The importance of ticks in Q fever transmission: what has (and has not) been demonstrated?

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The importance of ticks in Q fever transmission: what has (and has not) been demonstrated?

Olivier Duron, Karim Sidi-Boumedine, Elodie Rousset, Sara Moutailler, Elsa Jourdain

To cite this version:

Olivier Duron, Karim Sidi-Boumedine, Elodie Rousset, Sara Moutailler, Elsa Jourdain. The im-

portance of ticks in Q fever transmission: what has (and has not) been demonstrated?. Trends in

Parasitology, Elsevier, 2015, 31 (11), pp.536-552. �10.1016/j.pt.2015.06.014�. �hal-02637724�

Opinion

The Importance of Ticks in Q Fever Transmission: What Has (and Has Not) Been

Demonstrated?

Olivier Duron,

Karim Sidi-Boumedine,

Elodie Rousset,

Sara Moutailler,

and Elsa Jourdain

*

QfeverisawidespreadzoonoticdiseasecausedbyCoxiellaburnetii,aubiqui- tousintracellularbacteriuminfectinghumansandavariety ofanimals.Trans- missionisprimarilybutnotexclusivelyairborne,andticksareusuallythoughtto actasvectors.Wearguethat,althoughticksmayreadilytransmitC.burnetiiin experimentalsystems,theyonlyoccasionallytransmitthepathogeninthefield.

Furthermore,weunderscorethatmanyCoxiella-likebacteriaarewidespreadin ticksandmayhavebeenmisidentifiedasC.burnetii.Ourrecommendationisto improvethemethodscurrentlyusedtodetectandcharacterizeC.burnetii,and we propose that further knowledge of Coxiella-like bacteria will yield new insightsintoQfeverevolutionary ecologyandC.burnetiivirulencefactors.

QFever:AnAirborneZoonoticDisease

Qfeverisazoonosis(seeGlossary)foundworldwidethatiscausedbytheobligateintracellular bacteriumCoxiellaburnetii.Thispathogencaninfectawiderangeofvertebrates,including livestock(whicharethoughttobetheprimaryreservoir),avarietyofwildspecies,andhumans [1].TheclinicalsignsofQfevervarydramatically (Box1).In animals,infectionsareusually asymptomaticandarenotconsideredtobeaveterinaryproblem,exceptinruminantswhereQ feverisawell-recognizedcauseofabortion[2,3].Inhumans,C.burnetiiinfectionsvaryfrom self-limitingtosevere[4,5].Theacute formrangesfromcausing mildflu-likesymptomsto provokingpneumoniaorhepatitis,whichmayrequirehospitalization.Thediseasecanbecome chronicandresultinendocarditis,aneurysmal,valvular,orvascularinfections,chronicfatigue syndrome,premature birth,orabortion, andparticularlyfor individuals withrisk factorsof severity. Though rarely fatal, Q fever remains highly debilitating, even whentreated with antibiotics.Manysporadiccasesinhumansoccurannuallyworldwide,andoccasionalout- breaksare alsocommon.The2007–2010Q feverepidemic inTheNetherlands attracted attentionbecauseofitsexceptionalmagnitudeandduration:morethan4000humancases werereported[6,7].

C.burnetiiproducesspore-likesmallcell variantsthatareabletoresistharshenvironmental conditionsandarethusmorelikelytopersistintheenvironmentforlongperiodsoftime.Forthis reason,andbecausepublichealthmeasuresareparticularlydifficulttoimplementgiventhatthe diseaseisaeriallytransmitted,highlymorbid,anddifficulttodiagnose,C.burnetiiisclassifiedas a category B potential aerosolized biological weapon by the United States [8]. Infection commonlyoccursviatheinhalationofbarnyarddustcontaminatedwiththeexcretaofinfected

Trends

Qfeverisawidespreadzoonoticdis- ease causedbyCoxiella burnetii,an intracellular bacterium that infects humansand a widerange of verte- brates. Domestic ruminants are the mainreservoir.

C.burnetiiisfrequentlydetectedinticks, and laboratory experiments have revealedthatatleastsometickspecies arecompetentvectors.However,under naturalconditions,Qfeverisfarmore frequentlyairbornethanvector-borne.

