The multiple and complex and changeable scenarios of the Trypanosoma cruzi transmission cycle in the sylvatic environment

Accepted Manuscript Title: The multiple and complex and changeable scenarios of the Trypanosoma cruzi transmission cycle in the sylvatic environment Author: Ana M. Jansen Samanta C.C. Xavier Andr´e Luiz R. Roque PII: DOI: Reference:

S0001-706X(15)30066-8 http://dx.doi.org/doi:10.1016/j.actatropica.2015.07.018 ACTROP 3686

To appear in:

Acta Tropica

Received date: Revised date: Accepted date:

29-5-2015 13-7-2015 15-7-2015

Please cite this article as: Jansen, Ana M., Xavier, Samanta C.C., Roque, Andr´e Luiz R., The multiple and complex and changeable scenarios of the Trypanosoma cruzi transmission cycle in the sylvatic environment.Acta Tropica http://dx.doi.org/10.1016/j.actatropica.2015.07.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Themultiple,complexandchangeablescenariosoftheTrypanoso

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macruzitransmissioncycleinthesylvaticenvironment

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Ana M. Jansena,* Samanta C. C. Xavier a, André Luiz R. Roquea

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aLaboratoryofTrypanosomatidBiology,OswaldoCruzInstitute,FIOCRUZ,Av.Brasil4365,ZIPCODE210

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40-360,RiodeJaneiro/RJ,Brazil;

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e-mail:[email protected](AMJ),[email protected](SCCX),[email protected](ALRR)

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*Corresponding author. Tel: +55-21-2562-1416; Fax: +55-21-2562-1609

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E-mail address: [email protected] (A.M. Jansen)

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Abstract

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Inthisstudy,wereportanddiscusstheresultsgeneratedfromover20yearsofstudiesoftheTrypanosomacruzisy

26

lvatictransmissioncycle.Ourresultshaveuncoverednewaspectsandreviewedoldconceptsonissuesincludin

27

greservoirs,truegeneralistspecies,associationofmammalianspecieswithdistinctDiscreteTypingUnits-

28

DTUs,distributionofT.cruzigenotypesinthewild,mixedinfections,andT.cruzitransmissionecology.Usingp

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arasitologicalandserologicaltests,weexaminedT.cruziinfection

30

mammalianordersdispersedallover

31

cruziisolateswerecharacterizedbymini-exongenesequencepolymorphismandPCRRFLPtoidentifyDTUs.

32

InfectionbyT.cruziwasdetectedbyserologicalmethodsin20%oftheexaminedanimalsandisolatedfrom41%

33

ofthoseinfected,correspondingto8%ofalltheexaminedmammals.Eachmammaltaxonrespondeduniquelyt

34

oT.cruziinfection.Didelphisspp.

35

lastingparasitemias(positivehemocultures)causedbyTcIbutmaintainandrapidlycontrolparasitemiascause

36

dbyTcIItoalmostundetectablelevels.Incontrast,thetamarinspeciesLeontopithecusrosaliaandL.chrysomel

37

asmaintainlong-lastingandhighparasitemiascausedbyTcIIsimilarlytoPhilandersp.

38

ThecoatiNasuanasuamaintainshighparasitemiasbybothparentalT.cruziDTUsTcIorTcIIandbyTcII/TcIV(

39

formerlyZ3)atdetectablelevels.Wildanddomesticcanidaeseemtodisplayonlyashortperiodofreservoircom

40

petence.T.cruziinfectionwasdemonstratedinthewildcanidspeciesCerdocyon

41

brachyurus,and positive hemoculturewas obtainedinone hyper carnivorespecies (Leoparduspardalis),

42

demonstratingthatT.cruzitransmissionisdeeplyimmersedinthetrophicnet.T.cruziDTUdistributioninnature

43

didnotexhibitanyassociationwithaparticularbiomeorhabitat.TcIpredominatesthroughout(58%oftheT.cru

44

ziisolates);however,inspiteofbeingsignificantlylessfrequent(17%),TcIIisalsowidelydistributed.Concomi

45

tantDTUinfectionoccurredin16%ofinfectedmammalsofallbiomesandincludedarborealandterrestrialspec

46

ies,aswellasbats.TcI/TcIIconcomitantinfectionwasthemostcommonandwidelydispersed,withmixedTcI/

47

TcIIinfectionsespeciallycommonincoatisandinDidelphimorphia.Thesecondmostcommonpatternofconc

48

omitantinfectionwasTcI/TcIV,observedinChiroptera,DidelphimorphiaandPrimates.Takentogether,ourre

49

sultsdemonstratethecomplexityofT.cruzireservoirsystemanditstransmissionstrategies,indicatingthatthere

50

isconsiderablymoretobelearnedregardingecologyofT.cruzi.

51

in7,285mammalianspecimensfromnine theBrazilianbiomes.TheobtainedT.

areabletomaintainhighandlong-

thousandChrysocyon

52 53

Keywords: Trypanosomacruzi,DTU,Reservoirs,Transmissioncycles,Ecologyofparasites

54

1.”Everythingchangesandnothingstandsstill”(HeraclitusofEphesus)

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Inthisstudy,wepresentanddiscussaspectsofwhatconstitutestheenzootictransmissioncycleofTrypa

56

nosomacruzi(Kinetoplastida,Trypanosomatidae),presentingexamplesofthediverseanduniquescenariosin

57

whichtransmissionofT.cruzioccurs.Thesedataresultfrom20yearsofobservationandanalysesofT.

58

cruzitransmissionamong

59

rangingwildmammalsspanningsevenordersanddispersedamongthedistinctbiomesofBrazil.Furthermore,

60

wealsopresentasummaryofourkeyfindings,includingnewdataonthesubject.

free-

61 62

1.1.Generalaspects

63

TrypanosomiasisduetoT.cruziisprimarilyanancientandwidespreadenzootic

64

infectiondistributedthroughoutthemammalianfaunaoftheAmericas,reachingfromsouthernUSAtosouther

65

nArgentina.Humaninfectionmayresultin

66

Chagasdiseaseandmostlikelybeganassoonashumansarrivedonthecontinentapproximately15,000yearsbp(

67

Gul

68

2000).Untilrecently,itwasthoughtthatChagasdiseaseinBrazilwasundercontrol,nolongerposingapublichea

69

lthproblem.However,

70

threatenshumanhealthunderdistinctepidemiologicalscenarios,evenoutsidetheAmericas(Gascon et

71

2010).ThenumberofcasesandoutbreaksofChagasdiseasehaveincreased(or at least become more apparent)

72

overthelastdecadeduetoinfectionbytheoralrouteinLatinAmericaandduetobloodtransfusion,particularlyin

73

Spain(Andrade

74

2011).Chagasdiseasetransmittedbytheoralroutehasbeenprimarilyobserved

75

intheAmazon(inBrazilandneighboringcountries)andindicatesthattrypanosomiasisbyT.cruziisprimarilya

76

nenzootic

77

infection.ThisphenomenonmakesstudyingthetransmissioncycleofT.cruziinthewildoffundamentalimport

78

anceifpreventionofnewhumancasesistobeachieved.Additionally,thetransmissioncycleofthiskinetoplastid

79

inthewildenvironmentoffersanexceptionalstudymodelforunderstandingparasitism.

80 81

et

al,

trypanosomiasisbyT.cruzihas

et

al,

2014;

re-emerged

Tanowitz

andagain

et

al,

al,

ThemostpuzzlingfeaturesofT.cruzi,namely,itsextremeheterogeneityandbiologicalplasticity,mak ethestudyofitsecologybothinterestingandchallenging.

Infact,

someoftheissuesraisedbyCarlosChagas

82

remainunanswered

83

eventoday.Onesuchissueistheassociationofagivensubpopulationoftheparasitewithagiventriatominespeci

84

esreservoirsorhostspecies,aswellaswiththedistincthumandiseasecharacteristics.Morphologicalheterogen

85

eityhasalreadybeenrecognizedbyChagas(1909)andBrumpt(1913),bothofwhomhypothesized

86

roleplayedbytheslenderandstumpyformsoftheparasitethattheyobservedinbloodsmears, but currently this

87

feature

88

valued.Theincreaseintheanalyticalpowerofnewmethodologicaltoolsallowedinvestigationofintra-

89

speciesheterogeneitybybiological,biochemical,andmolecularmethods.Thesestudiesansweredmanyquest

90

ionsbutalsoraisedmanynewones(Burgos et al, 2007; Fernandes et al, 1998; Tibayrenc and Ayala, 1988;

91

Dvorak,

92

1977).TheseminalarticlesofMiles(1977)openedanimportantresearchfrontwithhispioneeringworkonthebi

93

ochemicalcharacterizationofT.cruziandmanyaspectsofitsdiversityandecology.Currently,sevengenotypes,

94

ordiscretetypingunits(DTUs),arerecognizedinthetaxon:TcI,TcII,TcIII,TcIV,TcV,TcVI,andTcbat,withthi

95

slatterbeingagenotypethatwillmostlikelyberecognizedasTcVIIinthenearfuture(Hamilton et al, 2012;

96

Zingales et al, 2009).

97

is

1984;

no

Miles

et

distinct

longer

al,

1980;

Theurgetoexplainabiologicalphenomenon,especiallywhenitisassociatedwithhumandisease,carri

98

estheriskofover-

99

generalizationbasedonalimitedunderstandingofthesubject.Itisimportanttorememberthatabiologicalphen

100

omenonhasnowell-

101

definedboundariesandshouldbestudiedasacomplexsystemasmuchaspossiblewithaholisticfocus.Studying

102

theindividualpartsofasystemdoesnotallowfortheunderstandingofthewholebecausethecomponentsexhibit

103

uniquepropertieswhenimmersedincompletescenarios.Complexsystemsincludedifferentorganizationalle

104

velsthatrangefromthesubatomicleveltoanimalpopulations(Mazzocchi,

105

2008).Similartoapuzzle,onepiecemeansnothingwhenisolatedfromtheset,andthepictureisnotclearuntilallt

106

hepiecesareputtogether.Thispointiswell-

107

illustratedbytheassumptionsthathavebeenconstructedanddeconstructedsequentially,overtheyears,aboutt

108

heecologyofT.cruziDTUs.

109

Classically,theparentalDTUTcIwasassociatedwiththesylvatictransmissioncyclebyseveralauthors

110

,mostlikelybecauseTcIprevailsamongtheisolatesobtainedfromwildmammals(Fernandes

111

1998).Inturn,TcIIwasclassicallyassociatedwiththedomiciliarytransmissioncycle,includinghumaninfecti

112

ons,andspecificallywiththe severe gastricformofhumandiseaseinthecentralwestregionofBrazil(Zingales

113

et

114

Theincreaseinfieldexcursions,theexpansionofthespectrumofexaminedmammalianspeciesandespeciallyt

115

helong-

116

termstudieshavedeconstructedseveraloftheformerlyproposedassociationsandprovidedsomeanswerstothe

117

manyquestionsonthissubject(Xavier et al, 2014; Ramírez et al, 2014; Enriquez et al, 2013; Maloney et

118

al,

119

2006).However,themainquestionregardingtheecologyofthelineagesofT.cruzistillremainsunanswered.

