ContentslistsavailableatScienceDirect
Veterinary
Parasitology
j o ur na l ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r
Taxonomy,
phylogeny
and
molecular
epidemiology
of
Echinococcus
multilocularis:
From
fundamental
knowledge
to
health
ecology
Jenny
Knapp
a,b,∗,
Bruno
Gottstein
c,
Urmas
Saarma
d,
Laurence
Millon
a,baDepartmentofChrono-environnement,UMRUFC/CNRS6249aff.INRA,UniversityofFranche-Comté,Besanc¸on,France bDepartmentofParasitology—Mycology,UniversityHospitalofBesanc¸on,Besanc¸on,France
cInstituteofParasitology,FacultyVetsuisse,UniversityofBerne,Berne,Switzerland
dDepartmentofZoology,InstituteofEcologyandEarthSciences,UniversityofTartu,Tartu,Estonia
a
r
t
i
c
l
e
i
n
f
o
Keywords:
Echinococcusmultilocularis
Molecularphylogeny Parasiteadaptationstrategies Taxonomy
a
b
s
t
r
a
c
t
Alveolarechinococcosis,causedbythetapewormEchinococcusmultilocularis,isoneofthemostsevere parasiticdiseasesinhumansandrepresentsoneofthe17neglecteddiseasesprioritisedbytheWorld
HealthOrganisation(WHO)in2012.Consideringthemajormedicalandveterinaryimportanceofthis
parasite,thephylogenyofthegenusEchinococcusisofconsiderableimportance;yet,despite
numer-ouseffortswithbothmitochondrialandnucleardata,ithasremainedunresolved.Thegenusisclearly complex,andthisisoneofthereasonsfortheincompleteunderstandingofitstaxonomy.Although tax-onomicstudieshaverecognisedE.multilocularisasaseparateentityfromtheEchinococcusgranulosus
complexandothermembersofthegenus,itwouldbeprematuretodrawfirmconclusionsaboutthe
taxonomyofthegenusbeforethephylogenyofthewholegenusisfullyresolved.Therecentsequencing ofE.multilocularisandE.granulosusgenomesopensnewpossibilitiesforperformingin-depth phylo-geneticanalyses.Inaddition,wholegenomedataprovidethepossibilityofinferringphylogeniesbased onalargenumberoffunctionalgenes,i.e.genesthattracetheevolutionaryhistoryofadaptationinE. multilocularisandothermembersofthegenus.Moreover,genomicdataopennewavenuesforstudying themolecularepidemiologyofE.multilocularis:genotypingstudieswithlargerpanelsofgeneticmarkers allowthegeneticdiversityandspatialdynamicsofparasitestobeevaluatedwithgreaterprecision.There isanurgentneedforinternationalcoordinationofgenotypingofE.multilocularisisolatesfromanimals andhumanpatients.Thiscouldbefundamentalforabetterunderstandingofthetransmissionofalveolar echinococcosisandfordesigningefficienthealthcarestrategies.
©2015ElsevierB.V.Allrightsreserved.
1. Introduction
Echinococcus multilocularis Leuckart 1863,commonly known as the fox tapeworm, is a cestode from the family Taeniidae, whichcontainsfourgenera:TaeniaLinnaeus1758andEchinococcus Rudolphi1801,HydatigeraLamarck,1816andVersteriaLavikainen, Iwaki,Haukisalmi,Konyaev,Oku,OkamotoandIto,2013(Nakao etal.,2013).Usingageological-eventcalibrationpointand relaxed-clockapproach,Knappetal.(2011)suggestedthattaeniidsstarted todivergeinthelateMiocene(11.2millionyearsago:Ma),while thegenusEchinococcusbegantodiversifysomewhatlater,about 5.8Ma,at theveryend of theMiocene(Fig.1).Inhumans,the metacestodestageofE.multiloculariscausesaninfectiousdisease calledalveolarechinococcosis,whichhasahighmortalityrateand
∗ Correspondingauthorat:DepartmentofParasitology—Mycology,University HospitalofBesanc¸on,Besanc¸on,France.Tel:+33364082274;Fax:+33363082232.
E-mailaddress:jenny.knapp@univ-fcomte.fr(J.Knapp).
isoneofthemostthreateningemerging zoonosesinEurasia.E. multilocularishasaHolarcticdistribution,encompassingregions locatednorthofthetropicofCancer,butisgenerallynotpresent inpolarregions(Davidsonetal.,2012;Nakaoetal.,2013);polar E.multilocularispopulationshaveonlybeenfoundintheSvalbard Archipelago,Norway(Henttonenetal.,2001).Thedistributionof theparasiteextendslongitudinallyfromNorthAmericatoEurasia andincludesseveralhighlyendemicareasinAsia(Russia,China).