Many Coxiella-like bacteria, closely relatedtobutgeneticallydistinctfrom C. burnetii, have been described in ticksand,veryoccasionally,inverte- brates.Theylikelybehaveasnon-viru- lentticksymbionts.Theirpathogenicity forvertebratesislargelyunknown.

Coxiella-likebacteriamayhavebeen misidentifiedasC.burnetiiinpastfield studies.Newmeansofdetectingand characterizing tick-borne C. burnetii andCoxiella-likebacteriaareneeded.

1LaboratoireMIVEGEC(Maladies InfectieusesetVecteurs:Ecologie, Génétique,EvolutionetContrôle), CentreNationaldelaRecherche Scientifique(CNRS)UnitéMixtede RechercheUMR5290,Université Montpellier1–UniversitéMontpellier 2–InstitutpourlaRechercheetle Développement,UnitédeRecherche UR224,Montpellier,France

2Anses,Sophia-AntipolisLaboratory, AnimalQfeverUnit,Sophia-Antipolis, France

536 TrendsinParasitology,November2015,Vol.31,No.11 http://dx.doi.org/10.1016/j.pt.2015.06.014

©2015ElsevierLtd.Allrightsreserved.

animals,suchasbirthproducts,whichmaycontainhighquantitiesofC.burnetii;otherinfection pathways(e.g.,sexual,oral,orcongenital)arethoughttoberare[2,4].

QFever:ATick-Borne Zoonosis?

The importance of ticks inQ fever epidemiology remains controversial even though major pioneeringstudieshavefocusedonC.burnetiiinticks [1,9].Itisnoteworthythatthehighly- virulentreferencestrain,NineMile,was isolatedfromaguineapiguponwhichDermacentor andersonitickshadfed[10].Furthermore,manyearlymicroscopicmorphologicalobservations suggestedthatover40tickspeciescarryC.burnetii[11].Nowadays,ticksarestillthefocusof manyfieldstudiesofQfeverepidemiology(TableS1inthesupplementarymaterialonline).The occasional reportsof unexpectedly high levels of C. burnetiiinfection inticks [12,13]raise questionsofwhetherticksplayanimportantroleinQfevertransmission.

Inthisarticlewereviewtheavailableliteraturetoassesstheimportanceofticksinnaturalcycles ofQfevertransmission.First,weexaminetheabilityoftickstoreadilytransmitC.burnetiiinboth experimentalandfieldsystems.Second,wehighlightrecentfindingsthatrevealthediversityof Coxiella-like bacteria,which aregenetically relatedto,butdistinctfrom,C.burnetii,andwe explorethereliabilityofthescreeningmethodscommonlyusedforticks.Wefurtherarguethat futureresearchmustfocusondevelopingmethodsthatbetterdetectandcharacterizetick- borneCoxiella.

TicksAre CompetentVectorsforC.burnetiiinExperimentalSystems

TheroleofticksinQfevertransmissionhasbeenextensivelystudiedeversincetheNineMile strainwasisolatedfromD.andersoniinthe1930s[10].Atleastsevenhardandsofttickspecies, including D. andersoni, have formally been shown to be competent vectors of C. burnetii (Table 1). For each species, three major traits related to vector competence have been experimentally confirmed: (i)theabilitytoacquireC.burnetiifrom aninfectedanimal, (ii)the trans-stadialtransmissionofinfectionfromlarvaetonymphsandfromnymphstoadults,and

3UMRBiologieMoléculaireet ImmunologieParasitairesetFongiques (BIPAR),LaboratoireSantéAnimale, ANSES,InstitutNationaldela RechercheAgronomique(INRA),Ecole NationaleVétérinaired’Alfort(ENVA), Maisons-Alfort,France

4Unitéd’EpidémiologieAnimale,UR 0346INRA,SaintGenèsChampanelle, France

*Correspondence:

elsa.jourdain@clermont.inra.fr (E.Jourdain)

Box1.QFever:AChallengingDiseaseforPublicandAnimalHealth

Qfeverisazoonoticdiseasethathasahighsocioeconomicburden[6]andisdifficulttodiagnose,prevent,andtreatin bothhumansandanimals[2,89].Itthereforepresentssignificantchallengesforbothpublicandanimalhealth.