120

et

al,

2010;

Meja-Jaramillo

et

al,

2012).

al,

2009;

Cortez

et

al,

WithrespecttotheecologyofTcII,Pinhoandcolleagues(2000)detectedtwoZ2infecteddidelphidmarsupial

121

species,DidelphisauritaandPhilanderfrenatus,collectedintheAtlanticcoastalrainforest.

122

Thereafter,TcIIwasfoundtoinfectseveralothermammaliantaxaindistincthabitatsandbiomes,includingthe

123

AmazonandalsoNewOrleans,USA(Herrera et al, 2015; Lima et al, 2014; Shikanai-Yasuda and

124

Carvalho, 2012).

125

TransmissionofT.cruziinnatureiscommonlyrepresentedasalinearandsequentialphenomenon.Not

126

hingcouldbemorereductionist.AnimalsareconstantlybeingexposedtoinfectionbydifferentT.cruziDTUsthr

127

oughsingle,mixed or multiple DTUsinoculae, whichcan occur once ormultipletimes.T. cruzi may also

128

access

129

themammalianhostbydistinctinfectiveforms(oralandcontaminative),whichcanoccuronceorrepeatedly.Ho

130

st-to-

131

hostdispersionisassuredbydozensoftriatominespecies,whicharehematophagousbutderivenourishmentfro

132

mvariousanimalspecies.Takentogether,thesetraitsresultinT.cruzibeingfoundinfectingmammalsinallwild

133

habitats,foreststrataandniches.Thus,theenzootictransmissioncycleofT.cruziinnatureshouldbereferredtoin

134

thepluraltransmissioncycles

135

becauseevenwithinthesameforestfragment,itmayornotbepresentinallstrata,and/oritmayoccurinseparatetr

136

ansmissioncyclesthatmayormaynotoverlap.

137

TransmissionofT.cruziisdeeplywovenintothefoodwebbecauseitisfrequentlytransferredtoanewho

138

stbytheoralroute,andbecauseitstriatominevectorsareconsideredmicropredators(Herrera

139

2011).Afoodnetiscomposedoftheorganismsthatareinterconnectedbytransferenceofenergy.Becausetheor

140

ganismswithinatrophicnetcommonlyfeedonmorethanonespecies,acomplexinteractionnetworkresults,en

141

ablingT.cruzitransmission.Indeed,eachhostspeciesexploitstheirexistinghabitatsandnichesindistinctways;

142

hence,theecologicalinteractionsandencountersamongthevectorandhostspeciesaremodulatedbythefaunal

143

composition,which,inturn,isevidentlyshapedbythelandscape.Consequently,transmissionofT.cruziinnatur

144

eoccurswithinahugenetworkinwhicheachnoderepresentsonespeciesandtheverticesrepresenttheirecologi

145

calinteractions.

146

Thisimpliesthattheremovalorinsertionofasinglenoderesultsinacompletelydifferentdesignofthisnetwork.I

147

nfact,theslightestchangewillresultintheemergenceofanewenzooticscenario,distinctfromtheprevious,mer

148

curial,butirreversible,asifitwereakaleidoscope.Theseaspectsareseldomconsideredwhencollectingandinte

149

rpretingdata.

150

et

al,

Acomplexsystemsimilartothisoneneedstobestudiedasawholebecauseitisimpossibletounderstand

151

thefunctionofsucha

152

onemustalsoconsidertheindividual

153

analyticalandtheholisticapproaches

154

hostparasitessuchasT.cruzi.

complex

network

focusingsolelyononepartofit.Paradoxically, componentsofthesystem.Reconcilingboththe isthegreatandinterestingchallengeofstudyingmulti-

155 156

2.T.cruzireservoirsandvectors:conceptualreview

157

Communicationrequiresthatobjects,livingthingsandnaturalphenomenareceivenames,areordered

158

andareclassified;however,whilea classificationsystem allowsforthe exchangeof information,experiences

159

andthoughts,thissameorderingsystemcanalsorestraintheadvanceoftheconceptualframework.

160

Theobservationofnaturedemandsadmittinguncertainty,instabilityandchangeability,alwayskeepinginmin

161

dthatnaturalphenomenaoccurin complexmatrices withelements thatare completelyinterdependent

162

(Mazzocchi,

163

donotdisplaydefinedcontoursorlimitsandareconstantlychanging,sometimesfasterthanourtheoretical(con

164

ceptualortaxonomical)boxesinwhichweinsistthatthesephenomenamustfit.Thisisexemplifiedbyourattem

165

ptstodefinereservoirs,anissuethathasbeenmodifiedovertime(Silva et al, 2005; Ashford 2003; 1996;

2012;

2008;

Montoya

et

al,

2006).Naturalphenomena

166

Haydon et al, 2002; Barreto and Ribeiro, 1979; Brumpt, 1936).

167

Thus,BrumptinhisseminalbookPrecisdeParasitologie(1936)definesareservoiras“Lestresquisont

168

susceptiblesdherbergerlesmmesparasites queHommeoules animauxdomestiquesconstituentles rservoirs

169

degermes”.

170

Underthisanthropocentricdefinition,theauthorconsidershumansasthetargetspeciesofparasitesfromothera

171

nimals.Domesticanimalsarealsocitedin

172

causeby infectingthem.

173

Carlos

thedefinition

duetothe

economiclosses

thatparasitesmay

174

Chagas(1912),inhisdescriptionofwhatcouldbetheT.cruzireservoir,referstothearmadilloTatusianovemcinc

175

ta(sic)asapossibledepositoryofTrypanosomacruziontheexternalworld.Thetermdepositoryreflectstheconc

176

eptthatunderliesthedefinitioninthosedayswhenanimalswerepresumedtoberatherstaticsystemsandnotlivin

177

gentitieswithintricateandpeculiarecologicalcharacteristics.

178

Actually,eachmammalianspeciesconstitutesacomplex,multivariateandchangeablelivingsystem,

179

exercisingdifferentselectiveforcesontheirparasitesinthedifferentenvironmentsoftheiroccurrence.Allofthe

180

hostspeciesselectsubpopulationsofT.cruziinauniquewayandpresentdifferentinfectionpatternsthatdepend

181

onnumerousmacroenvironmental(landscape)andmicroenvironmentalvariables(individualpeculiarities)(

182

Dvorak 1984; Deane et al, 1984a).Furthermore, fromanecological focus, humansare justonemore

183

hostspeciesin theT.cruzi transmission cycle,asareallotherspeciesofmammals.

184

Adefinitionthathasbecomewidelyacceptedsincethe1960sreferstoreservoirsastheanimalsthatares

185

ourcesofinfectionforhumansordomesticanimals(duetotheireconomicimportance).Moreover,itwasalsoac

186

ceptedasfactthatreservoirhostsdonotsufferdiseaseconsequencesfromthepresenceoftheparasite.Firstofall,

187

itisnowincreasinglyrecognizedthatbothvirulenceandpathogenicitymayrepresentfitnesstraits

188

thatcanenhancedispersion ofthe parasite(Woolhouse et al, 2001; Giorgio, 1995; Lenski and May,

189

1994).Furthermore,healthimpairmentina

190

trappedandthereforehighlystressedwildanimalisratherdifficulttodetect.Moreover,thelifeofananimalinthe

191

wildishighlycompetitive.Itislikelythatanimalswithsub-optimalhealthwillnotbeabletosurvive

192

longandmaybemoreeasilypreyedupon,enhancingparasite-i.e.,T.cruzi-dispersion.

193

Definingwhatisareservoirisatheoreticalandpracticalchallenge;moreover,identifyingreservoirsof

194

aheterogeneousandmulti-hostparasitesuchasT. cruziisspeciallypuzzlinganddifficult(Jansen and Roque,

195

2010).Wehavebeenconsideringareservoiraspossiblyincludingmorethanoneanimalspecies.Infact,weadopt

196

edaslightlymodifieddefinitionofAshford(1966),whostatedthatareservoirmaybeconsideredasacomplexec

197

ologicalsystemconsistingofoneormorespecies

198

innature.Thissystemshouldalwaysbeconsideredinasinglespace-timescale(Jansen and Roque, 2010).

responsibleformaintainingagivenparasitespecies

199 200

2.1. Thetriatominevectors

201

VectorsofT.cruziareincludedintheorderHemiptera,familyReduviidae,subfamilyTriatominae.Atot

202

alof142speciesdividedinto18generaconstitutethistaxon(Silva et al, 2012; Lent and Wygodzinsky,

203

1979).Thetriatominesconstituteapolyphyleticgroupthatdisplaysecologicalandbiochemicaldifferences,ev

204

enwithallrepresentativesbeingstrictlyhematophagous(Schofield

205

2009).Thus,asanexample,speciesofthegenusTriatomaareassociatedwithrockyenvironmentsandRhodnius

206

sp.withpalmtrees(Gaunt

207

notextendtoitshosts.Indeed,theyarespecialistsin

208

thetypeoftissuefromwhichtheyfeed(blood),althoughunderexperimentalconditions,therearedescriptionso

209

ftriatominethatcompletedtheircyclefeedingoncockroaches(Lazzari et al, 2013).

and

Miles,

and

2000).Thisassociationwith

Galvão,

agivenenvironmentdoes

210

Insidethegutofthevector,T.cruzimustfirstsurvivetheharshconditionsofthestomach;then,itmustmu

211

ltiplyandundergometacyclogenesis.Allthesecrucialstepsdependonthebiologicalandbiochemicalidiosyncr

212

asiesofthedifferent parasitesubpopulationsandvectorspecies (Meja-Jaramillo et al., 2009; Azambuja et

213

al, 2004; Mello et al, 1996).

