenceoftrematodeswaslowerthanthoseofnematodes,andtherewasoneinfectionpeakinApril.Theexcretionoftapewormeggsdidnotappeartofollowapattern.68 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable2-1:InformationonthesamplescollectedNumberofanimalsNumberofanimalsTimeFlocksFeedingmodeintheflockforcollectingsamplesJanuaryZG1halfgrazed5246FebruaryZG2halfgrazed10552MarchZG3halfgrazed5525AprilZG4halfgrazed5027MayZG5halfgrazed10050JuneZG2halfgrazed10555JulyZG6halfgrazed150100ZG7halfgrazed140102AugustZG8halfgrazed140108SeptemberZG6halfgrazed15092OctoberZG1halfgrazed5231NovemberZG3halfgrazed5523DecemberZG4halfgrazed5030Note:Fecalsamplesofgoats(6-12monthsofage)werecollectedfromoneselectedflockeverymonthfromJanuarytoDecemberin2014.AlltheeightflockssharethesamepastureduringthegrazingperiodfromApriltoOctober.Table2-2:MixedinfectionofHaemonchuscontortusandTrichostongylusin128fecalsamplesofgoatsbyspecies-specificPCRidentificationCo-infectionTrichostongylusTotalcondition＋－＋3878116H.contortus－5712Total4385128Note:“＋”meansthenumberofsampleswerepositiveforH.contortusorTrichostongylus;“－”meansthenumberofsampleswerenegativeforH.contortusorTrichostongylus69 HuazhongAgriculturalUniversityPhDDissertation2018PCR-basedsequencingofITS-2ampliconsderivedfromarepresentativesubsetofsampleswasusedtoestablishtheidentityofsomestrongylids(HaemonchusandTrichostrongylus).Todothis,genomicDNAwasextractedfrom128ofthe741faecalsamplesforsubsequentPCRusingprimerpairsHAE-NC2(H.contortus)andTRI-NC2(Trichostrongylus)(Bottetal.,2009).ForthesamplesusedforPCR,114of128samples(89.06%)testedpositivewithstrongylids.Intotal,116of128samples(90.6%)testedpositiveforH.contortus(Table2-2);10ampliconsweresequenced;theresultantsequences(231bp)wereidenticaltoH.contortus(GenBankaccessionno.AB908961.1).Ontheotherhand,43ofthesame128samples(33.6%)alsotestedpositiveforTrichostongylus(Table2-2).Fourampliconswererandomlyselectedandsequenced;allfoursequenceswereidentical(over221bp)toITS-2ofTrichostrongyluscolubriformis(accessionno.KC521378.1).Altogether,theprevalenceofH.contortuswashigherthanthatofTrichostongylus.MixedinfectionsofH.contortusandTrichostongyluswerefoundbymolecularmethods(Table2-2),38samples(29.69%)hadmix-infection,78samples(60.94%)werepositiveonlyforH.contortus,andfivesamples(3.91%)werepositiveonlyforTrichostongylus.Onlytwosamplestestedpositiveforstrongylideggs,butwerenegativeforH.contortusand/orTrichostongylusbyPCR;incontrast,8samplesthattestednegativeforstrongylidsbyfaecalexaminationwerepositiveforH.contortusand/orTrichostongylususingthePCR-basedmethod.Theprevalenceofnematodeswasthehighestthroughouttheyear.InZhanggang,farmersusuallygrazetheirgoatsonpermanentpastures,andseldomchangepastures.Thus,goatsarelikelyreadilyre-infectedcontinually.Notably,H.contortusisthecommonestnematode.InZhanggang,thewarmandhumidclimatethroughouttheyear(meanannualtemperatureis15.8°C;averageannualprecipitation:1.2m)isperfectlysuitedforthesurvivalanddevelopmentofH.contortus,explainingitshighprevalenceinZhanggang.Forthetrematodes,theprevalenceofgravidwormsingoatsseemedtopeakinApril.InZhanggang,thesnailintermediatehostsarecommononthepasturesalongtheHanRiver,thusprovidingasuitableenvironmentfortransmission.ThepeakdevelopmentofsnailsisbetweenAprilandSeptember,whichisinaccordwiththeprevalencepeak/sfortrematodes.70 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceFig.2-1:Theprevalenceofparasitichelminthsingoatsin2014alongtheHanRiverinZhanggang.71 HuazhongAgriculturalUniversityPhDDissertation20182.4DiscussionThisisthefirstsurveyofparasitichelminthsofgoatsalongtheHanRiverinHubei,China.Importantly,itwasfoundthattheprevalenceofhelminthswasveryhigh,eventhoughfarmersuseanthelmintics(includinglevamisole,benzimidazolesand/orivermectin)atupto2-5timestherecommendeddosage(personalcommunication).Giventhehighprevalenceofstrongylidnematodes,andthecommonover-usageand-dosageoftheseanthelmintics,itispossiblethataresistanceproblemisalreadylooming.Thisissueneedstobeinvestigatedinthenearfuture.WithincreasinglivingstandardsinChinaandaneedformoremilk,meatandleather,therehasbeenamassiveexpansioninthelivestockindustries,whichincludesgoatfarming.Therequirementfortheseproductshasacceleratedthebreedingofgoats,whichhasextendedintomanynewregions(suchastheHanRiverregion)inthepastdecadetoboostlocaleconomies.However,parasitichelminthssignificantlyrestricteconomicproductivity.Todate,helminthshavebeenreportedingoatsandsheepin24provincesofChina,butthereisstillverylimitedinformationontheprevalenceandburdensoftheseparasitesingoatsgenerally.Forthelastdecade,thegoatfarminghasdevelopedrapidlyalongtheHanRiver,whichisabranchfromYangtzeRiverandflowsthroughtwoprovinces(ShanxiandHubei),coveringanareaof159,000squarekilometers.Currently,thisregionisestimatedtocarry10milliongoats.ThepresentstudynowprovidesusefulbaselineinformationongoathelminthsinZhanggang,andastartingpointfortheimplementationofmonitoringandcontrolprogramsinthisregion.Moreover,withamassiveexpansionofthegoatindustryinChina,thepresentfindingsalsoemphasistheneedtoundertakeprevalencesurveysinotherregionsofChinawhereextensivefarmingpracticesareused,inordertoprovidecrucialinformationtounderpinfuturecontrolefforts.72 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceChapter3-InvestigationofhelminthsofgoatsalongtheDabieMountaininHubeiProvince3.1IntroductionWiththeimprovementoflivingstandardsinChina,substantiallybetterqualityandlargerquantitiesofmilk,meatandleatherareneededtosustainthehumanpopulation.Therefore,thereisaneedtopromoteanddevelopmentofthelivestockindustries,includingthoseinvolvinggoatproducts(e.g.,Ling,2015;Tuerxun,2015;Zhang,2015).Diseaseproblemslinkedwithparasitesingoatsarebecomingmoreandmoreapparent,whichareaffectingfarmproductivity(e.g.,Gu,2016;Yu,2016).Parasitichelminths,especiallygastrointestinalnematodes,arecommoninsmallruminants,andcanleadtosubstantialeconomiclosses.Animalsinfectedwiththeseparasitescanshowanaemia,emaciated,diarrhoea,respiratoryproblems,weightloss,reducedweightgain,orevendeath,andcausesignificanteconomiclossesinChina(e.g.,Chenetal.,2011;Li,2011;Pietal.,2014;Li,2016).Untilnow,helminthsofgoatshavebeenreportedinChina(e.g.,ShenandHuang,2004;Shen,2005;Maetal.,2014;Yangetal.,2016),aswellasabroadincludingBrazil(Vieiraetal.,2014),Denmark(Holmetal.,2014),Ethiopia(Sissayetal.,2007);India(Khajuriaetal.,2013),Italy(Zanzanietal.,2014),Malaysia(Chandrawathanietal.,2009),Mongolia(Sharkhuu,2001),Norway(Domkeetal.,2013),Pakistan(Ayazetal.,2013),Philippines(RupaandPortugaliza,2016),SouthAfrica(Tsotetsietal.,2012),Switzerland(Murrietal.,2014),Tanzania(Miranetal.,2015)andThailand(Ratanapobetak.,2012).Theprevalenceandintensityofhelminthsvariesconsiderablyamongdifferentregionsduetothedifferencesinclimate,managementandotherfactors(Maetal.,2014).Knowingtheprevalenceofhelminthsofgoatscanprovideafoundationforpreventingandcontrollinggoatparasites.LuotianislocatedintheeastofHubeiProvince.PlentyofriversoriginatinginmountainousregionsfeedpasturelandsinLuotianforgrazinganimals,especiallygoats,andalsoprovidesuitableenvironmentforthesurvivalofmanyparasites.Untilnow,therehasbeennoreportonparasitesofgoatsinLuotian,althoughunpublishedstudieshaveindicatedthatgastrointestinalnematodesarecommoningoatsatslaughter(personalcommunicationwithXiaoZX,ZhangSS,ZhangYHandZhouQZ,2016).Theaimofthe73 HuazhongAgriculturalUniversityPhDDissertation2018presentchapterwastoinvestigatethespeciesofgastrointestinalhelminthsofgoats,andprovideafoundationforthefuturecontrolofsuchparasites.3.2Materialsandmethods3.2.1StudyareaThisstudywasconductedinLuotian(Fig.3-1),HubeiProvince,China,from12thJanuaryto17thMayin2017(Table3-1).LuotianislocatedinthenortheastofHubeiProvince(115°40’E,30°50’N),andhasasubtropicalmonsoonclimate.Usually,SpringisfromFebruarytoApril;SummerisfromMaytoJuly;AutumnisfromAugusttoOctober;andwinterisfromNovembertoJanuary.DuetovarietyandtechnicalsupportfromtheJinxiuProfessionalCooperativesofForestryandAnimalHusbandryinLuotian,anincreasingnumberoffarmersarenowbreedinggoats.SinceLuotianisamountainousregion,goatsusuallyfeedongrassandshrubsnearhillsandmountainousareas,andfarmersalsoprovidetheirgoatswithhayorstraw.Fig.3-1:MapoftheHubeiareasfromwheresampleswereoriginatedinthisstudy.★Locationoftheslaughterhouse.74 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable3-1:Seasonaldistributionandthenumberofgoatsforpost-mortemexaminationSeasonaloriginsNo.ofgoatsPercentage(%)WinterJanuary12th1411.86SpringMarch7th2117.80March16th2521.19March26th108.47April21th3327.97SummerMay16th1512.713.2.2PostmortemexaminationAtotalof118goatsslaughteredintheabattoiratLuotianfromJan12thtoMay17thin2017wereexaminedforthepresenceofhelminthsbypostmortemexamination(e.g.,Liu,1983;Li,2006;Li,2011;Wang,2013;Maetal.,2014;Tayloretal.,2016).Inbrief,theabomasum,intestines,liver,pancreasandlungwereremovedandexaminedforthepresenceofwormsinindividualgoats(Fig.3-2).Forabomasum,theentirecontentandmucosawereexamined.Wormsintheabomasummucosawereisolatedandcounted.Gutcontentwasdilutedin1Lofwater,andanaliquot(250ml)wasexaminedforparasitesusingastereoscopicmicroscope(Li,2006;Wang,2013).Forthesmallintestine(first6meters),themethodforwormrecoverywasthesameasthatofabomasum(Li,2006;Wang,2013).Forlargeintestine,thewormswereisolatedafterthecaecumandcolonwereopenedandthecontentsgentlyremoved.Forliverflukes,thehepaticductsandgallbladderwereopenedandwormsrecovered.Forpancreasflukes,pancreaswascutdownalongthepancreaticductandopenedtorecoverworms.Forlungworms,thelungwascutopenalongtrachea,bronchiandbronchiolestocollectworms(Wang,2013;Tayloretal.,2016).Allthewormswerewashedthoroughlywith0.7%(w/v)sodiumchloride,andpreservedin70%(v/v)ethanolinlabelledbottlesandstoredat-20°Cuntiluse.Allwormswereidentifiedtogenusorspeciesusingacompoundmicroscopeorbynakedeye(e.g.,forTaeniahydatigenacysts,Fasciola),andidentifiedusingcharacteristicmorphologicalfeatures(e.g.,GibbonsandKhalil,2009;LancasterandHong,1990;Li,2006;LichtenfelsandHoberg,1993;Wang,2013;Tayloretal.,2016).75 HuazhongAgriculturalUniversityPhDDissertation20183.2.3IsolationofgenomicDNAfromparasitesTotalgenomicDNAwasextractedfromindividualadultwormsusingsodiumdodecyl-sulfate(SDS)/proteinaseKtreatment(Gasseretal.,1993),followedbyWizardDNAClean-Upcolumnpurification(Promega,USA)andelutionin50µlH20.3.2.4MolecularidentificationofgastrointestinalnematodesToconfirmtheidentityofsomenematodes,theITS-2region(~350bp)ofribosomalDNAwasamplifiedusingprimers:NC1(5’-ACGTCTGGTTCAGGGTTGTT-3’)andNC2(5’-TTAGTTTCTTTTCCTCCGCT-3’)(Gasseretal.,1993;Bottetal.,2009).PCR(50μl)wasperformedin1XPCRBuffer(TaKaRa,Japan),2.5mMMgCl2,250μMeachofdNTP,100pmolofeachprimerand1UTaqpolymerase(TaKaRa)underthefollowingconditions:initialdenaturationat94°Cfor5min,followedby30cyclesofdenaturationat94°Cfor30s,annealingat55°Cfor30sandextensionat72°Cfor1min,withafinalextensionof72°Cfor5min.AllPCRproductswereexaminedonagarosegels(1.5%)toverifythattheyrepresentedsinglespecificbands,andproductsweresequencedusingthesameforwardandreverseprimersasPCR.3.2.5MolecularidentificationoftrematodesToconfirmtheidentityofsometrematodes,theITS-2(~1000bp)ofribosomalDNAwasamplifiedbyprimersBD1(5’-GTCGTAACAAGGTTTCCGTA-3’)andBD2(5’-TATGCTTAAATTCAGCGGGT-3’)(Linetal.,2007;Lotfyetal.,2010).PCR(50μl)wasperformedin1XPCRBuffer(TaKaRa,Japan),1.5mMMgCl2,250μMeachofdNTP,25pmolofeachprimerand1UTaqpolymerase(TaKaRa)underthefollowingconditions:initialdenaturationat94°Cfor5min,followedby38cyclesofdenaturationat94°Cfor30sec,annealingat50°Cfor30sandextensionat72°Cfor1min15sec,withafinalextensionof72°Cfor5min.AllPCRproductswereexaminedonagarosegels(1.5%)toverifythattheyrepresentedsinglespecificbands,andproductsweresequencedusingthesameforwardandreverseprimersasPCR.3.3ResultsAtotalof12generaofparasitichelminthswerefoundin118goatsbypost-mortemexaminationinLuotian,HubeiProvince,China.Thegenus/species,parasiticlocation,76 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceprevalenceandintensityofinfectionarepresentedinTable3-2andFig.3-3.Approximately88%ofgoatswereinfectedwithoneormorehelminthspecies.Haemonchuscontortuswasthemostprevalenthelminthswithaninfectionrateof73%;Paramphistomumcerviwasthemostcommontrematodewiththeinfectionrateof8.5%;andTaeniahydatigenadominalcavity–serosalsurfacesorinliveroromentum)wastheonlycestodewithaprevalenceof43%.TheinfectionintensityofParamphistomumcervi,Haemonchuscontortus,Ostertagia,OesophagostomumasperumandEurytremapancreaticumwerehigh(1~1210)ininfectedgoats,whiletheintensityofinfectionwerelow(1~39)fortheotherspecies(datanotshown).Fasciolahepaticaisoneofthecommonestspeciesintheliversofanimals;however,onoccasions,F.hepaticawasfoundinthesmallintestineandpancreasofgoats.Eurytremapancreaticumwhichparasitesthepancreaswasalsofoundinthesmallintestine.3.4DiscussionPreviousstudiesonthesurveyofparasiteinfectioningoatsinotherregionsofHubeiProvincewereperformedbyLivestockSchistosomiasisControlStationofHubeiProvinceetal.(1981).LivestockSchistosomiasisControlStationofHubeiProvinceetal.(1981)investigatedthegoatparasitesfrommountainousareasinthewestofHubeiProvince,andfound15generaofparasitichelminthsingoats,anddetectedmoregenerathaninthepresentstudy.Thereasonsforlesstaxainpresentstudymightrelatetochangesingoatindustry(fromextensivetomoreintensive),husbandrypractices,goatvarieties,climateandenvironment(e.g.,Lietal.,2009;Li,2015;Ling,2015).Alimitationofthepresentstudyisthatskin,muscle,bloodandbrainwereexamined.It’sinterestingtofoundthatDicrocoeliumdendriticumandTrichurissppwereonlyfoundinthepresentstudy,thereasonwaslikelytobecausedbyimportinggoatvarietiesfromelsewheresincelong-distancetransportwasavailableandfarmerstendedtoimportgoodvarietiesfromotherregions.Altogether,17generaofparasitichelminthshavebeenfoundingoatsinHubeiProvince,whichwassimilartoreportsfromotherprovincesinChina,suchasGuizhou(Duetal.,1995),Hunan(Maetal.,2014),Shanxi(Zhang,1983),butlessthanthatofSichuan(Zhangetal.,1986)andZhejiang(Yang,1988).Clearly,differencesarelikelytorelatetosamplenumber,climate,goatbreedandanimalhusbandrypractices.77 HuazhongAgriculturalUniversityPhDDissertation2018Fig.3-2:Examinationoforgansandtissuesbypostmortemexamination.A-Jrepresentrumen,abomasum,largeintestine,abdominalcavity,liverandpancreas,respectively(redarrowsindicateParamphistomum,Haemonchuscontortus,Oesophagostomum,Taeniahydatigena,FasciolahepaticaandEurytremapancreaticum).78 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable3-2:Species,parasiticlocation,prevalenceandintensityofhelminthsofgoatsinLuotianPrevalenceInfectionintensitySpeciesParasiticlocationNo.ofgoatsInfectionrate(%)(mean+SD)NematodaHaemonchuscontortusAbomasum8672.8880+184OstertagiasppAbomasum1512.7198+94TrichostrongyluscolubriformisSmallintestine10.8539+0OesophagostomumcolumbianumSmallintestine10.8515+0OesophagostomumasperumLargeintestine1613.5632+41TrichurissppLargeintestine2420.345+4TrematodaParamphistomumcerviRumen108.47198+296FischoederiuselongatusRumen10.853+0HomalogasterpaloniaeLargeintestine32.5419+8FasciolahepaticaLiver,Smallintestine,65.086+5PancreasFasciolagiganticaSmallintestine10.851+0DicrocoeliumdendriticumLiver10.855+0EurytremapancreaticumPancreas,Smallintestine97.63190+165CestodaTaeniahydatigenaAbdomen5143.222+279 HuazhongAgriculturalUniversityPhDDissertation2018Fig.3-3:Helminthspeciesrecoveredbypostmortem.A-JrepresentHaemonchuscontortus,Trichostrongyluscolubriformis,Oesophagostomumcolumbianum,Dicrocoeliumdendriticum,Eurytremapancreaticum,Ostertagia,Trichuris,Paramphistomum,FasciolahepaticaandTaeniahydatigena,respectively.80 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable3-3:SeasonalprevalenceofhelminthsofgoatsinLuotianSeasonNo.ofNo.ofNematodesTrematodesCestodesgoatsgeneraSpeciesPrevalenceIntensitySpeciesPrevalenceIntensitySpeciesPrevalenceIntensityNo.Rate(%)(mean+SD)No.Rate(%)(mean+SD)No.Rate(%)(mean+SD)Winter149H.contortus964.2918+25P.cervi857.14242+315Ostertagiaspp214.2922+5F.elongatus17.143+0T.colubriformis17.1439+0H.paloniae321.4319+8O.asperum321.432+1F.hepatica642.866+5F.gigantica17.141+0E.pancreaticum535.71209+202Spring896H.contortus6775.2870+174E.pancreaticum33.37143+9T.hydatigena4449.442+2Ostertagiaspp1213.48119+94O.columbianum11.1215+0O.asperum1213.4840+44Trichurisspp2426.975+4Summer156H.contortus1173.33185+257D.dendriticum16.675+0T.hydatigena746.672+1Ostertagiaspp16.676+0E.pancreaticum16.6732+0O.asperum16.6713+0Note:Haemonchuscontortus-H.contortus;Trichostrongyluscolubriformis-T.colubriformis;Oesophagostomumcolumbianum-O.columbianum;Oesophagostomumasperum-O.asperum;Paramphistomumcervi-P.cervi;Fischoederiuselongatus-F.elongatus;Homalogasterpaloniae-H.paloniae;Fasciolahepatica-F.hepatica;Fasciolagigantica-F.gigantica;Eurytremapancreaticum-E.pancreaticum;Dicrocoeliumdendriticum-D.dendriticum;Taeniahydatigena-T.hydatigena.81 HuazhongAgriculturalUniversityPhDDissertation2018SeasonalvariationinprevalenceofparasitichelminthsofgoatsinLuotian,HubeiProvinceisgiveninTable3-3.Nematodeswereprevalenceinallseasonsstudied.ForHaemonchuscontortus,theinfectionintensityincreasedfromWintertoSummer,andpeakedinSummer;forOstertagiasppandOesophagostomumasperum,theinfectionintensityrosefromWinter,peakedinSpringandthendecreasedinAutumn;theremainingspecies(Trichostrongyluscolubriformis,OesophagostomumcolumbianumandTrichurisspp.)werefoundinoneseason.Trematodeswereprevalentinwinter,whileuncommoninspringandsummer.ForEurytremapancreaticum,theinfectionintensitypeakedinwinter,anddecreasedfromwintertoautumn;theothertaxa(Paramphistomumcervi,Fischoederiuselongatus,Homalogasterpaloniae,Fasciolahepatica,FasciolagiganticaandDicrocoeliumdendriticum)onlyoccurredinoneseason.Cestodeswereprevalentinspringandsummer,butabsentinwinter.Taeniahydatigena(i.e.larvalstageofT.hydatigena)wastheonlyspeciesdetecetedinthisstudy.ForTaeniahydatigena,theinfectionintensityinspringwassimilartothatinsummer.Yangetal.