E.multilocularisischaracterisedpredominantlybyitssylvatic lifecycle,whichinvolvestwomammalianhosts:rodentsas inter-mediatehosts (IH),and wildcanidsasdefinitivehosts(DH). In Europe,theredfox(Vulpesvulpes)isoneofthemaindefinitive hosts,whilevariousMicrotusandotherarvicolidrodents,but occa-sionallyalsolagomorphs,actasintermediatehosts(Davidsonetal., 2012;Vuittonetal.,2003).Regionally,E.multiloculariscanreach relativelyhighprevalencesinredfoxes,recordedalsoin newly-detectedareasinthelastdecade(Bagradeetal.,2009;Bruzinskaite et al., 2007;Moks et al., 2005).Followingtherelatively recent urbanisationofsomeredfoxpopulations,E.multilocularishasalso http://dx.doi.org/10.1016/j.vetpar.2015.07.030
90 100 98 100 65 62 95 99 68 99 99 99 99 99 100 100 100 100 99 85 95 50 100 99 11.2Ma (20.5~0.8) 25.7Ma (55.7~9.4) 15.7 Ma (38.0~5.2) 5.8Ma (9.2~2.5) 19.6Ma (40.7 ~ 1.2) 3.0 Ma 28.4 Ma (65.1~3.1) E. ortleppi E.canadensisG6 E. canadensisG7 E.canadensisG8 E. mullocularis E. shiquicus E.granulosuss.s. E.felidis E.equinus E. oligarthrus E. vogeli T. mustelae T. serialis T.madoquae T. mulceps T.saginata T.asiaca T.ovis T.solium T.twitchelli T.mars T. crassiceps T.lacollis T.hydagena T.taeniaeformis T.parva
*
Fig.1.Arelaxed-clockchronogramoftaeniidtapewormsreconstructedbyBayesianinference(BEAST)usinganexondatasetfromrpb2,pepckandpoldgenes.Valuesat eachnodeareposteriorprobabilities(%).Greyrectanglesindicateestimatedagesofrepresentativenodes(Ma,formillionyearsago),withthe95%HPD(thehighestposterior density)showninparentheses.AnasteriskdenotesthenoderepresentingthemostrecentcommonancestorofNeotropicalEchinococcusspp.,usedasacalibrationpointfor timeestimates(Knappetal.,2011).
beenreportedfromanumberofEuropeancities,prompting con-siderableconcernforpublichealth(e.g.Deplazesetal.,2004;Hofer etal.,2000;Plumeretal.,2014;Laurimaaetal.,2015).
TheintermediatehostsofE.multilocularisbecomeinfectedby ingestingparasiteeggs,afterwhich theparasitelarvaecolonise internal organs, usually the liver, where lesions develop. The parasitelesions cancause severe health problems due totheir tumour-likegrowthiftheyarenotdiagnosedandtreatedearly. WhilerodentsarethemainIH,humansrepresentaccidental inter-mediatehosts,asdocertainotherspecies,suchascaptivemonkeys, beaver,muskratandnutria(Gottsteinetal.,2014;Rehmannetal., 2005;Umhangetal.,2013;Vuittonetal.,2003).
Thediversityofmammalianhosts anditslargegeographical distributionhaveofteninspiritedtaxonomists,whohave histori-callyandmorerecentlyproposeddifferentspeciesdenominations orsub-speciesdescriptions.Here,wereviewthecurrentstateof knowledgeconcerningthetaxonomyand phylogeneticposition ofE.multiloculariswithinitsgenus.Empiricalobservationswere firstbasedonmorphologicalcriteria,buthavemorerecentlyalso includedgeneticdata.Thegeneticdiversityoftheparasiteasan indicatorofitsspatialandtemporaldynamicsisalsodiscussed. 2. WhatdefinesE.multilocularisasaspecies?