214

Theparasiteis

215

notalwayssuccessful.Infact,thesophisticatedimmunesystemofthetriatomineisahindrancethatmustbeover

216

comebytheparasite(Waniek

217

2011).Thus,somestrainsarenotabletosurviveinthedigestivetractofthebugorarenotabletotransformintothei

218

nfectivemetacyclic forms (Araujo et al, 2009; Azambuja et al, 2004; Mello et al, 1996).Another often-

219

overlooked

220

infectionoftriatominebugsbydifferentDTUs.Innature,hardlyatriatominewillnotcomeintocontactwithdiffe

221

rentDTUsbroughtbydifferentspecimensofmammalsonwhichtheinsectwillfeed.Experimentalstudiesonth

point

et

is

thefrequent

al,

concomitant

222

esubjectarelessfrequentbut

223

thatT.cruzigrowthinthesmallintestineofRhodniusprolixuswasmoresuccessfulinmixedTcI/TcIIinfectionst

224

haninsingleinfectionswiththesegenotypes.Additionally,theauthorsalsoobservedthatTcIseemedtobethepr

225

edominantisolateeveninmixedinfections(Araujo

226

2014).ItisworthmentioningthatT.cruziisolatesinonebiomemayinfectandestablishperfectlyintriatominebu

227

gsfromanotherbiome,includingthosepresentingtotallydifferentphysiognomiccharacteristics.Thiswasdem

228

onstratedbythesuccessfulexperimentalinfectionofTriatomabrasiliensis,atriatominespeciescommonlyfou

229

ndinsemi-aridareasofnorth-

230

easternBrazil,withoneT.cruzi(TcI)derivedfromaDidelphisauritacapturedintheAtlanticrainforest(Araujo

231

et al, 2007).

mightbehighly

informative.Recently,itwasobserved

et

al,

232

Oneofthekeydeterminantsofthesuccessofthecontaminativerouteofinfectionistheamountoftimebe

233

tweenthestartofthebloodmealbytheinsectanddefecation,whentheinfectivemetacyclicformsareeliminated.

234

Thus,itisclassicallythoughtthatspecieswithgoodvectorcapacityarethosewhosetimebetweenfeedingandde

235

fecationwastheshortest.Inthecurrentscenario,wheretheoraltransmissionthatoccursbyingestionoffoodcon

236

taminatedwithmetacyclicformsgainedgreaterimportance,thisdeterminantisnolongerthebestwaytodeterm

237

inethecompetenceofaspeciesoftriatomine.Infact,inthisscenario,anytriatominespeciescanbeconsideredop

238

timalbecauseitcontainsmetacyclicforms(Pereira et al, 2009).

239

ConsideringthateachofthedistinctBrazilianbiomes,includingAmazonia,Savannah,dryCaatinga,P

240

antanalandtheAtlanticForest,constituteidiosyncraticlandscapemosaics,onemustrealizethatthetransmissi

241

oncycleofT.cruziinnatureisacomplex,multivariable,nonlinearsystemthatdependsontheinterplayofseveral

242

stillunknownvariables.T.cruziisanextremelysuccessfulparasitespeciesthat

243

isabletooccupyalmostalltissuesofitshundredmammalianhostspecies,includingunorthodoxsitessuchasthe

244

stenchglandsofdidelphidmarsupials(Deane

245

1984b).ThefactthatT.cruzicanperformbothmultiplicativecycles(amastigoteandepimastigote)inthesamem

246

ammalianhost,theopossum,suggeststhatopossumsaretheonlymammalsthatcanactasareservoir,aswellasav

247

ectorofT.cruzi.Infact,opossumseliminatethecontentofthesescentglandswhenstressedandarethereforeable

248

todisseminatetheparasiteintotheirsurroundings.Thisfinding,observedonlyobserved70yearsafterthefirstd

249

escriptionofT.cruzibyCarlosChagas,indicatesthatotherecologicalfeaturesofthistrypanosomatidmightstill

et

al,

250

be unknown(Deane et al, 1984b).

251

T.cruzimaybeconsideredatruegeneralistparasitespeciesbecauseit

252

isabletoexploitthediverseresourcesofitshosts.Thereisanimportantissuewithintheparasitismphenomenon:

253

Whataretheevolutionaryadvantagesofaparasitebeinghighlyspecializedoverbeinggeneralist?Generalistsp

254

ecieshavetheabilitytoexploreabroadrangeofresourcesandarethereforemoreadaptabletoenvironmentalmo

255

dificationsinadditiontodisplayingahighercapacityofdispersion.Theconsequencesarethatamorevariableen

256

vironmentwillfavorgeneralistspecies(Fried

257

2010).Duetohumanactivity,wearecurrentlyexperiencingaperiodofprofoundclimaticchanges,includingani

258

ncreaseofglobalsurfacetemperaturesandanincreaseofCO2levels(Awmack and Leather, 2002; Percy et

259

al,

260

2002)additionaltothecontinuousinvasionofformerlywildareas.Theimpactofthesechangesontheecologyof

261

thetransmissioncyclesofT.cruziisunknown.However,therearevectorspeciesofT.cruziandseveralhighlyresi

262

lientmammalianspeciesinallBrazilianbiomes.Thus,thecurrenttendencyofincreasingdroughtandtemperat

263

uremayresultintheeliminationofseveraltriatomineandmammalianspecies;however,generalistspecieswillc

264

ertainlyadaptandevenbenefitfromthistypeofenvironmentalchange.Thatis,theenzootictransmissionofT.cr

265

uziwillcertainlynotbeeradicatedbythistypeofclimatechange.Onthecontrary,itismorelikelytoexpand.Infact

266

,theenzooticcycleofT.cruziisalreadybecomingmorerobustintheUSAandlocalauthoritiesarealreadyawareo

267

ftheproblem, especially concerning the Latin American immigrants(Bern et al, 2011).

268

et

al,

ThespectacularphenotypicplasticityofT.

269

cruziismostlikelytheresultofitslongevolutionprocess,whichincludeditsjumpfrom

270

absolutelycontrasting environments:the digestive tract ofinsectsto theintracellular environment

271

ofthetissuesofmammalianspecies(orviceversa).

two

272 273

3.

274

Thewildenvironmentalengineerscreatenewbiocenosesthatenhanceinterspeciesencountersandtrans-

275

missionofT.cruzi.

276 277

Duetotechnology,theenvironmentalimpactofhumanactionisamplifiedandmayberatherdramaticb ecauseofitsspeedandscale.However,

independent

ofman’saction,

the

environment

isconstantly

278

changingduetoseveralfactors,includingtheactivityofitsnon-

279

humaninhabitants.Naturalengineersmodifythenaturalenvironmentbybuilding

280

shelterstoprocreateorsimplytorest.Thesenests/sheltersfrequentlyresultintruebiocenosisbecause

281

theymaybeusedconcomitantlybyotherlivingbeings,includingtriatomines(Gaunt

282

2000).Oncetheyhavenofurtherusefortheirbuilders,suchshelterswillbeusedbyothertaxainaprocessthatincr

283

easestheecologicalinteractionofdistinctandunrelatedanimalspecies(Desbiez

284

2013).Onestrikingexampleincludesthegiantarmadillo(Priodontesmaximus).Thebehavioralpatternofthiss

285

peciesincludestheexcavation,useandabandonmentofdozensofholesduringitslifespan.Theseholes/tunnels

286

maysuccessivelybeusedbyotherspecies,suchasthecoatis(Nasuanasua),oncillas(Leopardustigrinus,alsok

287

nownasthelittlespottedcat),andanteaters(Myrmecophagatetradactyla).Foxes(Lycalopexvetulus)modifyth

288

esetunnelsadaptingthemforbreeding(Desbiez

289

Thesetunnelsareobviouslyhighlyfavorablesheltersfortriatominesandconsequentlyimportantforthemainte

290

nanceandspreadoftheparasite.Moreover,thesequenceofecologicalsuccessionbymammalianfaunaofagive

291

nhabitatisquiteunpredictableturningitratherunreliabletoestablishanyassociationhypothesisofaT.cruziDT

292

Uandamammalianhostspeciesbasedonitsuseofacertainhabitat.

and

and

and

Kluyber,

Miles,

Kluyber,

2013).

293

Anotherexampleoftherichnessofpossibilitiesofencountersbetweenmammalianspeciesisofferedb

294

ycoatinests.Nasuanasuabuildtheirnestsintreetopsatheightsbetween10and20m.Therearetwotypesofcoati

295

nests,restingandbreedingnests.Femalesraisetheiryounginthebreedingnestsduringthefirsttwoorthreemont

296

hsoftheirlife.Afterthistime,thefemaleandhercubsleavethenestthatwillbereusedbyotheranimals(Olifiers et

297

al,

298

2009).Recently,theuseofcameratrapstomonitorcoatisnestsrecordedthepresenceofaThrichomysfosteri,aca

299

viomorphrodentspeciesdescribedasbeingterrestrial.Insidethenest,triatomine

300

(andotherinsectspecies)

301

Thisterrestrialrodentspeciesreachedthecoatinestprobablybyexploringanaturalhighwayformedoftrunksof

302

fallentreesand/orlianasthattemporarilyconnectedtheforeststrata.

303

provided

bugs

anoptimalmicroenvironmentforT.cruzitransmission.

Theknowledgeofthepossibleencountersofeachhost/reservoirspeciesiscrucialifanunderstandingof

304

thisepizootiologyisintended.Indeed,itwouldberathernaivetotrytoassociateasubpopulationorgeneticlinea

305

geofaparasitewithaparticularanimalspeciesbasedonlyononestudyinoneoccasionwhiledisregardingthenu

306

mberandtypesofinteractionsthisanimalspecieshashadwithotherspeciesandhabitats.

307 308

4. Theinfectionroutes

309

T.cruziinfectionroutesforhumansareverywellknownandstudied,butthisisnotthecaseindomesticor

310

freerangingmammals;

311

thedifficultiesin-

312

herenttosuchfieldwork.ThecontaminativerouteisaratherproblematicstrategyfordispersionofT.cruzi.Nouv

313

elletandcolleagues(2013)concludedthattheprobabilityoftransmissionpercontactwithaninfectedtriatomin

314

eis6x104.Thisisevenmorestrikinginwildfree-

315

rangingmammalswhosedensefurmostlikelyrepresentsanimportantbarrierthathindersthecontactbetween

316

metacyclicformsandtheskin.

317

however,thisisanimportantissuetobeconsidered,thoughverydifficultgiven

Thecongenitalroute,which,incertaingeographicalareas,isanimportantrouteofhumaninfection(Ca

318

rlier

319

livingwildanimals.Basically,doingfieldworkisexpensiveandnoteasy,especiallyconsideringstudiesofcong

320

enitaltransmission.First,itisnoteasytoaccessnewbornanimalsinnature.Additionally,itisverystressfulforan

321

ewbornanimaltobemanipulatedandhaveitsbloodcollected.Furthermore,becauseofananthropocentricbias,

322

congenitaltransmissionpossibilitiesinmammalsareoftendiscussedasifitwereonesingletaxon,asisthecasew

323

ithhumans.However,mammalspresentvarioustypes of placentawith differentdegrees ofintimacy with

324

themother’s

325

likely,withdifferenttransmissionrates.Todate,theonlyobservationofcongenitalT.cruzitransmissioninfree-

326

rangingwildmammalswas reportedinbats(Añez et al, 2009).However, under experimental conditions,

327

thereareplentyof

328

Oliveira et al, 2013).