ChallengesforPublicHealth

Inhumans,initialexposuretoC.burnetiimayresultinasymptomaticormildinfectionbutalsoinacuteorchronicdisease [4,5].Theclinicaldiagnosiscanbeverydifficult.Thereasonsforthishighclinicalpolymorphismarelargelyunknown,even ifriskfactorsofseverity(e.g.,pregnancy,immunosuppression,preexistingcardiacvalvulopathy,vasculargrafts,and aneurysms)havebeendescribed.Althoughrarelyfatal,thediseasemayleadtosubstantialmorbidityandcanbehighly debilitating,evenundertreatment.Mosthumancasesresultfromtheinhalationofdustparticlescontaminatedby infectedlivestockoranimalproducts[2,4,89].C.burnetiiisalsoapotentialagentofbioterrorism[8].Preventionisdifficult toputinplacebecausetransmissionisessentiallyairborne.Althoughaneffectivevaccine(Q-VAX,CSLLimited,Parkville, Victoria,Australia)maybeusedinAustraliaforat-riskprofessions,itsuseinendemicareasisdifficultbecauseithas significantsideeffectsinpersonswhohavebeenexposedtoC.burnetii,thusrequiringpre-vaccinationscreening[8].

ChallengesforAnimalHealth

Indomesticanimals,Qfeverismostlyassociatedwithabortionpeaksinsmallruminantsandsporadicabortionsincattle [90].Thedifferentialdiagnosiswithotherinfectiousandnon-infectiouscausesofabortionmaybedifficult.Withinherds withQfeverabortions,thebacteriumisexcretedinlargequantitiesintheplacentaandfetalmembranesoffemales, whethertheyhaveabortedornot,andexcretioninvaginalmucusandfecesmayfurtherlastseveralmonths,which resultsinmassiveenvironmentalcontamination[2,91].Diseasemanagementisextremelydifficult:antibiotictreatmentis inefficientandnoenvironmentaldecontaminationprocedureshavebeenvalidated.Long-termcontroloptionsinclude segregatedbirthingareas,removalofabortion/birthmaterial,manuremanagement,andvaccinationofprimiparous females[2].DiseasemanagementisfurthercomplicatedbythefactthatQfevertransmissionisessentially,butnotonly, airborne,andalsothatmanyanimalreservoirsmaybeinvolvedintheepidemiologicalcycle.

TrendsinParasitology,November2015,Vol.31,No.11 537

Glossary

Enzootic:referstoadiseasethatisconstantlypresentinananimalpopulationwithlocaltransmissionevents;analogousto‘endemic’in

humans.Epizootic:referstoadiseasethatrapidlyspreadsinananimalpopulationwithinashortperiodoftime;analogousto‘epidemic’forhumandiseases.

Serologicaltest:adiagnostictestbasedonthedetectionofantibodiesintheserumofpatientsoranimals;specificantibodiesaresynthesizedinresponsetoaninfectionagainsta

givenmicroorganism.Transovarial:transmissionofamicroorganismfromafemaleticktoitsprogenythroughitsovaryandeggs.

Trans-stadial:transmissionofamicroorganismfromonetickstagetothenext.Vectorcapacity:ameasureofthetransmissionpotentialofavector

populationinfieldconditions;itcorrespondstothenumberofsecondarycasesarisingperdayfromoneinfectivehostinasusceptiblehostpopulationexposedtothe

vector.Vectorcompetence:theabilityofavectortoacquire,maintain,andtransmitamicrobialagentinlaboratoryconditions.

Zoonosis:adiseaseofanimalsthatcanbenaturallytransmittedtohumans.Zoonoticdiseasescanbeeitherenzooticorepizootic(seeabove).

