00292095

3902

3903

3904

60312295

9000003078326

9000006874417

Fujisaki, Kozo

Kawazu, Shin-ichiro

Kawazu, Takeshi

Panchadcharam, Chandrawathani

Sekizaki, Tsutomu

河津, 信一郎

English

OAK Obihiro University Archives of Knowledge

．J．P′β～βZβOJ．伽∫．．j．6イー（沼（J99jノ  C叩yrfg／汀◎Jタタj．月g∫gdrCムCどれ‘g′かP′♂‘βZOα〃〟oJgc血rJ／WⅧ邪心都  DevelopmentofDNA ProbesfortheDetectionand  Identi鰯cation ofβ瓜むe5fα0γαf瓜  SHIN－1CHIRO KAWAZUl，CHANDRAWATHANIPANCHADCHARAM2′   TAKESHIKAWAZUITSUTOMU SEKIZAKI－AND KOZO FUJISAKl一  ノ 他山闇JJ旧血Jgげ血i皿川gd帆乃〟毎れ伽′戚fj帆ノ岬d〃，2抽rgr上′叩触どα化ん  J融r〟re，車∂カブノヰ00，Pg和戚ル．〟d町∫隠JrんgC力e仰－∫e和一m即叩側血  伽M血＝郁血打批正叫仇棚靴血鋸恥旬朋  〟どこぞivd／／ノα仙〝プノタ9J／Acc甲J¢d 2イ∧prノJJ9タj   Keywords：DNAprobediagnosis，Babesiaovata  Babe扇a旦処isoneoftheimportantorganismthatpromotesanaemiainthe  presenceofmixedinfectionwithTheileriaspeCiesincattleoJIPaStureinJapan  （MinamiandIshihara1980）・ThepathogenicityofB・9ginfectionhasgenerally  beenfoundtobelowerthanthatofotherpathogenicspeCleSOf旦恕由suchas旦・  塾虚andB・蜘・However，Withincreasin芦1ntemationaltradeofcattlethereis  highpossibilitythatthelattertwopathogenlCSpeCies may spreadinthe cattle  POpulationofJapan，AnimalsdiagnosedaspositiveforB．如由or旦bi要eminaare  Culledtofacilitatethenationalcontrolanderadicationprogramme・Consequently，itis  皿perativethattheacctJrateidentificationofthecausativeorganistnsisdonerapidly．  Altbughdiagnosisofbabesiosisushgughtmicroscopyofstainedbloodsmearisone  Ofthemostreliabletechniques，itrequiresagkillfulnes5andexperience印Oughto  detectandidentifytheBabesiaparasiteatlowparasitaemiclevels（LarLaretal．1989）．  Serologicaldiagnosisofbabcsiosismayfai1todetectinfectionsandtoiderLtifythe  SpeCiesinsomecases，eSpeCiallytheearlystageofinfection（Minamietal．1979，  Fltiinagaetal・1980）・Thus，itislmportanttodevelopimprovedtechniqueswhichcan  diagnoseB・g芝生迫infectionforsurveillanceandepidemi010gicalstudiessothaueliablc  COntrOlmeasurescanbeimplementedtocontrolanderadicafethemorepathogenic  disease，旦・迦由and旦・bigemi血inf6ctiorLS．aSWellashelplnearlytreatmentofB．  ⊆些邑担infectionsin℃attle・Withtheadventof biotechnology，itism）WPOSSibletouse  DNAprobesasafaPidandsen＄itivetoolforthedetectionandidentificationofother  塑造墨由speCies（Bueningetal・1990，Petchpooetal・1992）・Inthispaperweatlempted  tousetheDNAprobe／tOdetectandidentuy塾．Qiこ虹旦．  ガ頭   β．Ol／ATノ1DNA PlミOBE   塾由堕近地（Miyakestock；Minamiandhhihara1980）wasusedinthisstudy・  BloodwascollectedfrornacalfexperlmenLallyinfectedwiththeparasitestoek，and  merozoiteswerepurificdbythenitrogencavitationmethod（Shimizuetal・1992）・  GenomicDNAoftheparasitewaspreparedrrompurifiedrnerozojtesbytreatmentwith  sodiumdodecylsulfate（ST）S）LPrOteinaseKsolution・andphenolextraction（Sambrook  etal・1989）・The声enOmicDNAofotherparasitesusedwerepreparedinthesam？  