329

et

al,

bodyand

2015)isnearlyunstudiedinfree-

therefore,most

seminalarticlesaboutcongenitaltransmissionindifferentmammalspecies(Alkimin-

Theoralroute(mostlikelytheearliestone)seemstousthemoreefficientdispersionstrategyofT.cruziin

330

thewild,andtheexperimentsofNobukoYoshidasupportthisview(Yoshida

331

2008).However,onemustconsidertheecologyofthedifferentgroupsofmammals.Thus,nomadicmammals,t

332

hosethatuseallforeststrataandthataregeneralistwithrespecttotheirdiet,aremorelikelyinfectedbythisroute.T

333

hesameistruefortheso-

2009;

334

calledhypercarnivores,mammalsthatpreyexclusivelyonothermammals,suchasthelargeFelidae.Smallrode

335

nts,onthecontrary,whicharemostlygranivoriusorherbivorousandtendtoremaininmorerestrictedareasthatm

336

aybesharedwithtriatominebugs,arethereforemorelikelytobeexposedtoinfectionbythecontaminativeroute.

337

Infact,infectionoffree-

338

rangingmammalianspeciesoccurindistinctwaysandbydistinctroutes,dependingontheirecology(Kribs-

339

Zaleta,

340

2014).Othervariablesthatdetermineinfectionroutesaretheratiobetweenthedensityofthevectorpopulationa

341

ndthedensityofhostpopulations(Pelosse and Kribs-Zaleta, 2012).

342

Underexperimentalconditions,wewereabletoinfectlaboratory-

343

rearedDidelphisauritaorally,byfeedingthemexperimentallyinfectedtriatominebugsandalsothroughexperi

344

mentallycontaminatedfooditemscontainingmetacyclicformsderivedfrominfectedscentglandmaterial(Jan

345

sen

346

1997)Theimportanceoftheoralrouteforcarnivoresandomnivoreswasalsosupportedbytheobservationthat

347

insectivorousCanidae showed higher positivity in serologicaltests compared to their non-

348

insectivorousrelatives(Rocha

349

2013a)Predationonmammalsasaneffectivetransmissionrouteisamatterofdebate(Roellig

350

2009);however,therecentisolationofT.cruziTcIfromapumaandpositiveserologicaltestsfromwild,free-

351

ranginghypercarnivorousFelidaeindicatethat

352

infectionrouteand

353

inatrophicnet.Thelargecarnivores(CanidaeandFelidae)arelong-

354

livinganimalsthatusehugelifeareas,comeintocontactwithalmostall existing fauna, andmay actasbio-

355

accumulators ofT.cruziDTUs (Rocha et al, 2013a; 2013b).

356

et

al,

et

predationof

al,

smallmammals

confirmsthe

insertionof

isalso

et

al,

aneffective T.cruzi

Mesopredatorssuchasthecoatisandraccoonsalsouselargelifeareasandalsopreyonsmallmammals(

357

Herrera

358

2011)Theyarefrequentlygeneralistfeeders,displaymuchlargerpopulationsincomparisontothegreatcarnivo

359

res,andusealldistinctforeststrata.Therefore,theymayalsoactasbio-

360

accumulatorsandasdispersersofT.cruziDTUs.Smallmammals(rodentsandmarsupials)thatareonalowerlev

361

elofthetrophicpyramiddisplaymuchlargerpopulationsthatundergoquickpopulation

et

al,

362

turnover.T.cruziinfectionoftheseshort-livingmammalsindicatesrecenttransmission.Inspiteof

363

beingmuchmorehabitatrestricted,rodentsalsomayactasdispersersofT.cruzsincetheyarefoodsourcesforani

364

ncrediblenumberofmammals.Marsupialspeciestendtobenomadicandaremuchmorehabitatandfoodgenera

365

listthanrodents,not

366

thebatsthatarecompetentflyersandconsequentlydisplayahighT.cruzidispersalcompetence.

forgetting

367 368

5. Diagnostictoolsandwhattheyindicateaboutinfectionprevalenceandinfectivitycompetence

369

ThediagnosisofT.cruziinfectioninwildanimalsisstilldifficultbecauseantibodiesarenotfoundinthes

370

pecificmarketforcombinedserologicaltestsformostwildmammalianspecies.Asaconsequence,mostofthea

371

uthorswhoworkwithwildanimalsuseonlyparasitologicalmethods(includingPCR)asdiagnosticmethodsof

372

detectingT.cruziinfection.Wecombineparasitologicalandserologicalmethodsbecausebothdeliverinformat

373

ionondifferentaspectsofinfection(Table1).Forexample,afreshbloodtestwithhighparasitemiaisindicativeof

374

highinfectivecompetence,butdoesnothaveenoughsensitivitytodetectparasitesbelow1x103.Lowerparasite

375

miathatissufficientlyhightoinfectthevector,i.e.,signalizinginfectivecompetence,isevidencedbybloodcult

376

ures

377

parasitemia).Xenodiagnosishasasimilarlevelofsensitivityasbloodcultures(Portela-Lindoso

378

Shikanai-Yasuda,

379

1966),butwepreferthelatterbecausethefirstmethodwouldinvolvethetransferandmaintenanceofliveinsectst

380

osmallanddistantstudyareasthatoffernoinfrastructuretoensuretheisolationofcontainerswiththetriatominei

381

nsectsfromthecuriosityoflocals.

382

(which

2003;

reflects

Chiari

host

and

and Brener,

PCRisahighlysensitiveandspecificmethodandunequivocallydemonstratesthepresenceoftheparas

383

iteintheanimal’sbody.However,thismethod

384

noinformationregardingtheinfectivecompetencebecauseitcannotdeterminetheviabilityoftheparasite.Add

385

itionally,itisimportanttorememberthatforthediagnosisofinfectionoftriatomines,almostalltheintestinalcon

386

tentisused,i.e.,atrulyrepresentativesample.Thediagnosisofinfectioninmammalsisbasedonlyonverysmallp

387

iecesofthetargettissuei.e.,non-representativesamples.Parasitedistributionintissuesisaggregated;thus,a

388

negativetestingfragmentdoesnotindicatethattheanimalisnotparasitized.Apromisingtechniqueintermsof

389

evaluatinginfectivitycompetence isRealTime-PCR (Melo et al, 2015; Moreira et al, 2013).However,this

gives

390

isa

391

trainedpersonnelandexpensiveequipment,whichhampersitsuseinpoorercountries,wheretheepidemiologi

392

calriskishigher.Additionally,thistechniqueisfarfrombeingstandardizedfordifferentmammalianhostspecie

393

s.

techniquethat

requireswell-

394

Antibodies,incontrast,arehomogeneouslydistributedintheseraandserologicaltestsareverysensitiv

395

e,thoughtheyfailsomewhatregardingspecificity.Moreover,thesignificantcontributionofserologicaltestsist

396

hattheycomplementtheenzootic

397

whoareinfectedbut presentlow infectivecompetence(undetectedparasitemia).

framework

beingstudiedbyidentifyingthe

individuals

398

Wehavealwayscombinedparasitologicalandserologicaltestingtoobtainamoreprecisediagnosisoft

399

heenzooticprofileofthestudyareatogaininformationonboththeprevalenceofinfectionandthetransmissibilit

400

ypotential,i.e.,thetransmissioncompetenceofaparticularanimal(Tables1and2).Theeffectofcross-

401

reactionscanbeminimizedbyassayingtheanimalseraagainstantigensfromphylogeneticallyrelatedparasites

402

,asourgrouproutinelydoes(Xavier et al, 2012; Rademaker et al, 2009).

403

Intheroutinelyusedserologicaltests(ELISAandIFA),weincludeinhouseintermediate

404

antibodyobtainedfromrabbitsimmunizedwiththetargetspeciesimmunoglobulin.Working

405

thisway,weincreasethespectrumofanimalspeciesthatcanbediagnosedandincreasetestsensitivity;however,t

406

hisapproachunfortunatelyalsoincreasesnonspecificreactions(Jansen

407

1985).Unfortunately,therearefewcommercialreagentsandkitsforsuchdiagnoses.Ourmethodofusinganinte

408

rmediateantibodyraisedinrabbitsorusingcommercialantibodiesfordomesticanimalsthatarephylogenetical

409

lyrelated(e.g.,anti-catforwildfelids)isfarfromideal.Evenso,therearemammaliantaxa

410

donothaveany possibilityof performingserologicaltests,suchasthe Cingulata, Pilosa and Chiroptera.

et

al,

forwhichwe

411

The need fora multidisciplinary approach for thestudy of health questions has

412

beenincreasinglyrecognized.TheOneHealthconcepthasbeengainingimpetus,andwithit,theneedfortoolsfo

413

rthediagnosisofparasiticinfectionsinwildanimals(Zinsstag

414

2011).Itislikelythatwewillhavemorereagentsinthenearfuturethatcanwidenserologicaldiagnosisforalarger

415

numberofspecies.

et

416 417

6. T.cruziparasitismassumespeculiarinfectionprofilesdependingonmammaliantaxa

al,

418

Dependingonthe

faunal

compositionofagiven

locality,

theenzootic

419

infectionwillhavedistinctscenarios.ThegeneralistcharacteristicsofT.cruziaremostlikelytheresultofthecon

420

servatismofphenotypiccharacteristicsacquiredduringitslongevolutionaryhistory.Trypanosomatidsofmam

421

malsareancienteukaryoticorganismsofdebatableorigin,althoughcurrently,thetrendistoaccepttheinsecthos

422

tastheprimevalone(Lukes

423

1904).Whethertheprimarysiteofcolonizationoftheseflagellateswasthedigestivetractofmammalsorinsects,

424

theprocessofadaptingtotheinternalenvironmentofboththemammalianhostand/orthevectormostlikelydem

425

andedalongadaptivetime.Irrespectiveofthesequenceofadaptiveevents(thedigestivetractofthemammalorv

426

ectorasthefirstcolonizationsite),eachnewniche(celltypeand/orextracellularenvironmentofmammalorinse

427

ct)requiredashift,aspilloverprocessoftheparasiteuntilitreachedandadaptedtothecirculatorysystemofthem

428

ammalianhost.

429

Theprocessofacquiringnewmammalianhostsmusthavehappenedcoincidingwiththearrivalofmammalians

430

peciesintheAmericasandtheconsequentincreaseinmammaldiversity(Carrillo

431

2015).ItisimportanttorememberthattheearlyfaunaofSouthAmericaconsistedprimarilyofCingulata,Pilosa

432

andDidelphimorphia(Voloch

433

2015).Allothermammaliantaxawereincludedandexpandedthetransmissioncyclewhentheyarrivedontheco

434

ntinent.Firstthecaviomorphrodentsandprimatesarrived,mostlikelybyislandhopping,35millionyearsbp(Fl

435

ynn and Wyss, 1998).Thisexplainswhywehaveneverfoundanyassociationbetween DTUT.cruziand

436

anyanimalspecies.Itislikelythatthedistinctpatternsofinfectionamongthedifferentspeciesofmammalsalrea

437

dybegantosettlethen.Therefore,theconstitutionofagivenenzooticscenarioaswellasthepredominantDTUw

438

illbegreatlydependentontheconstitutionofthelocaltrophicnet,whichisinconstantchange.Thetrophicnetevi

439

dentlyalsoincludesthetriatominevectorthatmayabsolutelybeconsideredamicropredator.Understandingthe

440

parasitetransferflowinagivenfoodwebisahighlydemandingtaskbecauseofthemanygapsintheknowledgeof

441

boththepossibilitiesofdifferentmammalianspeciesandvectorsmeetingandthelackofinformationaboutthec

442

ourseofinfectionofthemajorityofwildspeciesbythedifferentDTUs.

