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Ehren Snyder
Mentor: Dr. Gregory Adler
Transmission dynamics of Trypanosoma renjifoi in disturbed forest
Abstract
Throughoutmuchof the tropics,deforestationandurbanizationhave influenceddisease
transmission.Ashumansencroachintoforests,the probabilityof contactingnew disease agents
increases.Disease-causingparasitesincludePlasmodiumfalciparumand Trypanosoma cruzi(causal
agentsof malariaand Chagas disease,respectively)bothof whichinfectandcause disease inhumans.
Trypanosoma renjifoi,aparasite closelyrelatedto T.cruzi, was firstdescribedinfectingthe long-tailed
spinyrat (Proechimys longicaudatus) inBrazil.Recentstudieshave identifiedthisparasite in Proechimys
semispinosus (Central Americanspinyrat) incentral Panama,butits transmissioncycle islargely
unknown.Mystudyasks how T. renjifoipersistsinthe environmentandthe conditionsthatpromote its
transmission.Ihave beensampling P.semispinosus indegradedforestfragmentsincentral Panama.I
take bloodsamplesformoleculartestsof the parasite andbloodsmearsforvisuallydetectingthe
parasite usinglightmicroscopy. Ialsocombeach rat to collectfleas,whichare the putative vectors.I
hypothesizethatT.renjifoiwill be presentnearpeople, P.semispinosus isacompetentreservoir,and
the fleasare effective vectors.Preliminaryresultsshowedthatall 14 capturedrats were negative for
bothtrypanosome infectionandfleainfestationduringthe firstwetseasonsampling.Baseduponthese
results,subsequentfieldseasonswillfocusondifferentlocationswherefleasand T.renjifoiare more
likelytobe present.Thisstudymayillustratehow T.renjifoiismaintainedwithinanenvironmentand
may be a model forothervector-borne parasites.

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Introduction to Research Problem
Recently,global interestinidentifyingandpreventingdiseaseshascome tothe forefront.More
pathogensare makingthe leaptoinfecthumansbecause of deforestationand increasinghuman
encroachmentinto sylvatictransmissioncycles. Thiseffectisexacerbatedinthe tropicswhere there are
manytransmissioncyclesandpotential pathogensthatare yettobe documented. Some of the most
abundantpathogensinthese forestsare parasiteswhichare commonlyspreadbyvectors.Because of
the complexityof tropical ecosystems,the chance of comingincontact withthese organismsincreases.
In natural systems,these transmissioncyclesstaywithinthe wildpopulations,buthumanscan
unwittinglybecome apartof them.
Thiswas the case prehistorically withcurrentendemicdiseasessuchasmalariaand Chagas
disease (Rassi,2011).In these diseases,humansenteredthe transmissioncycleswhenthe vectorsfed
on themandcontinuouslyintroducedtheirparasitestohumans. Fromrepeatedhumancontactand
variousselectionpressures,the parasiteswere eventuallyable to utilizehumansasa definitive host
(Araujo,2009). The mainselectionpressureswere human-causedwhere the humanpopulation
increasedwhilesimultaneouslydecreasingthe natural hosts’abundance.Asthishappened,the
parasitesthatwere betterable to infecthumanshadan increasedlikelihoodof passingtheirgenes.This
same problemfaceshumanstodayas we continue tounwittinglybecomeexposedtodifferent
pathogens’life cycles.
In Central Panama,deforestationhasbecome awidespreadissue,and resultingendemic
diseasesalreadyexist (Gottdenker,2011).DiseasessuchasChagas disease,leishmaniasis,andmalaria
have alreadybecome entrenchedinthe Panamanianpopulation.Withthe continuation of
encroachment,otherpotential pathogensmayemerge.Myworkfocusesonthisproblemandhowto
potentiallyaddressit.Bydocumentingnumeroustransmissioncyclesandpotential bridgesforhuman