(2016)reportedtheprevalenceofparasitichelminthsofgoatsin2014alongtheHanRiverinZhanggang,HubeiProince.Theseauthorsfoundthatnematodeswereprevalentthroughouttheyear,andthattrematodesincreasedfromJanuary,peakedinApril,thendecreaseduntilOctober,theinfectionofcestodesdidnotshowanyseasonaldifferences.ThepresentresultscomplementthoseofYangetal.(2016)whichlackedinformationonwormspeciesandburdens.However,thepresentstudyisdifferentfromtheresultofHunanProvince(Maetal.,2014)adjacenttoHubeiProvince,inthattheinfectionpeakfornematodes,trematodesandcestodeswasinSummer,WinterandAutumn,respectively.Thereasonsforthismaybeduetodifferencesinsampling,seasons,climate,environment,goathusbandrypractices,goatbreedsandparasitesbetweenHubeiandHunanProvinces.Thesmallernumberofgoatsexaminedinthepresentstudymightalsocontributetodifferences.HubeiProvinceislocatedinthecentralChinawithhundredsofriversandlakes,abundantwaterresourcesandpastures,whichmakeitsuitableforfarmingandanimalindustries.WiththeincreasinglivingstandardsandhumanpopulationinChina,thereisanincreaseddemandforanimalproducts.Therefore,tooptimizetheproductivityofthegoatmeat,dairy,cheeseandleatherindustries,thereisaneedtoimprovemanagementpracticestoprevent,treatandcontrolinfectiousandnon-infectiousdiseases.Clearly,centraltothegoatindustryistheeffectiveandsustainablecontrolofparasites.Thebasis82 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceforcontrolisknowledgeoftheparasitesthatoccur,theirepidemiology(includingprevalence,distributionandtransmission)andtheextentofresistancetoanti-parasiticdrugs.Thisknowledgewouldunderpinfuturestrategicorintegratedparasitemanagementandcontrolstrategies,whichmightincludetheuseofnewandeffectivevaccines,anti-parasiticdrugsandreliablemolecular-diagnostictools.83 HuazhongAgriculturalUniversityPhDDissertation2018Chapter4-GeneticalcharacterizationofFischoederiuselongatususingmitochondrialgenomesequencing4.1IntroductionParamphistomesaredistributedworldwideandhavebeenreportedinmanycountries,suchasBulgaria,France,Poland,Hungary,Italy,India,Russia,SardiniaandYugoslavia(Horak,1971).Theparamphistomecaninfectfishes,reptiles,birdsandmammals,someofwhichcanleadtohugeeconomiclossesrelatedtoseriouslygastrointestinaldiseases,lowproducitivityordeathinruminants(Li,2011).InArumeruDistrict,theprevalencerateofparamphistomesisashighas56.7%incattle(Nzalawaheetal.,2014).Fischoederiuselongatusisanimportantmemberofparamphistomes,theparasiteusuallyinhabitstherumenofcattle,buffaloes,sheepandgoats.Ruminantsareusuallyinfectedbyingestingsnails,suchasLymnaeaacuminata,LymnaeasuccineaorGyrauluseuphraticus(Yamaguti,1975).RuminantsinfectedwithF.elongatusshowweakness,mentalfatigueandeventuallydeath.Moreseriously,F.elongatusmaybeazoonotictrematode,aChinesewomanfromGuangdongProvincewasreportedtobethefirsthumaninfectioncase(Li,1991),butitisstillunknownhowshewasinfected.Untillnow,themostcommondiagnosticmethodforF.elongatusisthemicroscopicalexamination,butit’stime-consuming,andhardtodistinguishwithotherparamphistomes.Asausefulmarker,mtgenomehasbeenwidelyusedforspeciesidentification(JaneandBernard,1990;Choietal.,2012;Gasseretal.,2012;Rameshetal.,2012;Ghatanietal.,2014).ThecompletemtgenomeofF.elongatuscanprovidealternativemolecularmarkersforthespeciesidentification,epidemiologyandgeneticdiversityofparamphistomes.Inthepresentstudy,wegotthefullsequenceandgenearrangementofmtgenomeofF.elongatusandcompareditwithselectedtrematodes.WefoundthatF.elongatushadtheclosestrelationshipwithP.cervi.84 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince4.2Materialsandmethods4.2.1ParasitecollectionandDNAisolationF.elongatusadultswerecollectedfromtherumenandreticulumofnaturallyinfectedcattleinZhanggang,Tianmen,HubeiProvince,PRChina,accordingtotheAnimalEthicsGuidelinesofHuazhongAgriculturalUniversity.Then,theadultwormswerewashedextensivelyin0.9%sodiumchloridesolution,andidentifiedthroughmorphologicalexaminations(Li,2011).Subsequently,onewormwasstainedforidentification(Wang,2013),andtherestwerefixedin75%alcohol(V/V)andstoredat−20°Cuntiluse(Liuetal.,2013).TotalgenomicDNAwasisolatedfromoneworm(Gasseretal.,2006).TheITS-2regionofF.elongatuswasamplifiedandsequencedasreportedpreviously(Itagakietal.,2003),itwas100%similartothatofF.elongatus(GenBankaccessionno.JQ688410.1).4.2.2AmplificationandsequencingofF.elongatusmtgenomeFirstly,wedesigned12oligonucleotideprimersaccordingtotheconservedregionsfromreportedmtgenomesequencesofF.hepatica(Leetal.,2001a),Clonorchissinensis(Shekhovtsovetal.,2010)andP.cervi(Yanetal.,2013)toamplifypartialfragmentsfromcox3,cytb,nad4,cox1,rrnSandnad5(Table4-1).PCRs(25µl)wereperformedinthefollowingreaction:10mMTris–HCl(pH8.4),50mMKCl,4mMMgCl2,200mMeachofdNTP,50pmolofeachprimer，2UTaqpolymerase(Takara)and2.5µlgenomicDNA.Reactionswererununderthefollowingconditions:94°Cfor5min,followedby35cyclesof94°C/30s,50°C/30sand72°C/1min.AmpliconsweresenttoSangonCompany(Shanghai,China)forsequencing.Then,12additionalprimers(Table4-1)weredesignedbasedontheobtainedsequencingresultstoamplifysixregionsfromgenomicDNA(~40-80ng)bylong-PCR.PCRs(50µl)wereperformedinreactionscontaining0.4mMeachofdNTPs,5μl10×LATaqbufferII(Mg2+Plus),2.5μMofeachprimer,2.5ULATaqpolymerase(Takara)and2.5µlgenomicDNA.Andthereactionswererununderthefollowingprogram:94°Cfor5min,followedby35cyclesof94°C/30s,50°C/30sand72°C/1-5min(dependingonthesizeofF.hepatica).AmpliconswereclonedintopGEM-T-Easyvector(Promega,USA)andthensequencedusingaprimer-walkingstrategy(Huetal.,2007).85 HuazhongAgriculturalUniversityPhDDissertation2018Table4-1:PrimersusedinthepresentstudyPrimercodesSequences（5’-3’）TargetgeneReferencesXCCOX3FAGYACDGTDGGDTTRCATTTcox31PresentstudyXCCOX3RCANAYATAATCMACARAATGNCAcox31PresentstudynxccobFATGTCWTWTTGRGCKGCBACNGTcytb1PresentstudynxccobRGADVCTCNGGRTGRCAVGCHCCcytb1PresentstudynxcND4FGAKTCBCCDTATTCDGARCGnad41Presentstudy1PresentstudynxcND4RACHCCNGCHGANANMCCRTGMCCnad41PresentstudyTXCCOX1FGGHTGAACHRTWTAYCCHCCcox11PresentstudyTXCCOX1RTGRTGRGCYCAWACDAYAMAHCCcox11PresentstudyXC12SFAAWAAYGAGAGYGACGGGCGrrnSXC12SRTARACTAGGATTAGATACCCrrnS1PresentstudyNxcND5FTGKTTGCBTCNCGNTTBGGNGATGnad51PresentstudyNxcND5RTAACACTTRCANAHMCCRTGHGTnad51Presentstudy3CF1TGCATGTAGTGATAGGTTTGGcox3-cytb2Presentstudy3CR1AACTAACGTAACATTTGTCACcox3-cytb2Presentstudy3CF2TTTGTTTTGTGGTTGCCTTCcytb-nad42Presentstudy3CR2AACGTAAATTAAACCTCCCCCcytb-nad42Presentstudy3CF3TGGCGTTTTTGAGTTTGTCTCnad4-cox12Presentstudy3CR3TCAACGAACTCAATATACTTGnad4-cox12Presentstudy3CF4TGGTTTCGGGGCTGTGAGACcox1-rrnS2Presentstudy3CR4ACCAAGCAAAGAAAATTCTACCcox1-rrnS2Presentstudy3CF5TGTTAAAAGGCTTTGGTGTGrrnS-nad52Presentstudy3CR5-1ACCAACCAAACCTACACATCrrnS-nad52Presentstudy3CF6-1TTACGTTAGTTGGGTTGTTGnad5-cox32Presentstudy3CR6TTACATCTTTATAAAACACTTTCnad5-cox32Presentstudy1shortregionsamplifiedbyPCRfromcox3(139bp),cytb(613bp),nad4(554bp),cox1(497bp),rrnS(500bp)andnad5(458bp).2largefragmentsthatwereamplifiedbylong-rangePCRfromcox3-cytb(724bp),cytb-nad4(1008bp),nad4-cox1(4675bp),cox1-rrnS(2198bp),rrnS-nad5(1981bp)andnad5-cox3(1718bp).86 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince4.2.3SequenceanalysesF.elongatusmtgenomesequenceswereassembledmanuallyandthenalignedwiththemtgenomesequencesofF.hepatica,C.sinensisandP.cerviusingtheprogramClustalX1.83(Thompsonetal.,1997).OpenreadingframeswereidentifiedbyORFFinder(http://www.ncbi.nlm.nih.gov/gorf/gorf.html)usingtheechinodermandflatwormmitochondrialcode.Initiationandterminationcodonsofthe12protein-codinggeneswereidentifiedasreported(Leetal.,2001a).The22tRNAgeneswerepredictedusingtRNAscan-SEormanualadjustments(LoweandEddy,1997;Huetal.,2002).ThetworRNAgeneswerepredictedbycomparisonwiththoseofF.hepatica(Leetal.,2001a),C.sinensis(Shekhovtsovetal.,2010)andP.cervi(Yanetal.,2013).Aminoacidsequencesof12protein-codinggeneswereinferredusingExPASyTranslatetool(http://web.expasy.org/translate/)usingtheechinodermandflatwormmitochondrialcodes,andalignedusingMEGA5.0withdefaultsettings(Tamuraetal.,2011).4.2.4NucleotidevariationanalysisThenucleotidevariationbetweenF.elongatusandP.cerviwasanalysedbyslidingwindowanalysisasreported(Yanetal.,2013).4.2.5PhylogeneticanalysisAminoacidsequencestranslatedfromindividualgenesofthemtgenomeofF.elongatuswerealignedwiththosepredictedfrommtgenomesofselectedtrematodes,includingC.sinensis(NC_012147)(Shekhovtsovetal.,2010),Dicrocoeliumdendriticum(NC_025280.1)(Liuetal.,2014),F.hepatica(NC_002546)(Leetal.,2001a),Haplorchistaichui(NC_022433.1)(Leeetal.,2013),Metagonimusyokogawai(KC330755.1),Opisthorchisviverrini(JF739555.1)(Caietal.,2012),P.cervi(NC_023095.1)(Yanetal.,2013),Schistosomahaematobium(NC_008074)(Littlewoodetal.,2006),Schistosomajaponicum(AF215860)(Leetal.,2001b),Schistosomamekongi(NC_002529)(Leetal.,2000),Schistosomaspindale(NC_008067)(Littlewoodetal.,2006),andthecestodeTaeniasolium(outgroup)(NC_004022.1)(Nakaoetal.,2003).TheaminoacidsequencesofselectedtrematodeswerealignedusingMEGA5.0(Tamuraetal.,2011),andphylogeneticanalysisofthealignedaminoacidsequenceswasconductedinMEGA5.0usingtheMaximumLikelihood(ML)method.87 HuazhongAgriculturalUniversityPhDDissertation20184.3Results4.3.1FeaturesofthemtgenomeofF.elongatusThecompletemitochondrialgenomeofF.elongatus(GenBankaccessionno.KM_397348)is14,120bpinlength.ThelengthoftheF.elongatusmtgenomeislargerthanthemtDNAgenomesofC.sinensis(13,875bp)andS.japonicum(14,085bp),butsmallerthanD.dendriticum(14,884bp),F.hepatica(14,462bp),H.taichui(15,130bp),M.yokogawai(15,258bp),S.haematobium(15,003bp),S.mekongi(14,072bp)andS.spindale(16,901bp).ThecircularmtgenomeofF.elongatusincludes12protein-codinggenes(cox1-3,nad1-6,nad4L,cytbandatp6),22tRNAgenes,tworRNAgenes(rrnSandrrnL)andtwonon-codingregions(SNRandLNR).Allthe12protein-codinggenesaretranscribedinthesamedirection(Fig.4-1),whichisthesameasinF.hepatica(Leetal.,2001a),C.sinensis(Shekhovtsovetal.,2010)andP.cervi(Yanetal.,2013).Thegenearrangementorderisasfollow:cox3-cytb-nad4L-nad4-atp6-nad2-nad1-nad3-cox1-rrnL-rrnS-cox2-nad6-nad5,whichisconsistentwithF.hepatica,O.viverrini,P.cervi,S.japonicumandS.mekongi,exceptforS.haematobiumandS.spindale(Littlewoodetal.,2006).OverlappingnucleotidesbetweenmtgenesofF.elongatusrangedfrom1to53bp(Table4-2).TheF.elongatusmtgenomehas26intergenicspacersrangingfrom1bpto148bpinlength(Table4-2).ThenucleotidecontentsofA,C,TandGinthemtgenomeare19.78%,9.62%,44.10%and26.50%,respectively(Table4-3),withTbeingthemostfavorednucleotide,followedbyG,AandC,whichisalsothesameasthemtgenomesofF.hepatica(Leetal.,2001a),C.sinensis(Shekhovtsovetal.,2010)andP.cervi(Yanetal.,2013).TheA+Tcontentof12proteincodinggenesand22rRNAgenesofF.elongatusrangedfrom59.65%(rrnS)to66.97%(cox3),andtheoverallA+Tcontentofthemtgenomeis63.88%.88 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceFig.4-1:ArrangementofthemitochondrialgenomeofFischoederiuselongatus.4.3.2Protein-codinggenesTheF.elongatusmtgenomehas12protein-codinggenes,includingcox3,cytb,nad4L,nad4,atp6,nad2,nad1,nad3,cox1,cox2,nad6andnad5.Fortheseproteincodinggenes,ATG(eightof12proteingenes)isthemostcommoninitiationcodon,followedbyGTG(fourof12proteingenes)(Table4-2),whichisthesameasothertrematodes,suchasF.hepatica(Leetal.,2001a),C.sinensis(Shekhovtsovetal.,2010),P.cervi(Yanetal.,2013),S.mekongi(Leetal.,2000).TAG(sevenof12proteingenes)orTAA(fiveof12proteingenes)aretheterminationcodons,thisisinagreementwithotherdigeneans,exceptforP.cervi(OnlyTAGwasusedasterminationcodons).Excludingtheterminationcodons,10,107nucleotidesencode3,369aminoacidsofprotein-codinggenesintheF.elongatusmtgenome.ThemostfrequentlyusedaminoacidisTTT(Phe),withthefrequencyof9.65%,followedbyTTT(Phe),TTG(Leu:8.61%),GTT(Val:5.25%)andTAT(Tyr:5.02%)(Table4-4).TheleastusedcodonsareAAC(Asn:0.06%)andGAC(Asp:0.06%).89 HuazhongAgriculturalUniversityPhDDissertation2018Table4-2:TheorganizationofthemitochondrialgenomeofFischoederiuselongatusSizeNumberofIni/Ter3Gene/regionPositions12AnticodonsIn(bp)aacodonscox31-645645215ATG/TAG0trnH648-71568GTG+2cytb717-18291113371ATG/TAA+1SNR1830-1892630nad4L1893-215626488ATG/TAG0nad42117-33971281427GTG/TAA-38trnQ3409-347163TTG+11trnF3486-354965GAA+14trnM3549-361264CAT-1atp63613-4128516172ATG/TAG0nad24133-5008876292GTG/TAG+4trnV5039-510264TAC+30trnA5109-517971TGC+6trnD5328-539770GTC+148nad15400-6296897299ATG/TAG+2trnN6314-637966GTT+17trnP6384-644764TGG+4trnI6449-651163GAT+1trnK6518-658265CTT+6nad36587-6943357119ATG/TAG+4trnS16955-701460GCT+11trnW7027-709165TCA+12cox17095-86361542514GTG/TAA+3trnT8646-870964TGT+9rrnL48710-97049950trnC9707-976761GCA+2rrnS49768-105187510cox210519-11100582194ATG/TAG0nad611046-11546501167ATG/TAG-53trnY11568-1163265GTA+21trnL111652-1171564TAG+19trnS211717-1178569TGA+1trnL211792-1185665TAA+6trnR11860-1192566TCG+3nad511926-135061581527GTG/TAG0trnG13510-1357465TCC+3trnE13587-1365165TTC+12LNR13652-141204690Theinferredlengthofaminoacidsequenceof12protein-codinggenes:1aminoacid;2initiationandterminationcodons;3intergenicnucleotides;4initiationorterminationpositionsofribosomalRNAsdefinedbyadjacentgeneboundaries.90 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable4-3:Nucleotidecontentsofgenesandthenon-codingregionwithinthemitochondrialgenomeofFischoederiuselongatus.GeneA(%)C(%)G(%)T(%)A+T(%)cox318.298.5324.5048.6866.97cytb18.968.8926.3345.8264.78SNR20.634.7631.7542.8663.49nad4L21.978.3325.3844.3266.29nad416.559.5225.4548.4865.03atp617.6410.0824.4247.8765.50nad215.647.9925.1151.2666.89nad116.397.4728.2147.9464.33nad315.977.8428.0148.1864.15cox118.8711.0224.5145.5964.46rrnL25.8310.3526.7337.0962.91rrnS24.3712.2528.1035.2959.65cox219.9311.1127.4941.5861.51nad617.448.6126.7147.2464.68nad516.328.2928.7846.6262.93LNR26.019.1726.4438.3864.394.3.3TransferRNAandribosomalRNAgenesTheF.elongatusmtgenomeencodes22tRNAs,andthelengthof22tRNAgenesrangedfrom60bpto71bp(Table4-2).Therearetwonon-codingregionsinF.elongatusmtgenome,rrnS(751bp)andrrnL(995bp)(Table4-2).ThelocationofrrnSisbetweentRNA-Cysandcox2andtherrnLisbetweentRNA-ThrandtRNA-Cys,whichisthesameasothertrematodes,suchasF.hepatica(Leetal.,2001a),C.sinensis(Shekhovtsovetal.,2010)andP.cervi(Yanetal.,2013).4.3.4Non-codingregionsManyflatwormshavenon-codingregions,it’scommontofindtwonon-codingregionsintrematodes:onelongnon-codingregion(LNR)andoneshortnon-codingregion(SNR).InF.elongatus,thereisashortnon-codingregion(SNR:62nucleotides),whichislocatedbetweencytbandnad4L.Inaddition,thereisalsoalongnon-codingregion(LNR:468nucleotides)betweentRNA-Pheandcox3(Table4-2),theLNRhastwoobviousfeatures,oneismicrosatellite-likesequences,suchas(TA)n(n<5);theotherishomopolymersequences,suchas(T)n(n<7).Peoplestilldon’tunderstandclearlywhythenon-codingregionsexist,andthefunctionofthem,peoplejustknewthenon-codingregionsmayparticipateinthereplicationofmitochondria(Littlewoodetal.,2006).91 HuazhongAgriculturalUniversityPhDDissertation2018Table4-4:Codonusagefor12protein-codinggenesinthemitochondrialgenomeofFischoederiuselongatusAminoFrequencyAminoFrequencyCodonNumberCodonNumberacid(%)acid(%)PheTTT3259.65IleATT1273.77PheTTC280.83IleATC60.18LeuTTA1674.96IleATA712.11LeuTTG2908.61MetATG1053.12SerTCT1183.50MetGTG1654.90SerTCC60.18ThrACT541.60SerTCA220.65ThrACC30.09SerTCG250.74ThrACA190.56TyrTAT1695.02ThrACG160.47TyrTAC110.33AsnAAT541.60StopTAA30.09AsnAAC20.06StopTAG90.27AsnAAA230.68CysTGT1123.32LysAAG501.48CysTGC90.27SerAGT922.73TrpTGA411.22SerAGC90.27TrpTGG722.14SerAGA310.92LeuCTT431.28SerAGG351.04LeuCTC30.09ValGTT1775.25LeuCTA170.50ValGTC120.36LeuCTG230.68ValGTA581.72ProCCT531.57AlaGCT952.82ProCCC40.12AlaGCC40.12ProCCA110.33AlaGCA130.39ProCCG150.45AlaGCG330.98HisCAT411.22AspGAT621.84HisCAC70.21AspGAC20.06GlnCAA130.39GluGAA170.50GlnCAG140.42GluGAG671.99ArgCGT451.34GlyGGT1654.90ArgCGC00GlyGGC160.47ArgCGA60.18GlyGGA220.65ArgCGG110.33GlyGGG511.514.3.5NucleotidevariabilitybetweenF.elongatusandP.cerviAslidingwindowanalysisofF.elongatusandP.cerviusingfullmtgenomesequencesreflectedthenucleotidediversity(π)foralltheprotein-codinggenes(Fig.4-2).Thehighestandlowestlevelofnucleotidevariabilitywaswithinnad6andcox3,respectively.Inourstudy,cox3andatp6arethemostconservedgenes,andnad6andcox2aretheleastconserved.Withslidingwindowanalysis,wecouldknowtheconservedregionsofmtgenomeamongspecies.92 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceFig.4-2:AslidingwindowanalysisofcompletemtgenomesequencesofFischoederiuselongatusandParamphistomumcervi.Theblacklineshowednucleotidediversityinawindowof300bp(10bpsteps).Nad4Landnad4,cox2andnad6areoverlappinggenes.Generegionsaremarkedingreyboxesandboundariesareindicated.4.3.6GeneticrelationshipsConcatenatedaminoacidsequencedatarepresenting12protein-codinggenesof11digeneanspecies(C.sinensis,D.dendriticum,F.hepatica,H.taichui,M.yokogawai,O.viverrini,P.cervi,S.haematobium,S.japonicum,S.mekongiandS.spindale)andonetapeworm(T.solium)wereusedforgeneticrelationshipanalysis(Fig.4-3).Inthetree,wecanfindtwolargecladeswithstrongsupport(100%):onecladeconsistsofeightmembersrepresentingfivefamilies(Heterophyidae,Opisthorchiidae,Fasciolidae,ParamphistomidaeandDicrocoeliidae);theothercladeisSchistosomatidae.Inthepresentanalysis,F.elongatushastheclosestgeneticrelationshipwithP.cervi(100%),followedbyFasciolidae,thisisconsistentwiththeirrelationshipintheclassificationofbiology.Atthesametime,wealsousedNJmethodanalysis(notshown),andtherewasnodifferencebetweenthesetwomethods.93 