Table 1. List of Studies Investigating the Transmission ofCoxiella burnetiiby Arthropods in Experimental Conditions^a Tick (or other arthropod)

species

Competent vector

Trans-stadial transmission

Transmission to vertebrate host (species)

Infection by engorgement (species)

Methods to confirm tick infection C. burnetiistrain Refs

L to N N to Ad Bite Feces Other Engorgement Inoculation Detection

Rhipicephalus microplus^b ? Yes^c Yes^d Yes (calf) Yes (GP) Yes^h Australian^e [92]

Dermacentor andersoni Yes Yes (GP) Yes^f Yes (GP) Nine Mile^g [93]

Yes Yes Yes (GP) Yes (GP) Yes (GP) Yes (GP) Nine Mile [15]

Yes^c Yes (GP) Nine Mile [14]

Haemaphysalis bispinosa ? Yes No Yes (GP) Yes (GP) Yes^h Australian [94]

Haemaphysalis humerosa Yes Yes Yes Yesⁱ(GP)/No^j Yes^c,k/No^l No^m Yes (GP) Yes (GP) Australian [16]

Hyalomma aegyptium Yes Yes Yes Yes (GP) Yes (GP) Yes (GP) Yesⁿ Nine Mile [95]

Hyalomma asiaticum Yes Yes Yes Yes (GP) No^o Yes (GP) Yes (GP) Yes (GP) Yes^h Ixodes II Luga^p [96]

Ixodes holocyclus Yes Yes Yesⁱ/No^j Yes (GP/bandicoot) Yes

(GP/bandicoot)

Yes (GP/bandicoot)

Yes (GP) Yes^h Australian [97]

Rhipicephalus sanguineus ? Yes Yes Yes (GP) Yes (GP) Yes^h Australian [98]

Yes^d,q Yes (dog) Yes (GP) Unknown^r [99]

Ornithodoros canestrinii ? Yes^d No^s Yes (GP) Grit [100]

Ornithodoros gurneyi No No No No (GP) No^t Yes (GP) Yes (GP) Yes (GP) Australian [94]

Ornithodoros hermsi Yes Yes Yes (GP) Yes (GP) Yes (GP) Yes (GP) Nine Mile [17]

Ornithodoros lahorensis ? Yes^d No^s/Yes^u Yes (GP) M44 and Grit [101]

Ornithodoros moubata Yes Yes Yes (GP) Yes (GP) Yes (GP) Yes (GP) Nine Mile [17]

Yes^d No^s Yes (GP) M44 and Grit [101]

Ornithodoros papillipes ? Yes^d Yes^u Yes (GP) M44 and Grit [101]

Yes^d Yes^u Yes (mice) Ixodes IV Luga^p [102]

Ornithodoros turicata No No No (GP) Yes^c Yes^d Yes (GP) Yes (GP) Yes (GP) Nine Mile [103]

538TrendsinParasitology,November2015,Vol.31,No.11

Table 1. (continued) Tick (or other arthropod) species

Competent vector

Trans-stadial transmission

Transmission to vertebrate host (species)

Infection by engorgement (species)

Methods to confirm tick infection C. burnetiistrain Refs

L to N N to Ad Bite Feces Other Engorgement Inoculation Detection

Ctenocephalides felis(flea) No No na Yes (mice) Yes (mice) Australian [104]

Aedes aegypti(mosquito) No No na No (GP) No^v Yes (GP) Yes (GP) Yes (GP) Nine Mile [14]

aAbbreviations: , not tested; Ad, Adults; GP, guinea pig L, Larvae; N, Nymphs; na, not applicable.

bRecently assigned to the genusRhipicephalusfrom the genusBoophilus.

cInoculation to guinea pig.

dIntraperitoneal inoculation of tick homogenates.

eAustralian:Coxiella burnetiiisolated fromHae. humerosa, successive passages in guinea pig (Queensland, Australia).

fNatural infection.

gNine Mile Strain:Coxiella burnetiiisolated fromD. andersoni, successive passages in guinea pig (Montana, USA).

hSmears.

iFemale.

jMale.

kDeposition of feces on abraded skin.

lDeposition of feces on unabraded skin.

mCutaneous transmission by deposition of tick homogenates on unabraded skin.

nPCR.

oTranspermal transmission from male to female during tick mating.

pIxodes II Luga:C. burnetiiisolated fromIxodes ricinusin Leningrad during Q fever epidemic.

qIntraperitoneal inoculation of crushed tick eggs.

rFetal membrane from infected sheep.

sIntracoelomic inoculation of tick homogenates.

tCutaneous transmission by deposition of tick coxalfluid on unabraded skin.

uEngorgement on artificial membrane.

vPercutaneous transmission during tick bite with interrupted feeding.