443

et

et

al,

al,

2014;

2013;

Leger,

et

Bond

al,

et

al,

ThegenusDidelphis,asitisnotablywell-known,isabletomaintainhighandlong-

444

lastingparasitemias(asexpressedbypositivehemocultures)andconsequentlyisanexcellentTcIdisperser(Ta

445

ble2).Moreover,DidelphisisabletorapidlycontrolandeveneliminateatleastsomesubpopulationsofTcII,aso

446

bservedbyDeaneandcolleagues(Deane

447

1984b)inT.cruziYstrainexperimentallyinfectedopossums.Incontrast,GoldenLionTamarinspeciesLeontopithecusro

448

saliaandalsoL.chrysomelasareabletomaintainlong-lastingandhighparasitemiascausedbyTcII(Lisboa

449

2015).Leontophitecusrosalia,andNasuanasuaareabletomaintainbothparentalT.cruziDTUsatdetectablelevels(Table

450

2).Incontrast,wildanddomesticcanidaeandhumansdisplayashortperiod(6-

451

8weeks)ofhighparasitemiaandconsequentlyashortperiodofinfectivepotential.Thesefindingsrepresentwh

452

atiscurrentlyknown,thoughthesefindingsonlyrepresentthetipoftheiceberg.

453

et

al,

et

al,

IfallthevariablesconcerningthespecificinteractionofT.cruziwithitsvertebrateandinvertebratehosts

454

andtheconstitutionofmammalianandtriatominefaunawereusedintheconstructionof

455

possiblearrangementsbycombinatorialanalysis,an

456

wouldemerge.Thiswouldnoteventakeintoaccounttheindividualhostvariablesrangingfrommixedinfections

457

withdifferentDTUsandmixedinfectionswithotherspeciesofparasites,hostgender,age,nutritionalstatus,am

458

ongothersand,veryimportantly,humanactivity.ThiscomplexmosaicexplainswhyoutbreaksofacuteChagas

459

diseasemayoccurinverydiversesituations,whichmakestheadoptionofstandardmeasuresofepidemiological

460

surveillanceandcontrolalmostimpossible(Roque

461

idiosyncrasies.

all

almostendlessrangeofepidemiologicalpossibilities

et

al,

2008).Infact,eachareadisplaysits

own

462

ApreviousstudyoftheT.cruzitransmissioncycleinthePantanalregion,locatedinthecentralpartofBra

463

zil,aratherpreservedareathatincludesallthecomponentsofthefoodweb,showedthatboththeoralrouteandcon

464

taminativeroutearelikelytooccur,dependingontheencounterpossibilitiesofthemammalsandvectors(Herrer

465

a

466

2011).Thus,theoralrouteismorepronetooccurbypredationoftriatominesorothermammals,especiallyincar

467

nivores.Agreaterpossibilityofacquiringtheinfectionbythecontaminativerouteispresentinmammalianshelt

468

ers,caves,treehollowsandotherrefugesusedmorepermanentlybytheanimals.Mesopredators,suchasthecarn

469

ivoreNasuanasuaandthenomadicDidelphismarsupialis,may

470

thecontaminative

471

studiedanimalgroupsarethecarnivoresthatarehighlydiverseandlong-

472

livedanimalsthatuselargelivingspaces,comeintocontactwithahugevarietyoffaunaandareatthetopofthetrop

473

hicpyramid.Infact,littleisknownregardingtheroleplayedbyNeotropicalwildcarnivoresintheT,cruzitransmi

et

andoral

al,

mostlikely

routes.Apropos,oneof

beinfected

byboth theless

474

ssioncycles.Ourpreviousstudies,whichincludedsixwild carnivore

475

wereinfected

476

fromwhatwasobservedinCanidae,respectively,redfoxes(Vulpesvulpes)andcoyotes(Canislatrans)intheUS

477

,whereonlytwocoyotesexaminedbyserologicalmethodshaveshowntobeinfectedbyT.cruzi(Rosypal et al,

478

2014).

byT.

cruzi(Rocha

et

al,

species,revealed

2013a).Our

resultsare

thatall

ofthem

quitedifferent

479

Moreover,thehighparasitemiasdetectablebyhemoculture,i.e.,thehighinfectivitycompetence,were

480

foundmainlyinProcyonidae.Thesemesopredators(ProcyoncancrivorusandNasuanasua)weredemonstrat

481

edtoharborTcI,TcII,TcIII/TcIVandTrypanosomarangeli,respectively,insingleandmixedinfections(Table

482

2).Moreover,itwasincoatis,anomadandgeneralist,thatweobservedT.cruziisolatesthatdisplayedoddbandpa

483

tternsintheMini-exonassay,reinforcingthenotionthatT.cruzidiversity

484

acknowledged.Additionally,

485

authorsconsistentlyobservedhighparasitemiasinMusteloideaspecies(Alves et al, 2011; Roellig et al,

486

2009;

487

1992).TcIwasalsoisolatedfromthefelidspeciesLeoparduspardalisaso-

488

calledhypercarnivorespecies,reinforcingtheoralroutebypredationofmammalsasimportantinthenaturalenv

489

ironment.Infact,thisfelidspeciesisnotinsectivorousatallandmostlikelyacquiredtheinfectionbypreyingonin

490

fectedmammals.

491

Herrera

et

mightbemuchhigherthancurrently other

al,

2008;

Pietrzak

et

al,

1998;

Karsten

et

al,

AninterestingexampleofhowknowledgeinecologycanhelptounderstandthetransmissionofT.cruzii

492

nthewildenvironmentispresentedbystudyingnestsofPhacellodomusrufifrons.Thisthornbirdspeciesbuilds

493

neststhathangfromthebranchesoftreesandthatare

494

easilydismantled.ThetriatominespeciesPsammolestescoreodesisassociatedwiththesebirdssuchthatahigh

495

numberofnestsinfestedbythistriatominespecieswereobservedinthearea(HerreraH,personalcommunicatio

496

n).However,noexampleofP.coreodeshasbeenfoundinfectedwithT.cruzi.Theexplanation?Itishighlyunlikel

497

ythatthefragileandpendantnestsofP.rufitronswillallowentryandpermanenceofsmallmammals;thus,noT.cr

498

uziinfectionwill occur, althoughP. coreodesissusceptibletoT.cruziinfection.

499

Whatvariables

500

determinetheinclusionofmanintheT.cruzitransmissioncyclecurrently?Peopleareathigherriskforinfection

501

whentheyspendmoretimeinthenaturalenvironment,forexample,byplayingsports,huntingorwhenbuildingi

502

nformerlywildareas,which

503

resultingreaterexposuretotheenzootictransmissioncycle.Thisexposurealsooccurswhentriatominesinvade

504

humandomiciles,attractedbylight,seekingfoodsourcesthattheforest,partiallydisturbedbyhumanaction,no

505

longeroffers.Itisknownthatexploratoryactivityofnaturecauseslossofbiodiversityand,consequently,lossoff

506

oodsourcesfortriatomines.

507

Itisnotnecessaryfortriatominestocolonizehumandwellingstoinfecthumans.Storing

508

orhandlingfoodininadequateconditionsfacilitatesitscontaminationbyinfectedtriatominebugs.Thisispartic

509

ularlystrikingintheAmazon,andinBrazilitislinkedmainlytotheingestionofaçaíorbacabajuice(Valente

510

al, 2002).IntheAmazon,theaçaí juice isprepared at leasttwice a day,atdusk and at dawn.Natural light,

511

still

512

makesthelocalsuseartificiallightinstalledjustabovethejuiceextractionequipment.Thelightmayattracttriato

513

minebugsthateventuallyfallinsidetheequipmentandarecrushedtogetherwiththefruitsandconsumed.Additi

514

onally,thefruit

515

collectionbasketsmayactasdispersersofinfectedtriatominebugsbecausethesemaybetransportedforconsu

516

mptioninremotelocationsand,ifthejuiceispreparedwithoutproperhygienicconditions,resultininfection(Xa

517

vier

518

2002).Inthesecases,Chagasdiseaseshouldbeconsideredasafoodbornedisease(Xavier

519

2014).Thus,thehigherdiseaseriskfactorformankindisignorance.Knowingthelocalenzooticscenarioandpas

520

singoninformationtothelocalpeopletodemonstrateriskbehaviorpatternsaretheonlyreallyeffectivemeasure

521

sofprevention.

scarce

et

atthese

al,

2014;

two

Valente

et

times,

et

al, et

al,

522 523

7.

524

Theconstitutionofthemammalianfaunamodelstheenzooticscenarioandfavorsorhampersthetransmissi

525

on of distinctT.cruziDTUs

526

ForT.cruzi,competenceforbeinginfectivetothevectordependsonparasitemia,i.e.,thenumberofpara

527

sitesingestedwiththebloodmeal.Thistrait,ofcourse,variesaccordingtothehostspeciesandT.cruziDTU.Mor

528

eover,infectivitycompetencemayvaryinspaceandtimeevenamongindividualsofthesamespeciesbecauseth

529

eabilitytocontrolparasitemiadependsonvariableslinkedtothehost(age,gender,infectionroute,nutritionand

530

healthstatus),aswellaswithconcomitantinfectionsbyotherparasitespeciesandthegenotypeofT.cruzi.Being

531

anefficientreservoiristhereforenotafixedattributeofagivenmammalianspecies.Thus,evenanuniversallyagr

532

eedmainreservoirspeciessuchastheopossumDidelphisauritamaynotactasamajorreservoireveninthesames

533

tateorinthesamebiome.Thus,theprevalenceofpositivehemocultures,i.e.,highparasitemia,inD.auritafrom6

534

distinctlocalitiesofRiodeJaneirostatevariedsignificantly,rangingbetween11%inItaguaand90%intheJagua

535

numisland.InSilvaJardim,isolationofT.cruzifromD.auritawaspossibleonlyin13%ofsamples(Fernandes

536

et al, 1999).Inthislocalityweobserveda robust and long-lastingtransmission cycle of TcIImaintainedby

537

L.rosalia,thegolden

538

upshowedthatthiscyclehasremainedstableandmaintainedthesameenzooticfeatures,with40%oftamarinsha

539

vingpositivebloodcultures(Lisboa

540

2015).Thisstabilityisfarfrombeingtherule.Infact,inonelocation,alsointherainforestofRiodeJaneiro,onesur

541

veyshowed40%ofopossumswithpositivebloodcultures,butshowedanentirelydifferentenzooticpictureduri

542

ngareevaluationtenyearslater,whenallanalyzedopossumswereshowntobeuninfected(Vaz et al, 2007;

543

Pinho

544

2000).Itislikelythatenvironmentaldisruptioncausedbytheincreaseinhumanintrusionresultedinacriticallos

545

sofbiodiversitythatimpairedT.cruzimaintenanceinthearea.Duetoitshighadaptabilitycompetence,D.aurita

546

stillresistedhumanactivityandremainedinthearea.

lionTamarin(Lisboa

et

al,

2000).A

ten-yearfollow-

et

al,

et

al,

547

OurstudiesofthecoatiNasuanasuainthesamePantanalregionexemplifyclearlyhowdynamictheenz

548

ooticscenarioinwhichthetransmissionofT.cruzioccursmaybe.Initially,thisProcyonidspecieswas shown to

549

betobeinfectedmainlybyT.evansi(Herrera

550

1999).Afewyearslater,webegantonoticeahighprevalenceofT.cruziinfection(singleormixedwithT.evansi).