HuazhongAgriculturalUniversityPhDDissertation2018Fig.4-3:ThephylogeneticrelationshipsofFischoederiuselongatusandothertrematodesbasedonconcatenatedaminoacidsequencedatarepresenting12protein-codinggenesbyMaximumLikelihoodanalysis,usingTaeniasoliumasanoutgroup.4.4DiscussionThepresentF.elongatusmtgenomecanprovideusefulinformationforthestudiesofepidemiology,speciesidentificationandgeneticdiversityofFischoederiusspp.Atthesame,itwillalsomakecontributiontothetaxonomystudyofFischoederiusspp.WiththefullmtgenomeofF.elongatus,wecanundertakeastudywithinF.elongatusfromdifferentregionsoramongFischoederiusspp.bycombiningthemorphologicalfeatureswithgeneticanalyses(withmolecularmarkersfrommitochondriaorribosome,suchascox1,nad4,18S,ITS-1andITS-2).Meanwhile,themtgenomeofF.elongatusmayalsoprovideinformationforthepreventionanddiagnosisofFischoederiusspp.andperhaps,thismtgenomeinformationmayassistinthenewdrug,sincemitochondriaisthetargetofsomedrugs,suchasdecoquinate.Inconclusion,thecompletemtgenomesequenceofF.elongatesisobtained,analysedandappliedforphylogeneticanalysisforthefirsttime.ThegenearrangementofF.elongatesisthesameasothertrematodes,suchasParamphistomumcervi.Phylogeneticanalysesusingconcatenatedaminoacidsequencesofthe12protein-codinggenesshowedthatF.elongateswascloselyrelatedtoP.cervi.ThecompletemtgenomesequenceofF.elongatesshouldprovideinformationforphylogeneticandepidemiologicalstudiesforF.elongatesandthefamilyParamphistomidae.94 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceChapter5-ComparativeanalysisofthefullmitochondrialgenomeofGastrothylaxcrumenifer5.1IntroductionParamphistomesconsistofagroupofover70species,someofwhichcancauseconsiderableeconomiclossestothelivestockindustry(Horak,1971;Rangel-Ruizetal.,2003).ParamphistomesareprevalentinAfrica,Asia,Australia,EasternEuropeandRussia(Horak,1971)andhaveleadedtoseveraloutbreaksamongyoungruminantsinIndia(Bawa,1939;KatiyarandVarshney,1963;PandaandMisra,1980;Pande,1935).It’sreportedthatG.crumenifer,P.cerviandP.epiclitumarecommoninruminantsinsomeregions(Guptaetal.,1987;Wangetal.,2006;Panyarachunetal.,2013).G.crumeniferisanimportantmemberofparamphistomesparasitizingtherumenand/orreticulumofruminants.AnimalsinfectedwithG.crumeniferusuallyshowmarasmus,weaknessorevendeath.Moreover,G.crumeniferhasobviousseasonalityineggproduction(Hannaetal.,1988),whichmakestheconventionalmicroscopicalexaminationmoredifficultduringthenon-reproductivestage.Therefore,alternativemethodsarerequiredforaccurateidentificationofG.crumenifer.AccurateidentificationofG.crumeniferwillprovidefoundationforthestudyofitsbiology,epidemiologyandgenetics.Althoughmorphologicalcharacteristicshavebeenusedinspeciesidentification,thesemethodsaretime-consumingandhardtoidentifytospecieslevel.Themitochondrialgenomehasbeenusedasanimportantmolecularmarkerforspeciesidentification,phylogeneticanalysisandsoon(Choietal.,2012;Gasseretal.,2012;Lvetal.,2012;Rameshetal.,2012;Shanetal.,2012;Chengetal.,2016;Liuetal.,2016).Besides,somestudiesindicatedthatthecompletemitochondrialgenomedatasetsmaybemoresuitableforexploringcryptic/siblingspecies(MattiucciandNascetti,2008).Todate,thereisstillnoreportaboutthemtgenomeofGastrothylaxtramatodes,despiteavailabilityofadvancedsequencingandanalysismethods.Thepurposeofthisstudywere(i)toobtainandanalysisthefullmtgenomeofG.crumenifer,whichisthefirstreportofmtgenomesofGastrothylaxtramatodes,(ii)tocomparethefullmtgenomewithothertrematodestoaccessthegeneticrelationshipofG.crumeniferandothertrematodes.95 HuazhongAgriculturalUniversityPhDDissertation20185.2Materialsandmethods5.2.1ParasitesandDNAisolationTheG.crumeniferadultswerecollectedfromtherumenofanaturallyinfectedcattleinTianmen,HubeiProvince,PRChina.Afterextensivelywashingwithphysiologicalsaline,thespecimenswereidentifiedaccordingtokeymorphologicalcharacteristics(Li,2011;Wang,2013).TotalgenomicDNAofG.crumeniferwasisolatedfromonespecimenusingakit(E.Z.N.A.®TissueDNAKit,D3396-01).Then,theITS-2regionofG.crumeniferwasamplifiedandsequencedformolecularidentification(Itagakietal.,2003),itwasidenticaltoasequenceavailableforG.crumenifer(GenBankaccessionno.JQ688412.1).5.2.2AmplificationandsequencingofG.crumenifermtgenomeTobeginwith,fivepairsofprimers(Table5-1)weredesignedbasedontheconservedregionsofmtgenomesofF.hepatica(Leetal.,2001a),O.viverrini(Caietal.,2012)andP.cervi(Yanetal.,2013)amplifyingshortfragmentsfromcox1,cox3,cytb,nad4,nad5andrrnS,respectively.PCRreactions(25µl)wereperformedin1×Taqpolymerasebuffer,0.2mMeachofdNTP,0.5µMofeachprimer,2UTaqpolymerase(Takara)and2.5µlgenomicDNAinathermocycler(Biometra)underthefollowingconditions:94°Cfor5min,followedby35cyclesof94°C/30sec,50°C/30s,72°C/1min,andafinalextensionof72°C/7min.Then,PCRproductswereidentifiedbyagarosegelelectrophoresisandpositiveampliconsweresequencedinbothdirections.Later,fiveadditionalpairsofprimersweredesignedbasedonthefiveshortfragmentstoamplifytheremainingfragmentstocoverthecompletemtgenomeinfiveLong-PCRreactions(Table5-1).TheLong-PCRreactions(25µl)wereperformedin0.8mMofeachdNTPs,2.5µl10×LATaqbuffer,0.5µMofeachprimer,2.5ULATaqpolymerase(Takara),and2.5µlgenomicDNAsample.PCRreactionswerecarriedoutunderthefollowingcondition:94°Cfor5min,then35cyclesat94°C/30s,andannealedat50°C/30s,followedbyextensionat72°C/1minper1kb,andafinalextensionof72°C/7min.Then,positivePCRampliconswereclonedintopMD19-Tvector(TAKARA)forsequencing(Huetal.,2007).96 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince5.2.3SequenceanalysesThefullG.crumenifermtsequenceswereobtainedbyassemblingthesequencesofeachfragmentmanually.GeneboundarieswereinferedusingtheprogramClustalX1.83(Thompsonetal.,1997)byaligningthemtgenomesequenceofG.crumeniferagainstthatofF.hepatica(Leetal.,2001a),O.viverrini(Caietal.,2012)andP.cervi(Yanetal.,2013).The12protein-codinggenes,22tRNA,tworRNAandtwonon-codingregionswereidentifiedasreportedbefore(Yanetal.,2013).ThenucleotidevariationbetweenG.crumenifer,F.elongatesandP.cerviwasaccomplishedbyslidingwindowanalysisusingDnaSP5.1(LibradoandRozas,2009).5.2.4PhylogeneticanalysisAll12protein-codinggenesofG.crumenifermtgenomeandotherselectedtrematodesweretranslatedandconcatenatedforphylogeneticanalysis.ParasitesusedintheanalysisincludedClonorchissinensis(NC_012147)(Shekhovtsovetal.,2010),Fasciolagigantica(NC_024025)(Liuetal.,2014),F.hepatica(NC_002546)(Leetal.,2001a),F.elongatus(KM_397348),Haplorchistaichui(NC_022433.1),Metagonimusyokogawai(KC330755.1),O.viverrini(JF729304.1)(Caietal.,2012),P.cervi(NC_023095.1)(Yanetal.,2013),Schistosomahaematobium(NC_008074)(Littlewoodetal.,2006),Schistosomajaponicum(AF215860)(Leetal.,2001b),Schistosomamekongi(NC_002529)(Leetal.,2000),Schistosomaspindale(NC_008067)(Littlewoodetal.,2006)andTaeniasolium(NC_004022.1)(NakaoandSako,2003).T.soliumwasusedasasanoutgroupcontrol.Concatenatedaminoacidsequencedatarepresenting12protein-codinggeneswerealignedusingtheClustalWinMEGAv.5.0(Tamuraetal.,2011).Thephylogeneticanalysiswasperformedbymaximumlikelihood(ML)methodsbasedontheTamura-Neimodel(Tamuraetal.,2011).Confidencelimitswereassessedbyusingbootstrapmethodwith1000replicationsforthetree,andtheremainingsettingsweredefaultvalues(Tamuraetal.,2011).97 HuazhongAgriculturalUniversityPhDDissertation2018Table5-1:PrimersusedinthepresentstudyPrimersSequences（5’-3’）TargetgeneNXCCOX3FTTTTGRTTRTTTWTDATGASKGAcox3NXCCOX3RACAWAATCHACAAAATGYCAATAcox3nxccobFATGTCWTWTTGRGCKGCBACNGTcytBnxccobRGADVCTCNGGRTGRCAVGCHCCcytBnxcND4FGAKTCBCCDTATTCDGARCGnad4nxcND4RAAVGCMARCCAHCGCTTVCCRTCnad4TCCOX1FGAYCCDTTRGGWGGWGGDGATCCcox1TCCOX1RACAMACWCGACGWGGYAAHCCcox1Insect12SFAAWAAYGAGAGYGACGGGCGrrnSInsect12SRTARACTAGGATTAGATACCCrrnSNxcND5FTGKTTGCBTCNCGNTTBGGNGATGnad5NxcND5RTAACACTTRCANAHMCCRTGHGTnad56CF6ACATGTGATTATAGGGTTGcox3-cytB6CR6AACCAACGTAACATTAGTCcox3-cytB6CF1TTTGTTCTATGATTGCCTTCcytB-nad46CR1ATCTACCTCAATTCAGAGACcytB-nad46CF2TGTTCACATTTTTTGTCTGGnad4-cox16CR2CAAATATGTCTTACAGCCCCnad4-cox16CF3AGCTTAAATAAGTATATGTTGCcox1-rrnS6CR3TTTTAAAGAACACACTCCCCcox1-rrnS6CF4GGTAGTAAATGCACTACTTTGrrnS-nad56CR4TCTTCCTCAATACAACCAACrrnS-nad56CF5AGAAGATTGTAGCTTTGTCCnad5-cox36CR5AAGGCAGCTCCAAAAACCTCGnad5-cox398 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince5.3Results5.3.1CharacteristicsoftheG.crumenifermtgenomeThefullmitochondrial(mt)genomeofG.crumenifer(GenBankaccessionNo.KM_400624)is14,801bpinlength,andincludes12protein-codinggenes(cox1-3,nad1-6,nad4L,cytbandatp6),22tRNAgenes,tworRNAgenes(rrnSandrrnL)andtwonon-codingregions(Fig.5-1).Thegenearrangementis:cox3-cytb-nad4L-nad4-atp6-nad2-nad1-nad3-cox1-rrnL-rrnS-cox2-nad6-nad5.ThemtgenomeofG.crumenifercontainsfouroverlappingregionsbetweengenesrangedfrom1to38bp(Table5-2).Thenucleotidecontentsinthemtgenomeare:19.82%(A),9.86%(C),43.69%(T)and26.63%(G).TheA+Tcontentofmitochondrialgenesrangedfrom56.06%(SNR)to67.13%(cox3),andtheA+Tcontentofthemtgenomeis63.51%.AT-skewsandGC-skewsofthecompletemtgenomewerecalculatedforG.crumeniferandotherselectedtrematodes(Table5-3).ThenucleotidecompositionofG.crumeniferwasinfavourofTcomparedwithA(ATskew=-0.376),andinfavourofGcomparedwithC(GCskew=0.460)(Table5-3).Fig.5-1:ArrangementofthemitochondrialgenomeofGastrothylaxcrumenifer99 HuazhongAgriculturalUniversityPhDDissertation2018Table5-2:TheorganizationofthemitochondrialgenomeofGastrothylaxcrumenifer.Gene/regionPositionsSize(bp)Numberofaa1Ini/Tercodons2AnticodonsIn3cox31-645645215ATG/TAG+2trnH648-71568GTG+1cytb717-18291113371ATG/TAG0SNR1830-1895660nad4L1896-215926488ATG/TAG-38nad42120-34001281427ATG/TAG+11trnQ3412-347362TTG+17trnF3491-355565GAA-1trnM3555-361864CAT+1atp63620-4135516172ATG/TAG+22nad24158-5015858286GTG/TAG+4trnV5020-508566TAC+530trnA5616-568772TGC+10trnD5698-576265GTC0nad15763-6665903301ATG/TAG+9trnN6675-674066GTT+4trnP6745-680864TGG+1trnI6810-687263GAT+5trnK6878-694467CTT-21nad36922-7299378126ATG/TAG+4trnS17304-736259GCT+3trnW7386-745065TCA+3cox17454-89951542514GTG/TAA+9trnT9005-907066TGT0rrnL49071-10060990+6trnC10067-1012559GCA0rrnS410126-108807550cox210881-11462582194ATG/TAG-5nad611456-11908453151ATG/TAG+17trnY11926-1199267GTA+29trnL112022-1208564TAG0trnS212086-1215368TGA+18trnL212172-1223665TAA+7trnR12254-1232067TCG0nad512321-139011581527GTG/TAG+11trnG13913-1398270TCC+7trnE13990-1405465TTC0LNR14055-148017470Theinferredlengthofaminoacidsequenceof12protein-codinggenes:1aminoacid;2initiationandterminationcodons;3intergenicnucleotides;4initiationorterminationpositionsofribosomalRNAsdefinedbyadjacentgeneboundaries.100 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable5-3:Compositionandskewnessinthemitochondrialgenomesofselectedtrematodes,includingGastrothylaxcrumeniferNucleotidefrequency(%)CompletegenomesequenceSpeciesSize(bp)AccessionnumberATCGA+T%ATskewGCskewGastrothylaxcrumenifer14,801KM_40062419.8243.699.8626.6363.51-0.3760.460Fischoederiuselongatus14,120KM_39734819.7844.109.6226.5063.88-0.3810.467Paramphistomumcervi14,014NC_02309518.4544.959.1027.5063.40-0.4180.503Fasciolahepatica14,462NC_00254616.0746.119.9427.8962.18-0.4830.474Fasciolagigantica14,478NC_02402515.2647.409.3827.9762.66-0.5130.498Haplorchistaichui15,130NC_02243319.5639.6712.4128.3259.23-0.3400.391Metagonimusyokogawai15,258KC33075517.7937.8914.2130.1155.68-0.3610.359Schistosomahaematobium15,003NC_00807429.0643.287.9719.6672.34-0.1970.423Schistosomaspindale16,901NC_00806730.5742.137.0620.2272.70-0.1590.482Schistosomajaponicum14,085AF21586024.8946.158.3720.5971.04-0.2990.422Schistosomamekongi14,072NC_00252925.9746.237.1820.6272.20-0.2810.483Opisthorchisviverrini13,877JF72930416.9242.4612.9727.6559.38-0.4300.361Clonorchissinensis13,875NC_01214717.2542.9012.3727.4760.15-0.4260.379101 HuazhongAgriculturalUniversityPhDDissertation20185.3.2Protein-codinggenesTheG.crumenifermtgenomehas12protein-codinggenes(Fig.5-1).ThenucleotidecompositionofG.crumeniferwasbiasedtowardGandT(Table5-4),whichissimilartothatofC.sinensis,F.hepatica,O.viverriniandP.cervi,butisslightlydifferentfromS.haematobium,S.japonicum,S.mekongiandS.spindale(biasedtowardAandT).Inthe12protein-codinggenes,usageofCislessfavored(9.86%,onaverage)butTisstronglyfavored(43.69%,onaverage).TheoverallA+Tcontentis63.51%.Fortheseproteincodinggenes,themostcommonlyusedinitiationcodonisATG(nineof12proteingenes)andGTGisusedfortherestgenes(threeof12proteingenes)(Table5-2),whichissimilartootherspecies(ThemostcommonisATG,followedbyGTG,e.g.,F.hepatica(Leetal.,2001a),O.viverrini(Caietal.,2012),P.cervi(Yanetal.,2013),S.spindaleandS.haematobium(Littlewoodetal.,2006).TheterminationcodonisTAG(11of12proteingenes)orTAA(oneof12proteingenes).The12protein-codinggenesencode3,372aminoacidsexcludingtheterminationcodons.ThecodonusageisshowninTable5-5.CodonscomposedofAandTwereusedmorefrequently,indicatingthepreferenceforAandT.Forinstance,themostfrequentlyusedaminoacidisPhe(TTT)withthefrequencyof9.43%,followedbyPhe(TTT),Leu(TTG:8.36%),Val(GTT:5.16%),Gly(GGT:4.92%),Tyr(TAT:4.86%)andCys(TGT:3.35%).TheleastusedcodonsareArg(CGA:0.15%),His(CAC:0.12%)andArg(CGC:0.03%).5.3.3TransferRNAgenes,ribosomalRNAgenesandNon-codingregionsTheG.crumenifermtgenomeencodes22tRNAgenes.Thelengthofthe22tRNAgenesrangedfrom59to72bp(Table5-2).IntergenicandoverlappingnucleotidesarefoundbetweenadjacenttRNAgenes.ThesizeofrrnSandrrnLwere755bpand990bp,respectively(Table5-2).ThelocationofrrnSisbetweentRNA-Cysandcox2andtherrnLislocatedbetweentRNA-ThrandtRNA-Cys,andtheirA+Tcontentswere61.85%and62.32%,respectively(Table5-2).102 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable5-4:Comparisonsofnucleotidecontentsof12protein-codinggenes,tworRNAgenes,andtwonon-codingregionsofthemitochondrialgenomeofGastrothylaxcrumenifer.GeneA(%)C(%)G(%)T(%)A+T(%)cox318.298.6824.1948.8467.13cytb18.7810.2425.6145.3764.15SNR22.739.0934.8533.3356.06nad4L21.977.5825.3845.0867.05nad416.0810.4625.6047.8563.93atp615.899.8824.6149.6165.50nad215.738.5125.7650.0065.73nad116.948.6428.1346.2963.23nad316.938.9926.9847.0964.02cox119.1311.6724.5144.6863.81rrnL25.6610.2027.4736.6762.32rrnS25.0312.0526.0936.8261.85cox221.4812.5426.2939.6961.17nad617.008.3927.1547.4664.46nad515.888.2928.6547.1963.06LNR22.899.6429.4538.0260.91Therearetwonon-codingregionsinthemtgenomeofG.crumenifer,oneshortnon-codingregion(SNR:66bp)andonelongnon-codingregion(LNR:747bp)(Table5-2).PreviousstudiespredictedthattheAT-richnon-codingregionmightbeinvolvedintheinitiationofreplication(Yanetal.,2013).TheLNRofG.crumenifercontainsshorthomopolymertracts(<7nucleotides)andshortmicrosatellite-liketracts(e.g.,(AT)n,n<6),butnolongrepeats,whichissimilartoothertrematodes(Caietal.,2012;Shekhovtsovetal.,2010;Yanetal.,2013).103 HuazhongAgriculturalUniversityPhDDissertation2018Table5-5:Codonusagefor12protein-codinggenesinGastrothylaxcrumenifermitochondrialgenomeAminoFrequencyAminoFrequencyCodonNumberCodonNumberacid(%)acid(%)PheTTT3189.43IleATT1273.77PheTTC300.89IleATC110.33LeuTTA1604.74IleATA671.99LeuTTG2828.36MetATG1103.26SerTCT1193.53MetGTG1484.39SerTCC80.24ThrACT481.42SerTCA160.47ThrACC60.18SerTCG330.98ThrACA170.50TyrTAT1644.86ThrACG160.47TyrTAC100.30AsnAAT521.54StopTAA10.03AsnAAC100.30StopTAG110.33AsnAAA240.71CysTGT1133.35LysAAG511.51CysTGC80.24SerAGT932.76TrpTGA421.25SerAGC150.44TrpTGG732.16SerAGA230.68LeuCTT501.48SerAGG421.25LeuCTC30.09ValGTT1745.16LeuCTA120.36ValGTC160.47LeuCTG341.01ValGTA581.72ProCCT601.78AlaGCT992.94ProCCC80.24AlaGCC50.15ProCCA50.15AlaGCA120.36ProCCG100.30AlaGCG361.07HisCAT421.25AspGAT561.66HisCAC40.12AspGAC80.24GlnCAA140.42GluGAA300.89GlnCAG160.47GluGAG561.66ArgCGT451.33GlyGGT1664.92ArgCGC10.03GlyGGC210.62ArgCGA50.15GlyGGA260.77ArgCGG110.33GlyGGG411.22104 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince5.3.4NucleotidevariabilityAslidingwindowanalysisindicatedthatthehighestlevelofnucleotidevariabilitywaswithincox2,andthelowestwaswithincox3(Fig.5-2).Inthepresentstudy,cox3andnad4Larethemostconservedgenes,andcox2andnad5aretheleastconserved.Fig.5-2:AslidingwindowanalysisofcompletemitochondrialgenomesequencesofGastrothylaxcrumenifer,FischoederiuselongatusandParamphistomumcervi.Theblacklineinthepictureshowednucleotidediversityintheslidingwindowanalysis(windows=300bp;steps=10bp).Therearetwooverlappinggenesintheprotein-codinggenes,oneisbetweenNad4Landnad4,andtheotherisbetweencox2andnad6.Allthe12protein-codinggenesareindicatedusinggreyboxes.5.3.5PhylogeneticanalysesIt’sreportedthatthefullmtsequenceismorereliableforphylogeneticanalyses(Waeschenbachetal.,2012).Therefore,weusedconcatenatedaminoacidsequencedataofthe12protein-codinggenesofG.crumenifer,othereighttrematodes(C.sinensis,F.hepatica,O.viverrini,P.cervi,S.haematobium,S.japonicum,S.mekongiandS.spindale)andonetapeworm(T.solium,asanoutgroup)forthephylogeneticstudy.TherelationshipofG.crumeniferwithselectedspecieswasinvestigated(Fig.5-3).Thetreecontainstwolargecladeswithverystrongsupport(100%),onecontainsfivemembers105 HuazhongAgriculturalUniversityPhDDissertation2018fromthreefamilies(Fasciolidae,ParamphistomidaeandOpisthorchiidae),andtheothercontainsfourmembersfromSchistosomatidaefamily.ThetreeshowedthatG.crumeniferhastheclosestrelationshipwithF.elongatus(100%),whichisinaccordancewiththeirrelationshipintaxonomy.However,additionaldigeneanmtDNAareneededforphylogeneticanalysesforfurtherunderstandingofthephylogenticrelationship.Fig.5-3:ThephylogeneticrelationshipsofGastrothylaxcrumeniferandothertrematodesbasedonconcatenatedaminoacidsequencedatarepresenting12protein-codinggenesusingMaximumLikelihoodanalysis,usingTaeniasoliumasanoutgroup.5.4DiscussionManyparasitescanleadtoconsiderableimpacttothehealthofhumanandanimalsworldwide.Asignificantexampleisparamphistomes(Horak,1971).G.crumeniferisacommonspeciesinsmallruminantsthatcancauseimportanteconomiclossestothebreedingindustry(Li,2011).Despitetheimprovementanddevelopmentoftechnology,thestudyofepidemiology,molecularbiologyandgeneticsislimited.Inthisstudy,weobtainedthecompletemtgenomeofG.crumenifer,whichwillprovidenewmolecularmarkersforthestudyofspeciesidentification,epidemiologyandmoleculargenetics.Inaddition,itwillprovideusefulinformationforsupportingtaxonomicstudiesofparamphistomesofruminantsandotheranimals,alsoassistinginidentifyinglarvaebymolecularmethods.It’sthefirsttimetoreportfullmtgenomeofGastrothylaxtrematodes.Thelength(14,801)ofG.crumenifermtgenomeisdifferentfromotheravailabletrematodes,suchasParamphistomumcerviandFasciolahepatica.Thisismainlycausedbythefractionofnon-codingsequenceandintergenicsequences(Xieetal.,2011).Thegenearrangementis106 