Thestudyoftaxonomyisanessentialdisciplinepermittingthe identification,namingandclassificationoftaxa–organismswith commoncharacters–andspeciesisthemostfundamentaltaxon inthesystematicclassification.Thequestionofwhetherthetwo typesofechinococcosis,cysticandalveolar,werecausedbythe variantsofthesame ortwo differentspecies,remaineda mys-teryforalongperiodoftime(Tappeetal.,2010).Inthemiddleof 19thcentury,RudolfVirchowdescribedthemorphologicalfeatures oftheE.multilocularislarval stageinhumanpatients(Virchow, 1856).ItdifferedfromEchinococcusgranulosusintheappearance ofthealveolarcolloid,its smallvesiclescontaininggelatin, and thepresenceofrelativelyfewprotoscoleces,namedinVirchow’s description“younganimals”.The“multitudeofsmallechinococcal vesicles”observedbyVirchowprovidedthebasisfortheterm
“mul-tilocular”todescribetheappearanceofthelesionsandcontrast withtheunilocularmetacestodestagedevelopmentinE. granulo-sus.However,atthattime,alveolarechinococcosiswassuspectedto becausedbyanaberrantEchinococcussp.,andwasonlydescribed asadistinctspeciesin1863,thankstoLeuckart’sinvestigations, whichprovidedthenameE.multilocularis.
In 1954,RauschandSchillerdescribed a newspecies which infectedsledgedogsandpolarfoxes(Vulpeslagopus),and micro-tinerodentsonSaintLawrenceIsland(Alaska)intheBeringSea (RauschandSchiller,1954).Theauthorsnamedthisnewspecies E.sibiricensisandsuggestedthatasthelarvaeresemblesclosely thealveolarlarvaofEchinococcussp.(=E.multilocularis)described insouthernEuropeandRussia.Thetaxonwaslaterrelegatedby Vogeltothestatusofapossiblesub-speciesE.multilocularis sibiri-censis,duetoslightdifferencesinmorphologyandlifecycle(Vogel, 1957).However,RauschandVogelwerelaterabletoprovethe con-specificstatusofE.multilocularisandEchinococcussibiricensisby exchangingeggsoftheparasiteandinfectingthenatural interme-diatehostsoftheirresearchareas.ThenameintroducedbyLeuckart forthelarvalstagewasgivenpriority(reviewedinTappeetal., 2010).However,molecularanalysisofisolatesfrommetacestodes originatingfromadultwormsinfectingarcticfoxesinAlaskaand Svalbard(seepart4)haveagainraisedthepossibilityofa“polar species”(Knappetal.,2007,2012).
In 1961 Shults described another subspecies Alveococcus (=Echinococcus)multilocularis kazakhensis, from Kazakhstan and Russia,whichexhibitedmultiloculardevelopmentbutinfectedthe domesticdogasDH,andsheepandpigsasIH,thusdifferingfrom thenominalE. multiloculariswhich infects rodentsas IH (from
KumaratilakeandThompson,1982).However,RauschandNelson
(1963)suggestedthattheparasitecouldbeE.granulosus.
More recently, a further subspecies, E. m. russicensis, was described in corsac foxes (Vulpes corsac) from InnerMongolia, China (Tang, 2007), exhibiting slight morphological differences to the uterine structure in comparison with the nominal E.multilocularis.
Proposals have been made to place E. multilocularis into a separate genus Alveococcus, but these have not gained
gen-Fig.2.MorphologicaldifferencesbetweenfourEchinococcusspecies,(A)E.vogeli, (B)E.granulosuss.s.,(C)E.multilocularisand(D)E.ortleppi.Thearrowsindicatethe genitalpores(adoptedfromThompsonandMcManus,2002).
eral acceptance; the following reasoning is cited from Rausch
and Nelson(1963): “Because oftheunusualmorphological and
biologicalpeculiaritiesofthiscestode,aseparategenus, Alveococ-cusabuladze,1959,hasbeenproposed(LukashenkoandZorikhina, 1961).Whilethisproposalwouldseemtohaveatleastasmuch meritastheestablishmentofseparategeneraforspeciesofTaenia s.l.,itcanberejectedonsimilargrounds(Rausch,1963). Morpho-logicaldifferencesinthestrobilarstagearenotsignificantabove thespecieslevel;thestructureofthelarvaisdistinctive,but,in viewofthepleomorphismexhibitedbythelarvalE.granulosusand oftheinadequateknowledgeofthelarvalstagesof someother speciesofEchinococcus,itseemsprematuretoerectanewgenus onthisbasis.”Nonetheless,thenameAlveococcusmultilocularishas notvanishedcompletelyandhasbeenusedinafewpublications.