551

Overtime,T.cruzibecamepredominantintheseanimals(Herrera

552

2005).Soon,wewerenolongerabletodetectT.evansiandthecoatisappearedto

553

T.cruzionly.Recently,

554

isolatedT.cruzibutstartedisolatingT.rangeli.

wereturned

et

al,

2004;

et

tothe

Silva

et

al,

2011; beinfectedby

Pantanal,andnolonger

555 556 557

al,

8. AreT.cruziDTUsassociatedwithmammalianhostspeciesorwithgeographicalareas? Asmentionedearlier,sevendiscretetypingunits(DTUs)inthetaxonarecurrentlyrecognized.

558

Nevertheless,allattemptsthathavebeenundertakensincethefirstresearchersattemptedtocorrelatethedistribu

559

tionandpossibleassociationoftheT.cruzisubpopulationswithitsmammalianhostsspecies,triatominespecies

560

orwithhumandiseasehaveledtocontroversialresults.However,certainassociationsofT.cruzigeneticlineages

561

togeographicalregionsareconsistentlybeingobserved(Llewellyn

562

2009).Theseobservations,addedtotheabsenceofrobustdataontheDTUsassociationwith

563

animalspecies,haveledauthorstoproposethatthediversificationprocessofT.cruziDTUshashappenedbyecol

564

ogicalfittingmorethanbyaco-evolutionaryprocess(Llewellyn et al, 2009).Evenso,therearestillmanygaps

565

intheknowledgeoftheecologyandbiologyofthe7T.

566

cruziDTUs:(1)Onemajordifficultyisthatwearemostlikelynotworkingwithtrulyrepresentativesamplesofth

567

epopulationsofT.cruzi.Certainly,oursamplesarenotrepresentativeofthevastdiversityofthisparasitetaxon;th

568

us,wearemostlikelyunderestimatingthenumberofT.

569

cruzivariants.(2)Eachhostspeciesactsasabiologicalfilter,positivelyselectingdistinctclonesoftheparasitesu

570

chthattheclonalassemblageoftheparasitediffersineachhostspeciesandeveneachindividual,accordingtothe

571

natureandkineticsoftheselectionprocessduringinfection.(3)Therearehostspeciesandhabitatsthathave

572

beeneither

573

notyetbeensampledatall.(4)WearenotconsideringandstudyingtheDTUsthatinfectthoseanimalsthatdispla

574

yonlypositiveserologicaltestsbutnegativeparasitologicaltests(themajority),asobservedinTable1.Therefor

575

e,wearelosingimportantinformationbecausewedonotevenknowbywhichDTUtheseanimalsareinfected.(5

576

)Conductingfollow-upstudiesoffree-

577

rangingwildmammalsisquitedifficult,andthemajorityofthestudiesincludeonlyonehemoculture

578

orxenodiagnosisofeachanimal.Oneevaluationrepresentsonlyafragment,asnapshot

579

ofthetotalityoftheparasiticinfrapopulationthatinfectsagivenspecimen.Certainly,itdoesnotincludethewhol

580

esetofclonesthatareinfectingthisgivenanimal.

581

(6)Theselectiveforcesexertedbytheisolationandamplificationmethodsoftheparasitealsohinder

582

thepossibilityofharvestingallclonespresent

583

Thedifficultiesoffieldworkandthebroaddispersionoftheparasitehostsalsolimitareliablesampling.(8)Finall

584

y,thereisnoclearerexamplethantherecentfindingofTcIIandhybridT.cruziinfectingtriatominebugsandmam

585

malsintwodifferentareasoftheBrazilianAmazon,abiomealwaysbeforequotedasfreefromTcII(Lima et al,

et

al,

under-sampledorhave

inonesinglespecimen.(7)

586

2014).

587

Overall,

thelikelihoodisthatweareonlyseeingapartofthewhole,thetipoftheiceberg,andthatnew

588

DTUsmaybereportedasmorestudiesonT.cruzitransmissioninsylvaticenvironmentsareperformed.Reinfor

589

cingthisprobabilityarethedescriptionsofoddpatternsofT.cruziisolatesthatcouldnotbeincludedinanyofthec

590

urrentlyacceptedDTUsderivedfrominfected coatis(Rocha et al, 2013a).

591 592

9. DistributionofT.cruziDTUsinBrazil:stateoftheart

593

Figure1showsthedistributionofT.cruziDTUsinwildmammalsofBrazilianbiomes.Thismapalsosho

594

wsthattherearelargeareasthathavenotbeensampledatallasyet.TheparentalDTUsarerepresentedbythepurpl

595

e(TcI)andbythelightblue(TcII)dots,andshowsthat,asinothersitesintheAmericas,isolationofTcIprevailsthr

596

oughoutinthesylvaticcycle.TcII,formerlyassociatedexclusivelywithhumaninfectionanddiseaseandsubse

597

quentlyassociatedwitharmadillos

598

2005),displaysthesecondlargestdistribution.TcIIalsodisplaysabroadwildhostrangeasithasbeenisolatedfro

599

mrodents,marsupials,primatesandcarnivoresinallofthebiomeswehavestudied(Table1).Moreover,thisDT

600

Uisisolatedfromasmallernumberofanimals,suggestingthatTcIIpresentsadistincttransmissionand

601

maintenancestrategy fromTcI inthe wildenvironment.

602

Like

(Yeo

matryoshkadolls,toolswith

et

al,

increasinganalyticalpower

have

603

demonstratedthatthereisalsointraDTUdiversity.ThisisthecasewithTcI,andassociationsofcertainhaplogro

604

ups ofTcI withwild ordomestic transmissioncycleshavealready beenrecognized (León et al, 2015;

605

Ramírez

606

2012).Moreover,asignificantdiversityofintraTcIhasalreadybeennotedbymicrosatelliteapproach,eveninth

607

esameisolatederivedfromonehostindividual,theopossumDidelphisaurita(Llewellyn

608

2009).RemarkableheterogeneityinintraDTUTcIIhasalsobeenobservedbyourgroup(VSLima,unpublished

609

data).

et

al,

et

al,

610

Our data include the examination of a total of 7,213 mammalian specimens from nine

611

mammalian orders by parasitological and serological diagnostic tests (Table 1). The obtained T. cruzi

612

isolates were characterized by miniexon gene sequence polymorphism and PCR RFLP

613

sequencepolymorphismandPCRRFLP(Lima

et

al,

2014;

Rocha

et

al,

614

2013a).InfectionbyT.cruziwasdetectedbyserologicalmethodsin20%(n=1413)oftheexamined

615

animals,andisolationoftheparasitebyhemocultures

616

wasachievedin547(39%)ofthese,correspondingto8%ofalltheexaminedmammals

617

(Table1).Consideringthatpositivehemoculturesindicatehighparasitemia

618

competence

619

inferthatdispersionandmaintenanceofT.cruziinthewildiswarrantedby40%ofinfectedmammals.

intransmitting

the

parasiteto

the

and

feeding

consequently, vectors,

we

high can

620

T.cruziinfectedmammalshavebeenfoundinallBrazilianbiomes(Tables1and2,Figures1and2).Inspi

621

teoftheparticipationofalmostallmammaliantaxaintheT.cruzireservoirsystem,Primates,Carnivora,andDid

622

elphimorphiapresentedhigherparasitemia,as

623

2,Figure3).Interestingly,

624

rodentsthatarehighlysusceptibletoT.cruziinfectionsinexperimentalconditionsapparentlyplayonlyasecond

625

aryroleinmaintenanceofT.cruziinthewild(Tables1and2,Figure3).Infactonly2.3%outofthe3,857examinedr

626

odentsdisplayedhighparasitemiaasexpressedbypositivehemocultures(Table1).Twopossiblescenariosmay

627

explainthisfact:(i)rodentsdonotsurviveT.cruziinfectioninthewild;and(ii)rodentsdisplayashortlifecyclean

628

dhomerangeandarethereforelessexposedtoT.cruziinfection.Thefirstscenariodoesnotseemveryplausiblebe

629

causeonly8.6%ofthecollectedrodentswere

630

demonstratedtobeinfected(seropositivity),aratiomuchlowerthanobservedforothertaxa.

631

expressedby

positivehemocultures(Table

ThesefindingsareapparentlyincontradictiontothehypothesisofOstfeldandcolleagues(Ostfeld

et

632

al,

633

2014),whopredictthatkeyreservoirsofmultihostparasitesarethosespeciesthatdisplayhighpopulationdensit

634

iesandsmallbodies.Actually,thelifehistorytraitsandpeculiaritiesthatdeterminethecompetenceofagivenani

635

malspeciesasareservoirofageneralistparasitespeciessuchasT.cruziisfarfrombeingknown.Oneofthesetraits

636

seemstobethegeneralisttraitofthehost.Infact,thespeciesthatdemonstratedhigherreservoircompetencewere,withex

637

ceptionofthegoldenLionTamarin,generalistconcerningtheirfeedinghabitsaswelltheircapacitytouseforeststrata:Dide

638

lphisspp.,Nasuanasua,Phylostomussp,CebuslibidinosusandPhilandersp.(Table2,Figure3).