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceinaccordancewithotherdigeneans,butdifferentfromthoseofS.haematobiumandS.spindale,sincerearrangementhappenedinS.haematobiumandS.spindale(Littlewoodetal.,2006).Theprotein-codinggenesofG.crumeniferwerepredictedtostart/endwithnormalinitial/stopcodon,whichisthesameasothertrematodes(Littlewoodetal.,2006;Caietal.,2012;Yanetal.,2013).Alltheselectedtrematodesshowedstrandasymmetry(WithATskewrangedfrom-0.159to-0.513,GCskewrangedfrom0.359to0.503).Assistedbyslidingwindowanalysis,weknewtheconservedregionamongparamphistomes,andthiswillhelpprimersdesignforPCR.ThephylogeneticanalysesindicatedspeciesinParamphistomoideawerecloselyrelatedtothatofFasciolidae,followedbyHeterophyidae,OpisthorchiidaeandSchistosomatidae.ThisfullmtgenomeofG.crumeniferenrichesthemitochondrialgenomedataofparamphistomums,andalsoprovidesalternativemolecularmarkersforbiology,epidemiology,populationvariation,diagnosticsandphylogeneticanalysesofG.crumeniferandotherdigeneanspecies.Now,thecompletemtgenomeofG.crumeniferisavailable,it’sinterestingtoconductastudytounderstandtheepidemiologyandecologyofG.crumeniferindifferentstagesandhostscombinedwithmorphologicaldata.Inaddition,conductingtargetedmtgeneticanalyseswillalsohelpustoanalysissequencevariabilityintraditionalmolecularmarkersforspeciesidentificationoftrematodes.Inconclusion,ourstudydeterminedthecompletemtDNAsequenceofG.crumenifer,andcompareditwithotherselecteddigeneans.ThenewmtDNAinformationwillprovideusefulmarkersforstudyingthemolecularepidemiologyandpopulationgeneticsofG.crumeniferandotherParamphistomidae.Italsoprovidesusefulinformationforthediagnosis,preventionandcontrolofParamphistomosisinruminants.107 HuazhongAgriculturalUniversityPhDDissertation2018Chapter6-CharacterizationofthecompletemitochondrialgenomesequenceofHomalogasterpaloniae6.1IntroductionGastrodiscidaespeciesareimportantparamphistomesparasitinginthelargeintestineofsmallruminantssuchasgoats,sheepandcattles,andHomalogasterpaloniae(Poirier,1883)isoneofthemostcommonspecies(Tayloretal.,2007;Li,2011).Asaneglectedpathogen,H.paloniaehavebeenreportedinBurma,China,Formosa,India,Indonesia,Japan,PhilippinesandThailand(Yamaguti,1971).H.paloniae(Poirier,1883)usuallyinhabitsinthecaecumofanimals.Matureeggsareexpelledwithfaecesfromtheintestineintotheenvironment,andwilldevelopintomiracidiumafterseveraldaysunderfavorconditions.Subsequently,miracidiumwillinvadeintofreshwatersnails(intermediatehost).Miracidiumwilldevelopintocercaria,andescapefromthefreshwatersnails,thendevelopintometacercariaonthewaterplants.RuminantsinfectH.paloniaebyintakingwaterplantspollutedbymetacercaria.AlthoughanimalsinfectedwithH.paloniaeusuallydon’tshowobvioussymptoms,itcancauseconsiderablelossestothebreedingofsheepandcattleunderheavyburden(Li,2006).Sincenoeffectivevaccineisavailable,applicationofchemicaldrugsisthemainmethodsforthepreventionandcontrolofH.paloniaeandotherparamphistomes.AccuratediagnosisofH.paloniaeinfectionisessentialforthepreventionandcontrolofthisspecies.Traditionalmorphologicalmethodshavebeenwidelyusedforalongtime,however,thesemethodsaretime-consumingandinaccurate(Bottetal.,2009).Basedontheserestrictedfactors,molecularmethodsbasedonPCRweredevelopedforspeciesidentification(MorganandBlair,1995;Itagakietal.,2003).Recently,themitochondrialgenomehavebeenusedforspeciesidentification,populationdiversity,phylogeneticanalysisandsoon(Choietal.,2012;Liuetal.,2012;Liuetal.,2015;Chengetal.,2016).Besides,advancesinPCR-coupledsequencingtogetherwithbioinformaticsanalysishavebeenprovedtobeusefulforthemolecularstudyofspecies(Jexetal.,2010).Inthepresentstudy,weaimed(i)tocharacterisetheH.paloniaemtgenome,andthisisthefirstreportaboutfullmtgenomeofGastrodiscidae;(ii)toassessthephylogeneticrelationshipbetweenGastrodiscidaeandotherfamily;(iii)toprovideusefulinformationforfurtherstudyofspeciesidentification,biology,populationgeneticsandphylogeneticanalysis.108 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince6.2Materialsandmethods6.2.1ParasitesandDNAextractionAdultflukeswerecollectedfromthecaecumofanaturallyinfectedblackgoatpost-morteminMacheng,HubeiProvince,PRChina.Aftersufficientlywashedwithphysiologicalsaline,thespecimenswerefixedin75%(V/V)ethanolandpreservedat-20°Cuntiluse.TheseflukeswereidentifiedtobeHomalogasterpaloniaebasedonkeymorphologicalcharacteristicsandparasiticpositions(Li,2006;Li,2011).Subsequently,totalgenomicDNAofH.paloniaewasisolatedfromsinglewormbyakit(E.Z.N.A.®TissueDNAKit,D3396-01).Thethesecondinternaltranscribedspacer(ITS-2)regionofH.paloniaewasamplifiedandsequencedforfurthermolecularidentification(Itagakietal.,2003),thesequencewas100%similaritywithasequenceavailableforH.paloniae(GenBankaccessionno.KM281535.1).6.2.2PCR-coupledsequencingofH.paloniaemitochondrialgenomeFirstly,sevenpairsofprimers(Table6-1)weredesignedandsynthesisedtoamplifyshortfragmentsfromnad1,nad4,cox1,cox2,nad5,rrnSandcytbbasedontheconservedregionsofthemtgenomesofF.hepatica(Leetal.,2001a)andP.cervi(Yanetal.,2013).PCRreactions(25µl)wereasfollow:1×Taqpolymerasebuffer,0.2mMeachofdNTP,0.5µMofeachprimer,2UTaqpolymerase(Takara)and2.5µlgenomicDNAinathermocycler(Biometra)underthefollowingconditions:94°Cfor5min,followedby35cyclesof94°C/30sec,50°C/30s,72°C/1min,andafinalextensionof72°C/7min.Subsequently,PCRproductswereidentifiedbyagarosegelelectrophoresisandpositiveampliconsweresentforsequencinginbothdirections.Later,sevenpairsofprimersweredesignedbasedontheobtainedsevenshortfragmentstoamplifytheremainingsequencesofthecompletemtgenomeinsevenLong-PCRreactions(Table6-1).TheLong-PCRreactions(25µl)wereperformedin0.8mMofeachdNTPs,2.5µl10×LATaqbuffer,0.5µMofeachprimer,2.5ULATaqpolymerase(Takara),and2.5µlgenomicDNAsample.PCRreactionswerecarriedoutunderthefollowingcondition:94°Cfor5min,then35cyclesat94°C/30s,andannealedat50°C/30s,followedbyextensionat72°C/8min,andafinalextensionof72°C/7min.Then,positivePCRampliconswerepurifiedandclonedintopGEM-Tvector(Promega,USA)forsequencingusingaprimer-walkingstrategy.109 HuazhongAgriculturalUniversityPhDDissertation20186.2.3Assembly,annotationandbioinformaticsanalysesThecompleteH.paloniaemtsequenceswereassembledbybioinformaticsanalysisandmanuallyadjustment.GeneboundariesofthemtgenomesequenceofH.paloniaewerepredictedbyaligningagainstthatofofF.hepatica(Leetal.,2001a)andP.cervi(Yanetal.,2013)usingthesoftwareClustalX1.83(Thompsonetal.,1997).Theprotein-codinggenes,transferRNA,ribosomalRNAandnon-codingregionswereannotatedasreported(Yanetal.,2013).NucleotidecomparisonofthecompletemtgenomeofH.paloniaeandother17selecteddigeneanswasconducted.Meanwhile,theAT-skewandGC-skewwerecalculatedaspreviousstudy(Baeketal.,2014;Yanetal.,2014).6.2.4SlidingwindowanalysisofnucleotidevariationPairwisealignmentofthecompletemtgenomeofH.paloniae,Fischoederiuselongates,Paramphistomumcervi,GastrothylaxcrumeniferandOgmocotylesikaewasaccomplishedbyMEGAv6.0topredictvariablenucleotidesites(Tamuraetal.,2013).Subsequently,aslidingwindowanalysisofH.paloniae,F.elongates,P.cervi,G.crumeniferandO.sikaewasaccomplishedusingDnaSPv.5.0toassessthenucleotidevariationdiversityforthe12protein-codinggenesamongthesefiveparamphistomes(LibradoandRozas,2009).6.2.5PhylogeneticanalysisAll12protein-codinggenesofH.paloniaemtgenomeandother17selecteddigeneansweretranslated,concatenatedandalignedforphylogeneticanalysis,includingClonorchissinensis(NC_012147)(Shekhovtsovetal.,2010),Fasciolagigantica(NC_024025)(Liuetal.,2014),Fasciolahepatica(NC_002546)(Leetal.,2001a),F.elongatus(KM_397348)(Yangetal.,2015a),G.crumenifer(KM_400624),Haplorchistaichui(NC_022433.1)(Leeetal.,2013),Hypoderaeumconoideum(KM111525)(Yangetal.,2015b),Metagonimusyokogawai(KC330755.1),Ogmocotylesikae(KR006934)(Maetal.,2015),Opisthorchisfelineus(EU_921260)(Shekhovtsovetal.,2010),Opisthorchisviverrini(JF729304.1)(Caietal.,2012),Paramphistomumcervi(NC_023095.1)(Yanetal.,2013),Schistosomahaematobium(NC_008074)(Littlewoodetal.,2006),Schistosomajaponicum(AF215860)(Leetal.,2001b),Schistosoma110 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincemekongi(NC_002529)(Leetal.,2000),Schistosomaspindale(NC_008067)(Littlewoodetal.,2006)andTrichobilharziaregent(NC_010976)(Websteretal.,2007).AndTaeniasolium(NC_004022.1)(NakaoandSako,2003)wasincludedasanoutgroupcontrol.TheaminoacidsequenceswerealignedandsubjectedtophylogeneticanalysisbyMaximumLikelihoodMethodsusingMEGAv.6.0withdefaultsettings(Tamuraetal.,2013).6.3Results6.3.1GenomecontentandorganizationThecompletemitochondrial(mt)genomeofH.paloniae(GenBankaccessionno.KT266674)is14,490bpinlength(Fig.6-1),andcontains12protein-codinggenes,22tRNAgenes,tworRNAgenes(rrnSandrrnL)andtwonon-codingregions(Table6-2).Allthegenesaretranscribedinthesamedirection,whichisinaccordancewithotherdigeneans(Leetal.,2001a;Yanetal.,2013).ThegenearrangementissimilarwithotherdigeneansexceptforS.haematobiumandS.spindale(Littlewoodetal.,2006).Asforthenucleotidecomposition,H.paloniaemtgenomeisobviouslyfavorinT(Table6-3).Thenucleotidecontentsinthecompletemtgenomeare:21.92%(A),9.28%(C),43.21%(T)and25.6%(G).AndtheA+Tcontentofmtgenesrangefrom63.23%to71.04%,totalA+Tcontentis65.12%.6.3.2AnnotationofH.paloniaemtgenomeTheH.paloniaemtgenomehas12protein-codinggenes(Fig.6-1).Forthesegenes,themostcommonlyusedstartcodonisATG(nineof12proteingenes)andGTGisusedbytheremaininggenes(threeof12proteingenes)(Table6-2),whichisinagreementwithotherdigeneans(Leetal.,2001a;Littlewoodetal.,2006;Caietal.,2012;Yanetal.,2013;Yangetal.,2015a).TheterminationcodonisTAAfornad5,andTAGfortherestgenes.NoincompletecodonsareusedinthemtgenomeofH.paloniae.111 HuazhongAgriculturalUniversityPhDDissertation2018Table6-1:PrimersusedforamplifyingthemitochondrialgenomeofHomalogasterpaloniaePrimersSequences（5’-3’）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-cytbPFXCR1ACAAAGGATTTTTAGAACCCCCnad5-cytbPFXCF2TGTTCTTTGGTTACCGACAGcytb-nad4PFXCR2AATGGGGGTAACGGTATCTTTGcytb-nad4PFXCF3GCCTTTGGGGCTGTTTTGTGnad4-nad1PFXCR3ATTGAACCTAACAACCTAGnad4-nad1PFXCF4TGGGGTTATTAGTCACATTTGTGnad1-cox1PFXCR4CTTAATACCTGTTGGTATACCnad1-cox1PFXCF5ATTCGATGGTACATGATACGTGcox1-rrnSPFXCR5GGACGAACTTCATCGGCTGCcox1-rrnSPFXCF6CCAGGTCTTTGTGCTGCTGArrnS-cox2PFXCR6CATCCGCAGACGTCACCArrnS-cox2PFXCFF7GTGATTTTGTGGGTGGTGTGcox2-nad5PFXCFR7CTACTTTATTACACGTTGATAACGcox2-nad5112 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceFig.6-1:ArrangementofthemitochondrialgenomeofHomalogasterpaloniaeGenesorganizationof12protein-codinggenes,22tRNAgene,twoRNAgenesandtwonon-codingregionsinthemitochondrialgenomeofHomalogasterpaloniaeThe12protein-codinggenesencode3,359aminoacidsexcludingtheterminationcodons(Table6-4).Amongalltheaminoacids,Phe(TTT:10.00%)isthemostused,followedbyLeu(TTG:7.09%)andLeu(TTA:6.91%).TheleastusedcodonisArg(CGC:0.12%),followedbyLeu(CTC:0.15%)andArg(CGG:0.15%).AsforthetRNAgenesandrRNAgenes,thelengthofthe22tRNAgenesrangedfrom59to72bp(Table6-2).ThesizeofrrnSandrrnLwere741bpand984bp,respectively(Table3).ThelocationofrrnSisbetweentrnCandcox2andrrnLisbetweentrnTandtrnC,andtheirA+Tcontentwas63.70%and65.14%,respectively(Table6-3).IntheH.paloniaemtgenome,twonon-codingregionswererecognizedbasedontheirAT-richfeaturesandlocations(Yanetal.,2013),oneshortnon-codingregion(SNR;71bp)andonelongnon-codingregion(LNR:1022bp)(Table6-2).ThelocationofSNRisbetweencytbandnad4L,andLNRislocatedbetweentrnEandcox3.113 HuazhongAgriculturalUniversityPhDDissertation2018Table6-2:ContentoftheHomalogasterpaloniaemitochondrialgenomeSize1Ini/Ter3Gene/regionPositionsNumberofaa2AnticodonsIn(bp)codonscox31-645645215ATG/TAG0trnH647-72175GTG+1cytb724-18451122374ATG/TAG+2SNR1846-1916710nad4L1917-218026488ATG/TAG0nad42141-34211281427ATG/TAG-40trnQ3423-348664TTG+1trnF3499-356365GAA+12trnM3564-362764CAT0atp63628-4137510170ATG/TAG0nad24149-5021873291ATG/TAG+11trnV5028-509164TAC+6trnA5113-517967TGC+21trnD5183-524866GTC+3nad15249-6151903301GTG/TAG0trnN6154-622370GTT+2trnP6227-629165TGG+3trnI6292-635463GAT0trnK6360-642566CTT+5nad36426-6782357119ATG/TAG0trnS16785-684460GCT+2trnW6851-691565TCA+6cox16919-84601542514GTG/TAG+3trnT8484-855269TGT+23rrnL48553-95369840trnC9537-960165GCA0rrnS49602-103427410cox210343-10924582194ATG/TAG0nad610918-11370453151GTG/TAG-7trnY11386-1144863GTA+15trnL111456-1151964TAG+7trnS211524-1158966TGA+4trnL211601-1166363TAA+11trnR11666-1173368TCG+2nad511734-133141581527ATG/TAA0trnG13325-1339066TCC+10trnE13400-1346869TTC+9LNR13469-1449010220Theinferredlengthofaminoacidsequenceof12protein-codinggenes:1aminoacid;2initiationandterminationcodons;3intergenicnucleotides;4initiationorterminationpositionsofribosomalRNAsdefinedbyadjacentgeneboundaries.6.3.3ComparativeanalysesofthemtgenomesNucleotidecomposition,ATskewsandGCskewsofthemtgenomeofH.paloniaeandotherdigeneanswerepresentedinTable6-5.Allthe18digeneansmtgenomesarerichinA+T.ThenucleotidecompositionofH.paloniaeisbiasedtoTcomparedwithA(ATskew=-0.327),andbiasedtoGcomparedwithC(GCskew=0.468),whichisinaccordancewiththatofotherdigeneans.114 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince6.3.4NucleotidevariabilityTheslidingwindowanalysiswasshowedinFig.6-2,thehighestlevelofnucleotidevariabilitywaswithincox1,andthelowestwaswithincox3.Inourstudy,cox3andcytbarethemostconservedgenes,andcox1andnad6aretheleastconserved.Fig.6-2:AslidingwindowanalysisofcompletemtgenomesequencesofH.paloniae,Fischoederiuselongates,Paramphistomumcervi,GastrothylaxcrumeniferandOgmocotylesikaeTheblacklineinthepictureshowednucleotidediversityintheslidingwindowanalysis(windows=300bp;steps=10bp).Therearetwooverlappinggenesintheprotein-codinggenes,oneisbetweenNad4Landnad4,andtheotherisbetweencox2andnad6.Allthe12protein-codinggenesareindicatedusinggreyboxes.Table6-3:NucleotidecompositionofthemitochondrialgenomeofH.paloniae.GeneA(%)C(%)G(%)T(%)A+T(%)cox322.028.3722.4847.1369.15cytb19.619.9825.1345.2864.88SNR29.585.6330.9933.8063.38nad4L23.488.7123.8643.9467.42nad418.359.4524.8247.3865.73atp617.2510.0025.6947.0664.31nad217.418.4824.0550.0667.47nad119.387.7528.1344.7464.12nad318.216.7225.2149.8668.07cox119.9710.8925.8143.3263.29rrnL28.6610.5724.2936.4865.14rrnS26.8611.2025.1036.8463.70cox223.029.6227.1540.2163.23nad616.347.7326.7149.2365.56nad518.288.6727.6445.4163.69LNR31.906.4622.5039.1471.04Total21.919.2825.6043.2165.12115 HuazhongAgriculturalUniversityPhDDissertation2018Table6-4:Codonusagefor12protein-codinggenesinHomalogasterpaloniaemitochondrialgenomeAminoFrequencyAminoFrequencyCodonNumberCodonNumberacid(%)acid(%)PheTTT33710.00IleATT992.94PheTTC160.47IleATC50.15LeuTTA2336.91IleATA732.17LeuTTG2397.09MetATG1063.14SerTCT1063.14MetGTG1534.54SerTCC80.24ThrACT300.89SerTCA371.10ThrACC70.21SerTCG330.98ThrACA240.71TyrTAT1594.72ThrACG230.68TyrTAC150.44AsnAAT441.31StopTAA10.03AsnAAC60.18StopTAG110.33AsnAAA260.77CysTGT1063.14LysAAG521.54CysTGC140.42SerAGT972.88TrpTGA551.63SerAGC60.18TrpTGG641.90SerAGA361.07LeuCTT330.98SerAGG441.31LeuCTC50.15ValGTT1524.51LeuCTA150.44ValGTC120.36LeuCTG290.86ValGTA992.94ProCCT561.66AlaGCT862.55ProCCC20.06AlaGCC70.21ProCCA130.39AlaGCA220.65ProCCG130.39AlaGCG230.68HisCAT421.25AspGAT581.72HisCAC80.24AspGAC30.09GlnCAA140.42GluGAA320.95GlnCAG150.44GluGAG551.63ArgCGT421.25GlyGGT1504.45ArgCGC40.12GlyGGC130.39ArgCGA80.24GlyGGA371.10ArgCGG50.15GlyGGG531.57116 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceTable6-5:Comparisonsofnucleotidecompositionofthefullgenomeofselecteddigeneans,includingHomalogasterpaloniae.SpeciesNucleotidefrequency(%)CompletegenomesequenceATCGA+T%ATskewG+C%GCskewClonorchissinensis17.2542.9012.3727.4760.15-0.42639.850.379Opisthorchisviverrini16.9242.4612.9727.6559.38-0.43040.620.361Opisthorchisfelineus17.2042.6512.3827.7659.85-0.42540.140.383Dicrocoeliumchinensis18.0944.019.9827.9162.10-0.41737.90.473Fasciolagigantica15.2647.409.3827.9762.66-0.51337.340.498Fasciolahepatica16.0746.119.9427.8962.18-0.48337.820.474Hypoderaeumconoideum18.9242.4611.7126.9161.38-0.38438.620.394Haplorchistaichui19.5639.6712.4128.3259.23-0.34040.770.391Metagonimusyokogawai17.7937.8914.2130.1155.68-0.36144.320.359Fischoederiuselongatus19.7844.109.6226.5063.88-0.38136.120.467Homalogasterpaloniae21.9143.219.2825.6065.12-0.32734.880.468Paramphistomumcervi18.4544.959.1027.5063.40-0.41836.600.503Gastrothylaxcrumenifer19.8243.699.8626.6363.51-0.37636.490.460Ogmocotylesikae22.4644.038.7424.7666.49-0.32433.510.478Trichobilharziaregenti22.0046.547.6823.7868.55-0.35831.450.512Schistosomahaematobium29.0643.287.9719.6672.34-0.19727.660.423Schistosomajaponicum24.8946.158.3720.5971.04-0.29928.960.422Schistosomamekongi25.9746.237.1820.6272.20-0.28127.80.483Schistosomaspindale30.5742.137.0620.2272.70-0.15927.30.482117 HuazhongAgriculturalUniversityPhDDissertation20186.3.5PhylogeneticanalysesWaeschenbachandcolleaguesreportedthatthecompletemtsequencesaremorereliableforphylogeneticanalyses(Waeschenbachetal.,2012).Basedonpreviousstudy,theconcatenatedaminoacidsequencedataofthe12protein-codinggenesofH.paloniaeandother17digeneans(C.sinensis,F.gigantica,F.hepatica,F.elongatus,G.crumenifer,H.taichui,H.paloniae,H.conoideum,M.yokogawai,O.sikae,O.felineus,O.viverrini,P.cervi,S.haematobium,S.japonicum,S.mekongi,S.spindaleandT.regent)andonetapeworm(T.solium,asanoutgroup)wereusedforthephylogeneticstudy.TherelationshipofH.paloniaewithselecteddigeneanswasshowedinFig.6-3.Thephylogenetictreecontainstwocladeswithsignificantlystrongsupport(100%),onecontains13membersfromeightfamilies(Opisthorchiidae,Heterophyidae,Echinostomatidae,Fasciolidae,Notocotylidae,Gastrodiscidae,ParamphistomidaeandGastrothylacidae),andtheothercontainsfivemembersfromtheSchistosomatidaefamily.ThetreeindicatedthatH.paloniaewastogetherwithotherparamphistomesincludingF.elongatus,G.crumenifer,O.sikaeandP.cerviinonesub-clade,butseparatedfromF.giganticaandF.hepaticafromFasciolidae,andH.paloniaehastheclosestrelationshipwithmembersfromParamphistomidaeandGastrothylacidaethatinhabitinginsmallruminants.Nevertheless,moremtgenomefromdigeneansareneededforfurtherphylogeneticanalysesinthefuture.Fig.6-3:ThephylogeneticrelationshipsofHomalogasterpaloniaeandotherdigeneansbasedonconcatenatedaminoacidsequencedatarepresenting12protein-codinggenesusingTaeniasoliumasanoutgroup118 