Basedonmorphology,fourspeciesarecurrentlyrecognisedin thegenus Echinococcus: E. multilocularis, E.granulosus, E.vogeli and E. oligarthrus (Eckert et al., 2001). Additional species cur-rentlyproposedareE.shiquicus(Xiaoetal.,2006),andE.felidis, E.ortleppi,E.equinus,E.canadensisandE.intermedius,whichare currentlyclassifiedasE.granulosussensulato(s.l.)(Thompsonand
McManus,2002a,b;Hüttneretal.,2008;Thompson2008;Saarma
etal.,2009;Nakaoetal.,2010;Nakaoetal.,2013). Morphologi-cally,theEchinococcusspecieshaverelativelysimilaradultstages. However,theadultstageofE.multilocularisissmallerthanthatof E.granulosussensustricto(s.s.),andE.vogeli(Fig.2),thoughE. oli-garthrus,andtheE.multilocularisbothexhibitalargenumberof segmentsandavaselikeuterus.Thelarvalormetacestodestage allowsE.multilocularistobemoreeasilydistinguishedfromother species.E.multilocularishasamultiloculardevelopment,E. granu-losusaunicysticdevelopmentandE.vogeliandE.oligarthrus(the Neotropicalspecies)apolycysticdevelopment(Eckertetal.,2001). IncontrasttoE.granulosus,whichonlyhasendogenous prolifera-tionofthegerminallayerofthemetacestode,E.multilocularishas bothendogenousandexogenousproliferation(Eckertetal.,2001;
KumaratilakeandThompson,1982).Insummary,the
characteris-ticsthatseparateE.multilocularisfromotherEchinococcusspecies are:itslargenumberofsegmentsandavaselikeuterusintheadult stage;amultilocularormultivesicularmetacestodedevelopment; anendogenousandexogenousproliferationofthegerminallayer; andalifecycleinvolvingwildrodents.
Toconclude,onecansaythatthefirsttaxonomyconsiderations aboutE.multiloculariswereestablishedonmorphologicalcriteria. Onlyacentury afterVirchow’sdescription,E.multiloculariswas fullyrecognisedasthecausativeagentofalveolar echinococco-sisandtheexperimentsconductedbyRauschandothersonthe differentstagesofEchinococcusspecies havepermittedtoavoid multiplicationofspeciesdescriptions.Thus,thetaxonstatusofE. multilocularisisundeniable.
3. PhylogeneticpositionofE.multilocularisinthegenus Next to taxonomy, phylogenetics is another, more recent disciplineinvestigatingrelationshipsamongorganisms.Both disci-plineshavelargelyconvergedbynow.Phylogeneticreconstruction isanessentialtoolforunderstandingparasiteevolutionandgenetic diversity,andforrecognizinganddiagnosingdifferentEchinococcus parasites.
ThemajorquestionconcerningthepositionofE.multilocularis inthegenusiswhetheritisaseparateentityfromtheE.granulosus complex(i.e.genotypesG1-G10)–assuggestedbytaxonomic stud-ies–ornot.Todate,thereisnounequivocalanswertothisquestion. Ononehand,anumberofstudieshaveplaceditseparatelyfrom theE.granulosuscomplexandarethusinaccordancewithclassical taxonomy.Ontheotherhand,somestudieshaveplacedE. multi-locularisinthemidstoftheE.granulosusgenotypes,renderingthe E.granulosuscomplexparaphyletic(Fig.3A–C)andcontradicting theclassicaltaxonomyofthegenus.Intheearlydaysof Echinococ-cusphylogenetics,analysesusingmitochondrialDNA(mtDNA)data (Bowlesetal.,1992;BowlesandMcManus,1993)indicatedthatE. multiloculariswasdifferentfromE.granulosus.Manyrecent stud-iesbasedonmtDNA(e.g.Lavikainenetal.,2006;Leetal.,2002; McManus,2002;Moksetal.,2008)andnucleardata(Bartetal., 2006;Haagetal.,2009;Lavikainenetal.,2003;Saarmaetal.,2009) supportthisview.However,therearealsostudiesthatpostulatea paraphyleticrelationshipbetweenE.multilocularisandE. granulo-susbasedbothonmtDNA(Hüttneretal.,2008;Lavikainenetal., 2003;Nakaoetal.,2007;Nakaoetal.,2013;Obwalleretal.,2004; Thompsonetal.,2006),andnucleardata(e.g.Knappetal.,2011). Evenanalysesrelyingondataderivedfromalargeportionofthe mitochondrial genome(Nakaoetal.,2007), and morethanfive thousandbasepairsofnuclearDNA(Saarmaetal.,2009)havebeen unabletoresolvetheconflict.Basedonanalysisof12mitochondrial genes(Nakaoetal.,2007)E.multiloculariswaspositioned,together withE.shiquicus,inthemiddleofdifferentE.granulosusgenotypes (Fig.3A).Paraphylyhasalsobeenobservedinotherstudies involv-ingnucleargenes(Knappetal.,2011;Nakaoetal.,2013).However, adifferentphylogenywasinferredbySaarmaetal.(2009)basedon 5nucleargenes(5086bp)whichplacedE.multilocularisclearly sep-aratefromtheE.granulosuscomplex(i.e.notparaphyletic)(Fig.3B). Thus,itappearsthatfurthergeneticanalysisisneededtoresolve thephylogenyofEchinococcusandthepositionofE.multilocularis inthegenus.