TrendsinParasitology,November2015,Vol.31,No.11539

(iii)theabilitytotransmitinfectiousC.burnetiitoanuninfectedanimal.Obviously,manymoretick speciesmaybecompetentvectorsofQfever.Othertickspecieshavebeenfoundtotransmit thepathogen,buttheirvectorcompetencehasnotbeenfullydemonstrated(forinstance,trans- stadialtransmissionhasnotbeenshown;Table1).Ofallthetickspeciesexaminedthusfar,only twohavebeenexperimentallyshownto beincompetent vectors(Table1).Consequently,in experimentalsystems,mosttickspeciesseemtobeabletotransmitC.burnetiitouninfected animals.

Inlaboratory-infectedticks,infectionistypicallysystemic;C.burnetiihasbeendetectedinthe midgut,hemolymph,Malpighiantubules,salivaryglands,andovaries[1].Tickshavealsobeen foundtoexcretelargenumbersofinfectiousC.burnetiiintheirbodyfluidsandfeces–upto10¹⁰ organisms per gramof feces [14].This finding underscores the potentialriskof tick-borne infectionposedbytickexcreta,throughinhalation(e.g.,duringsheepshearing),directcontact (e.g., whilecrushingatickwith one's barehands), ortick bites.Furthermore,transovarial transmission–thetransmissionofC.burnetiifromafemaleticktoheroffspring–hasalsobeen observedinthreetickspecies[15–17],whichshowsthatC.burnetiicanbemaintainedbytick hostsacrossseveralgenerationswithoutneedingtoinfectvertebrates.Asaresult,thispatho- genmaybetransmittedbothtransovariallyandviabloodmealsinseveraltickspecies.

TheVectorCapacityofTicksintheFieldRemainsUnknown

Fieldstudiesareessentialtoevaluatingthepotentialoftickstovectorpathogensundernatural conditions.ThenaturalabilityofaticktotransmitC.burnetii(i.e.,itsvectorcapacity)dependson severalfactorsbesidesvectorcompetence,includingtickpopulationdensity,hostpreference, bitingrate,andecologicalconstraints.Consequently,evenifticksarecompetentvectorsunder laboratoryconditions,theymayinefficientlytransmitdiseaseinnatureiftheirvectorcapacityis low.ThismightbethecaseforC.burnetii.

Todate,mostfieldstudiesexaminingtheroleofticksinQfeverepidemiologyhaverestricted themselvestodescribingC.burnetiiprevalence.TheobservedpercentageofC.burnetii-positive ticksistypicallylow(<5%)butprevalencelevelsgreaterthan5%oreven10%arealsoreported (TableS1).Theselevelsareconsistentwiththoseobservedforstrictlytick-bornepathogens, suchasbacteriafromthegenusAnaplasma[18].Therefore,asylvaticcyclebasedonC.burnetii tick-bornetransmissionseemstobesustainable.ThefactthatC.burnetiihasoccasionallybeen isolatedfromtickssampledfromwildlife[12]orwildlifeburrows[19]supportsthishypothesis.

However,directtransmissionlikelyalsotakesplaceamongwildlifespeciesbecauseC.burnetii hasbeenreportedinthefeces[20–22],placenta[23–25],andvaginalmucus[26]ofdiverse wildlifespecies.Interestingly,withinorinthevicinityoffarmswhereQfeverhasbeenknownto circulate,C.burnetiiprevalenceinticksmaybeloworseeminglyabsent[27,28].Conversely,a strongcorrelationbetweentheseropositivityindomesticruminantsandtheirinfestationwithC.

burnetii-infectedtickshasbeenreported[29],andseveralstudieshaveidentifiedthepresenceof ticksasariskfactorforseropositivityinlivestock[30–32].Thus,thevectorcapacityofticksto transmitC.burnetiiremainsunclear.Inhumans,limiteddatasupporttheoccurrenceoftick- borneC.burnetiitransmission,includingoccasionalreportsofC.burnetiiinfectionsinpatients bittenbyticks[33–37],orconcomitantlyinfectedwithtick-bornepathogens[38,39],orpositive forQfeverbyserology[40].However,inthesecases,exposuretoinfectionsourcesotherthan ticks(particularlyviatheaerialroute)generallycannotbeexcluded.