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince6.4DiscussionAsanimportantparamphistome,H.paloniaecanleadtoconsiderableeconomiclossestothebreedingindustryofsmallruminantsunderheavyburden.Althoughthedevelopmentofadvancesintechnology,knowledgeaboutepidemiology,biologyandgeneticsisstilllimited.ThepresentstudyfirstlycharacterizedthemtgenomeofH.paloniae.Thegenecontentandorganizationarethesameasotherdigeneans.KnowledgeoftheH.paloniaemtgenomeshouldprovideusefulforcomparativestudyofthisspeciesandotherdigeneans.AsforthecompletemtofH.paloniae,thegenearrangementisthesameasotherdigeneansexceptforS.haematobiumandS.spindale(Littlewoodetal.,2006).Alltheprotein-codinggenesusecompletecodons,whichisinaccordancewithotherselecteddigeneans.Amongallthe18digeneans,allspeciesshowstrandasymmetry(ATskew=-0.513~-0.159;GCskew=0.359~0.512).Withtheaccomplishmentofslidingwindowanalysis,cox1geneisthemostconservedregionamongthesefourparamphistomes,thisisinaccordancewithpreviousstudies,whichindicatedtheconservedcharacteristicsofcox1gene(Rollinsonetal.,2009;Chibwanaetal.,2013;Pérez-del-Olmoetal.,2014).PhylogeneticanalysescanprovideabasicunderstandingoftherelationshipofH.paloniaewithotherdigeneans.AlthoughH.paloniaeisclosertoFasciolidaeinshape,phylogeneticanalysisbasedonthecompletemtgenomeofH.paloniaeandotherdigeneansindicatedthatH.paloniaeiscloselyrelatedtoparamphistomes,thisisinaccordancewiththeirrelationshipintaxonomy.Now,theH.paloniaemtgenomeisavailable,thisshouldprovideusefulinformationforthestudyofepidemiology,biology,speciesidentification,populationgeneticandphylogeneticanalyses.Inconclusion,ourstudyfirstlyreportedthecompletemtgenomesequenceofH.paloniae,andcomparedthemtgenomeofH.paloniaewithotherselecteddigeneans.TheH.paloniaemtgenomeisthefirstmtgenomeavailableforGastrodiscidae.KnowledgeofmtgenomeofH.paloniaeshouldenrichthemtgenomedatabasesofdigeneansandalsoprovideusefulinformationforthestudyofepidemiology,biology,populationgenetics,aswellasphylogeneticanalyses.119 HuazhongAgriculturalUniversityPhDDissertation2018Chapter7-Developmentandevaluationofaloop-mediatedisothermalamplification(LAMP)assayforthedetectionofHaemonchuscontortusingoatfecalsamples7.1IntroductionHaemonchuscontortusisoneofthemostsignificantstrongylidnematodes,withaworldwidedistribution,thatinfectssmallruminants(Tayloretal.,2007).AnimalsinfectedwithH.contortuscanhaveanemia,edema,diarrhea,weakness,emaciationorevendeath,especiallyfortheyoungones(Li,2006;Tayloretal.,2007).Haemonchuscontortushasadirectlifecyclewithoutanyintermediatehosts.Theeggscontainingembryoniccellswereproducedfromfemalewormsandthendischargedwiththegoatfecesintotheenvironment.Theseeggsdevelopintoinfectivethird-stagelarvae(L3)within7daysunderfavourableconditions,andL3cantolerateharshenvironment,astheyhaveasheathcoveringtheirbodies.RuminantsgetinfectedthroughintakingfoodorwaterpollutedwithL3.Itisreportedthatindividualfemalesatpregnantstagecanproducemorethan5000eggsperday,whichincreasethelarvaeburdenheavilyinashortamountoftimeandseriouslythreatenthehealthoftheanimals(Li,2006;Tayloretal.,2007).H.contortusisprevalentthroughouttheworldanddrugresistanceofthisparasiteisaseriousglobalproblem,thereforeaccuratediagnosisofH.contortusinfectionhasimportantimplicationsforthestudyofbiology,epidemiology,controlstrategyanddrugresistance.Traditionaldiagnosticmethodsrelyonexaminationofthemorphologyusingmicroscopy,whichistime-consumingandinaccurate(Van-Wyketal.,2004;Bottetal.,2009;Wang,2013).Forexamples,itisdifficulttodistinguishH.contortuseggsfromthoseofotherstrongylidnematodesastheyareverysimilarinmorphology(Demeleretal.,2012).Toovercomethedisadvantagesofmorphologicalmethods,moleculartechniquesbasedonpolymerasechainreaction(PCR)havebeenusedforidentificationanddiagnosisofkeygastrointestinalnematodesincludingH.contortus(Bottetal.,2009;Roeberetal.,2013a).PCRassayshavegoodspecificityandhighsensitivity(Bottetal.,2009;Roeberetal.,2013b),buttheirapplicationsarelimitedtothelaboratoryastheyneedexpensivePCRthermocycler.Therefore,thedevelopmentofaconvenient,rapidandcost-effectivemethodisneeded,especiallyfordetectionofclinicalsamples,andloop-mediatedisothermal120 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceamplification(LAMP)wasasuitablecandidate.TheLAMPmethodwasfirstlyreportedfordetectionofHIVviralDNAclonedintopBR322vector(Notomietal.,2000),theestablishedLAMPmethodhastheabilitytodetectsixcopiesoftheHBVtargetwithin45min.Also,areversetranscription-coupledLAMPmethodcanbeusedtodetecttheprostate-specificantigen(PSA)mRNAinmixtureswithsinglePSA-expressingcell(Notomietal.,2000).Asanoveltechnology,theLAMPmethodissuperiorinsomeaspectscomparedwithPCRasLAMPiseasytoperform,rapid,cost-saving,specific,sensitiveandsuitableforclinicaldetection.Untilnow,LAMPhasbeenappliedindiagnosisofmanyparasites,includingparasiticprotozoanssuchasBabesiaspp.(Guanetal.,2008),Eimeria(Barkwayetal.,2011),Toxoplasmagondii(Huetal.,2012)andLeishmania(Sriworaratetal.,2015);parasitictrematodesincludingFasciolahepaticaandFasciolagigantica(Aietal.,2010)andparasitictapewormTaenia(Sakoetal.,2013).Recently,LAMPhasalsobeenestablishedfordetectionofparasiticnematodesincludingHaemonchuscontortus(Melvilleetal.,2014),Strongyloidesstercoralis(Wattsetal.,2014)andNecatoramericanus(Mugambietal.,2015).AlthoughaLAMPmethodfordetectingH.contortusinfectioninsheephaspreviouslybeenreported(Melvilleetal.,2014),itsapplicationinclinicalsampledetectionwasnotassessed.ThepresentstudywasaimedatdevelopingaLAMPmethodforthedetectionofH.contortusingoatsandtoevaluateitsusefulnessindiagnosingclinicalfaecalsamples.7.2Materialsandmethods7.2.1ParasitesandgenomicDNAisolationAdultH.contortuswormswerecollectedfromanaturallyinfectedgoatinTianmen,HubeiProvince,China.OthereightspeciesofadultwormsofcommonhelminthsinfectinggoatswerecollectedandusedasthenegativecontrolstoassessthespecificityoftheLAMPmethod,includingBunostomumphlebotomum,Cooperiaoncophora,Oesophagostomumasperum,Teladorsagiacircumcincta,Trichostrongyluscolubriformis,Fasciolahepatica,ParamphistomumcerviandMonieziaexpansa.Allsamplesofadulthelminthswereidentifiedtospeciesusingmorphologicalandmolecularmethods.MorphologicalidentificationofwormswasconductedinaccordancewiththekeyspublishedbyLi(2011)andTayloretal.(2007).Formolecularmethods,firstly,totalgenomicDNAwasisolatedfromasinglewormorsectionsoftheworm(forMoniezia121 HuazhongAgriculturalUniversityPhDDissertation2018expansa)usingSDS/ProteinaseKtreatmentandcolumnpurification(Gasseretal.,1993).Then,thefirstandsecondinternaltranscribedspacerregionsofribosomalDNA(ITS-1/ITS-2)oftheadulthelminthswereamplifiedandsequencedforspeciesidentificationasfollows:fornematodes(B.phlebotomum,C.oncophora,H.contortus,O.asperum,T.circumcinctaandT.colubriformis),ITS-2(~350bp)wereamplifiedusingprimersNC1(5’-ACGTCTGGTTCAGGGTTGTT-3’)andNC2(5’-TTAGTTTCTTTTCCTCCG-CT-3’)(Bottetal.,2009).Fortrematodes(F.hepaticaandP.cervi)ITS-2(~1000bp)wereamplifiedbyprimersBD1(5’-GTCGTAACAAGGTTTCCGTA-3’)andBD2(5’-TATGCTTAAATTCAGCGGGT-3’)(Linetal.2007;Lotfyetal.2010).Fortapeworm(M.expansa),ITS-1(~800bp)wasamplifiedbyPCRusingprimersITS1-F(5’-GCTGCTACCCGCATGATGTT-3’)andITS1-R(5’-GGCAAGCCTATAGCCGCAAT-3’)(Nguyenetal.2012).PCR(25μl)wasperformedin2.5μlof10XPCRBuffer(TaKaRa,Japan),2μlof25mMMgCl2,200μlof2.5mMdNTPmix(TaKaRa,Japan),25pmolofeachprimerand0.5UofTaqDNApolymerase(TAKARA,Japan)underthecyclingconditionsreviouslyreportedfornematodes(Bottetal.2009),trematodes(Aietal.,2007;Lotfyetal.,2010)andtapeworm(Nguyenetal.,2012).AllthepositiveampliconswithonlyonebandonagarosegelweresenttoSangonBiotech(Shanghai,China)fordirectsequencingusingbothforwardandreverseprimers.GenomicDNAofhelmintheggswasisolateddirectlyfrom94goatfaecalsamplesusingSoilDNAkit(OMEGA,USA)followingthemanufacturer’sprotocol.AlltheDNAsampleswerestoredat-20°Cuntiluse.7.2.2LAMPprimerdesignFourLAMPprimers(twoouterprimers:F3andB3;twoinnerprimers:FIPandBIP)weredesignedbasedontheH.contortusITS-2region(GenBankaccessionNo.X78803.1)usingthePrimerExplorerV4softwarepackage(http://primerexplorer.jp/elamp4.0.0/index.html).ITS-2genewasselectedasithadbeenrecognizedasthespecies-specificmarkerandusedinspecies-specificPCRdevelopment(Bottetal.,2009).TheprimersequenceswerevalidatedusingBLASTsoftware(http://www.ncbi.nlm.nih.gov/BLAST)toensurethattheywerespecifictoH.contortus.TargetsequencesforLAMPprimersarepresentedinFig.7-1andprimerinformationislistedinTable7-1.122 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceFig.7-1:Targetsequenceforloop-mediatedisothermalamplification(LAMP)primers.Forwardouterprimer(F3),backwardouterprimer(B3),forwardinnercompositeprimer(FIP;F1c-F2),backwardinnercompositeprimer(BIP;B2c-B2).c=reversecomplement.Table7-1:LAMPprimersforHaemonchuscontortusamplificationPrimernameSequence(5’-3’)LengthF3ACCATATACTACAATGTGGCTAA23bpB3GATAAAAGAACATCGTCGCC20bpFIPTCACGTTGCATGTATATGTTCTTGACAACATTGTTTGTCAAATGGC46bpBIPACATCCCTGAATGATATGAACATGTGTCAATCTCAATATTCGCTGAG47bp7.2.3LAMPreactionTooptimizetheLAMPmethod,LAMPreactionswereperformedunderdifferenttemperatures(rangingfrom59°Cto64°C)andprimerconcentrations(F3/B3=2.5~7.5pM,FIP/BIP=10~40pM).TheoptimaltemperaturefortheLAMPmethodwas63°Candtheoptimalprimerconcentrationswere5pMforF3andB3,40pMforFIPandBIP,respectively.TheLAMPassayswereperformedin25μlreaction.Inbrief,2μlDNAwasheatedat95°Cfor5mintodenatureDNA,thenchilledoniceimmediately.Later,theremainingmixture(23μl)containing1μlBstDNApolymeraselargefragment(NewEnglandBiolabs,USA),2.5μlof10XThermoPolReactionBuffer(NewEnglandBiolabs,USA),4μlof2.5mMdNTPmix(TaKaRa,Japan),4μlof5Mbetaine(Ruibio,China),5pmoleachofF3andB3,40pmoleachofFIPandBIP,and2μlof25mMMgCl2wasaddedtothedenaturedDNA.Subsequently,thetotal25μlmixturewereincubatedat63°Cfor1hourfollowedbyheatingat80°Cfor10mintoterminatethereaction.AfterLAMPreaction,thepositivesampleshowedladder-likebandsonagarosegelafterelectrophoresis.Tovisualizethesampleswitheyes,twomicrolitersofSYBRGreenIdye(CodeNo.EP0801,Aidlab,China)wereaddedtoindividualsamplesandpositivesamplesshowedgreen-yellowcolourundernormallight,whilenegativesamplesremainedorange.123 HuazhongAgriculturalUniversityPhDDissertation20187.2.4PCRassayHaemonchuscontortusspecies-specificPCRwasperformedtoassessthesensitivityoftheLAMPmethod.PartialITS-2(265bp)wasamplifiedbyaspecies-specificprimerHAE(5’-CAAATGGCATTTGTCTTTTAG-3’)andauniversalprimerNC2(5’-TTAGTTTCTTTTCCTCCGCT-3’)(Bottetal.,2009).EachPCR(50μl)wasperformedin10μlof5Xreactionbuffer,4μlof25mMMgCl2,5μlof2.5mMdNTPmix,1μlof5%TritonX-100,50pmolofeachprimerand1.25UofGoTaqDNApolymerase(Promega,USA)underthefollowingconditions:initialdenaturationat94°Cfor5min,followedby35cyclesofdenaturationat94°Cfor30sec,annealingat55°Cfor30secandextensionat72°Cfor30sec,withafinalextensionof72°Cfor5min.7.2.5AssessmentofnonspecificamplificationandpotentialinhibitionTotestforpotentialnonspecificreactionsintheLAMPassay,LAMPproductswereusedastemplatetoamplifyITS-2ofH.contortusbyouterprimerF3andB3.PCRreaction(25μl)wasperformedin2.5μlof10XPCRBuffer,200μlof2.5mMdNTPmix,10pmolofeachprimer,1.25UofTaqDNApolymerase(TAKARA,Japan)and1μlof10folddilutionofLAMPproductunderthefollowingconditions:initialdenaturationat95°Cfor5min,followedby35cyclesofdenaturationat94°Cfor30sec,annealingat50°Cfor30secandextensionat72°Cfor30sec,withafinalextensionof72°Cfor10min.AndtotestforpotentialinhibitionintheLAMPassay,genomicDNAfromthenegativesamplespikedwithlimitedH.contortusgDNA(10pg)wasusedastempletforLAMPdetectionagain.7.2.6StatisticalanalysisStatisticalanalysiswasperformedbySPSSStatisticsv.17.0.StatisticallysignificantdifferencesinpositiveratesofH.contortusbetweenthespecies-specificPCRandLAMPwereaccomplishedusingChi-squaretestinSPSSStatisticsv.17.0.124 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince7.3Results7.3.1SpeciesidentificationForthemolecularidentificationofninespeciesofhelminthsusedtoassessthespecificityofLAMPmethods,allthepositiveampliconsinPCRforeachspeciesweresentforsequencing.AndthesequencesresultsindicatedtheninespeciesofhelminthswereidenticaltoreferencesequencesavailableforB.phlebotomum(GQ859497.1),C.oncophora(AB245040.2),H.contortus(AB908961.1),O.asperum(AB971665.1),T.circumcincta(KC998710.1),T.colubriformis(KC521378.1),F.hepatica(KX198630),P.cervi(KJ459936)andM.expansa(AB367793),respectively.7.3.2SensitivityofLAMPmethodToassessthesensitivityoftheLAMPmethod,H.contortusspecies-specificITS-2PCRwasperformedatthesametime.Todoso,totalgenomeDNAisolatedfromsingleH.contortusadult(50ng/μl)wasusedinbothLAMPandPCRwith10-106folddilution(50ng-5x10-5ng/μl).TheleastvisiblebandshownonagarosegelwasfromPCRproductamplifiedfrom1pggenomicDNA(Fig.7-2A),andtheLAMPresultsstillshowedstrongbandamplifiedfrom1pggDNA(Fig.7-2B),indicatingthesensitivityofLAMPwasequaltothatofPCR.Fig.7-2:Sensitivitycomparisonbetweenthespecies-specificPCR(A)andtheLAMPassay(B)forthedetectionofHaemonchuscontortususingserialdilutionsoftemplateDNAfromsingleH.contortusadult.M:DNAmarker;lane1:100ng;lane2:10ng;lane3:1ng;lane4:100pg;lane5:10pg;lane6:1pg;lane7:100fg.125 HuazhongAgriculturalUniversityPhDDissertation20187.3.3SpecificityofLAMPmethodGenomicDNAfromH.contortusandfromothereightspeciesofparasitichelminths(B.phlebotomum,C.oncophora,O.asperum,T.colubriformis,T.circumcincta,F.hepatica,P.cerviandM.expansa)commonlyfoundingoatsanddistilledwaterwereappliedinLAMPtoassessitsspecificityforH.contortusdetection.TheresultsrevealedthatonlytheproductofLAMPamplificationfromH.contortusgDNAshowedladder-likebandsbyelectrophoreticidentification(Fig.7-3A),andgreen/yellowcolourundernormallightafterstainedwithSYBRGreenI(Fig.7-3B).TheresultindicatedthatLAMPwashighlyspecificandhadnocross-reactionwithanyoftheeighttestedspeciesofparasitichelminthscommonlyfoundingoats.Fig.7-3:SpecificityoftheLAMPassayforthedetectionofHaemonchuscontortususingcommonhelminthsingoatsanddoubledistilledwaterascontrols(A)ElectrophoreticexaminationoftheLAMPproductsM:DNAmarker;lane1:H.contortus;lane2:T.colubriformis;lane3:T.circumcincta;lane4:B.phlebotomum;lane5:C.oncophora;lane6:O.asperum;lane7:F.hepatica;lane8:P.cervi;lane9:M.expansa;lane10:doubledistilledwater.(B)StainedexaminationwithSYBRGreenIofLAMPproducts1:H.contortus;2:T.colubriformis;3:T.circumcincta;4:B.phlebotomum;5:C.oncophora;6:O.asperum;7:F.hepatica;8:P.cervi;9:M.expansa;10:doubledistilledwater.126 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince7.3.4AssessmentofnonspecificamplificationandpotentialinhibitionNonspecificamplificationandinhibitionwhichsometimeshappenedwhenusingfaecalsampleswereassessedinthepresentstudy.Totestfornonspecificamplification,10folddilutionofLAMPproductsfromthefoursamplesthatwerepositiveinLAMPassaybutnotbyPCRwereusedastemplatebyPCRusingouterprimersF3andB3.Afterelectrophoresis,PCRproductsamplifiedfrompositivesamplesshowedaspecificbandwiththeexpectedsizeof220bp,whilenonspecificamplificationdidn’tshowanyband.Testfornonspecificamplificationindicatedthatallthefoursamplesshowedaspecificband(Fig.7-4),thusnonspecificamplificationwerenotinvolvedintheLAMPassay.InordertotestforpotentialinhibitionintheLAMPtest,limitingamounts(10pg)ofH.contortusDNAwerespikedintothesamplepreviouslytestednegativeforbothPCRandLAMP.ThespikedsamplepreparedinthisfashionwasagainamplifiedbytheLAMPassaywhichyieldedpositiveresults,indicatingthatnegativeresultswerenottheresultofinhibitionoftheLAMPassay(Fig.7-5).Fig.7-4:PCRtestforpotentialnonspecificamplificationintheLAMPassayforthedetectionofHaemonchuscontortusM:DNAmarker;lane1:Positivecontrol;lane2:Negativecontrol;lane3-6:PositivesamplesforH.contortusinLAMP.127 HuazhongAgriculturalUniversityPhDDissertation20187.3.5ClinicalsampledetectionNinetyfourfaecalsamplespositiveforstrongylidsbyfaecalexaminationwereusedinLAMPandPCR.Amongthese94clinicalsamples,93(98.94%)wereH.contortuspositivebyLAMPand89(94.68%)wereH.contortuspositivebyPCR.TheLAMPmethoddetectedmorepositivesamples(4)thanPCR.Asindicated,theLAMPmethodhadhigherpositiveratethanthespecies-specificPCR.However,therewasnostatisticallydifferencebetweenthepositiveratesofLAMPandthoseofPCRmethods(χ2=17.991,P=0.053)bythechi-squaretestinSPSSStatisticsv.17.0.Fig.7-5:LAMPtestforpotentialinhibitioninnegativesamplesinLAMPassaysbyspikinglimitedH.contortusgenome(10pg)withnegativesamplesM:DNAmarker;lane1:Positivecontrol;lane2:Negativecontrol;lane3:NegativesamplesspikedwithlimitedH.contortusgenome.7.4DiscussionInthepresentstudy,arapidandreliablediagnosticLAMPmethodwhichhashighsensitivityandspecificityfordetectingH.contortusinfaecalsamplesfromgoatswasestablished.ThisLAMPmethodwasH.contortusspecificsincefourprimerstargetingsixregionsofITS-2ofH.contortus,andthespecificitytestalsodemonstratedthatithadgood128 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincespecificityasitdidn’tdetectotherparasitichelminthscommonlyfoundinsmallruminants,suchasT.colubriformis,T.circumcinctaandO.asperumetc.Forthesensitivity,theresultshowedequalsensitivitytothatofthespecies-specificPCR.Atthesametime,theLAMPassaydidnotneedPCRthermocyclers,onlyawaterbathwasenough.Therefore,theLAMPmethodismoresuitablefordiagnosisofH.contortusinclinicalpractice.Meanwhile,94faecalsampleswereexaminedtoassesstheapplicationofLAMPmethodinclinicaldetectionofH.contortusinfection.TheresultsshowedthattheLAMPmethodhadhigherpositiveratethanPCR,andnonspecificamplificationswerenotinvolvedintheLAMPassays,however,nosignificantdifferencewasobservedbetweentheLAMPandPCR.Wilson(1997)andRådströmetal.