marmer．TheparaslteStOCksusedwereasfo1lows＝旦垣虫（Australianstock；Minaml  ＆Tshihara1980）；旦bigemim（Kochindastock；Fujinagaetal・1980）；Theileria  星型呈（Ikedastock；Fujisakietal・1992）；王・地主（Warwickstock；Fujisakietal・  1992）；工．0rientalis（Essexstock；Fujisakietal・1992）；Anaplasmamarginale  （Kochindastock；Nakamumetal．1991）；皇．centrale（Aomoristock；Nakamuraetalr  1991）；Epcrvthrozoon塑聖迎皇（Naturalhfection；Kawazuetal・1990）・Thegenomic  DNAof旦．迦（2．7pg）wasdigestedwith80unitsof旦拉RIat370Cfor2h・The  digestedDNAwasthenfractionatedbythesizefractionationcolumn（Amersham  Japan，Japan），andfracLjonscontainingthefragmentsofnlOrethanlkilobase（kb）were  p001ed・TheDNAfragmentswcreconcentratedbyprecIPltationunderethanolat－80OC  wjthO．3Msodiumacetate．andwereinsem元＝nto入ZAPⅢ旦⊆旦RIarms（Stratagene，  U．S．A．）．The．ibrarysizepackagedinvitrowasl・1x107・Approximately99・7％ofthe  resultingplaqueswererecombinantphagesasshownor15－bromo－4ヾh10rO－3－indolyl－b－  r）－galactosideindicatorplatc・Atotalof894recombinantphageswereplatedoutoヮ  EschcrichiasQiiXLIJBlue（AmershamJapan，Japan）withlOO－2OOplaquesperpetrl  dish，andgrownat370Covernight・DNAfromphagcplaqueswereabsorbedo？tO  nybnfilters（Hybond－N＋；AmershamJapan．Japan）inreplicate，ardscreenedbyusmg  B．姐and旦．塾虫genomicDN＾ashybridizationprobs・TheDNAwaslabelled  byrandomhexanucleotideprimingTnCthodwithdigoxigenin（DIG）－dUTP（DNA  labelinganddetectionkit，nOnradioactlVe；BoehringerMannheim－YamanouchiJapan）■  HybridizationandimmunologlCaldetectionofhybridizedDIG－labelledprobewere  alsoperformedwiththiskit・Twophageclones，nanlely，BOZAP6andBOZAP7that  showedintensivesignalwhenhybridizedto塾・地genomicDNAbutnotdetectable  to旦．址genomic DNA were selected as possiblc candidates for probe・  RccombinatedpBluescrlPtPlasmidwasexcisedoutfromthephageDNAbytheinvivo  excisiontechnique（Stratagene）．BOZAP6andl∃OZAP7had3・6kband5・Okbinsert，  respectively（data notshown）・Theplasmid DNAswerepreparedinlarge－SCale  （Sambr00ketal．1989），andapproxirnatelylmgofDNAwaslabelledwithDTG－dUTP・  Toinvestlgatethegenomiccrrganizationofclonedsequences・Weperformed  Southern hybrjdization usingtwoprobes・Thegenomic DNAsof旦▲型旦！旦，旦・  bigemina．andB．出立weredigestedwith塾迫RTandelectrophoreticallyfractionated  inaO・8％agarosegelinduplicate・1≠eDNAsweretmnsferredontonylonfilters  （Nytran－N；Schleicher＆Schuell，F・R・G・）andhybridizedwithBOZAP6ardBOZAP7  PrObes．