Overall, therefore, theabilityofticks to vectorC. burnetiiseemslimited: althoughthey may occasionally transmit thebacterium to vertebrateanimals and humans, thisroute isclearly secondarycomparedtoairbornetransmission.Nonetheless,ticksmayserveasanecological bridge for C. burnetii transmission between wild anddomestic animal hosts [12,41,42]. In crossingthesespeciesbarriers,C.burnetiimaybeexperiencingincreasedselectionforgenomic

540 TrendsinParasitology,November2015,Vol.31,No.11

plasticity andenhancedgeneticdiversity, promotingitsdiversityofvirulence andresistance factors[43,44].

Coxiella-like BacteriaAreCommoninTicks

C. burnetiiis theonly speciesthat has beenformally describedinthe Coxiellagenus [45], althoughanotherputativespecies(C.cheraxi)hasbeenreportedincrayfishes[46].Interestingly, inthemid-1990s,theadventofsimplePCRassays,togetherwithextensive16SrRNAgene sequencing,ledtothedescriptionofCoxiella-likebacteriainthreetickspecies[47].Thesenovel Coxiella-likebacteriawerecloselyrelatedto(butgeneticallydistinctfrom)C.burnetii,revealing thatanoverlookeddegreeofdiversitymayactuallyexistwithintheCoxiellagenus[48].Wenow knowthatCoxiella-likebacteriaareexceptionallydiverseandwidespreadinticks.Inarecent study,Duronetal.[49] identifiedCoxiella-likebacteriafrom40of58examinedtickspecies, suggesting that more than two-thirds of tick species may be infected. Overall, molecular evidencebasedon16SrRNAgenesequencesshowedthatatleast52tickspeciesareinfected, with aninfectionfrequencycloseto 100%inmany cases(Table 2).Inaddition,afewother Coxiella-likebacteriahavesporadicallybeenfoundindomesticbirds[46,50–52].Inmostcases, these newly described bacteria have been characterized solely by their 16S rRNA gene sequences.Therefore,althoughothergenesmayprovetodiscriminatewellbetweenC.burnetii andCoxiella-likebacteria, theyarecurrentlymainlydefinedbasedonphylogenetic analyses consideringthe16SrRNAgene(Figure1).MultilocusDNAsequencingfurtherindicatesthatthe Coxiellagenusissubdividedintofourhighly-divergentgeneticclades(A–D;Figure1),withC.

burnetiibelongingtotheAclade[49].Remarkably,phylogeneticinvestigationsalsoconvergeto supportthe hypothesisthatone ofthe Coxiella-like bacteria,belonging to theA cladeand primarilyhostedbysoftticks,hasservedastheprogenitorofC.burnetii[49].

Despitetheirgeneticrelatedness,tick-borneCoxiella-likebacteriaandC.burnetiiareecolog- icallydistinctfromeachother.Inparticular,someCoxiella-likebacteria,suchasthosedetected inAmblyommaamericanum,A.cajennense,andOrnithodorosrostratus,displayprevalencesof 100% inallthe life-stagesoftheir hosts;theinfection ismaternallytransmitted,via theegg cytoplasm,andmaintainedtrans-stadially,ratherthanbeingacquiredthroughbloodfeedingon infected vertebrates [49,53–55]. Accordingly, when the Coxiella-like bacterium found in A.

americanumwasrecentlysequenced[56],norecognizablevirulencegeneswerefound,which indicatesthatthisbacteriumislikelynotapathogen.Bycontrast,itsgenomeencodesmajor vitaminandcofactorbiosynthesispathways,whichsuggeststhatitmaybeavitamin-provision- ingendosymbiontinstead.Remarkably, eliminatingthisbacteriumfromA.americanum ticks usingantibioticsreducedtickfecundityandviability[57],whichfurthersupportsthehypothesis thatCoxiella-likebacteriaareengagedinmutualisticsymbiosesinthistickspecies(Box2).