TheavailabilityofnearlycompletegenomesofE.multilocularis andE.granulosus(Tsaietal.,2013)offersthepossibilityofinferring theEchinococcusphylogenywithamuchlargernumberofgenes thanhaspreviouslybeenused.The115megabasegenomeofE. mul-tiloculariscomprises9chromosomes,ofwhich10345geneshave beenidentified.AsinothersequencedtapewormssuchasE. gran-ulosus,Taeniasolium,Hymenolepismicrostomaandthetrematode Schistosomamansoni,thegenerepertoireformetabolicfunctionsis highlyreducedinE.multilocularis,whereasthecapacitytoabsorb nutrientsfromthehostis increased.Concerning theprocessof reproductionincestodesandtrematodes,thetwochromosomesof E.multiloculariscorrespondtotheZsexchromosomeinS.mansoni (aspeciesrepresentinguniquesexualdimorphismintheflatworm group),promptingdiscussionaboutthedivergenceandoriginof sexualdimorphismandhermaphroditisminflatworms.
Withtheavailabilityofnextgenerationsequencing technolo-gies(NGS),hugeamountsofgeneticdatacanbeobtainedasnever before.FuturesequencingprojectsfocusingonotherEchinococcus speciesgenomesandgroupsoftargetedgenes(e.g.genesinvolved inparasitedevelopment,survivalandmetabolicfeatures)canopen thewaytodeeperinvestigationsofEchinococcusphylogenyand manyotheraspectsoftheevolutionaryhistoryoftheseparasites.
E. mullocularis E. granulosuss.s. E. granulosuss.s. E. granulosuss.s. E. canadensisG6 E.canadensisG7 E. canadensisG8 E.canadensisG6/G7 E.canadensisG8/G10 E. canadensisG6 E. canadensisG7 E. canadensisG8 E. ortleppi E. ortleppi E. ortleppi E. shiquicus E.shiquicus E. shiquicus E. mullocularis E.mullocularis E. equinus E. equinus E. equinus E.felidis E. felidis E. felidis E. vogeli E. vogeli E. vogeli E. oligarthrus E.oligarthrus E. oligarthrus
A
B
C
Fig.3. ThesummaryofrecentpublicationsdescribingphylogeneticrelationshipsamongEchinococcusspp.(A)AphylogenybasedontheDNAsequencesofmitochondrial genes(Nakaoetal.,2007;Hüttneretal.,2008).(B)AphylogenybasedontheDNAsequencesofnuclearprotein-codinggenes(Saarmaetal.,2009).(C)Aphylogenybasedon exonsequencesofnuclearprotein-codinggenes(Knappetal.,2011).
