The Greater White-Toothed Shrew (Crocidura russula)

17 Pages·2016·2.55 MB·English

RESEARCHARTICLE
Emerging Infectious Disease Implications of
Invasive Mammalian Species: The Greater
White-Toothed Shrew (Crocidura russula) Is
Associated With a Novel Serovar of
Pathogenic Leptospira in Ireland
JarlathE.Nally1*,ZbigniewArent2,DarrellO.Bayles1,RichardL.Hornsby1,
ColmGilmore3,SiobhanRegan4,AllanD.McDevitt7,JonYearsley5,Se´amusFanning6,
a11111
BarryJ.McMahon4
1 InfectiousBacterialDiseasesResearchUnit,NationalAnimalDiseaseCenter,AgriculturalResearch
Service,UnitedStatesDepartmentofAgriculture,Ames,Iowa,UnitedStatesofAmerica,2 UniversityCentre
ofVeterinaryMedicineJU-UAK,UniversityofAgriculture,Krakow,Poland,3 OIELeptospirosisReference
Laboratory,VeterinarySciencesDivision,AFBI,Belfast,NorthernIreland,UnitedKingdom,4 UCDSchoolof
Agriculture&FoodScience,UniversityCollegeDublin,Belfield,Dublin,Ireland,5 UCDSchoolofBiology&
EnvironmentalScienceandUCDEarthInstitute,UniversityCollegeDublin,Belfield,Dublin,Ireland,6 UCD
CentreforFoodSafety,SchoolofPublicHealth,Physiotherapy&SportsScience,UniversityCollegeDublin,
OPENACCESS
Belfield,Dublin,Ireland,7 EcosystemsandEnvironmentResearchCentre,SchoolofEnvironmentandLife
Citation:NallyJE,ArentZ,BaylesDO,HornsbyRL, Sciences,UniversityofSalford,Salford,UnitedKingdom
GilmoreC,ReganS,etal.(2016)Emerging
InfectiousDiseaseImplicationsofInvasive *[email protected]
MammalianSpecies:TheGreaterWhite-Toothed
Shrew(Crocidurarussula)IsAssociatedWitha
NovelSerovarofPathogenicLeptospirainIreland. Abstract
PLoSNeglTropDis10(12):e0005174.
doi:10.1371/journal.pntd.0005174
Thegreaterwhite-toothedshrew(Crocidurarussula)isaninvasivemammalianspeciesthat
Editor:AnaLTONascimento,InstitutoButantan, wasfirstrecordedinIrelandin2007.Itcurrentlyoccupiesanareaofapproximately7,600
BRAZIL km2ontheisland.C.russulaisnormallydistributedinNorthernAfricaandWesternEurope,
Received:September1,2016 andwaspreviouslyabsentfromtheBritishIsles.Whilstinvasivespeciescanhavedramatic
Accepted:November8,2016 andrapidimpactsonfaunalandfloralcommunities,theymayalsobecarriersofpathogens
facilitatingdiseasetransmissioninpotentiallynaivepopulations.Pathogenicleptospiresare
Published:December9,2016
endemicinIrelandandasignificantcauseofhumanandanimaldisease.From18trapped
Copyright:Thisisanopenaccessarticle,freeofall
C.russula,3isolatesofLeptospirawerecultured.However,typingoftheseisolatesbystan-
copyright,andmaybefreelyreproduced,
distributed,transmitted,modified,builtupon,or dardserologicalreferencemethodswasnegative,andsuggestedan,asyet,unidentified
otherwiseusedbyanyoneforanylawfulpurpose. serovar.Sequenceanalysisof16SribosomalRNAandsecYindicatedthatthesenoveliso-
TheworkismadeavailableundertheCreative
latesbelongtoLeptospiraalstonii,auniquepathogenicspeciesofwhichonly7isolates
CommonsCC0publicdomaindedication.