Coxiella-like BacteriaMayBeCommonlyMisidentifiedasC.burnetii

ThediscoverythattickscarrybothC.burnetiiandCoxiella-likebacteriaunderscorestheneedto beabletoclearlydistinguishbetweenthetwo.NumerousC.burnetiidetectionmethodsarein use(TableS1)and,insomecases,theyproduceclearevidencethatticksareinfectedbyC.

burnetiiratherthanbyCoxiella-likebacteria,asshowninFigure1forthebacteriadetectedinthe tick speciesD. andersoniiandA. trigrinum. Forinstance, inthe noteworthy case-study by Pachecoetal.(2013),C.burnetiiinfectionintickswasconfirmedusinganimpressivearrayof detection methods, includinghemolymph tests, isolation inVero cells, andmultilocus DNA sequencing.However,manyotherstudiesaimingtoestimateC.burnetiiprevalenceintickshave notbeenasrigorous,andmayhavemisidentifiedCoxiella-likebacteriaasC.burnetii.

Historically, and until the late 1990s, ticks were essentially screened for C. burnetii using morphologicalobservations,staining,andimmunodetectiontechniquesbecausethisobligate intracellularbacteriumisdifficulttoculture.However,therecentdiscoveryofsomanytick-borne

TrendsinParasitology,November2015,Vol.31,No.11 541

Table 2. List of Tick Species Infected byCoxiella-like Bacteria

Tick species Countries or

regions

Prevalence of Coxiella-like bacteria

Targeted genes by molecular assays

Infected stages Examined

organs

Refs

Hard ticks (Ixodidae)

Amblyomma americanum USA 75–100% 16S rRNA gene,rpsF,rpsG,

dnaK, andFusA

Eggs, larvae, nymphs, adults

Midgut, ovaries, salivary glands

[49,55,60–62, 105,106]

Amblyomma cajennense Brazil 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Eggs, larvae, nymphs, adults

Midgut, ovaries, salivary glands

[49,54]

Amblyomma loculosum Indian Ocean 64–100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

n.d.^a n.d. [49,107]

Amblyomma variegatum Indian Ocean n.d. 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Bothriocroton auruginans Australia 100% 16S rRNA gene andIS1111 Adult females n.d. [67]

Dermacentor silvarum China 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Eggs, larvae, nymphs, adults (males and females)

Ovaries, malpighian tubes

[49,108]

Dermacentor marginatus France 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Haemaphysalis hystricis Thailand 17% 16S rRNA gene Adults n.d. [109]

Haemaphysalis concinnae Russia 100% 16S rRNA gene,gltAand

ompA

Adult females n.d. [110]

Haemaphysalis falva Japan 100% 16S rRNA gene andIS1111 Adults Salivary glands [63,111]

Haemaphysalis lagrangei Thailand 39% 16S rRNA gene Eggs, larvae, nymphs,

adults

n.d. [109,112]

Haemaphysalis longicornis Korea, Japan 2% 16S and 23S rRNA genes,

Com1

Adults Ovaries,

malpighian tubes

[47,65]

Haemaphysalis obesa Thailand 47% 16S rRNA gene Adults n.d. [109]

Haemaphysalis shimoga Thailand 58% 16S rRNA gene Eggs, larvae, nymphs,

adults

n.d. [109,112]

542TrendsinParasitology,November2015,Vol.31,No.11

Table 2. (continued)

Tick species Countries or

regions

Prevalence of Coxiella-like bacteria

Targeted genes by molecular assays

Infected stages Examined

organs

Refs

Haemaphysalis punctata England 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ixodes hexagonus France 100% 16S and 23S rRNA genes,

groEL, and rpoB

Adults n.d. [49]

Ixodes ovatus Japan 95% 16S rRNA gene Adults Salivary glands [63]

Ixodes persulcatus Japan 20% 16S rRNA gene Adults Salivary glands [63]

Ixodes ricinus France, Austria n.d. 16S and 23S rRNA genes,

groEL, and rpoB

Adults n.d. [49]