639

T.cruziDTUdistributioninnaturedidnotdemonstrateanyassociationwithbiomeorhabitat(Table1;Fig

640

ure1).TcIpredominatesthroughout(58%oftheT.cruziisolates).Inspiteofbeingsignificantlylessfrequent(17

641

%),TcIIisalsowidelydistributed.AquestionthatemergesconcernstheecologyofthelessprevalentT.cruziDT

642

Ussuch

643

asthehybrids,whichinBrazildemonstratedtobeextremelyrare.AlsoTcIIIandTcIVarewidelydistributed,but

644

occuratsignificantlylowerrates(Figure1).Therearethreenon-

645

mutuallyexclusiveexplanations:(i)theseDTUsdependonsimultaneousinfectionwithotherDTUsorparasite

646

stobetransmitted;(ii)theyaremaintainedinnatureinverylowparasitemias,undetectablebyourmethods;and/

647

or(iii)wedidnotsucceedinsamplingtheirsuitablereservoirspecies.

648 649

10. MixedT.cruziDTUinfections

650

Veryrarely,ifithappens,animalsareparasitizedbyasinglespeciesorvarietyofagivenparasitictaxon.

651

WedetectedmixedDTUinfectionsin57(16%)ofhemoculture-

652

positivemammals,andthemostfrequentcombinationwasTcIwithTcII(Figures2and4).Thisisfarfrombeingc

653

omparabletothe48%mixedinfectionsobservedinhumanand

654

TcITcVI indogsin theGran Chaco(Argentina)(Monje-Rumi et al, 2015).

655

dogsamples.TcV/TcVIin

humansand

Itis

656

notdifficulttopresumehowwildmammalsacquireconcomitantinfectionsbyT.cruzigenotypes.Infact,wildan

657

imalsarealwaysbeingexposedtoinfection,andbothsequentialandsimultaneousinfectionsbydifferentDTUs

658

andinfectionroutesarehighlylikely.AsequentialinfectionispossiblebecauseitisknownthatpriorT.cruziinfec

659

tiondoesnotprotectagainstasubsequentinoculum,butonlypreventsanewacutephase(Guerreiro

660

2015).Thesecondpossibilitycaneasilyoccurbypredationontriatominebugswithmixedinfections,aconditio

661

nthatisnotuncommonlyobserved.Concerningmixedinfections,thecongenitalrouteseemslesslikely.

et

al,

662

ThemammalsthatdisplayedthelargestdiversityofDTUsonconcomitantinfectionswereProcyonida

663

e(Nasuanasua),Phyllostomidae(ArtibeusspandPhyllostomussp),Didelphidae(Monodelphissp.,Gracilina

664

nussp.,Philandersp.,andDidelphissp.)

665

andCebidae(Cebussp.)(Figures2and4).Themammaliangenerainwhichweobservedthehighestrateofmixed

666

infectionswereNasuasp.andDidelphissp.(Figure2).Theseresultsconfirmgeneralisttaxaasbioaccumulators

667

andprobabledispersersofT.cruziDTUsinnature, andsuggestthatone can make a fairly accurateassessment

668

ofDTU

669

areafocusingonthesemammaliantaxa.Furthermore,thereareseveralquestionsthatneedtobeevaluated:(i)for

diversity

ofT.cruzi

inacertain

670

howlongcanthesedistinctparasiteDTUs

671

DTUs?ItistemptingtospeculatethattheparentalDTU,namelyTcI,maybethemostcompetitiveDTU.

672

co-existwithinthehost?Whatarethemorecompetitive

Allmammalsthatdisplayedmixedinfectionshaveasacommontrait-

673

thattheyaregeneralistsinrelationtotheirdietandtheirhabitatandthattheyincludeinsectsintheirdiet.Ecologic

674

alpeculiaritiesofeachmammalspeciesmayturnthemmorepronetogetinfected.Thisis thecase of thenomad

675

andgeneralist

676

vastlivingareasandoftheChiropteransthatbesidestheirhighpossibilityofdisplacementalsohavealargelifesp

677

an.LessknownistheroleplayedbythehypercarnivoresthatareonthetopoffoodchaininrelationtomixedDTUi

678

nfection.Theseanimalsoccurinfewernumbersinthewild.Thisandtheinherentdifficultiesinhandlingthemres

679

ultedinoursamplesizebeingfairlysmall.However,weexpectthemtobehighlyexposedtoconcomitantDTUinf

680

ections.

didelphids,the

mesopradatorsNasua

nasuathat

alsouse

681

Theimpactofconcomitantinfectionsonthefitnessoffreelivingmammalsisabsolutelyunpredictable

682

becausetheseoccurinanimalsthatveryprobablydisplayinfectionsalsobyotherparasitetaxathatmayinteract.

683

RegardingsolelyconcomitantinfectionswithT.cruzidifferentDTUs,ithasalreadybeendescribedinexperime

684

ntalconditionsthatconcomitantinfectionswithisolatesthatdisplaydistinctbiologicalpropertiesresultedinan

685

ewinfectionpatternsuggestingtheoccurrenceofinteractionbetweentheseisolates(Ragone

686

2015).EvidenceofinteractionbetweendifferentgenotypesofT.

687

cruziatamolecularlevelhasbeenobservedbyMachinetal.(2014).We

688

arecertainlyunderestimatingthosemixedinfectionsthatmustbemuchmorefrequentinnaturethanwehavebee

689

nabletodetect.Indeed,weareonly

690

showingtheDTUsthatwesucceededinisolatingbyhemoculture,i.e.,thosethatwerepresentinsignificantamo

691

untsintheperipheralbloodattheexactmomentinwhichwedrewtheblood.Foritshighfrequencyinnaturalcondi

692

tions,probableresultsin

693

pathogenesis,hostfitnessandfinallytheepidemiologyofthedisease,mixedinfectionsdeservespecialattentio

694

n.

infective

competenceof

et

al,

reservoirs,

695 696 697

11. WherearetheotherT.cruziDTUshidden? OurdataclearlyshowthatthemajorityoftheT.cruziisolatesoffreerangingwildmammalsaretheparent

698

alDTUsTcIandTcII(Figure1).TheotherDTUsaremoresporadicallyisolated,buttheirwidedistributionandh

699

ostrangeshowthattheyaresuccessfullydispersedinthewild(Table2).

700

ItisworthquestioningwhatisthemaintenancestrategyofthemorerarelyisolatedDTUs,wheredothey

701

hideandhowtheypersistinnature.

702

ThefactthattheseDTUspersistinnatureinmammalsthatpresentlowparasitemiaisaquestionforwhichwehave

703

noanswer.PerhapstheseDTUsdependonthesimultaneouspresenceofotherparasitetaxaintheirhoststoachiev

704

eparasitemialevelssufficienttoinfectthefeedingvector,orperhapstheyonlycausetransientparasitemias(buts

705

ufficientastoensuretransmission)intheirmammalianhosts.Infact,highparasitemia(asexpressedbypositive

706

hemoculture)occursinlessthan10%oftheexaminedanimals(lessthan40%oftheinfectedones).Intheotherani

707

mals,theinfectioncouldonlybeevidencedbythepresenceofspecificantibodies(Table1).

708

TherecentfindingofLimaetal.(2014)onTcIIandahybrid(TcVandTcVI)intheAmazonbrokeadecad

709

e-oldparadigmthatthisDTUwasnon-

710

existentinthatbiome.AnotherveryimportantpointthatistherecoveryofparasiteDNAdirectlyfromserumfor

711

minimizingtheselectionpressuresinherenttotheamplificationoftheparasitepopulationsbytheparasitologic

712

almethods.Besides,serologicaltestsforwildanimalswere

713

demonstratedtobeextremelyusefultoolstoindicatesub-patentparasiticinfectionandshowthatobtaining

714

DNA

715

fromseraopensanunexploreduniverseofpossibilitiesforstudyofDTUdistribution,insingleormixedinfectio

716

ns,thathavebeenlargelydisregarded.

717 718 719

12. Openquestionsandfinalremarks ThevariablesthatruletheT.cruzitransmissioncycleinthewildarediverseanduniquetoeachecological

720

scenario.

721

Atleastparasiteandhostspeciesidiosyncrasies,environmentalcharacteristics,existingvectorspecies,chance

722

sfornumberandsortofencounters,

723

fromunderstandingtheinterplayofthesevariablesandthefactorsthatmodifythem.

724

Wewonderifitisnottimetoabandontheexpectationtosucceedinestablishingstrictassociationsbetweenhuma

725

ndisease,hostspecies(vectorormammalian)and a particular genotypeof T. cruzi.Parasitism by this

amongothers,

certainlymodulatetransmission.Wearestill

far

726

parasitespecies

727

phenomenon,andallattemptstoestablishepidemiologicalgeneralizationstodatewereratherunsatisfactory.T

728

hisisnottoinvalidateordisparagecontinuingeffortstodeepenthestudyofthisfascinatingtaxon.Itonlymeansth

729

atlowerexpectationsconcerningthechancesofdeterminingassociationwithhumandiseaseormammalhostm

730

ustbeheldandthatconsiderationoftheuniquenessofeachepidemiological/enzooticscenariomustbealwaysta

731

kenintoaccount.

732

constitutes

a

complexand

multivariate

Theprediction of transmission efficiency and epidemiologicalrisk of sucha complex

733

systemrequires

734

ofallvariablescollectivelyatthesametime,animpossibletask.Therefore,atleastweshouldtrytoassessriskfact

735

orsunderamorecomprehensivetask.Therefore,basingepidemiologicalsurveillanceanddefininganysanitary

736

measurebasedonlyon the presence/absence of triatominesis oversimplifying.For this purpose,

737

mathematical modeling provides arobustandpromisingtool(Nouvellet et al, 2015).

measurement

738

Inthelasttwodecades,anewconceptinhealthsciencesarosethatrecognizesadeepinterdependencebe

739

tweenenvironmentalhealth,animalhealth(wildanddomestic),andhumanhealth.Withinthisnewconcept,ter

740

medOneHealth,itisessentialtointegrateeffortsofexpertsofthesedifferentareasasawaytoachieveasustainabl

741

edevelopmentinourglobalizedworld(Lerner

742

2015).Moreandmoreitbecomesclearthatpreventingandcontrollingparasiticdiseasesinthecontemporarywo

743

rld,whichisincreasinglyandquicklychangingandwherethebordersofhuman-

744

animalinterfaceecosystemsarebecomingblurred,willonlybepossiblewiththejointcollaboration

745

ofallbranchesofknowledgeofthenaturalsciences.

746

and

Berg,

Asmentionedabove,thereisstillmuchtounderstandconcerningthisfascinatingmodel.Whatwewoul

747

dliketore-emphasizeisthattheparasiticphenomenon,especiallywhenitisamulti-

748

hostparasitesuchasT.cruzi,shouldbeaddressedusingamultidisciplinaryfocus.Thisisparticularlyimportant

749

whenthisparasitecancause

750

itisessentialtointegrateecologists,cartographers,anthropologists,mathematicians,andothersinsolvingthis

751

puzzle.