havebeendescribedtodate.EarlierisolationswerelimitedgeographicallytoChina,Japan
DataAvailabilityStatement:Allrelevantdataare
andMalaysia,andthisleptospiralspecieshadnotpreviouslybeenculturedfrommammals.
withinthepaperanditsSupportingInformation
Restrictionenzymeanalysis(REA)furtherconfirmsthenoveltyofthesestrainssinceno
files.TheannotatedassemblyforL.alstoniiserovar
Room22strainGWTS#1isavailableinGenBank similarpatternswereobservedwithareferencedatabaseofleptospires.Aswithotherpath-
undertheaccessionnumbersCP015217 ogenicLeptospiraspecies,theseisolatescontainlipL32anddonotgrowinthepresenceof
(ChromosomeI)andCP015218(ChromosomeII).
8-azagunaine;howevernoevidenceofdiseasewasapparentafterexperimentalinfectionof
Funding:ADMacknowledgesfundingfromthe hamsters.TheseisolatesaregeneticallyrelatedtoL.alstoniibuthaveanovelREApattern;
IrishResearchCouncil(grant:PD/2011/2093),the
theyrepresentanewserovarwhichwedesignateasserovarRoom22.Thisstudy
HeritageCouncil,Ireland(grant:R02511),a
HeredityfieldworkgrantawardedbytheGenetics
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InvasiveSpeciesasCarriersofLeptospires
Society,andtheVincentWildlifeTrust.Thefunders demonstratesthatinvasivemammalianspeciesactasbridgevectorsofnovelzoonotic
hadnoroleinstudydesign,datacollectionand pathogenssuchasLeptospira.
analysis,decisiontopublish,orpreparationofthe
manuscript.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
AuthorSummary
Leptospirosisisaglobalzoonoticdisease.PathogenicspeciesofLeptospiraareexcretedin
urinefromasymptomaticcarrierhostswhichfacilitatesdiseasetransmissiontonewhosts.
Todate,thereare10speciesofpathogenicleptospireswhichcomprisemorethan200ser-
ovars.Diseasetransmissionofthesestrainsismaintainedbyawiderangeofdomesticand
wildanimalspecies.Inthiswork,wediscoveredthataninvasivemammalianspecies,the
greaterwhitetoothedshrew,whichwasfirstidentifiedinIrelandin2007,actsasacarrier
foraspeciesofleptospiresneverbeforeidentifiedinIreland.Resultsdemonstratethat
invasivemammalianspeciesactasbridgevectorsofnovelzoonoticpathogenssuchas
Leptospira.
Introduction
Thegreaterwhite-toothedshrew(Crocidurarussula)isanexoticspeciestoIrelandfirst
recordedin2007[1],andnowclassifiedasaninvasivemammalianspecies[2].Accordingto
recentstudies,thisspeciesisrapidlyspreadingwithradialexpansionestimatesofapproxi-
mately5.5km/yr[2].ThesourceofthisinvasivepopulationisfromEuropeasopposedto
NorthAfrica[3],andevidencesuggeststhatthegreaterwhite-toothedshrewisassociatedwith
thelocalextinctionofindigenouspopulationsofthepygmyshrew(Sorexminutus)[2].How-
ever,acomprehensiveinvestigationontheOneHealthimplicationsofthisinvasivespecies
hasyettobeperformed.
PathogenicspeciesofLeptospiracauseleptospirosis,abacterialzoonoticdiseasewitha
globaldistributionaffectingoveronemillionpeopleannually[4,5].Leptospirescolonizethe
renaltubulesofreservoirhosts,fromwheretheyareexcretedviaurineintotheenvironment
andsurviveinsuitablemoistconditions.Contactwithinfectedurine,orcontaminatedwater
sourcescanresultindiseasesincepathogenicleptospirescanpenetratebreachesoftheskin,or
mucosalsurfaces,anddisseminatehaematogenouslytocausearangeofclinicalsymptoms
frommildfever,toictericWeil’sdiseaseandpulmonaryhemorrhagesyndrome.Indeveloped
countries,leptospirosisisprimarilyarecreationaldisease,oroccupationaldiseaseoffarm
workers,veterinarians,andslaughterplantworkers.Indevelopingcountries,itisasocioeco-
nomicdiseaseperpetuatedbyrapidurbanization,rodentinfestationandtransmissionviacon-
taminatedwatersourcesassociatedwithlimitedinfrastructuresandsevereweatherevents.