(2004)reportedthatfaecalsamplescancontaininhibitorysubstances,suchasbilesalts,haemeandhumicacids,whichmayreduceorinhibitamplification.Todetectpossibleinhibition,H.contortusnegativesamplesweretestedbyLAMPamplifyingtemplatecontainingbothgDNAextractedfromnegativesamplesandthosefromH.contortus,fromwhichLAMPshowedpositiveresult.Therefore,nopotentialinhibitionwasinvolvedintheLAMPassays.Basedonthepresentresults,combinedsensitivetestwithclinicalsampledetectionshowedthatthepresentLAMPwasmoresuitablethanPCRinclinicalfaecalsamplesdetection.ThisiscausedbytheapplicationsofBstDNApolymeraseandLAMPprimers:ononehand,BstDNApolymeraseusedinLAMPreactionislesssusceptibletoinhibitorsthanotherDNApolymeraseusedinPCRreaction,suchasfrequently-usedTaqDNApolymerase(Poonetal.,2006).Inaddition,BstDNApolymerasefunctionsathightemperature(60°C~65°C),whichhelpsreducenonspecificamplifications(Nagamineetal.,2002).Onanotherhand,fourLAMPprimerstargetingsixregionsstrengthenedthespecificityandsensitivityoftheamplification(Nagamineetal.,2002).Asforthe94faecalsamplesusedforclinicaldetectionofH.contortus,onesamplewaspositiveofstrongylidsinfaecalexaminationbutnegativeofH.contortusinLAMP,thiswasnottheresultofinhibitionoftheLAMPsample.Probably,thesamplewasinfectedwithotherstrongylids,suchasTrichostongylusorOstertagia.Recently,aLAMPmethodwasreportedforthedetectionofH.contortusinsheepfaecalsamples(Melvilleetal.,2014),whichisconvenient,rapid,specificandsensitive.However,thepresentLAMPmethodisdifferentfromthatreportedmethodinseveralaspects.Firstly,thepresentmethodwasbasedontheITS-2regionofthenuclearribosomalDNA,whileMelville’smethodtargetedtheITS-1region.Asausefulmolecularmarker,129 HuazhongAgriculturalUniversityPhDDissertation2018ITS-2hasbeenwidelyusedforspeciesidentificationinPCR,real-timePCRandmultiplexedtandemPCR(Bottetal.,2009;Roeberetal.,2013a;Roeberetal.,2013b;Roeberetal.,2015),whiletheuseofITS-1islimited.Meanwhile,thepresentmethodissuitableforthedetectionofH.contortusinfaecalsamplesofgoatsinChinaasitdidn’thaveanycross-reactionwiththespeciesofparasitichelminthscommonlyfoundingoatsinChinaastheselectionofthespeciesspecificallyusedinthespecificitytestofthepresentLAMPmethodwasbasedonapreviousepidemiologicalstudyofgastrointestinalhelminthsingoatsinChina(Yangetal.,2016).WhileinMelville’smethod,theytestedspecificityoftheLAMPprimerswithparasiticspeciescommonlyfoundinsheepwhichdidn’tincludeP.cerviandM.expansa.Meanwhile,thepresentLAMPmethodwasalsoappliedintoclinicaldetectionofgoatfaecalsamplesanddetectedmorepositivesamplesthanPCR.Therefore,thepresentLAMPmethodisasuitablemethodforthedetectionofH.contortusinfaecalsamplesofgoatsbasedontheabove-mentionedadvantagescomparedwithapreviouslyreportedmethod(Melvilleetal.,2014).Untilnow,manydiagnosticmethodsincludingcoproscopic,clinical,immunologicalandmolecularmethodshavebeenappliedinthedetectionofH.contortusinfectioninsmallruminants(Demeleretal.,2012).CoproscopicandclinicalmethodsarebasedonmorphologicalcharacteristicsofH.contortuseggsandanimalhealthstatus,includingflotationmethods(Tayloretal.,2007),McMastertechnique(Tayloretal.,2007),FAMACHAscoreandBCS(Torresetal.,2014).FAMACHA(FAffaMAlanCHArt)scoreisaparasiticanaemiaindexusedtoidentifyanimalsneededforanthelmintictreatmentbyevaluatingocularmucosacomparedwithastandardchart;andBCS(BodyConditionScoring)isusedtohelpidentifyanimalsneededforanthelmintictreatmentbyassessingtheirhealthcondition.Althoughtheyhavebeenusedforalongtime,thesemethodsaretime-consumingandinaccurate(Bottetal.,2009).Inaddition,immunologicalmethodssuchasELISA-basedmethodshavealsobeendescribedforthedetectionofH.contortus(Lietal.,2007;Prasadetal.,2008).However,serologicalapproachesneedspecificantigenorantibodyandcan’tdistinguishpreviousinfectionsfromtherecentones.Molecularmethodsbasedoninvitroamplificationoftargetedgenes,suchasPCR,real-timePCRandmultiplexedtandemPCR(Bottetal.,2009;Roeberetal.,2013a;Roeberetal.,2013b;Roeberetal.,2015),havebeenusedwidelyinrecentyears.Thesemethodsarespecificandsensitive,butneedsexpensivemachinesandlimitedforuseinlaboratory.Incontrast,thepresentLAMPmethodisagoodalternativemethodfordetectionofH.130 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincecontortusinruminants,especiallyfortestingfieldsamplessinceitiseasytoperform,economic,sensitive,specificandsuitableforclinicaldetectionofH.contortus.Inconclusion,wehaveestablishedaLAMPmethodforthedetectionofH.contortusingoats.Ithasbeenproventhatthemethodisspecificandsensitive,andappliedinclinicalfaecalsamplesdetection.Thus,theLAMPmethodcouldprovideanalternativemethodforthedetectionofH.contortusandmoresuitableforclinicaldiagnosis.131 HuazhongAgriculturalUniversityPhDDissertation2018Chapter8-AninvestigationofanthelminticresistanceinwormsofgoatsalongtheHanRiverinHubeiProvince8.1IntroductionGastrointestinalnematodesarecommonlyfoundinsmallruminants,costingovertwobilliondollarsperannumworldwidetocontrolthem(Heetal.,2011).Duetoalackofeffectivevaccinesformostnematodespecies,themainmethodforcontrolofgastrointestinalnematodesofgoatsisthetreatmentwithchemicals(anthlemintics),includingbenzimidazoles,macrocycliclactones,imidazothiazoles,amino-acetonitrilederivativesandcyclooctadepsipeptides(Roeberetal.,2013b;Besieretal.,2016).However,withlong-termanduncontrolleduse,anthelminticresistancehasemergedasamajorproblem(Kaplan,2004;KaplanandVidyashankar,2012),withmultipledrugresistancereportedinregionsincludingAfrica(Van-Wyketal.,1999),Australia(BesierandLove,2003;Love,2007;KaplanandVidyashankar,2012),Brazil(Cruzetal.,2010;Sczesnyetal.,2010),Europe(Sargisonetal.,2007;Sargisonetal.,2010),NewZealand(Leathwicketal.,2000;Waghornetal.,2006;Wrigleyetal.,2006;McKenna,2010),Trinidad(Georgeetal.,2011)andUnitedStates(Howelletal..,2008;KaplanandVidyashankar,2012).WiththeneedformoreanimalproductsandsupportfromtheChinesegovernment(e.g.,Gu,2016;Ouyangetal.,2016),thelivestockindustryhasdevelopedsubstantiallyinrecentyears,withover148milliongoatsin2015accordingtothenationalbureauofstatistics(http://data.stats.gov.cn/index.htm).Withthedevelopmentofgoatbreedingindustry,goatparasites,especiallygastrointestinalnematodes,spread,developandcausehugeeconomiclossestotheindustry(eg.Xiaoetal.,2011;Gu,2016;Yu,2016).Tocontrolandpreventgastrointestinalnematodes,benzimidazoles,macrocycliclactonesandimidazothiazoleshavebeenappliedwidelyinChinafordecades.Untilnow,benzimidazoleresistancehasbeenreportedinHeilongjiang(Heetal.,1999a),InnerMongolia(Zhangetal.,2016),Guangxi(Zhangetal.,2016),Jiangsu(Guetal.,1999),Liaoning(Zhangetal.,2016),Ningxia(Caietal.,2007),Shaanxi(Fengetal.,2016),Shanghai(Heetal.,1999b)Xinjiang(Zhaoetal.,2010)andYunnan(Zhangetal.,2016)inChina,butknowledgeofresistancetootherchemicalsislimited(Zhenetal.,1995;Fengetal.,2016).Thus,thereisaneedtoinvestigatedrugresistancetoprovidea132 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincefoundationfortheeffectiveapplicationofanthelmintics.Inthepresentstudy,weinvestigatedbenzimidazole,macrocycliclactoneandimidazothiazoleresistancesingastrointestinalnematodesofgoatsonselectedfarmsalongtheHanRiverinZhanggang,HubeiProvince;andprovideinformationondeworminginZhanggangandregionswithsimilarconditions.8.2Materialsandmethods8.2.1StudyareaHanRiverisanimportantbranchoftheYangtzeRiver,whichflowsthroughHubeiandShaanxiProvinces.TherearerichpasturesalongtheHanRiver,whichprovidenaturalpasturesforgoatbreedingintheseareas,includingZhanggang.InZhanggang,farmersusuallygrazetheirgoatsonpasturesalongtheHanRiverfromApriltoOctober,andfeedtheirgoatswithplantstrawasrequired.8.2.2AnimalgroupingThetestingforanthelminticresistanceofgastrointestinalnematodesingoatswasperformedintwoanimalflocksalongtheHanRiverinZhanggang,HubeiProvince(112°33’E,30°22’N)fromOctobertoNovember2015forflockA,andfromMarchtoAprilin2016forflockB(Fig.8-1).Fig.8-1:MapoftheHubeiareasfromwheresampleswerecollectedinthisstudy.(★Studyfarms)133 HuazhongAgriculturalUniversityPhDDissertation2018ForflockA,atotalof80goats(oneyearold;30-50kg;witheartagoneachanimal)withaveragefaecaleggcountofover150eggspergram(EPG)wererandomlydividedintofourgroups(20animalspergroup),includingBZ(orallytreatedwithbenzimidazoleatadoseof10mg/kg),LEV(treatedwithlevamisoleatadoseof0.15ml/kgbysubcutaneousinjection),IVM(treatedwithivermectinatadoseof0.02ml/kgbysubcutaneousinjection)andCT(control-withouttreatment).Alloftheanimalsintreatmentgroupswereadministeredaccordingtothebodyweightoftheheaviestgoatinagroup.Theexperimentwasconductedoveraperiodof14days;groupsBZ,LEVandIVMweretreatedonday0.ForgroupBZ,LEV,IVMandCT,faecalsampleswerecollectedfromgoatsondays0and14(Colesetal.,1992).ForflockB,atotalof100goats(oneyearold;30-50kg;witheartagoneachanimal)withaveragefaecaleggcountof>150EPGwererandomlydividedintofivegroups(20animalspergroup),includingBZ(orallytreatedwithbenzimidazoleatadoseof10mg/kg),LEV(treatedwithlevamisoleatadoseof0.15ml/kgbysubcutaneousinjection),IVM(treatedwithivermectinatadoseof0.02ml/kgbysubcutaneousinjection),BZplusLEV(orallytreatedwithbenzimidazoleatadoseof10mg/kgandwithlevamisoleatadoseof0.15ml/kgbysubcutaneousinjection)andCT(withouttreatment).Alltheanimalsintreatedgroupswereadministeredaccordingtothebodyweightoftheheaviestgoatinagroup.Theexperimentwasconductedoveraperiodof14days;groupsBZ,LEV,IVMandBZplusLEVweretreatedonday0.ForgroupBZ,LEV,IVM,BZplusLEVandCT,faecalsampleswerecollectedfromgoatsondays0and14(Colesetal.,1992).8.2.3Faecaleggcountreductiontest(FECRT)Faecaleggcountreductiontest(FECRT)wasperformedusingthemethodrecommendedbytheWorldAssociationfortheAdvancementofVeterinaryParasitology(W.A.A.V.P.)(Colesetal.,1992).Freshfaecalsamples(>4g)werecollectedfromtherectumofeachgoatintoindividualplasticbagsandmarkedwiththeeartagnumber,andsenttotheVeterinaryParasitologyLaboratoryinHuazhongAgriculturalUniversityforFECsundercoolandanaerobicconditionassoonaspossible(Nielsenetal.,2010).FECswerecarriedoutusingtheMcMasterslideaccordingtothestandardprotocol(Colesetal.,1992).Inbrief,2gofindividualsampleswerehomogenisedusingagrinderusing60ml134 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceofsaturatedsodiumnitrate(specificgravity:1.3).Then,0.15mlofthesuspensionwereaddedtoeachchamberoftheslideforeggcountingafter10min,andtheeggspergram(EPG)werecalculatedbymultiplyingthemeaneggsofthetwochambersby100.FECreductionwascalculatedusingtheformula:FECRT=100X(1-Xt/Xc),wheretandcmeantheaverageofEPGfortreatedanduntreatedgroupatthesametime.ResistanceisconfirmedwhentheFECreductionis<95%,andthelower95%confidencelevelis<90%,accordingtoW.A.A.V.P.protocol(Colesetal.,1992).8.2.4Post-mortemexaminationPost-mortemexaminationwasaccomplishedtoconfirmthepresenceorabsenceofgastrointestinalnematodesaftertreatmentforeachgroup,includingthecontrolgroup,inbothflocks.Foreverygroupineachflock,onegoatwiththehighestEPGaftertheFECRTtestwasselectedforpost-mortemexamination(e.g.,Liu,1983;Li,2006;Wang,2013;Maetal.,2014;Tayloretal.,2016).8.2.5DewormingassaysForflocksAandB,themostsensitiveanthelminticmethod(accordingtoFECRT)wasappliedinthedewormingofthewholeflocktotesttheanthelminticeffectforthetwoinvestigatedflocksfollowingFECRTandpost-mortemexamination.8.3ResultsForflockA,resistancesagainstbenzimidazole,ivermectinandlevamisoleweretestedbyFECRT(Table8-1).TheFEC(mean+SD)decreasedto205+256,985+766and15+48aftertreatmentforBZ,IVMandLEVgroups,respectively,andtherewasstatisticallysignificantdifference(P<0.0001)forFECaftertreatmentamongtheBZ,IVMandLEVgroups,andsignificantdifferenceswerefoundbetweenBZandIVMgroups(P<0.0001)andbetweenIVMandLEVgroups(P<0.0001),butnosignificantdifference(P=0.4267)wasfoundbetweenBZandLEVgroupsbyone-wayANOVAanalysisinSPSSV.17.0.TheFECRTforBZ,IVMandLEVgroupswere79%,-11%and99%,respectively,andtheresultsindicatedthatgastrointestinalnematodeswereresistanttobenzimidazoleandivermectinsincetheFECRTofbothBZandIVMgroupswerelessthan95%(79%forBZand-11%forIVM),andthelowerlimitof95%confidence135 HuazhongAgriculturalUniversityPhDDissertation2018intervalswerelessthan90%(73%forBZand0forIVM).Bypost-mortemexamination(Table8-2),nowormwasrecoveredfromLEVgroup,and116,204and250ofHaemonchuscontortuswerefoundintheBZ,IVMandCTgroups,respectively.OesophagostomumandTrichuriswerefoundonlyingroupCT.TheresultsindicatedthatresistanceagainstbenzimidazoleandivermectinmightbeinHaemonchuscontortus,andthatOesophagostomumasperumandTrichurisweresusceptibletotheanthelminticstested.Table8-1:Faecaleggcountreductionrate(FECR%),95%confidenceintervals(UpperCI%andLowerCI%)andresistancestateagainstbenzimidazole,ivermectinandlevamisoleinflockAGroup(No.ofFEC(mean+SD)FECR%UpperCI%LowerCI%Resistancegoats)statusPre-Post-treatmenttreatmentTreatedgroupBZ(20)985+728205+256799473RIVM(20)890+576985+766-11660RLEV(20)930+56915+489910095SUntreatedgroupCT(20)1000+7181325+1530N/AN/AN/AN/AR=resistant,S=susceptible;N/A=notavailableTable8-2:Post-mortemexaminationresultsinflockAGoatno.GroupaEPGAbomasumLargeintestineHaemonchuscontortusOesophagostomumasperumTrichuris170BZ120011600125IVM21002040063LEV0000191CT4200250310Resultsoftotalwormcountforfourgoatsafteranthelminticresistanceassay.aGroupsofgoatsassignedasBZ(benzimidazole),IVM(ivermectin),LEV(levamisole)andCT(control).ForflockB,benzimidazole,ivermectin,levamisoleandbenzimidazolepluslevamisoleweretestedintheassay(Table8-3).TheFEC(mean+SD)decreasedto84+114,358+464,42+88and58+104forBZ,IVM,LEVandBZplusLEVgroupsaftertreatment,respectively,andtherewasstatisticallysignificantdifference(P=0.0006)forFECaftertreatmentamongtheBZ,IVM,LEVandBZplusLEVgroups,andsignificantdifferenceswerefoundbetweenBZandIVMgroups(P=0.0079)andbetweenIVMand136 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceBZplusLEVgroups(P=0.0029)andbetweenIVMandLEVgroups(P=0.0016),butnosignificantdifferenceswerefoundbetweenBZandLEVgroups(P=0.9567)andbetweenBZandBZplusLEVgroups(P=0.9888)andbetweenLEVandBZplusLEV(P=0.9975)byone-wayANOVAanalysisinSPSSV.17.0.TheFECRTforBZ,IVM,LEVandBZplusLEVgroupswere87%,46%,94%and91%,respectively,theresultsindicatedthatgastrointestinalnematodeswereresistanttobenzimidazole,ivermectin,levamisoleandbenzimidazolepluslevamisolesincetheFECRTofBZ,IVM,LEVandBZplusLEVgroupswerelessthan95%(87%forBZ,46%forIVM,94%forLEVand91%forBZplusLEV)andthelowerlimitof95%confidenceintervalswerelessthan90%(72%forBZ,0%forIVM,81%forLEVand77%forBZplusLEV).Post-mortemexaminationresults(Table8-4)revealedHaemonchuscontortusinallthegroups;groupsBZandIVMandCTgrouphadmorewormscomparedwithgroupsLEVandBZplusLEV,andtenOesophagostomumasperumwerefoundinIVMgroup.ParamphistomumcerviandTaeniahydatigenawerealsofoundinmostgroups.AftertheFECRTassays,allanimalsinflocksAandBweretreatedwithBZplusLEV(orallytreatedwithbenzimidazoleatadoseof10mg/kgandwithlevamisoleatadoseof0.15ml/kgbysubcutaneousinjection).Sevendayslater,faecalsampleswererandomlycollectedfrom50ofthe120goatsinflockA,and97ofthe200goatsinflockB.Byfaecaleggcounttest,theaverageEPGforflockAandBdecreasedfrom1110+1257to45+102andfrom895+1027to33+85,respectively.Table8-3:Faecaleggcountreductionrate(FECR%),95%confidenceintervals(UpperCI%andLowerCI%)andresistancestateagainstbenzimidazole,ivermectin,levamisoleandbenzimidazolepluslevamisoleinflockBGroup(No.ofFEC(mean+SD)FECRUpperCI%LowerCI%Resistance%statusgoats)Pre-Post-treatmenttreatmentTreatedgroupBZ(20)295+23784+114879472RIVM(20)369+302358+46446750RLEV(20)286+30042+88949881RBZplusLEV(20)753+57858+104919777RUntreatedgroupCT(20)719+508668+595N/AN/AN/AN/AR=esistant,S=susceptible;N/A=notavailable137 HuazhongAgriculturalUniversityPhDDissertation2018Table8-4:Post-mortemexaminationresultsofeachgroupafterFECRTinflockBGoatGroupaEPGRumenAbomasumLargeLiver,hepaticno.intestineserosaandmesenteryP.cervibH.contortuscO.asperumdT.hydatigenae173BZ1200110001149IVM2100432310080LEV5000440378BZplusLEV50004101163CT2700073602Resultsoftotalwormcountforfivegoatsafteranthelminticresistanceassay.aGroupsofgoatsassignedasBZ(benzimidazole),IVM(ivermectin),LEV(levamisole),BZplusLEV(benzimidazolepluslevamisole)andCT(control);bParamphistomumcervi;cHaemonchuscontortus;dOesophagostomumasperum;eTaeniahydatigena8.4DiscussionThepresentstudyreportedanthelminticresistanceingastrointestinalnematodesingoatsalongtheHanRiverinZhanggang,HubeiProvince.Inthepresentstudy,levamisolewasonlyeffectiveinflockA,andbenzimidazoleandivermectinresistancewasindicatedbyFECRTinthetwoflocks.HaemonchuscontortuswascommonlyfoundinBZ-treated,IVM-treatedandcontrolgroupsandmaybethepredominantspeciescontributingtoanthelminticresistance.AlthoughH.contortuswasalsofoundinLEV-treatedandBZplusLEV-treatedgroupsinflockB,thenumberofH.controtusindividualswaslessthaninBZ-treated,IVM-treatedandcontrolgroups.Byconsultingwithfarmersoftheinvestigatedfarms,BZandIVMweretwocommonlyusedanthelminticsfordeworming,andfarmersoftenusedseveraltimestherecommendeddosesofanthelminticsfordewormingduetothedecreasedeffectoverlongperiods.However,farmersseldomusedLEVfordeworming(personalcommunication).Thus,theresultssuggestthatBZandIVMresistanceisestablishedinbothflocks,butthatLEVisstilleffectiveinflockA.InflockB,combinedtreatmentwithBZandLEVwasapplied,whichwasproposedtoachieveagoodeffectongastrointestinalnematodes,especiallyforHaemonchuscontortusinnorthareasofChinabasedoncommunicationswithpharmaceuticalscompany.Theresultsindicatedthatcombinedtreatmentachievedsimilareffectwithsinglemedication(BZorLEV)intreated-group.Subsequently,afterFECRTandpost-mortemexamination,combinedmedicationofBZplusLEVwasappliedfordeworminginbothflocksAandB,andachievedgoodresultswiththeFECRTof95.95%and96.31%forflocksAandB,respectively.138 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceUntilnow,aseriesofmethodshasbeenusedforsurveysofanthelminticresistance,includingfaecaleggcountreductiontest(Woodetal.,1995),controlledtest(Tayloretal.,2002),egghatchassay(Golesetal.,1992),larvaldevelopmenttest(Golesetal.,1988),larvalparalysisandmigrationtests(MartinandJambre,1979)andPCR-basedmethods(Lacey,1988).Amongthesemethods,FECRTisausefulmethodrecommendedbyW.A.A.V.P.