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infection,these jumpscanbe predictedorprevented.Althoughthisiswell withinthe meansof
possibility,notmuchresearch hasbeendedicatedtotakingproactive measurestostoppathogens.
A goodway to beginthisprocessistodocumentthe pathwayswiththe highestprobabilityof
infectinghumans (Krasnov,2005).To dothis, the areas of interestneedtobe identifiedalongwiththeir
respective methodological techniques. Thisstudyfocusesonanundocumentedtransmissioncycle in
Central Panamainvolvingthe spinyrat(Proechimyssemispinosus),aflea(Polygenisklagesi),anda
nonhumanparasite (Trypanosoma renjifoi).Itwill involveworkingcollaborativelywiththe Gorgas
Institute of ParasitologyinPanamaCityandcolleaguesfromthe Universityof Georgia.
Research Objectives& Methodology
The objective of thisstudyistodocumentthe transmissioncycle of Trypanosomarenjifoiandto
provide researchmethodsforlinkingavectortoits associatedparasite. Since the definitivehosthas
alreadybeenidentified,findingthe vectorforitconnectsthe full cycle (Mello,1978). Thisstudywill be
carriedout indegraded forestin Trinidadde lasMinas, Central Panama.Thisislocatedaboutfifty
minutesfromPanamaCityandwill require rentingavehicle. Spinyrats(Proechimyssemispinosus)will
be capturedusingTomahawktraps baitedwithplantain,peanutbutter,andvanillaextract. Fiftytoone
hundred trapswill be placedeitheronasingle longtransectoron multiple paralleltransects(depending
on forestfragmentsize).Trapswill be setdailyfortwotendaytrappingperiods,andwill be examined
on a daily basiswhendrivingouttothe fieldsite.Capturedratswill thenbe processedbytakingablood
sample fromthe metatarsal veinfollowedbycombingeachratfor fleas. Eachrat ishandledasquickly
and carefullyaspossibleandthenreleasedatthe exact place itwas captured. The fleasandbloodwill
bothbe collectedand usedforDNA amplificationviaPCRorqPCR where theywill be testedfor
leishmaniasisandtrypanosomes (Allender,2004 & Henry,2007). The Gorgas Institute andcolleagues
fromUniversityof Georgiaalreadyhave the primersspecificforthese procedures. Evenif the proposed

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researchdoesnotleadto an identificationof aflea-borne trypanosome,itcanserve as the basisfora
publicationonspinyratparasite loads.
StudentInvolvement
The studentwill carryout the researchprocedure includingtrapsettingandchecking,rodent
processing,PCRamplification, anddataanalysis. The studentwillbothprepare forthe fieldseasonalong
withnecessitiesduring.The dataanalysiswill be performedbothinPanamaat the Gorgas Institute and
back inthe UnitedStates.The mentorwill provide suggestionsandvaluableinsighttoincrease the
probabilityof success.The mentorwillassistinthe fieldforthe firsttwoweeksforbothtrainingand
studyestablishment.Afterwards,the studentwillworkalone togatherdata,analyze them, andprepare
it forthe resultingmanuscript(s).
StudentEducation
Performingthisstudywillallow the studenttolearnunique skillsbothinthe fieldandin the
laboratoryina unique setting.The tropical forestallowsforacomplete experience of aforeign
ecosystem,foreignculture,andachance to rely on one’sself.Studyingthisparasiticrelationshipwill be
invaluable forfuture workwithdiseasesregardingeitherdisease modelsorcontrol efforts.This
knowledge maybe usedeitherforaresearchpositionorPh.D. candidacyfollowingcompletionof this
Master’sprogram. Followingthe time spentatUW Oshkosh,the studentwill be able tobothcontinue
hiseducationandto pursue greaterapplicationsof disease research.
Associationsto Future Studies
Withthe contentinthisstudy,future researchmayencompassmore pathwaysof
nonpathogenicorganisms.Mappingmanydifferentpathwayscanleadtofaster identificationof future