Bothrodentsanddomesticfarmanimalspeciescanserveasreservoirhostsofinfectionand
sourcesofdiseasetransmissiontohumans.
LeptospirosisisendemicinIreland[6–12].Themeanannualincidencefor2009was5.6per
millioninhabitantsperannum,comparedtothatof1.4permillionacrosstheEU[13].Thepre-
dominantserovarsassociatedwithhumaninfectionwereserovarsIcterohaemorrhagiaeand
Hardjo,indicativeofrodent/recreationalandoccupationalexposurerespectively.Ratsareres-
ervoirhostsforserovarIcterohaemorrhagiaewhilstcattleactasreservoirhostsforserovar
Hardjo[14].Over80%ofIrishbeefsucklerherds,andmorethan40%ofindividualbeefpro-
ducinganimals,showevidenceofexposuretoleptospires[15].Similarly,79%ofunvaccinated
dairyherdswerepositiveforantibodiestoLeptospirabybulktankmilktesting[16].
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InvasiveSpeciesasCarriersofLeptospires
Leptospirosiscontinuestobealeadingcauseofbovineabortion[17].Otherdomesticanimals
speciesthatshowevidenceofexposuretopathogenicleptospiresinIrelandincludepigs,
sheep,horsesanddogs[18–26].
Thereisclearevidencethatinvasivespeciesactasvectorsforpathogensandparasites
thatcanhaveenvironmentalconservation,andhumanhealth,implications.Globalization
hasfacilitatedthemovementofexoticandinvasivespecies,andarangeofassociatedpatho-
gense.g.mosquitoesandWestNileVirus[27].Thecombinationofinvasivespeciesanddeg-
radationofecosystemspresentsasubstantialthreatinrelationtoemerginginfectious
diseases[27,28].Novelpathogenscanhavedevastatingeffectsonnaivecommunities;exam-
plesincludetheinvasivegreysquirrel(Sciuruscarolinensis)whichcarriessquirrelpoxvirus
thatseverelyadverselyaffectednativeredsquirrels(Sciurusvulgaris)inBritainandIreland
[29,30];theintroducedraccoondog(Nyctereutesprocyonoides)inEurope,whichhasan
expandingrange,andwhichcanfacilitatethespreadofinfectiousdiseasesincludingechino-
coccosis,trichinellosisandrabies[31].Inthisstudy,weidentifiedthatarecentlyintroduced
mammalianspecies(C.russula)inIrelandisareservoirhostforanovelstrainofpathogenic
Leptospira.
Materials&Methods
Greaterwhite-toothedshrews
Greaterwhite-toothedshrews(GWTS)werelive-trappedandeuthanizedbycervicaldisloca-
tion.Allanimalexperimentalprocedureswereperformedinaccordancewithrelevant
guidelinesandregulations,andasapprovedbytheNationalParksandWildlifeService
(NPWS)inIrelandandtheAnimalResearchEthicsCommitteeinUniversityCollegeDublin
(AREC-13-24).