ThepattemsofhomoIogoushybridizationbetweerltheprobesand旦・旦処  gcnomesuggestedthat旦・型二旦迫genomeCOntahedmultiplecopiesofthesequence  （Fig．1）．TherewasnodetectablehybrldizatiorlbetweentheprobesandtheDNAof  OthertwoBabesiaspeCjes（Fig．1）・   8．OV八丁A DNA PROBE  Fig・1 Agarosegelelectrophoresisof宣型Rl－digestedgenomicDNA（0．3膵／1ane）  fromBabesia班旦払（1anel），旦・bigemina（1ane2）and旦．』辻由（1ane3）；（panelA）．  HybridizationpattemsofSouthernblotsofpanelAwithBOZAP6probe（parlelB）and  BOZAP7prol光（panelC）t Filterswere ． kilobases（kb）ontheleft．   ThespecificityOftheseprobeswithlOOngofDNAfromvariousorganismsthat  maycontaminateintoafieldsampleandfrombovinewhitebloodcellswereassessed  bydot／blothybridization・TheDNAwasb10ttediLlduplicateontotheHybond，N＋  filtersbytheBio－Dotappara［uS（Bio－RadLaboratoriesJapan，Japan）・AsshowninFig・  2，StrOnghybridizationslgnalisnotedonlywith旦．週睦DNA．   The旦・旦処DNAwasdotblottedinduplicateontotheHybond－N＋filtersin  Serialdilutions and hybridizedwith BOZAP6and BOZAP7probes・Thelimit  detection立1thisassaywasat62pgandat125pgforBOZAP6andBOZAP7probes，  respeCtively（Fig．3）．  ぶβ   β．OVATA DNA PROBE   1 2 3 4 5 6 7 8 9 10 A ●  B ●  Fig・2・ReactivityofclonedgeTmicDNAof塾恕由gnLa・BOZAP6（columnA）  andI】OZAP7（columnB）withvanousorganismsandbovinewhiteblodcellDNAat  lOOng・TheDNAsamplesarealignedasfo1lows：1anel，Babesia旦竺旦睦；lane2，旦，  地；lane3，旦bigemina；lane4，Theileriasergenti；1ane5，工．地Iane6，工．  Orientalis；1ane7，Anat）1asmamargirLale；1ane8，A．centraLe；1ane9，Eoervthrozoon  WenyOni；1anelO，bovinewhitebloodcell．Filterswerewashedinthesamecondition  那Figu陀1・   1 2 3 4 5 6 7 8  A ● ● 密†   B ● 尊  Fig・3・ SensitivityofclonedgenomicDNAofBabesiaQ竺旦垣，BOZAP6（columnA）  andBOZAP7（columnB）todecreasingamouTltSOfpurified塾．9竺旦迫genomicDNA．  TheamountofDNAblottedareasfollows；lanel・10ng；lane2・1ng；1ane3，500pg；  1ane4・250pg；1ane5，125pg；1ane6・62pg；1ane7，31pg；1ane8，15pg・Filterswere  WaShedinthesameconditionasFigurel・  These resultsindieate that the selected clones，BOZAP6and BOZAP7were  Suitableforprobesinthediagnosisof旦・建過払infection・Theseprobesarealsocapable  OfdistinguishingthecloselyrelatedspeCieswithcertainty．However，theusefulnessof  theseprobesshouJdbeevaluatedtotestsamplesfromthefieldaswellastofollow  experimenta11yinducedparasitaemiasincattleespeC】a11ywheretheparasitaemiclevels  arelow・TheseexperimentsarenowiLlprOgreSSinourlaboratorytoestablishaDNA－  baseddiagnosisof旦．塑旦迫infections．Thiswi11aidintheeffectivecontrolofthe  diseaseswhichwillintumreducelossesincurredthroughbabesiosiscausedby旦．  Q3こ堕旦，B・b塾虫andB，bigemina．   