4. Geographicalgenotypesandmolecularepidemiology 4.1. DiversityofmtDNA
Theintra-specificdiversityofE.multiloculariswasfirstdescribed bysequencingcodingornon-codingfragmentsofmtDNA.These analysesrevealedverylowdiversityamongstgeographically dis-tinctisolates:nucleotidediversityinE.multiloculariswasestimated tobeabout10timeslowerthaninE.granulosuss.l.(Haagetal., 1997).Fromearlymitochondrialstudies,twogeographical geno-types(haplotypes)weredescribedamongEuropeanisolates(M1) andChinese,Japanese,AlaskanandNorthAmericanisolates(M2)
(Bowlesetal.,1992;BowlesandMcManus,1993;Okamotoetal.,
1995).Basedonsequencingof3mitochondrialtargets,Nakaoand co-workershavesincedescribed17regionalhaplotypesfrom76 isolatessampledinEurope(5haplotypes),Asia(10haplotypes)and NorthAmerica(2haplotypes)(Nakaoetal.,2009).Moreover,one NorthAmericanandtwoAsianhaplotypesco-existonStLawrence IslandintheBeringSea;ageographicalborderbetweenAsiaand NorthAmericathatcouldbeconsideredapassagewayforE. multi-locularis.Suchgeographicaldiversityanddifferentiationamongst E.multilocularispopulationscouldbeexplainedbythemovements ofmammalsduringthePleistocenealong withlocalorregional isolationoftheparasiteinglacialrefugiaduringmultiple glacia-tionevents(Nakaoetal.,2009)andbottleneckeffects(observed forE.shiquicusontheQinghai-TibetanPlateau)(Maetal.,2012). Nakaoandco-workerssuggestedanintroductionofE. multilocu-larisbyfoxesintoEuropeinthelatePleistoceneperiod,between 130,000and10,000years agoand itsspreadin AsiaandNorth AmericaacrosstheBeringSeawasestimatedtohaveoccurred dur-ingtheHoloceneperiod(<10,000yearsago).InEuropethecore areaof E.multilocularisisin theAlpinearchregion,comprising Switzerland,southernGermany,easternFranceandAustria and thishasbeenproposedastheparasite’shistoricalendemicarea (Eckert andDeplazes,2004).However,totracebackthespatial dynamicsoftheparasitefromthishistoricalendemicregion,more discriminantmolecularmarkersarerequired.
4.2. Diversityaccordingtonucleardata:theEmsBmicrosatellite TheEmsBmicrosatellitewasselectedfromamong17E. granu-losuslocitoassessgeneticdiversityinE.multilocularis(Bartetal.,
Fig.4.ElectropherogramofthemicrosatelliteEmsBfluorescentPCRproducts,with profilesfrom(A)anAlaskansample,(B)aChinesesampleand(C)aFrenchsample.
2006).Thismulti-locusmicrosatelliteishighlypolymorphicamong geographicallydistinctsamples(Fig.4)comparedtoothermarkers described,providingadistinct(CA)n(GA)npattern.EmsBandthe single-locusEmsJ,EmsKandNAK1microsatelliteswereassessed forpolymorphismusing76E.multilocularisisolatesfrom differ-entlocations(Europe,NorthAmericaandAsia)(Knappetal.,2007; Nakaoetal.,2003).HighestpolymorphismwasobtainedwithEmsB (29profiles)incomparisontoNAK1(7genotypes)andEmsJ/EmsK combined together(3 genotypes). Thediscriminatory power of EmsBwasassessedatdifferentspatialscales(micro-local,localand regionalscales).Thecirculationofasingleprofilewasdescribed amongrodentsfromsize-limitedfields,inSwitzerlandandAlaska (Knappetal.,2007),allowingconclusionstobedrawnaboutthe dis-criminatorypowerofthemarkerwithoutoverestimatingdiversity atthisspatialscale.
Onastudiedareaof900m2,locatedinanendemicregionin France, where fox density is 3 to4 individuals per km2
(win-ters2003–2004and2005–2006),6EmsBprofilesweredescribed among79E.multilocularispositivefoxes(Knappetal.,2008).At thetime,thiswasthefirstdescriptionofgeneticdiversityin E. multiloculariswormsisolatedatalocalscale.
Thegeneticdiversityoftheparasitehasalsobeenassessedmore widelyinEuropeusingEmsB(Knappetal.,2009).Thisallowedthe expansionoftheparasitetobetracedfromitshistoricalendemic area(intheAlpineArch),wheregeneticdiversityisrelativelyhigh, tonewlydescribedendemicareas(e.g.northernPoland,Slovakia, CzechRepublic),wherediversityislower.Fivefociinthehistorical endemicareasand4fociinthenewendemicareaswere inves-tigatedbygenotyping571wormsfrom123redfoxes,resulting inthedescriptionof32EmsBprofiles.Thisstudyrevealeda pre-dominanceofcertainEmsBprofilesatlocalscales–mostlyinnew endemicregions–duetofounderevents.