Cultures
KidneyswereremovedfromGWTSattimeofeuthanasiaandimmediatelyprocessedforthe
cultureofleptospires[32].Inbrief,asinglekidneywasasepticallyremovedusingadisposable
forcepsandscalpelandplacedin5ml1%BovineSerumAlbumin(BSA).Thekidneywassub-
sequentlymaceratedwithscalpelsandtheresultingmixturehomogenizedbypassingit
througha10mlsyringe(withoutneedleattachment).Eachtissuehomogenatewasserially
diluted10-fold(toafinaldilutionof10−3)into1%BSAand500μlofthismixturewasusedto
inoculatethesurfaceof10mlEMJHmediumcontaining200μg5-Fluoruraciland0.2%noble
agar.Culturesweretransportedbacktothelaboratoryandmaintainedat29˚C.Cultureswere
examinedatweeklyintervalsbydark-fieldmicroscopy.
L.alstoniiSerogroupRanarumSerovarPingchangStrain80–412andL.alstoniiSerogroup
UndesignatedSerovarSichuanStrain79601weresourcedfromtheWHO/OIELeptospirosis
ReferenceLaboratoryattheRoyalTropicalInstitute,TheNetherlands.L.alstoniistrains
MS267,MS311andMS316werekindlyprovidedbyDepartmentofBacteriology,Facultyof
MedicalSciences,KyushuUniversity,Japan.
Growthassessmentinthepresenceof8-azaguaninewasperformedaspreviouslydescribed
[33];inbrief,leptospireswereculturedinEMJHmediumwith1%rabbitserumand225μg/ml
8-Azaguanine(A52848-Azaguanine,Sigma,St.Louis,MO).Duplicatetubeswereinoculated
withtheshrewisolateswhileLeptospirabiflexa(ATCC123582™)wasusedasapositivecon-
trol.Cultureswereincubatedat30˚Cfor14days.Thecultureswerecountedbydark-field
microscopyatdays1,3,5,7and14usingaCellometer1disposablecellcountingchamber
(NexcelomBioscience).
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InvasiveSpeciesasCarriersofLeptospires
Serologicaltypingofisolates
Serologicalstrainidentificationwasinitiallyattemptedbycross-agglutination.Inthisproce-
dure,theMicroscopicAgglutinationTest(MAT)wascarriedoutusingapanelof19reference
antiseraagainstthe17majorpathogenicLeptospiraserogroups[34–36].TheLeptospiraser-
ogroupstestedincludedAustralis(serovarsAustralisandBratislava),Autumnalis,Ballum,
Canicola,Celledoni,Cynopteri,Grippotyphosa,Hebdomadis,Icterohaemorrhagiae,Javanica,
Louisiana,Mini,Pomona(serovarPomonaandAltodouro),Pyrogenes,Sejroe,Semaranga
andTarassovi.Inaddition,rabbitserageneratedagainsteachofthethreeshrewisolateswere
thentestedagainstthepanelofLeptospiraantigensfromthe17serogroupsmentionedabove,
andadditionallyagainstapanelof9antigensfromserogroupscomprisedof:Andamana,
Semaranga,Hursbridge,Sarmin,Lyme,Louisiana,Shermani(serovarShermaniandAqua-
runa),Bataviae,Ranarum,andagainstoneundesignatedserogroup(serovarSichuan).
Restrictionenzymeanalysis
FourhundredmlculturegrownfromeachshrewisolateofLeptospirawasharvestedand
wholecellleptospiralDNApurifiedaspreviouslydescribed[18].DNAconcentrationwasesti-
matedafterspectrophotometricmeasurementusingaNanophotometerPearl(Implen).
RestrictionendonucleasedigestionwithEcoRI,electrophoresisandgelanalysiswerecarried
outaspreviouslydescribed[18].
Generationofantiserum
Rabbitserawerepreparedaspreviouslydescribedwithslightmodification[34]andaslicensed
undertheAnimals(ScientificProcedures)Act(1986).Inbrief,rabbitswereinjectedintraperi-
toneallyatweeklyintervalswithliveleptospiresatadensityof2x108perml.Theweekly
injecteddoseswere5,10,15,and20mlrespectively.Rabbitswerebledbycardiacpuncture
oneweekafterthelastinjection.