鼠 OVATA DNA PROB召  ACKNOWmGMm：  Thi＄WOrkwascorLductedincollabominbetweenNatiozlalInstituteofAnimal  Health・Japan（NIAH）andVeterinaryResearchInstitute／ASEANPoultryDisease  Re聞arChandTrainiqgCentre（WDRTC）・W・Mahysiawithcomphmentsad  馳PPOrtffOmJapaneseIntcrnatio血CooperationAgency（JICA）．Theauthorsare  thankfultoDrs・GanCheeHiong，ChristopherRajamickamandChikaraKuniyasuof  VRI／APDRTC，W．Malaysiafortheirvaltmblecoordination．Thanksarealsoduet。  Dr・Y・NakamtmofNIAHforthesupplyofAnaDlasmacentr＊andA．rmrginale  DNA，  REWCE5：  Buening，G・M・，Bafbet，A・，Myler，PりMahan．S．，Nene，V．＆McGuire，T．C．1990．   Charactedzatior10farepetitiveDNAprobeforBabesiabigemina．迦Parasi血．   並ニ11－20．  FLdinaga・T・・Minami，T・＆Ishihara，T・1980・SeroIoglCalrelationshipbetweena   lafge辿foundinJapanesecattleand．辿由如，旦，bigemirmand旦   出．旦堕．逆堕呈出．三塑：23♭234．  Fltjisaki，K・1992・AreviewofthetaxonomyofTheileriasergent地庭師entalis  groupparasiteincattle．LPfOtOZCd．塾堕．呈：B7－96．  Kawazu・S・，Nakamura，Y・・Kami0，T＝叫isakilK・＆Minami・T・1990・Enzymeて   1inkedimmunosor血tassayfordeteetionofantibodiestoEDerVthrozoonx担  h∽山e．迦．よ迦．迦皇．盟：12卯－1300．  Lanar・D・E・・McLaughlin・G⊥・，Wirth・D・F”Barker，RJ・，Zolg，J・W・＆Chulay，J・D・   1989・Comparisonofthickfilms・invitroculttdeandDNAhybridizationprobes   fordetectingPlasmodiumfalciparummalariai血塾・1．迦．辿迫虻裏．坐‥3－6．  Minami・T・・Yamabe，K，Hayashi・S・＆Isbihara・T・1979・Serologicalrelationshipofa   Japanese迦塾鎚由speCi讐and旦恕血塾匹由bythecomplementfixationand   CaPi11ary－tubeagglutinatlOnteStS．迦Parasitol．皇：29r38．  Minami，T・＆Ishihara，T・1980・呈出由堕旦』sp皿isolatedfromcattleinJapan・泣出・   出・迦由弘也地史．也．）迦：101－113．  Nakamura，Y・，Kawazu・S・＆Minami，T・1991・Armlysisofproteincomposlt10nSand   SurfaceproteinepitopeSOfAnaplasma生血辺』andAnaplasm旦marginale．i．支出．  迦王立皇．裏：73－79．  Petchp00・W・－Tan－ariya・P・・Boon鎚eng・Ⅴ・・BrockelrnarllC・R・，Wilairat，P・＆Pa   S・1992・AspeCificDNAprobewhichidentifiesBabesiabQd5．inwhQlebloo止  逝迫parasitoL盟：189－198．  Sambrook－JリFritsch・E・F・＆Maniatis，T・1989・MolecularCloning，ALaboratory   Manual，2nded・，ColdSpriLlgHarborLabratoryPress，NewYork・  Shimizu，S・，Shimtm，K・．Ito，Si＆Minami，T．1992．Babesiaovata：isolationfrom   erythr∝yteSanddevelopmentofanenzyme－1ikedimmunogotbentassayfor  detectionofantibcdies．Parasitol．旦生むZ8：684－6舶．  紺   

Development of DNA Probes for the Detection and Identification of Babesia ovata

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Development of DNA Probes for the Detection and Identification of Babesia ovata

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OAK Obihiro University Archives of Knowledge