Thecontaminationofnewendemicregionscouldthusbe inves-tigatedthankstothedatacollectedonEmsBpolymorphism by
Bart et al. (2006) and the different research teams using the
EmsBmarker.OntheSvalbardarchipelago,wheretheparasitewas describedin1999,acircumpolarorigin–presumablyduetothe introductionofaRussianrodentinthe1960s(Henttonenetal., 2001)–rather than aEuropeanorigin involvingarcticfoxwas firsthighlightedbyEmsBdata(Knappetal.,2012).InCanada,a metacestodelesioninadogwasgenotyped,andaEuropean ori-ginfortheparasitewassuggestedbyanalysisofmtDNA(Jenkins etal.,2012).However,anEmsB-basedgenotypingstudyshould beperformedto confirmthesefindings. In France,which is an endemic areain Europe,a total of 383worms from 128foxes wereanalysedinthehistoricalendemiceasternpartand2new fociinthenorthernandthewesternparts(Umhangetal.,2014). Theauthorshypothesisedthattherangeoftheparasitemayhave expanded in two episodes of dispersal: first from the eastern parttothenorth,andsubsequentlyfromthewesternparttothe west.
Thankstothese studies,a collectionof EmsBdatahasbeen established,and alargenumber ofgenotypedand geo-localised samples are now available for furtherinvestigation. Studies of geneticdiversity among strainsinfectingdifferentintermediate hosts–primarilyhumans,butalsorodentsandaberranthostssuch ascaptivemonkeysor dogs–areurgentlyrequiredinorderto betterunderstandroutesofcontaminationandtolinkpathogenic strainswithdominantprofiles.Tocomplementthelargenumberof genotypingstudiesalreadyperformed,furtherinvitroandinvivo studiesondifferentE.multilocularisstrainssampledfromhumans andotheranimals,representingvariouspathogenicphenotypes, shouldbeperformedtoshedlightonclassificationsaccordingto pathogenicity(Barteletal.,1992).
Theepidemiologicalcontrastbetweenareasknowntobe hyper-endemic,suchasChina(Torgersonetal.,2010),andthosewithlow humaninfectionrate,suchasNorthAmerica(Yamasakietal.,2008) providesagoodopportunitytoinvestigatedifferencesinE. multi-locularispathogenicity.Epidemiologicalstudiesshouldbebasedon theestablishmentofnationalreferencecentrestorecorddetailed informationforallalveolarechinococcosiscases,perform system-aticstraingenotypingandepidemiologicalinvestigationssuchas establishingtheriskofinfectionassociatedwithactivitiessuchas agriculture,owningadomesticgarden,contactwithwildanimals, domesticanimaldewormingandothers(Kernetal.,2003). 4.3. Copro-sampleanalysis
EmsBgenotypinghasusuallybeenperformedonadultworms ormetacestodesamples.However,this requireshostssampling and necropsy, which is time-costly, administratively laborious andoftenconsideredtobeunethical.E.multilocularisisrelatively easytodetectinfaecalsamplesofdefinitivehostsusingvarious
moleculartechniques,e.g.standard-PCR(Laurimaa etal.,2015), multiplex-PCR(Trachseletal.,2007),real-timePCR(Knappetal., 2014) or real-time multiplex-nested PCR (Dinkel et al., 2011). Recently, a newly developed non-invasive geneticmethod was publishedbyLaurimaaetal.(2015)whichallowsboth E. multi-locularisanditshostspeciestobeidentifiedfromcarnivorefaecal samples.Themethodisalsohighlysensitive:thepresenceofE. multiloculariscanevenbedetectedwhenonlyasingleparasiteegg ispresentinthesample.
CurrentPCR-baseddetectionmethodsonlypermitthepresence orabsenceofE.multilocularistobeestablishedanddonotallow parasitediversitytobeinvestigated.However,studiesbasedon isolationofeggsandtotalparasitecopro-DNAextractioncan pro-videinformationaboutparasitediversityandthisisofimportance becausewormswithdifferentEmsBprofileshavebeendescribed fromtheintestinesofindividualfoxes(Knappetal.,2009;Knapp etal.,2008).Micromanipulationsarenecessarytoisolateindividual andintacteggsusingmicroscopy.SubsequentDNAamplification followedbygeneticanalysisonthissmallquantityofDNAis cur-rentlytheonlywaytoobtainaccurategeneticdiversitydatafrom copro-samples.Insuchanalyses,theidentityoftheparasitehost shouldalso beevaluated, especially in cases of positive copro-samplesfromdomesticanimalend-hostslivinginclosecontact withhumans. In addition tothe PCR-based methods described above,microsatelliteanalysisandDNA-barcodingcanalsobe per-formedinthiscontext(e.g.Bensonetal.,2012;Galanetal.,2012).