Genomesequencing
GenomesequencingwasperformedbytheCentreforGenomicResearchattheUniversityof
Liverpool.GenomicDNAmaterialwaspurifiedwith1xcleanedAmpurebeads(Agencourt)
andthequantityandqualitywasassessedbyNanodropandtheQubitassay.Inaddition,the
FragmentAnalyser(usingahighsensitivitygenomickit)wasusedtodeterminetheaverage
sizeoftheDNAandtheextentofdegradation.Thisprocedurewasalsousedatthestepsindi-
catedbelowtodetermineaveragefragmentsizeoftheDNA.DNAwasshearedusingCovaris
Gtubesbycentrifugationat7,000rpminanEppendorf5415Rcentrifuge.Thefragmentsize
wascheckedasbefore.DNAwaspurifiedwith0.5xampurebeadsandtreatedwithExonucle-
aseVIIat37˚Cfor15minutes.TheendsoftheDNAwererepairedasdescribedbyPacificBio-
sciencesprotocol.Eachsamplewasincubatedfor20minutesat37˚CwithDNADamage
RepairMixsuppliedintheSMRTbelllibrarykit(PacBio).Thiswasfollowedby5minutes
incubationat25˚CwithEndRepairMix.DNAwascleanedusing0.5xampureand70%etha-
nolwashes.DNAwasligatedtoadaptersequencesovernightat25˚C.Ligationwasterminated
byincubationat65˚Cfor10minutesfollowedbyexonucleasetreatmentfor1hourat37˚C.
TheSMRTbelllibrarywaspurifiedwith0.5xampurebeads.Thequantityoflibraryandthere-
foretherecoverywasdeterminedbyQubitassayandtheaveragefragmentsizedeterminedby
FragmentAnalyser.SMRTbelllibrarywasannealedtosequencingprimeratvaluespredeter-
minedbytheBindingCalculator(PacBio)andacomplexmadewiththeDNAPolymerase
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InvasiveSpeciesasCarriersofLeptospires
(P6/C4chemistry).ThecomplexwasboundtoMagbeadsandthiswasusedtosetup3SMRT
cellsforsequencing.Sequencingwasdoneusing240minutemovietimes.
Phylogeny
The16SrRNAgenesequenceidentifiedwithinthenewlysequencedorganismdescribed
hereinwasusedtoretrieve108similarsequencesfromtheRibosomalDatabaseProject(RDP)
viatheSeqMatchtool[37].SequenceswerealignedwithMUSCLE[38],anddivergentand
ambiguouslyalignedalignmentblockswereremovedwithGblocks[39].ThemodelTestfeature
ofPhangorn[40]wasusedtocalculatetheBayesianInformationCriterion(BIC)foravariety
ofmodels,andguidedtheselectionoftheHKYmodel.Themodelparametersforcomputing
themaximumlikelihoodofphylogenywereoptimizedusingoptim.pml,andbootstrap.pml
wasusedtoperformabootstrapanalysis[40].Thephylogeneticreconstructionwithboot-
strappedvaluesassignedtotheedgeswasgraphicallyrenderedwithTreeDyn[41].
ThesecYgenesequenceidentifiedwithinthenewlysequencedorganismdescribedherein
wascomparedwithothersequencesofsecYfromthegenusLeptospira,asretrievedfromGen-
Bank[42].SequencesofsecYwerealignedwithCLUSTALW[43].Phylogenicanalysiswascon-
ductedwithMEGA4[44]andthemaximumlikelihoodsmethodwasusedforestimationof
distanceofalignedsequences[45].
Experimentalinfectionofhamsters
GoldenSyrianhamsterswereinoculatedbyintraperitoneal(IP)injectionaspreviously
described[46].Groupsofthreehamsterseachreceived107ofGWTSisolate#1,#2or#3IP
respectively.Threehamstersactedasnegativecontrolsandreceivedmediaalone.Allanimal
experimentalprocedureswereperformedinaccordancewithrelevantguidelinesandregula-
tions,andasapprovedbyUSDAInstitutionalguidelines.