752 753

Acknowledgments

fatalities

oratleastadebilitatingdiseasesuchasis

Chagasdisease.Thus,

754

TheauthorsthankCarlosArdéandMarcosAntôniodosSantosLimafortechnicalsupport.Weofferspe

755

cialthankstoDr.Paulo

756

D’AndreaforhistechnicalsupportindatacollectionandtoDr.VeraBongertzformanyhelpfulcommentsonthe

757

Englishversionofthemanuscript.ThisstudywasfundedbyFundaçãoOswaldoCruz-

758

FIOCRUZ;VicePresidênciadePesquisaeLaboratóriosdeReferênciaVPPLR/FIOCRUZforinvestinginourr

759

esearch;Bio-

760

Manguinhos;ChagasDiseaseNationalControlProgramoftheBrazilianHealthMinistry.LaboratóriodeBiolo

761

giaeParasitologiadeMamíferosSilvestresReservatórios(LBPMSR/IOC);theEuropeanUnionSeventhFram

762

eworkPro-

763

gramGrant223034ChagasEpiNet.AllisolatesinthepresentstudyoriginatedfromCOLTRYP/IOC-

764

FIOCRUZ.

765

thankstoallworkersfromPDA/ICMBiooffice,totheGoldenLionTamarinConservationProgram,Pró-

766

carnívorosInstitute,WildlifeConservationSocietyOWOH2008-

767

001.NationalResearchCenterfortheConservationofNaturalPredatorsCENAP/ICMBio.ConselhoNaciona

768

ldeDesenvolvimentoCientíficoeTecnológico(CNPq);FundaçãodeAmparoa

769

PesquisadoEstadodoRiodeJaneiro(FAPERJ);CAPES.Thefundershadnoroleinstudydesign,datacollection

770

andanalysis,decisiontopublish,orpreparationofthemanuscript.Theauthorshavedeclaredthatnocompetingi

771

nterestsexist.

772

Sérgio

We

also

offer

773

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1091 1092

1093

FIGURE LEGENDS:

1094 1095 1096

Figure1.MapofthespatialdistributionofTrypanosomacruziDTUsintheBrazilianBiomes:AmazonForest,A

1097

tlanticForest,Caatinga,CerradoandPantanal.

1098

Figure2.ThedistributionofTrypanosomacruzimixedDTUsamonginhostsintheBrazilianBiomes:Amazon

1099

Forest,AtlanticForest,Caatinga,CerradoandPantanal.

1100

Figure 3. Map of the infective competence of Trypanosoma cruzi(based on positivity of

1101

hemocultures) and DTU diversity in naturally infected hosts in the wild environment and in Brazilian

1102

Biomes: Amazon Forest, Atlantic Forest, Caatinga, Cerrado and Pantanal.

1103

Figure 4. The distribution of Trypanosoma cruzi mixed DTUs infections among in naturally infected

1104

hosts in the wild environment in Brazil.

1105

Table 1. Prevalence of Trypanosoma cruzi infection by IFAT and hemocultures among

1106

free ranging order of the wild mammals in the Brazilian Biomes: Amazon Forest,

1107

Atlantic Forest, Caatinga, Cerrado and Pantanal.

Biomes

Order

Amazon Artiodactyla Forest

Atlantic Forest

Genera Species specimens

Positive IFAT (%)

Positive Hemocultures (%)

Parasite characterization

1

1

11

8 (72)

0

Chiroptera

25

26

278

n.d

34/278 (12)

Cingulata

2

2

4

1/4 (25)

Didelphimorphia

9

9

220

n.d 113/220 (51)

Lagomorphia Pilosa

1 1

1 1

1 3

n.d n.d

0 2/3 (67)

Primates

2

3

57

18/57 (32)

28/57 (49)

Rodentia 8

15 56

8 51

117 691

29/117 (25) 168 (24.31)

8/117 (7) 130 (19)

TcI (5); TcI+T. rangeli; TcI+TcIV (1)

Carnivora

5

6

10

5/10 (50)

1/10 (10)

TcIII (1)

Chiroptera Cingulata

16 1

22 1

219 3

n.d n.d

6/219 (3) 0

TcI+TcIII/TcIV (1)

57/220 (26)

TcI (4); TcI+TcIII/TcIV (2); TcIV (1); TcI+T. range TcI+TcIV (1) TcIV (1) TcI (39); TcII (1); TcI+T. rangeli (2); TcI+TcIII (1 T. rangeli (2); TcI+TcIV (1); TcI+TcIII/TcIV TcI (1); TcI+T. rangeli (1)

TcI+TcIV (2); TcI (2); TcI+T. rangeli (10); T. rangel

Didelphimorphia

9

14

555

Lagomorphia

1

1

1

Primates

3

5

344

24 59 1 2 2 2

20 69 1 2 1 2

1396 2528 1 4 5 53

131/484 (27) n.d 272/344 (79) 52/1396 (4) 460 (19) 0 0 n.d n.d

Didelphimorphia

5

5

273

82/273 (30)

Rodentia

13

11

774

25 1 10 4 3 10 2 3 22 55 2

22 1 11 3 3 12 2 3 30 65 2

1110 1 253 20 9 515 5 45 1211 2059 41

Carnivora

4

4

380

264/380 (69)

91/380 (24)

Chiroptera cingulata Didelphimorphia Rodentia 6 Carnivora Didelphimorphia Primates Rodentia 4

2 4 4 7 27 3 1 1 5 10

2 4 4 6 28 3 1 1 4 9

3 30 49 285 788 7 26 2 74 109 7285

n.d n.d 3/49 (6) 35/285 (12) 330 (42) 4/7 (15) 8/26 (31) 0 4/74 (5) 16 (15) 1413 (20)

3/3 (100) 3/30 (33) 10/49 (20) 14/285 (5) 122 (15) 0 0 0 0 0 586 (8)

Rodentia 7 Caatinga Artiodactyla Carnivora Chiroptera Cingulata

6 Cerrado Artiodactyla Carnivora Chiroptera Cingulata Didelphimorphia Pilosa Primates Rodentia 8 Pantanal Artiodactyla

Pampa

Total 1108 1109

149/774 (19) 231 (21) 0 75/253 (30) n.d n.d 56/515 (11) n.d 12/45 (27) 65/1211 (5) 208 (10) 0

102/555 (18)

TcI (35); TcII (4); TcIV (2); TcI+TcII (3); TcI+TcI TcI + T. rangeli (2); TcIII/TcIV (1)

0 65/344 (19)

TcI (4); TcII (46); TcI+TcII (1)

5/1396 (0.4) 179 (7) 0 0 0 1/53 (2)

TcI (3)

56/273 (20) 42/774 (5) 99 (9) 0 5/253 (2) 8/20 (40) 0 23/515 (4) 0 0 20/1211 (2) 56 (3) 1/41 (2)

TcIII (1) TcI (35); TcII (1); TcIV (1); TcI+T. rangeli (2); TcII+V/VI (2); T. rangeli (1)

TcI (11); TcII (1); TcIII (1); TcIV (2); TcV (1); TcI+T (1); TcII/V/VI (1)

TcI (2); TcIII (2) TcI (1); TcII (2); TcI+TcII (2); T. rangeli (2) TcI (13); TcIII (1)

TcI (15) TcIII (1)

TcI (38); TcII (9); TcI+TcIII/TcIV (1); TcI+TcII (5 TcIII/TcIV (1); TcI+T. rangeli (4); TcII+T. rangeli ( T. rangeli (3) TcII (2); TcII+TcIII/TcIV (1) TcIV (1) TcI (6); TcI+TcII (2); TcI+TcIV (1) TcI (6); TcIV (1)

1110 1111 1112 1113

Table 2. The distribution infective competence of Trypanosoma cruzi in naturally infected hosts from the Brazilian Biomes: Amazon Forest, Atlantic Forest, Caatinga, Cerrado and Pantanal. BIOMES

ORDER

Amazon Forest

CHIROPTERA

>INFECTIVE COMPETENCE (+HC/Specimen N) Phylostomus hastatus (8/13) 61.5%

DIDELPHIMORPHIA Didelphis marsupialis (24/58) 41% Philander opossum (18/57) 31.5% PRIMATES Cebus libidinosus (25/46) 54% CINGULATA Dasypus novemcinctus (1/1) RODENTIA Hylaemus sp. (2/10) Proechimys sp. (3/55) Atlantic Forest

CARNIVORA CHIROPTERA

Caatinga

Cerrado

Pantanal

1114 1115

Galictis vittata (1/1)

T. cruzi DTU TcI; TcI + T. rangeli TcI; TcII; TcI + TcIII; TcI + T. rangeli; T. rangeli TcI; TcI + T.rangeli; T. rangeli TcI; TcI + T. rangeli; T. rangeli TcIV TcI TcI; TcI + TcIV TcIII

Artibeus planirostris (1/19) Carollia perspicillata (2/57) Micronycteris microtis (1/3) TcI + TcIII/TcIV DIDELPHIMORPHIA Didelphis aurita (66/258) 25.5% TcI; TcIII/TcIV; TcI + TcII; TcI + TcIV; TcI + T. rang Philander frenatus (19/66) 29% TcI; TcII PRIMATES Leontopithecus chrysomela (28/74) 38% TcI; TcII Leontopithecus rosalia (34/266) TcI; TcII; TcI + TcII RODENTIA Nectomys squamipes (1/28) TcI CINGULATA Dasypus novemcinctus (1/2) TcIII DIDELPHIMORPHIA Didelphis albiventris (49/134) 36.5% TcI; TcI + T. rangeli; TcII; TcII + TcV/TcVI; T. rang RODENTIA Thrichomy laurentius (23/519) 4% TcI; TcIII; TcIV; TcV ;TcI + TcIV; TcII/V/VI Rattus rattus (15/71) TcI; TcII CARNIVORA Leopardus pardalis (2/3) TcI Lycalopex vetulus (3/65) TcIII CHIROPTERA Phylostomus hastatus (8/10) 80% TcI; TcII; TcI + TcII; T. rangeli DIDELPHIMORPHIA Didelphis albiventris (9/101) TcI RODENTIA Cerradomys subflavus (5/15) TcI ARTIDACTYLA Sus scrofa (1/9) TcIII TcI; TcII; TcI + TcIII/TcIV; TcI + TcII; TcIII/TcIV; T CARNIVORA Nasua nasua (86/235) 36.5% + T. rangeli; TcII + T. rangel; T. rangeli CHIROPTERA Artibeus sp. (1/1) TcII + TcIII/TcIV Phyllostomus sp. (2/2) TcII CINGULATA Euphractus sexcinctus (1/15) TcIV Dasypus novemcinctus (2/2) DIDELPHIMORPHIA Gracilinanus agilis (4/15) TcI; TcI + TcII RODENTIA Oecomys mamorae (6/6) TcI

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1116 Formatted: Font: (Default) Times New Roman

1117 1118 1119

3

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1120 1121 1122

4

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1123 1124 1125

GA

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1126 1127 1128