(Woodetal.,1995),andhasbeenwidelyusedindetectionofanthelminticresistancearoundtheworld(eg.VanWyketal.,1999;Lover,2007;Barrereetal.,2013).InChina,anthelminticresistanceingastrointestinalnematodesingoatshasbeenreportedinGuangxi(Zhangetal.,2016),Heilongjiang(Heetal.,1999a),InnerMongolia(Zhangetal.,2016),Jiangsu(Guetal.,1999),Liaoning(Zhangetal.,2016),Ningxia(Caietal.,2007),Shaanxi(Fengetal.,2016),Shanghai(Heetal.,1999b),Xinjiang(Zhaoetal.,2010)andYunnan(Zhangetal.,2016).MoststudiesassessedbenzimidazolesagainstH.contortus(Heetal.,1999b;Fengetal.,2016;Zhangetal.,2016),andinonecaseivermectinwasevaluated(Zhangetal.,2016).AlthoughthereislimitedinformationonanthelminticresistanceingastrointestinalnematodesofgoatsinChina,resistancetochemicalssuchasbenzimidazoleandivermectin,whichhavebeenusedfordecades,islikelytoberelativelywidespread.However,futurestudiesshouldexploreanthelminticresistanceinmoreregionsofChinatounderstandthecurrentstateofanthelminticresistanceingoats,andtoprovidefoundationformanagingthedevelopmentofanthelminticresistance,andeffectivelycontrollinggastrointestinalnematodes.Managementofgoatparasites,especiallygastrointestinalnematodes,iscriticalforthedevelopmentofthegoatindustry(Gu,2016;Yu,2016).Knowledgeabouttheanthelminticresistancestatusofgastrointestinalnematodesingoatsisessentialforpreventingandcontrollingthem.ForAustralia,forexample,theupdatesforanthelminticresistancestatusofinternalparasitesoflivestockisavailableforknowingthedevelopmentofanthelminticresistance(https://wormmailinthecloud.wordpress.com/).ForEngland,theSCOPS(SustainableControlofParasitesinSheep)guidelineswereappliedinthemanagementofanthelminticresistanceinsheep(Abbottetal.,2012).Asforanthelminticresistanceingastrointestinalnematodesofgoats,referencecanbemadetotheexperiencesfromAustraliaandEngland.Inthefuture,investigationsofwidescopeforanthelminticresistanceingoatsneedtobeconductedtocomprehensivelyunderstandthestatusandmanagetheproblem.Inconclusion,thischapterinvestigatedthebenzimidazole,ivermectinandlevamisoleaswellasbenzimidazolepluslevamisoleresistancesingastrointestinal139 HuazhongAgriculturalUniversityPhDDissertation2018nematodesofgoatsalongtheHanRiverinHubeiProvince,andinferredbenzimidazole,ivermectinandlevamisoleresistance,Haemonchuscontortusmaybethepredominantresistantspecies,butfurtherworkisneededtoconfirmthisconclusion.ThepresentstudyprovidessomebaselineinformationforanthelminticresistanceingastrointestinalnematodesofgoatsalongtheHanRiver.140 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceChapter9-DetectionofanthelminticresistanceingastrointestinalnematodesofDabieshanBlackGoatsinLuotian,HubeiProvince9.1IntroductionHelminthsarecommonlyfoundinruminants,especiallygastrointestinalnematodes.Gastrointestinalnematodesofruminantscanleadtosignificanteconomiclossesduetothediseasestheycauseandcostoftreatmentandcontrol(Zairepamu,2016).Duetolackofeffectivevaccines,themainmethodforcontrollinggastrointestinalnematodesisbasedontheapplicationofchemicaldrugs,includingbenzimidazoles,imidazothiazoles,macrocycliclactonesandamino-acetonitrilederivatives(e.g.,Roeberetal.,2013b;Besieretal.,2016).However,duetolong-termandwideuseofthesechemicaldrugsfordeworming,drugresistanceofgastrointestinalnematodeshasbeenreportedinChinaandabroad(e.g.,Zhaoetal.,2010;KaplanandVidyashankar,2012).Excessiveanduncontrolleduseofanthelminticsacceleratesthedevelopmentofdrugresistance(Kaplan,2004).LuotianislocatedintheeastofHubeiProvince.Thereareextensivepasturelandsforgrazinganimals,especiallyforgoatbreeding,andprovidessuitableenvironmentforthedevelopmentandtransmissionofparasites.StudieshaveindicatedthatgastrointestinalnematodesarecommonandaseriousproblemingoatsinLuotian.Benzimidazoles,levamisoleand/orivermectinhavebeenusedfordewormingfordecades,andthedosagesofivermectinincreaseyearbyyear.Somefarmersuseseveraltimestherecommendeddosageofivermectintotreatagainstgastrointestinalnematodes(personalcommunicationwithfarmersinLuotian–ZhangShuisong,ZhangYanhuaandZhouQizhan,2016).Theaimsofthepresentstudywere:(i)tosurveybenzimidazole,levamisoleandivermectinresistanceingastrointestinalnematodesand(ii)toidentifyspeciesthataredrugresistant.141 HuazhongAgriculturalUniversityPhDDissertation20189.2Materialsandmethods9.2.1ExperimentaldesignThepresentstudywasperformedonafarm(flockLT1)inLuotian(115°40’E,30°50’N),HubeiProvince(Fig.9-1)inMarch2017;ethicsapprovalwasfromtheHuazhongAgriculturalUniversity(permitnumber:4200695757).Ingeneral,goats(n=50;lessthan1yearofage;30-50kg;witheartagidentification)wereusedforfaecaleggcountreductiontesting(FECRT)andwereshowntohavemeanfaecaleggcounts(mean+SD)of500+354.Goatswererandomlydividedintofivegroups(10pergroup),includingBZ(benzimidazole-treated),LEV(levamisole-treated),IVM(ivermectin-treated),BZplusLEV(benzimidazole-andlevamisole-treated)andCT(controlgroup;withouttreatment).Allthethreegroupswerekeptonthesamepasture.Benzimidazolewasadministeredorallybyaqualifiedveterinarianatadoseof10mg/kgandivermectinwasadministeredbysubcutaneousinjectionatadoseof0.02ml/kg,accordingtothebodyweightoftheheaviestgoatingroupsBZ,LEVandIVM,respectively.Theexperimentwasconductedoveraperiodof14days;goatsingroupsBZ,LEV,IVMandBZplusLEVweretreatedonday0.ForgroupsBZ,LEV,IVM,BZplusLEVandCT,faecalsampleswerecollectedondays0and14(Colesetal.,1992).Fig.9-1:MapoftheHubeiareasfromwheresampleswerecollectedinthisstudy.(★Studyfarms)142 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvince9.2.2FaecaleggcountreductiontestInbrief,freshfaecalsamples(>4g)werecollectedfromtherectumofindividualgoatsintoplasticbags,andtransferredtotheparasitologylaboratoryatHuazhongAgriculturalUniversityundercoolandanaerobiccondition,andstoredat4°Cforlessthan1week(Nielsenetal.,2010).Then,faecaleggcountswerecarriedoutusingtheMcMasterchamberaccordingtoastandardprocedure(Colesal.,1992).Inbrief,2gofindividualsampleswerehomogenisedin60mlofsaturatedsodiumnitrate(specificgravity:1.3).Then,0.15mlofthesuspensionwasaddedtoeachchamberoftheslideforeggcountafter10min.FECwascalculatedbymultiplyingthemeannumberofeggsinthetwochambersby100asperinstructions.9.2.3StatisticalanalysisThereductioninEPGwascalculatedusingtheprogramRESOFECRTv4.0(http://www.vetsci.usyd.edu.au/sheepwormcontrol/index.html).ApopulationofstronglidnematodeswasdefinedasresistanttoananthelminticifthereductioninEPGwas<95%andthelowerconfidencelimitofthepercentageofreductionwas<90%(Colesetal.,1992).9.2.4DewormingassaysPreviouswork(unpublished)indicatedthatthecombinedtreatmentwithabenzimidazolepluslevamisolehasabettereffectagainstgastrointestinalnematodesingoatsthantheapplicationofbenzimidazole,levamisoleorivermectinalone.Therefore,theusageofbenzimidazolepluslevamisolefordeworminggastrointestinalnematodeswasperformedinalltheanimalsofthewholeflockLT1afterFECRTaswellastwootherflocks(flockLT2andLT3)withsimilarcondition.Beforedeworming,faecalsampleswererandomlycollecteddirectlyfromtherectumof50goatsforeachflockandtheFECscalculated.Sevendaysaftertreatment,faecalsampleswererandomlycollecteddirectlyfromtherectumof50goatsforeachflockandtestedagaintocomparetheFECbeforeandaftertreatment.143 HuazhongAgriculturalUniversityPhDDissertation20189.2.5Post-mortemexaminationDuringtheperiodforFECRT,wealsoexamined56goatsatalocalslaughterhousetoinvestigatetheprevalenceofhelminthsduringthatperiod.Theproceduresusedforpost-mortemexaminationandtheidentificationofhelminthsaregiveninChapter3.9.3ResultsAspresentedinTable9-1,anthelminticresistancewasfoundinallfourgroupstested;especiallyintheivermectin-treatedgroup,theFECincreasedconsiderably14daysafterdewormingcomparedwithbeforedeworming.TheFEC(mean+SD)decreasedto200+232,1080+724,50+50and110+266forBZ,IVM,LEVandBZplusLEVgroupsaftertreatment,respectively,andtherewasstatisticallysignificantdifference(P<0.0001)forFECaftertreatmentamongtheBZ,IVM,LEVandBZplusLEVgroups,andsignificantdifferenceswerefoundbetweenBZandIVMgroups(P=0.0003)andbetweenIVMandBZplusLEVgroups(P<0.0001)andbetweenIVMandLEVgroups(P<0.0001),butnosignificantdifferenceswerefoundbetweenBZandLEVgroups(P=0.8591)andbetweenBZandBZplusLEVgroups(P=0.9645)andbetweenLEVandBZplusLEV(P>0.9889)byone-wayANOVAanalysisinSPSSV.17.0.Theresultindicatedthebenzimidazolepluslevamisole-treatedandlevamisole-treatedgroupsshowedbetteranthelminticeffectthanthebenzimidazole-treatedgroup,buttheivermectin-treatedgroupshowednoanthelminticeffect.Table9-1:Faecaleggcountreductionrate(FECR%),95%confidenceintervals(UpperCI%andLowerCI%)andresistancestateagainstBZ,IVM,LEVandBZplusLEVGroupFEC(mean+SD)FECRUpperCI%LowerCI%Resistance%status(No.ofgoats)Pre-Post-treatmenttreatmentTreatedgroupBZ(10)410+151200+23255870RIVM(10)690+3751080+724-145150RLEV(10)510+45950+50899763RBZplusLEV(10)430+265110+26675960RUntreatedgroupCT(10)460+364440+599N/AN/AN/AN/AR=resistant,S=susceptible;N/A=notavailable144 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceAfterfinishingFECRT,allthegoatsintheflockLTwereadministratedwithbenzimidazolepluslevamisole;theaverageFEC(mean+SD)decreasedfrom500+354to40+57.Combinedtreatmentwithbenzimidazolepluslevamisolewasperformedinothertwoflocks,flockLT2andflockLT3,andtheaverageEPG(mean+SD)decreasedfrom2903+2365to275+224forflockLT2,andfrom852+841to117+37forflockLT3.Thissuggeststhatthedewormingstrategyusingbenzimidazolepluslevamisolecanreducethewormburdenofgastrointestinalnematodesinareaswithmultipleresistanceagainstanthelminticsinthisregion.However,strategiesconsideringgoodmanagementalsoneedtobetakenintoconsideration(BissetandMorris,1996;WoolastonandBaker,1996;Colvinetal.,2008;Kellyetal.,2009;Spickettetal.,2012;Maqbooletal.,2017).9.4DiscussionThepresentstudyisthefirsttoreportanthelminticresistanceingastrointestinalnematodesinLuotian,HubeiProvince.Theresultsindicatethatgastrointestinalnematodesingoatsofthisregionareresistanttoivermectin,benzimidazolesandlevamisole.ComparedwithpreviousstudyinZhanggang,HubeiProvince(seeChapter8),benzimidazoleandivermectinresistanceexistsingastrointestinalnematodesingoatsbothregions,althoughnematodesofgoatsinZhanggangwerestillsusceptibletolevamisoleandlevamisoleplusbenzimidazole.Benzimidazoleisoneofthetraditionaldrugsappliedindewormingsincetheearly1960s.Sincethattime,benzimidazoledrugswithbroadspectrum,highefficiency,lowtoxicity,includingalbendazole,fenbendazoleandoxfendazole,havebeensynthetizedandappliedinpractice(Brownetal.,1961).Thefirstcaseofbenzimidazoleresistancewasreportedinasheepflockundertheapplicationofthiabendazoleforaboutthreeyearsin1964(Conway,1964).Sincethen,duetolong-termandexcessiveusageofbenzimidazoles,anthelminticresistanceemergedworldwide(Kaplan,2012).Untilnow,themechanismofbenzimidazoleresistanceisunderstood,andisreportedtobecausedoneormoreofthreenucleotidepolymorphismsintheisotype-1beta-tubulingene,namelyF167Y(SilvestreAandCabaret,2002),E198A(Ghisietal.,2007)andF200Y(Kwaetal.,1994).BenzimidazoleshavebeenusedfordecadesinChina.Inrecentyears,benzimidazoleresistancehasbeenreportedinGuangxi(Zhangetal.,2016),Heilongjiang(Heetal.,1999a),InnerMongolia(Zhangetal.,2016),Jiangsu(Guetal.,1999),145 HuazhongAgriculturalUniversityPhDDissertation2018Liaoning(Zhangetal.,2016),Ningxia(Caietal.,2007),Shaanxi(Fengetal.,2016),Shanghai(Heetal.,1999b),Xinjiang(Zhaoetal.,2010)andYunnan(Zhangetal.,2016).Ivermectinisalsoawidelyusedanthelminticfordewormingingoatsandsheep;resistancetoivermectinhasbeenreportedinAfrica(VanWyketal.,1999),Australia(BesierandLove,2003),Brazil(Sczesnyetal.,2010),Europe(Sargisonetal.,2007),NewZealand(Waghornetal.,2006),Trinidad(Georgeetal.,2011),theUSA(KaplanandVidyashankar,2012)aswellasinChina(Fengetal.,2016).IvermectinresistanceispoorlyunderstoodbutappearstoinvolvemutationsintheP-glycoproteingene(Kerboeufetal.,2003).Levamisoleresistancehasbeenwidelyreportedthroughouttheworld(Kaplan,2012),buttherearestillnoreportsinChina.Thisprobablyrelatestothefactthatlevamisoleisrarelyusedfordeworminginpractice,sincesomefarmersarelikelytoapplydrugsinexcessivedoses,whichmaycauseserioussideeffectsincludingataxiaorevendeath(Wen,1996).AlthoughtherearelimitedreportsaboutanthelminticresistanceinChina,thepresentworksuggeststhatresistancemaybeprevalent,especiallyforbenzimidazolesandivermectin,whichhavebeenexcessivelyusedforalongtimebyfarmers(personalcommunication).However,thisproposalneedstobetested.AnewdrugcaninduceresistanceinHaemonchuscontortuspopulationswithintenyears(KotzeandPrichard,2016).Clearly,morestudiesneedtobeconductedtocomprehensivelyunderstandthecurrentstatusofanthelminticresistanceingastrointestinalnematodesofgoatsandotherlivestockanimalsinChina.Post-mortemexaminationatalocalslaughterhouseinLuotianinMarch,2017(Table9-2)revealedHaemonchuscontortus,Ostertagiaspp,Oesophagostomumasperum,Trichurisspp,Paramphistomumcervi,EurytremapancreaticumandTaeniahydatigenain56goats.Haemonchuscontortuswasthepredominantspecieswiththeinfectionrateof68%,followedbyTaeniahydatigenawithaprevalenceof52%.H.contortus,Ostertagiaspp,O.asperumandTrichurissppfoundduringtheinvestigationmightcontributetoanthelminticresistanceinLuotian,butthisproposalneedstobetestedinfurtherstudies.Theintensityofinfection(fromhightolow)was:Ostertagiaspp>O.asperum>H.contortus>Trichurisspp.AlthoughtheintensityofH.contortusinfectionwaslowercomparedwiththatofOstertagiaspp.andO.asperum,thedamagecausedbythisspecieswasnotnegligible(Tayloretal.,2016).ThelowintensityofH.contortusinfectioningoatsduringtheinvestigatedperiodsmightbecausedbyarresteddevelopmentinwinterwhichappearstocontributetothespringriseinthenextyear(BlitzandGibbs,1971a;BlitzandGibbs,1971b).Otherinvestigationsofgoat146 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincehelminthsinLuotianseemedtoconfirmthis,sincethewormburdenforH.contortusincreasedsignificantlyinwarmer,humidperiodsoftheyear(seeChapter3).Table9-2:ParasitesandtheirprevalenceandinfectionintensityingoatsinLuotianInfectionrateInfectionintensityGroup/speciesParasiticlocationNo.ofgoatsPrevalence(%)(mean+SD)NematodaHaemonchuscontortusAbomasum3867.8622+29OstertagiasppAbomasum1119.64115+97OesophagostomumasperumLargeintestine1017.8648+45TrichurissppLargeintestine1526.796+5TrematodaParamphistomumcerviRumen11.7935+0EurytremapancreaticumPancreas,small35.36143+9intestineCestodaTaeniahydatigenaSurfaceofliver,serosa2951.792+1and/ormesenteryInconclusion,thepresentchapterreportsivermectin,benzimidazoleandlevamisoleresistanceingastrointestinalnematodesofgoatsinLuotian,withH.contortus,Ostertagiaspp,O.asperumlikelycontributingtotheresistanceproblem,sincethesenematodeswerefoundinpost-mortemexaminationofgoatsduringthesameperiodinLuotian.However,morestudiesareneededtoprovidefurthersupportfortheseproposals.Inthefuture,strategiesneedtobeputinplacetomanageanthelminticresistanceingastrointestinalnematodesofgoatsandotherlivestockanimalsinChina.147 HuazhongAgriculturalUniversityPhDDissertation2018Chapter10-GeneraldiscussionThegoatindustryinChinaisthrivingduetosupportfromthecentralandlocalgovernments(HanandLin,2012;Liuetal.,2012;LvandZhang,2015;Gu,2016;Ouyangetal.,2016;Liu,2017a).ThenumberofgoatsinChinaincreasedtomorethan144millionin2014(http://data.stats.gov.cn/index.htm).However,thedevelopinggoatindustryalsopromotesthespreadofgoatparasites(Gu,2016;Yu,2016);transportationofgoatsacrossthecountryexacerbatesthespreadofparasites(Liu,2017b).Inrecentyears,therehavebeenconsiderableeconomiclossesduetogoatparasites.Forexample,outbreaksofparasiteproblems(e.g.,coccidia,cryptosporidiaandtrichostrongylids)haveledtomortalitiesof8.67-13.23%inewesand27.52-48.13%inkidsonagoatfarminShanghaifrom2006to2011(Chenetal.,2011);in2014,parasiteinfectionsingoatsresultedinthedeathof31goatsandaneconomiclossofoverRMB100,000onafarminGuizhouProvince(Pietal.,2014);in2016,onecaseofoutbreakofFasciolahepaticaingoatsledtoclinicaldiseasein27%ofgoatsanddeathin35%themfrom2010to2015(Li,2016).Therefore,attentionmustbepaidtoavoidingeconomiclossesinthefuture.Helminthsareamajorproblemingoatsandmustbeeffectivelycontrolled.Throughhardworksincethe1950s(Chen,1956),informationonhelminthsingoatswasavailablefor28provincesinChina(seeChapter1).Fromthereporteddata,helminths,especiallygastrointestinalnematodes,wereprevalentinall28provincesinvestigated(ShenangHuang,2004;Shen,2005).FromthesurveysalongtheHanRiverinHubeiProvince(seeChapter2andChapter3),theresultsindicatedthatgoathelminthswerecommonandprevalent,especiallyforHaemonchuscontortusinZhanggangandLuotian,regardlessofgoodorpoormanagementonfarms.Thus,inbothregionsgoatfarmsthecontrolofgoatparasitesmustbeenhanced.Diagnosticmethodsshouldbeimprovedaswell.Currently,thereislimiteduseofmolecularmethodsinChina,despitemolecularmethodstargetingribosomalDNA(ITS-1,ITS-2and18S)andmitochondrialDNA(cox1andnad4)regionshavingbeenwidelyusedelsewhere(Gasseretal.,1993;Aietal.,2007;Linetal.,2007;Lotfyetal.,2010;Shekhovtsovetal.,2010;Wangetal.,2011;Nguyenetal.,2012).Toaddressthisissue,mitochondrialgenomesofimportanttrematodesofsmallruminantwerecharacterizedtoprovidemolecularmarkersforspecificidentification(Chapters4,5and6).Inaddition,aloop-mediatedisothermalamplification(LAMP)methodwasestablishedtodetectH.148 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincecontortusinfectioningoats(Chapter7).FortheinvestigationofhelminthsinthefutureinChina,morepracticalmolecularmethodsneedtobeestablishedandconductedinpractice,suchasthosedevelopedbyBottetal.(2009)andRoeberetal.