Microscopicagglutinationtest
ThemicroscopicagglutinationtestwasperformedaspreviouslydescribedaccordingtoOIE
guidelines[47].
Fluorescentantibodytest
Thefluorescentantibodytestwasperformedaspreviouslydescribed[32].
Accessionnumbers
TheannotatedassemblyforL.alstoniiserovarRoom22strainGWTS#1isavailableinGen-
BankundertheaccessionnumbersCP015217(ChromosomeI)andCP015218(Chromosome
II).
Results
CultureandserologicalclassificationofGWTSisolatesofleptospires
Cultureofleptospireswasattemptedfromasinglekidneyineachof18trappedGWTS.Kid-
neysfromthreeoftheGWTSwereculturepositiveasconfirmedbydark-fieldmicroscopyand
theisolateswerenamedGWTSIsolate#1,#2and#3respectively.
EachGWTSisolateofLeptospirawastestedagainstastandardpanelofreferenceantisera,
representing19serovarsfrom17serogroupsandrepresentativeofthegeographicallocale,for
typingpurposes,Table1.NosignificantreactivitywasdetectedbetweenanyGWTSisolate
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InvasiveSpeciesasCarriersofLeptospires
Table1. MATtitresofGWTSIsolates1,2&3withreferenceantisera.
Referenceantisera Antigen
Serogroup serovar GWTS-1 GWTS-2 GWTS-3
Australis Australis(Ballico) 0 0 0
Australis Bratislava 0 0 0
Autumnalis Autumnalis 0 0 0
Ballum Ballum 0 0 0
Canicola Canicola 0 0 0
Celledoni Celledoni 0 0 0
Cynopteri Cynopteri 0 0 0
Grippotyphosa Grippotyphosa 0 0 0
Hebdomadis Hedbomadis 0 0 0
Icterohaemorrhagiae Icterohaemorrhagiae 0 0 0
Javanica Poi 0 0 0
Louisiana Louisiana 0 0 0
Mini Mini 0 0 0
Pomona Pomona 0 0 0
Pomona Altodouro 0 0 0
Pyrogenes Pyrogenes 0 0 0
Sejroe Hardjo 0 0 0
Semaranga Patoc 0 0 0
Tarrassovi Tarrassovi 1:30 1:30 0
EachGWTSisolatewastestedforagglutinationbythemicroscopicagglutinationtest(MAT)againstapanelofreferenceantiserarepresentativeof19
serovarsand17serogroupsofleptospires.Titresareasindicated.Nosignificantreactivitywasdetected.
doi:10.1371/journal.pntd.0005174.t001
andanyreferencesera.InafurtherattempttotypeeachGWTSisolate,rabbitantiseraspecific
foreachGWTSisolatewasthenpreparedandtestedagainstanadditionalpanelofreference
strainsofLeptospira,representing9serogroups,oneundesignatedserogroup,and13serovars,
Table2.SlightreactivitywasdetectedbyantiseraspecificforGWTSisolate#1&#2againstser-
ovarShermani,whichbelongstoLeptospirasantarosai.However,thelackofaconsistently
highMATtitredetectedbetweenGWTSisolate-specificantiseraandreferenceantigenindi-
catedaninconclusiveserologicaltypingclassificationofanyoftheGWTSisolates,andsug-
gestingthattheywereofanasyetunidentifiedserovar.