5. Conclusion
WhileclassicaltaxonomyplacesE.multilocularisclearly sepa-ratefromotherspeciesintheEchinococcusgenus,includingtheE. granulosuscomplex,itsphylogeneticpositioninthegenusrequires furtherstudieswithanextendedrepertoireofnuclearmarkersand sub-specieshavetobefurtherstudiedinthiswaywith compar-isontoecologicaland epidemiological datatovalidatethemas validtaxaornot.Therecentlypublishednearlycompletegenome sequencesof E.multilocularisand someof itsrelatives,suchas E.granulosusandT.solium(Tsaietal.,2013),holdgreatpromise forfinetuningitsphylogeneticposition,andbetter understand-ingitsevolutionaryhistory,includingspecificadaptationstoits hostspecies.Detoxificationmechanismsandthecapacitytoevade intermediate hostimmune systems with partialcontrol of the infectionrepresentinterestingtargetsforfindingbettersolutions fortherapy(VuittonandGottstein,2010).Froman epidemiolog-icalperspective,itisinterestingtonotethatE.multilocularisisa taxonwithrelativelylowgeneticdiversity.Thischaracteristicis dueononehandtothespecies’recentevolutionaryhistoryand ontheothertoitshermaphroditereproduction,whichiscommon totheentireTaeniidae familyandreflectsself-fertilisationwith autogamyorgeitonogamy,whengametesarefromseparatedbut geneticallyidenticalorganisms(Haagetal.,2007).Thespeciesalso presentscontrastingepidemiologicalcharacteristicsthroughoutits range,withahyper-endemicfocusinChinaandrareoccurrence inNorthAmerica inhumans,eventhoughtheadulttapeworms arehighlyprevalentincarnivoresinboth areas(Storandtetal., 2002;Vaniscotteet al.,2011).Eventhoughgenotypingis easily performedontheEmsBmicrosatellitebyfragmentsizeanalysis, additionalhighlypolymorphicmicrosatellitesorSNPmarkersare neededtoassessgeneticdiversity.Themethodologyforanalysis oftheselocishouldbestandardised,sothatdatafromdifferent sourcescanbeeasilycompared.Moreover,accumulateddata espe-ciallyonEmsB,shouldbegatheredintoacommonpublicdatabase inordertoadvanceinternationalcooperationinepidemiological studiesofthisdangerouszoonoticdisease.Ontopofgenotyping studies,epidemiologicaldatashouldbecollectedforeachhuman
case,basedontherulessetbytheexistingnationalreference cen-tres.TheFrancEchino-AlveolarEchinococcosisNationalReference CentreinBesanc¸on,France,ortheRobertKochInstituteinBerlin, Germanyhavealready conductedhealth monitoringforseveral decades.Thesereferencecentresinvestigatepopulationrisk fac-tors,whichwerehighlightedbyEuropeancollaborationprograms suchastheEurEchinoRegproject,basedoncollaborationof west-ernandcentralEuropeancountries(Kernetal.,2003).Moreover, eco-epidemiologicalprojectsassociatedwithgenotypingprograms inEurope(especiallyinnewlyendemicareasofEasternEurope) andAsiaareneededtofurtherstudythediversityoftheparasitein ordertoinvestigateitsspatio-temporalpatternsandemergingor re-emergingstatus.Inthisway,lesionphenotypes,geneticaspects andepidemiologicalcontextcouldbestudiedinmultidisciplinary andlong-termmonitoringprojects(Lindenmayeretal.,2011),to betterunderstandtheepidemiologyofalveolarechinococcosisand steadilymovetowardeffectivecontrolofthemultifaceted tape-wormparasite.
Acknowledgments
WeareverygratefultoBenoîtValotandGéraldUmhangfor complementarystudiesontheEmsBmicrosatellite,andJohn Davi-sonforlanguageediting.ThisworkwassupportedbytheSwiss NationalScienceFoundation(grantno.31,003A141039/1); insti-tutionalresearchfunding(IUT-2032)fromtheEstonianMinistry ofEducationandResearch;bygrantESF-8525fromtheEstonian ResearchCouncil;bytheEuropeanUnionthroughtheEuropean RegionalDevelopmentFund(CentreofExcellenceFIBIR);bythe FrenchNationalInstituteforHealthSurveillanceandtheFrench MinistryofHealththroughtheNationalReferenceCentreFor Alve-olarEchinococcosis.
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