(2012,2013a,b).Untilnow,althoughmoleculessuchasH11(e.g.,Knoxetal.,2003;Zhouetal.,2010),H-gal-GP(e.g.,Sunetal.,2007),disorganizedmusclefamilymember1(Dim-1)(e.g.,Yanetal.,2013)andactin(e.g.,Yanetal.,2014)havebeenassessedasvaccinecandidatesagainstHaemonchuscontortus,thereisstillnovaccinecommerciallyavailableinChinaforpreventingH.contortusinfection,althoughBabervaxR(gutderivedantigenswithbothH11andH-gal-GP)issoldinAustralia(Nisbetetal.,2016).Therefore,currently,themainmethodforcontrollinggoathelminthsinChinaistheuseofanthelminticsincludingbenzimidazoles,imidazothiazoles,andmacrocycliclactones(Caietal.,2007;Fengetal.,2016).Nevertheless,thesedrugshavebeenexcessivelyusedfordecadesandhaveinducedresistanceingastrointestinalnematodes(Chapters8and9).InvestigationsofanthelminticresistanceofgoatsalongtheHanRiverinZhanggang(Chapter8)andLuotian(Chapter9),HubeiProvinceindicatedbenzimidazole,ivermectinandlevamisoleresistancewerecommoninbothtwoareas,andHaemonchuscontortusmightapredominantspeciescontributingtotheresistanceproblem(becauseofitshighreproductivepotential;Li,2011).AlthoughfarmmanagementinLuotianappearstobebetterthanthatinZhanggang,theanthelminticresistancestatusingastrointestinalnematodesofgoatsinLuotianisworsethanthatinZhanggang.Severalaspectsmaycontributetothisfinding.Firstly,limitedanimalsandflockswereinvestigatedwhichmaybenotsufficienttoreflectrealsituationinbothregions.Secondly,managementwaspoorintheinvestigatedflocksinLuotianandtherewasignoranceamongfarmersregardingparasitemanagementandcontrolandtheneedtoreduceproductionlossescausedbyparasites.Inthefuture,morestudiesneedtobeundertakentocomprehensivelyunderstandtheanthelminticresistanceproblem.ThereisalsoalikelyconsiderableproblemwithanthelminticresistanceingoatsinotherregionsofChina,sincedrugsfrombenzimidazoles,imidazothiazoles,macrocycliclactoneshavebeenusedfordecadesandbecausefarmersarelikelytousedrugsatseveraltimestherecommendeddose(personalcommunicationwithXuHeping,ZhangQinglong,2014;ZhangYanhua,ZhangShuisong,ZhouQizhan;2016).Nowadays,anthelminticresistanceofgastrointestinalnematodesisaseriousglobalproblemforthebreedingindustryofsmallruminant,especiallyinsheepandgoats(KotzeandPrichard,2016).Toaddressthisproblem,integratedparasitemanagementstrategies149 HuazhongAgriculturalUniversityPhDDissertation2018mustbetakenintoaccount(Abbottetal.,2012;McMahonetal.,2013;Chiejinaetal.,2015;Maqbooletal.,2016).Therearefivemainwaysthatanthelminticresistantnematodepopulationsmightbetackled:First,althoughgastrointestinalnematodesareresistantagainstcommonlyuseddruginsomeregions(e.g.,Fengetal.,2016;Zhangetal.,2016),combinedadministrationoftwoormoreclassesofdrug,androtationofdifferentclassesofdrugeveryonetotwoyearscouldbeagoodwayofmanaginggastrointestinalnematodes,whichmightprolongtheeffectivelifeofanthelminticsforuseagainstgastrointestinalnematodes(e.g.,Abbottetal.,2012;Leathwick,2013;Maqbooletal.,2016).Tocontrolsensitivewormsinarelativelylowportionofthepopulation,oncandelaythedevelopmentofanthelminticresistancebyusinga“refugiastrategy”,whichcanbeachievedbytargetedtreatmentofsomeanimalsbasedonthefaecaleggcounts,FAMACHAand/orbodyconditionscore(Van-Wyk,2001;Van-Wyketal.,2006;Pérez,2014).Comparedwithwholeflockdeworming,targetedtreatmentisusefulfordelayingthedevelopmentofanthelminticresistance(e.g.,Abbottetal.,2012;Fieletal.,2017),butmanagementfactorsarealsointegral.Second,goodmanagement,inrelationtograzing,nutritionandbreeding,isalsoessentialforwormcontrol.Criticalisalsotoavoidimportingresistancewormsontofarms;thisrequiresconfirmationthatimportedanimalsdonothavewormsoraredewormedandexaminedcoprologicallybeforeintroducedwithotheranimals(Abbottetal.,2012).Forgrazingmanagement,pasturerestingisessential(Bargeretal.,1994),andtheperiodshouldbebasedonthelocalclimate,rotationalgrazingwithrestingofpastureassistingincontrollinggastrointestinalintropicalandcoldclimaticzones(Chandrawathanietal.,2004;Colvinetal.,2008).Meanwhile,sequencingofgrazingwithdifferentspeciesofanimals,suchascattleandsheep,mightbeausefulstrategytocleansepastures(SouthcottandBarger,1975).Besides,supplementinganimalswithplantswithanthelminticpropertiesongastrointestinalnematodesisalsoanalternativewayofdealingwithanthelminticresistance(Stepeketal.,2004;Anthonyetal.,2005;Hosteetal.,2006).Fornutritionalmanagement,supplementationwithadditionalproteinmaynotreduceinfectionofgastrointestinalnematodes,butcandecreasepathophysiologicalsymptomscausedbyinfections(CoopandHolmes,1996),andalsodecreaseFECsandimproveresilienceaswellasgrowthrateandwoodproductioncomparedwithanimalsnotreceivingsupplements(Steel,2003).Othermanagementaspectsmightincludebreedinganimalsthathaveresiliencetogastrointestinalnematode150 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincechallenge(BissetandMorris,1996;WoolastonandBaker,1996)-breedingforresistanceisaneffectivestrategytocontrolanthelminticresistance(Gill,1991;Windon,1996;Gray,1997).Third,biologicalcontrolcouldbeanotherwayofcontrollinganthelminticresistantnematodes.Nematophagousfungi,suchasArthrobotrysoligospora,Cystopagelateralis,Stylopagehadra,DrechmeriaconiosporaandMeristacrumasterosperum,havebeentestedtoshowtheirabilitytokillparasiticnematodelarvae(e.g.,WallerandLarsen,1993;Yangetal.,2004;Kellyetal.,2009),butthisstillneedstobetestedonalargescaleandindifferentregionsoftheworld.Fouth,discoveryofnewcompoundsfordewormingwouldalsobeuseful(e.g.,Kaminskyetal.,2008a;Burkeetal.,2009;Learmountetal.,2012;Prestonetal.,2016).Asanimportantparasitewithveterinarysignificance,Haemonchuscontortushasbeenusedforamodelforthediscoveryofnewdrug(Geary,2016;Prestonetal.,2016).Forinvitroscreenmethod,H.contortuslarvaehavebeenusedforthediscoveryofnewcompounds,andwasappliedinthediscoveryofpyrantel(Austinetal.,1966),levamisole(Thienpontetal.,1966)andivermectin(Campbell,2012).Usually,themethodisaccomplishedbyexaminationofmobilityofexsheathedthirdstagelarvae(L3)exposedtodrugsforover24hours(Boisvenueetal.,1983)orlarvaldevelopassay(LDA)(IbarraandJenkins,1984).Gearyetal(2015)reportedthatmostoriginal,prototypeanthelminticsonsaleswerediscoveredbyscreeningininfectedanimalstreated.Fifth,thedevelopmentanduseofavaccinewouldbeadvantageous.Untilnow,importantvaccinecandidatemoleculeshavebeenidentifiedinH.contortus;theseincludeH-gal-GP(Smithetal.,1994)andH11(NewtonandMunn,1999).VaccinesofnativeH-gal-GPandH11canresultinasubstantialreductionof70-82%inwormburdenand>90%inFECs(Smithetal.,1994;NewtonandMunn,1999;Smithetal.,2003).In2014,BarbervaxR,avaccinederivedfromnativegutmembraneproteinscontainingbothH-gal-GPandH11ofH.contortusisolatedfromsheep,wasregisteredandonsaleandcansignificantlyreduceFECsinMerinosheep(Nisbetetal.,2016),andhavebeenappliedinsomeflocksinAustralia,Brazil,Merida,SouthAfrica,SwitzerlandandTanzania(www.moredun.org.uk).However,thevaccine’sefficacytocontrolH.controtusneedstobeverifiedindifferentclimaticzonesandhostanimalsonalargescale.AsforthecontrolofanthelminticresistanceinChina,themosteffectivestrategywouldcurrentlyconsistofimprovedmanagementandapplicationofanthelmintics.InChina,mostownersofgoatflockslackknowledgeandtechniquestosupportbuilding151 HuazhongAgriculturalUniversityPhDDissertation2018flocksinagoodmanagementenvironment.Alternativepasturesandrestingperiodforpasturearenotpracticed,thusthere-infectionofgastrointestinalnematodesiscommonandserious.Meanwhile,benzimidazoleandivermectinarewidelyusedindewormingfordecadesevenwithseveraltimesrecommendeddosage;alternatetreatmentsareseldomused,andknowledgeaboutdrugresistanceislackingamongfarmers(personalcommunicationandclinicalobservation).Basedonthissituation,improvementofmanagement,breedingandnutritionaswellasanthelmintictreatmentstrategiesareneeded.Experiencedveterinariansareneededinpracticetoguideparasitetreatmentandcontroltoreduceproductivitylossescausedbyhelminthsandotherparasitesingoats.Regulartrainingoffarmersinthetreatmentofparasitesandmanagementofanthelminticresistancearealsoneededtoensureeffectivecontrol.Recently,insomeregions,somefarmershaveestablishedcooperativestosharetheirexperiencesandimprovefarmingpracticesandproductivityontheirfarms.Thisseemstobeagoodstart.Inanycase,farmersneedtostrivetowardeffectivemanagementandcontrolpracticestoalleviatetheburdenofparasitesingoatsandotherlivestockanimalsinChina.ConclusionInconclusion,thisthesishascontributedtotheknowledgeoftheepidemiologyofgoatparasitesandhasmolecularlycharacterised,forthefirsttime,importantparasitictrematodesanddetectedanthelminticresistanceinhelminthsofgoatsinHubeiProvince.Firstly,epidemiologicalinvestigationsofgoatparasitesinpartsofHubeiProvincewereconductedbasedonmorphologicaltechniquesaswellasPCR-basedsequencingmethods,theresultsindicatedhelminthswerehighthroughouttheyear,particularlyforgastrointestinalnematodes.AndParamphistomumandHaemonchuscontortuswereprevalentinalmostallofthefarmsinvestigated.Secondly,mitochondrialgenomeswerecharacterisedforsomeoftheparasitesidentified,includingFischoederiuselongatus,GastrothylaxcrumeniferandHomalogasterpaloniae.Allthethreespeciessharedthesamegenearrangementformtgenome,andhadarelativelycloserelationshipwithParamphistomumcervibasedonthemtgenome,andtheobtainedmitochondrialgenomecanprovidegeneticmakersforfuturemolecularepidemiological,populationgeneticandphylogeneticstudies.Thirdly,aloop-mediatedisothermalamplification(LAMP)methodwasdevelopedtospecificallydetectH.contortusinfectioningoats,andthemethodprovedtobeasensitive,specificandtime-savingapproachforspecificdetectionanddiagnosis.152 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceFourthly,studieswereconductedinZhanggangandLuotiantoinvestigateanthelminticresistanceusingfaecaleggcountreductiontesting(FECRT)andpostmortemexamination.Theresultsshowedtheexistenceofbenzimidazole,levamisoleandivermectinresistancesinZhanggangandLuotian,andHaemonchuscontortusmaybethepredominantresistantspecies,butfurtherworkisneededtoconfirmthisconclusion.ThefindingsofthisthesishaveimplicationsforfuturestudiesandforthecontrolofgoatparasitesinHubeiProvince.Althoughthefocusofthethesiswaspredominantlyonparasitichelminthsofgoats,theoutcomesachievedalsohaveimplicationsforstudyingonthehelminthsofotherlivestockanimalsinChina.153 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MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceAcknowledgementsThisthesisisanaccumulationofsixyearsofwork,whichwouldnothavebeenpossiblewithouttheguidanceandassistanceofmysupervisors,theassistanceofmycolleaguesandthesupportfrommyfamilyduringthisjourney.Iamsincerelygratefultomyprincipalsupervisors,Prof.RobinB.GasserandProf.HuMin,fortheirkeeninterest,constructivecomments,auspiciousguidanceandforboostingmymoraleduringdiscussionsaboutresearchprogress.Prof.RobinB.Gasserisaworldfamousscientistmajoringinmolecularparasitology,hisrigorousscholarship,criticalattitude,enthusiasmandinterestinscientificresearchreallyinspiremegreatlyforthetimetocome.Prof.HuMinisagreatteacher,andalsoafriendtome;notonlydidsheguidemetoberigorous,criticalandpassionateinmystudies,butsheencouragedmetotakeadvantageofthetimeandopportunitiestodothingswhileIamyoung.SincerethanksalsogotoDr.WangTaoforhisconstructivesuggestionsandselflessassistanceduringmystudies.ThanksalsotoProf.ZhaoJunlong,Prof.ShenBang,AssociateProf.ZhouYanqin,AssociateProf.FangRuiandAssociateProf.HeLanfortheirconstructivesuggestionsonmywork.IamverygratefultoFanYingying,LeiWeiqiang,TanLi,WangBo,HeLi,LiFacai,DiWenda,ZhuKaixiang,QiMingwei,ZhangZongze,WangChunqun,GaoChong,ZhouCaixian,ZhangTing,YanXingrun,YinFangyuan,YuanWang,ZhaoLu,MudassarNiazMughal,ZhouHuan,LiuXiaofang,ZhangHongrun,HuJinyang,ZhangYing,LiXuesong,ZhuTao,LiaoCong,LiTingting,LiuLuandShanJianan,mycolleaguesattheStateKeyLaboratoryofAgriculturalMicrobiology,DepartmentofPreventiveVeterinaryMedicine,fortheirtechnicalguidanceandassistanceduringthisstudyperiod.The“SpecialFundforAgro-scientificResearchinthePublicInterest”(Grantno.201303037)and“HuazhongAgriculturalUniversityScientific&Technological211 HuazhongAgriculturalUniversityPhDDissertation2018Self-innovationFoundation”(Grantno.2015RC005)aresincerelyacknowledgedforsupportingmefinancially.Last,butbynomeansleast,Iamhighlyindebtedtomygrandparents,parentsandyoungerbrotherwhohavegiventheirunequivocalsupport.YangXinJune,2018212 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvinceCurriculumvitaeYANGXINEDUCATIONALPROFILE:HuazhongAgriculturalUniversity,2012.9-2018.6PhD,PreventiveveterinarymedicineZhongnanUniversityofEconomicsandLaw,2008.9-2010.6B.S.Mgt.Sci.,ManagementHuazhongAgriculturalUniversity,2006.9-2011.6BVSc,VeterinarymedicineINTERNATIONALPEER-REVIEWEDPUBLICATIONS:1.YangX,GasserRB,FangR,ZengJR,ZhuKX,QiMW,ZhangZZ,TanL,LeiWQ,ZhouYQ,ZhaoJL,HuM.FirstsurveyofparasitichelminthsofgoatsalongtheHanRiverinHubeiProvince,China.ActaParasitologica.2016,61(3):602-606.(Chapter2)2.YangX,ZhaoY,WangL,FengH,TanL,LeiW,ZhaoP,HuM,FangR.AnalysisofthecompleteFischoederiuselongatus(Paramphistomidae,Trematoda)mitochondrialgenome.Parasites&Vectors.2015,8:279.(Chapter4)3.YangX,WangLX,ChenHM,FengHL,ShenB,HuM,FangR.ThecompletemitochondrialgenomeofGastrothylaxcrumenifer(Gastrothylacidae,Trematoda)andcomparativeanalyseswithselectedtrematodes.ParasitologyResearch.2016,115:2489-2497.(Chapter5)4.YangX,WangLX,FengHL,QiMW,ZhangZZ,GaoC,WangCQ,HuM,FangR.CharacterizationofthecompletemitochondrialgenomesequenceofHomalogasterpaloniae(Gastrodiscidae,Trematoda)andcomparativeanalyseswithselecteddigeneans.ParasitologyResearch.2016,115:3941-3949.(Chapter6)213 HuazhongAgriculturalUniversityPhDDissertation20185.YangX,QiM,ZhangZ,GaoC,WangC,LeiW,TanL,ZhaoJ,FangR,HuM.Developmentandevaluationofaloop-mediatedisothermalamplification(LAMP)assayforthedetectionofHaemonchuscontortusingoatfecalsamples.JournalofParasitology,2017.103(2):161-167.(Chapter7)6.YangX,GasserRB,WangL,ZhuK,ChenL,FengH,HuM,FangR.MitochondrialgenomeofHypoderaeumconoideum-comparisonwithselectedtrematodes.Parasites&Vectors.2015,8:97.7.ZhaoYY,YangX,ChenHM,WangLX,FengHL,ZhaoPF,TanL,LeiWQ,AoY,HuM,FangR.ThecompletemitochondrialgenomeofOrthocoeliumstreptocoelium(Digenea:Paramphistomidae)forcomparisonwithotherdigeneans.JournalofHelminthology.2016.6:199-206.8.ZhangZZ,YangX,AhmadAA,LeiWQ,WangCQ,DiWD,ZhouYQ,ZhaoJL,FangR,HuM.Developmentoftetra-primerARMS-PCRfordetectingtheE198ASNPintheisotype-1β-tubulingeneofHaemonchuscontortuspopulationsinChina.VeterinaryParasitology,2018,252:127-130.9.ZhangZZ,GasserRB,YangX,YinFY,ZhaoGH,BaoM,PanBL,HuangWY,WangCR,ZouFC,ZhouYQ,ZhaoJL,FangR,HuM.Twobenzimidazoleresistance-associatedSNPsintheisotype-1β-tubulingenepredominateinHaemonchuscontortuspopulationsfromeightregionsinChina.InternationalJournalforParasitology:DrugsandDrugResistance.2016,6:199-206.10.TanL,LiY,YangX,KeQ,LeiW,MughalMN,FangR,ZhouY,ShenB,ZhaoJ.GeneticdiversityanddrugsensitivitystudiesonEimeriatenellafieldisolatesfromHubeiProvinceofChina.Parasites&Vectors,2017,10:137.11.GaoY,ZhangY,YangX,QiuJH,DuanH,XuWW,ChangQC,WangCR.MitochondrialDNAevidencesupportsthehypothesisthatTriodontophorusspeciesbelongtoCyathostominae.FrontiersinMicrobiology.2017,8:1444.12.WangCQ,LiFF,ZhangZZ,YangX,AhmadAA,LiXR,DuAF,HuM.RecentresearchprogressinChinaonHaemonchuscontortus.FrontiersinMicrobiology.2017,8:1509.13.ChenL,FengY,ChenH,WangL,FengH,YangX,MughalN,FangR.MitochondrialgenomeanalysisofClinostomumcomplanatumanditscomparisonwithselecteddigeneans.ParasitologyResearch.2016,115:3249-3256.14.YinF,GasserRB,LiF,BaoM,HuangW,ZouF,ZhaoG,WangC,YangX,ZhouY,ZhaoJ,FangR,HuM.PopulationstructureofHaemonchuscontortusfromseven214 MolecularandepidemiologicalinvestigationsofkeyparasitichelminthsofgoatsinpartsofHubeiProvincegeographicregionsinChina,determinedonthebasisofneutralmicrosatellitemarkers.Parasites&Vectors.2016,9:586.15.YinF,GasserRB,LiF,BaoM,HuangW,ZouF,ZhaoG,WangC,YangX,ZhouY,ZhaoJ,FangR,HuM.GeneticvariabilitywithinandamongHaemonchuscontortusisolatesfromgoatsandsheepinChina.Parasites&Vectors.2013,6:279.CHINESECOREJOURNALPUBLICATIONS:1.YangX,LeiWQ,DiWD,WangCQ,ZhouCX,ZhangZZ,FangR,ZhouYQ,ZhaoJL,HuM.Investigationofbenzimidazoleresistance-associatedSNPsintheisotype-1β-tubulingeneinHaemonchuscontortuspopulationsinYili,Xinjiang.ChineseJournalofVeterinaryMedicine,2018.(InChinese;Accepted)2.QiM,YangX,FangR,HuM.Coccidiaandcoccidiosisingoats.HeilongjiangAnimalScienceandVeterinaryMedicine,2016,06:91-95.(InChinese)3.QiM,YangX,FangR,WangKS,HuM.InvestigationandmorphologicalexaminationofCoccidiaofgoatsinHubeiProvince.ChineseJournalofVeterinaryMedicine,2017,53(7):7-9.(InChinese)ADVANCEABSTRACTS:1.YangX,ZhuKX,QiMW,ZhangZZ,FangR,ZhouYQ,ZhaoJL,HuM.EstablishmentofaLAMPmethodforthedetectionofHaemonchuscontortus.Proceedingsofthe13thSymposiumonVeterinaryParasitology.2015.(InChinese)2.YangX,WangT,HuM,GasserRB.IdentifyingpriorityareasforresearchongoatparasitesinChina.TheAbstractsofthe7thInternationalSymposiumonParasitology.2017.PATENTS:1.YangX,HuM,FengH,FangR,ZhuK,QiM,ZhangZ,TanL,LeiW,ZhouY,ZhouJ,“EstablishmentofaloopmediatedisothermalamplificationkitforthedetectionofHaemonchuscontortus”,ChinaPatent,No.ZL201510025191.1.2.HuM,ZhangZZ,YangX,FangR,GaoC,WangCQ,ZhouCX,HeL,ShenB,ZhouYQ,ZhaoJL,“PrimersandapplicationofanthelminticresistancerelatedgenemutationsiteforHaemonchuscontortus”,Patentapplicationnumber,201710700846.X.215 HuazhongAgriculturalUniversityPhDDissertation2018EXPOSURETOINTERNATIONALFORUMS:1.The13thAcademicSymposiumonVeterinaryParasitologyofChinaanimalhusbandryandVeterinaryAssociation,2015inHarbin,PRC.2.The14thAcademicSymposiumonVeterinaryParasitologyofChinaanimalhusbandryandVeterinaryAssociation,2016inBeijing,PRC.3.The7thInternationalSymposiumonParasitology/the16thBiennialMeetingoftheChineseSocietyofParasitology,2017inJiujiang,PRC.HONOURS:1.SocialPracticeActivistAward,HZAU,20152.NationalScholarshipforDoctoralGraduateStudents,MOE,20163.TheThirdPrizeofExcellentAcademicPapers,HBAV,2014-20164.ExcellentDoctoralStudentFundingProgram,HZAU,20185.TheTopTenStarforInnovationandEntrepreneurship,HZAU,2018216 华中农业大学博士学位论文ＰｈＤＤＩＳＳＥＲＴＡＴＩＯＮ
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