MolecularclassificationofGWTSisolatesofleptospires
TheinabilitytoserologicallytypetheGWTSLeptospiraisolatesusingreferenceantiseraand
referenceantigensindicatesthattheGWTSLeptospiraisolatesareatypicalcomparedtothose
previouslyidentifiedinWesternEurope.Therefore,wholegenomesequencingwasperformed
onasinglestrain,GWTSisolate#1.Thegenesequencefor16SrDNAwasextractedfromthe
completegenomeandcomparedto10816SrDNAsequencesavailableforLeptospirafromthe
RibosomalDatabaseproject(https://rdp.cme.msu.edu/).Phylogeneticanalysisindicatedthat
GWTSisolate#1clusteredamong4strainsofLeptospirarecentlyisolatedfromsoilsamplesin
Fukuoka,Japan(designatedasMS267,MS306,MS311,andMS316respectively[48]),Fig1and
S1Fig.These,inturn,clustermostcloselywithLeptospiragenomospecies1,whichhasrecently
beenrenamedL.alstonii,andiscomprisedoftwoserovarsofLeptospirathatwereoriginally
isolatedfromfrogsinChina[49];serogroupRanarumserovarPingchangandserogroup
UndesignatedserovarSichuan.Similarly,thesequenceforsecYwasextractedfromthegenome
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InvasiveSpeciesasCarriersofLeptospires
Table2. MATtitresofreferenceserogroupantigenswithantiseraspecificforeachGWTSIsolates1,2&3.
Referenceantigens Antisera
Serogroup serovar α-GWTS-1 α-GWTS-2 α-GWTS-3
Andamana Andamana 1:10 0 0
Bataviae Bataviae 0 1:10 0
Hebdomadis Kremastos 0 0 0
Hursbridge Hursbridge 0 0 0
Lyme Lyme 0 0 0
Louisiana Louisiana 0 0 0
Louisiana Orleans 0 0 0
Ranarum Pingchang 0 0 0
Sarmin Cuica 0 0 0
Sarmin Weaveri 0 0 0
Shermani Aquaruna 1:100 1:30 0
Shermani Shermani 1:1000 1:3000 0
Undesignated Sichuan 0 0 0
AntiseraspecificforeachGWTSisolatewastestedbythemicroscopicagglutinationtest(MAT)againstapanelofreferencestrainsofLeptospira
representativeof9serogroupsand11serovars.Titresareasindicated.
doi:10.1371/journal.pntd.0005174.t002
andphylogeneticanalysisperformed;thesecYsequenceofGWTSisolate#1alignedmost
closelywiththatofL.alstoniiserovarPingchangandL.alstoniiserovarSichuan,Fig2.How-
ever,rabbitantiserumspecificforGWTSisolate#1,2or3,failedtoagglutinatewitheitherof
thesetwoserovarsrepresentativeofL.alstonii,Table2.Nucleotidesequencefor16SrDNA
andsecYofGWTS#1isprovided(S2Fig).
RestrictionenzymeanalysiswasperformedonDNApurifiedfromeachGWTSisolate#1,2
&3forcomparisonwith5ofthe6availableisolatesofL.alstoniithathavebeenculturedto
date,Fig3.ResultsindicatethatGWTSisolate#1and#3haveanidenticalREApatternthat
differedslightlyfromthatofGWTSisolate#2.ResultsalsoindicatethattheREApatternsare
significantlydifferenttothatofanyoftheL.alstoniiisolates.AnalysisofREApatternscom-
paredwithareferencedatabaseofLeptospirastrainsheldintheOIEReferenceLaboratory
(AFBIStormont,NorthernIreland)didnotidentifyanysimilarREApatterns.
Collectively,theseresultsprovideevidenceoftheuniqueandnovelmolecularattributesof
eachoftheGWTSisolates,whichwedesignateasL.alstoniiserogroupUndesignatedserovar
Room22.
PathogenicityofGWTSIsolates
LeptospiraalstoniiisconsideredtobeamemberofthepathogeniccomplexofLeptospira,as
definedbyDNA-DNArelatedness,16SrDNAandsecYsequence.Inadditiontothesecriteria,
thegenomesequenceofGWTS#1containslipL32,whichtodatehasonlybeenidentifiedin
pathogenicleptospires(S2Fig).EachoftheGWTSisolateswasalsotestedforgrowthinthe
presenceof8-azagunaine;aswithallpathogenicleptospires,noneoftheshrewisolateswere
abletogrowinthepresenceof8-azaguanine.