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pathogensorpreventingselectionpressuresthatmayresultinpathogenesis.If the primaryobjectiveis
successful,the transmissiondynamicsof T.renjifoiwill be documented.Furtherresearchcouldthenbe
done to determine its relatednessto T. cruzi (Chagasdisease),the impactithason P. semispinosus
populations,anditsrole withinthe ecosystem.Thisresearchwill consequentlyleadtomore
documentationon P.semispinosus parasite loads,andpossiblyawayto combat T. cruzi. Withmore
research,the co-infectivityof bothtrypanosomesmaybe addressed.If acoinfectionisnotpossible,it
can presenta wayto preventreservoirsfrombecominginfectedwith T.cruzithus reducingthe
prevalence of Chagasdisease.
Feasibility
Thisstudyis highlyfeasiblebecause the fieldsite hasalreadybeenestablishedandbothratsand
theirfleasare presentinthe area. Previoussamplingperiodshave shownahightrapsuccessrate and a
highpercentage of fleainfestation (Unpublisheddata).The trapsare alsoalreadyinthe collaborators’
possessionandneedonlytobe broughttothe fieldsite.The collaboratingGorgasInstitute hasthe
necessarylaboratoryequipmenttoperformboththe bloodsamplingandthe consequentDNA
amplification.The bloodsampleswill be keptcoldandthendeliveredtoGorgasdailyto ensure the DNA
isviable.Bothfleasandbloodsamplescanbe analyzedatGorgas while stillinPanama,andany
remainingsamplescanbe broughtback to the UnitedStates.The primaryobjective’ssuccessis
dependentuponinfectionratesof fleasandtheirtrypanosomes,butthe secondaryobjectiveisvery
likelytobe successful.Spinyratsare knownto carry veryhighparasite loadsincludingleishmania
species, T.cruzi,filarial worms,mites,ticks,fleas,andlice (Krasnov,2004 & Gottdenker,2011). Because
of this,itisverylikelythe workwill resultinsignificantamountof dataregardingtheirecto- and
endoparasites.Thisisaugmentedwiththe helpandexpertiseofferedbycolleaguesfromthe University
of Georgiaandthe Gorgas Institute whoare doingrelatedworkinthe same area.

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ExpectedOutcomes
There are several outcomesthatare anticipatedfromthiswork
 assistance withfuture publications bythe GorgasInstitute of Parasitology
 furtherthe knowledgeof trypanosomesforcollaboratorsfromthe Universityof Georgia
 data will contribute totwopossiblemanuscripts,authoredbythe student,mentor,and
collaboratorsforsubmissioninpeer-reviewedliterature
 data to be includedinfuture workbythe mentor’ssuccessivestudents
Time Period Activity
May 30-31 Travel to Panama
June 1-15 Researchestablishment andinitial collection (withassistance of mentor)
June 16-25 Data collection
June 26-July5 Data analysis
July6-July15 Data collection
July16- July30 Data analysis
July31 ReturnfromPanama
Budget
Expense Amount($)
Travel (flightandvaccines) $1000
Roomand board $500
Car rental (tostudysite) $400
Total $1900

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Literature Cited
Allender,C.J.,Easterday,W.R., VanErt, M. N.,Wagner,D. M., andKeim, P.(2004) High-throughput
extractionof arthropodvectorand pathogenDNA usingbeadmilling. BioTechniques.37:730-
734.
Araujo,A.,Jansen,A.M., Reinhard,K.,andFerreira,L.F. (2009) Paleoparasitologyof Chagasdisease –A
Review. Mem.Inst.Oswaldo Cruz.104: 9-16.
Gottdenker,N.L.,Calzada,J.E.,Saldana,A.,andCaroll,C.R.(2011) Associationof anthropogeniclanduse
change and increasedabundance of the chagasdisease vectorRhodniuspallescens inarural
landscape of Panama. The American Society of Tropical Medicineand Hygiene.84(1): 70-77.
Henry,K. M., Jiang,J.,Rozmajzl,P.J.,Azad,A.F., Macaluso,K. R.,and Richards,A.L. (2007) Development
of quantitativereal-timePCRassaystodetect Rickettsia typhiand Rickettsia felis, the causative
agentsof murine typhusandflea-borne spottedfever. Molecularand CellularProbes.21: 17-23
Mello,D.A. (1978) Sobre o encontrodo Trypanosoma (Herpetosoma) renjifoiDeane 1961, infectando
Proechimyslongicaudatus (Rodentia-Echimyidae). Revista deSaudePublica.12:523-525.
Krasnov,B.R.,Shenbrot,G.I.,Khokhlova,I.S.,andPoulin,R.(2004) Relationshipsbetweenparasite
abundance andthe taxonomicdistance amonga parasite’shostspecies:anexample withfleas
parasiticon small mammals. InternationalJournalforParasitology.34: 1289-1297.
Krasnov,B.R.,Stanko,M., Miklisova,D.,andMorand, S.(2005) Distributionof fleas(Siphonaptera)
amongsmall mammals:meanabundance predictsprevalenceviasimpleepidemiological model.
InternationalJournalforParasitology. 35:1097-1101.
Rassi,A.J., Rassi,A,and Marin-Neto,J.A.(2010) Chagasdisease. Lancet.375: 1388-1402.