TofurtherassessvirulencepropertiesofGWTSisolates,3groupsofthreehamsterswere
experimentallyinoculatedwith107leptospiresofGWTSisolate#1,#2and#3respectively.No
hamstershowedanysignofacutediseaseasdeterminedbyweightgainwhichremainedcom-
parabletonon-infectedcontrolsatalltimes.Allexperimentallyinfectedhamstersserocon-
verted,Table3,asdeterminedbyapositiveMATtitreonseracollectedat3weekspost-
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Fig1.Phylogenybasedon16SrDNA.Phylogeneticreconstructionbasedonmaximumlikelihoodestimation.Branchlengthsareproportional
tothenumberofsubstitutionspersiteandbranchvaluesarethebootstrapvaluesassignedtotheedges(i.e.thebranchsupportvalues).
doi:10.1371/journal.pntd.0005174.g001
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InvasiveSpeciesasCarriersofLeptospires
Fig2.PhylogenybasedonsecY.Phylogeneticreconstructionwasinferredusingthemaximumlikelihoodmethod.Thetreeisdrawn
toscale,withbranchlengthsmeasuredinthenumberofsubstitutionspersite.
doi:10.1371/journal.pntd.0005174.g002
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InvasiveSpeciesasCarriersofLeptospires
Fig3.RestrictionEnzymeAnalysisofGWTSisolatesofLeptospira.GenomicDNAfromGWTSisolates
#1(1),#2(2)and#3(3)werecomparedbyREAtothatofL.alstoniiisolatesofserovarPingchang(4),serovar
Sichuan(5),MS267(6),MS311(7)andMS316(8).L=DNAMarker.
doi:10.1371/journal.pntd.0005174.g003
inoculation.Serafromexperimentallyinfectedhamsterswereonlyreactivewiththechallenge
isolate;nocross-reactingMATtitresweredetectedwhentestedagainstanMATpanelrepre-
sentativeforIreland,andwhichincludedserogroupBratislava,Canicola,Grippotyphosa,
Hardjo,IcterohaemorrhagiaeorPomona.Kidneysfromexperimentallyinfectedhamsters
wereculturenegativeforleptospires.
Table3. MATresultsofhamstersinfectedwithGWTSisolates.
Challengeisolateand GWTS#1 GWTS#2 GWTS#3 B Ca G H Co P
Animalnumber
GWTS#1 1 1:800 1:800 1:800 neg neg neg neg neg neg
2 1:400 1:800 1:400 neg neg neg neg neg neg
3 1:800 1:1600 1:800 neg neg neg neg neg neg
GWTS#2 4 1:1600 1:1600 1:800 neg neg neg neg neg neg
5 1:800 1:800 1:400 neg neg neg neg neg neg
6 1:800 1:400 1:400 neg neg neg neg neg neg
GWTS#3 7 1:800 1:800 1:1600 neg neg neg neg neg neg
8 1:800 1:800 1:800 neg neg neg neg neg neg
9 1:800 1:800 1:800 neg neg neg neg neg neg
AntiserafromhamstersinfectedwithGWTSisolate#1(animalnumbers1,2&3),GWTSisolate#2(animalnumbers4,5&6)orGWTSisolate#3(animal
numbers7,8&9)wastestedagainsteachchallengeisolateoragainstastandardMATpanelasindicated;B=serovarBratislava,Ca=serovarCanicola,
G=serovarGrippotyphosa,H=serovarHardjo,Co=serovarCopenhageniandP=serovarPomona.Serafromnegativecontrolhamstersdidnotreact
withanyantigen.neg=notreactive.
doi:10.1371/journal.pntd.0005174.t003
PLOSNeglectedTropicalDiseases|DOI:10.1371/journal.pntd.0005174 December9,2016 10/17