Ixodes uriae Canada 0–50% 16S and 23S rRNA genes,

groEL, and rpoB

Adults n.d. [49,113]

Ixodessp. 1 Ivory Coast 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ixodessp. 2 Ivory Coast 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Rhipicephalus annulatus Burkina-Faso, Benin 100% 16S and 23S rRNA genes, groEL,rpoB, anddnaK

Adults n.d. [49]

Rhipicephalus australis New Caledonia 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Rhipicephalus bursa Italia 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Rhipicephalus decoloratus Africa 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Rhipicephalus evertsi Zimbabwe 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Rhipicephalus geigyi Burkina-Faso, Benin 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

TrendsinParasitology,November2015,Vol.31,No.11543

Table 2. (continued)

Tick species Countries or

regions

Prevalence of Coxiella-like bacteria

Targeted genes by molecular assays

Infected stages Examined

organs

Refs

Rhipicephalus microplus USA, Africa 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Eggs and adults Ovaries [49,64]

Rhipicephalus pusillus France n.d. 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Rhipicephalus sanguineus Switzerland, France, USA

12–100% 16S and 23S rRNA genes Eggs, larvae, nymphs, adults

Ovaries, malpighian tubes

[47,49,106, 114–116]

Rhipicephalus turanicus Europe 23–100% 16S rRNA gene Nymphs and adults Ovaries,

malpighian tubes

[49,114–116]

Rhipicephalussp. 1 Ivory Coast 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Rhipicephalussp. 2 Ivory Coast 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Soft ticks (Argasidae)

Argas monolakensis USA 53% 16S rRNA gene,mucZ, and

gltA

n.d. n.d. [70]

Argas monachus Argentina 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros amblus Peru 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros brasiliensis Brazil 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros capensis Various tropical and temperate regions

46–100% 16S rRNA gene,icd,sod, pyrG,gltA,mucZ,groEl (htpB), etc

Eggs, nymphs, adults n.d. [49,68,69,

107,117]

Ornithodoros denmarki Unknown 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

544TrendsinParasitology,November2015,Vol.31,No.11

Table 2. (continued)

Tick species Countries or

regions

Prevalence of Coxiella-like bacteria

Targeted genes by molecular assays

Infected stages Examined

organs

Refs

Ornithodoros erraticus North Africa 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros kairouanensis North Africa 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros maritimus Mediterranean Islands

100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Eggs, adults n.d. [49]

Ornithodoros marocanus North Africa 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros moubata n.d. n.d. 16S and 23S rRNA genes n.d. Ovaries,

malpighian tubes

[47]

Ornithodoros occidentalis North Africa 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros peruvianus Chile 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros rostratus Brazil 100% 16S rRNA gene,pyrG, and

Cap

Eggs, nymphs, adults n.d. [49,53]

Ornithodoros rupestris North Africa 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros sonrai North Africa 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodoros spheniscus Chile 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

Ornithodorossp. Cape Verde 100% 16S and 23S rRNA genes,

groEL,rpoB, anddnaK

Adults n.d. [49]

aAbbreviation: n.d., not defined.

TrendsinParasitology,November2015,Vol.31,No.11545

Coxiella-like bacteriausingmoleculartechniques puts theresults of thesepast studiesinto question.ThecaseofA.americanumisillustrative:whileC.burnetiiisrepeatedlyreportedto occur in this species in the older literature [58,59], recent studies using sequence-based methodshave foundthatA. americanum actuallyharborsaCoxiella-like bacterium [60,61].

Next-generation sequencing (NGS) approaches, which provide new means to exhaustively describethebacterialcommunitiesfoundinticks,havealsorevealedthatCoxiella-likebacteria, andnotC.burnetii,predominateinmosttickspeciesinvestigatedthusfar[62–64].Ittherefore seemsreasonabletoassumethatsomeofthestrainsinitiallyidentifiedvisuallyasC.burnetiiwill bereclassifiedasCoxiella-likebacteria.Hence,thehistoricanddogmaticassertionthatover40 tickspeciesareinfectedbyC.burnetii[11]maybeerroneous,andshouldbereevaluatedwhen appropriatemoleculardatabecomeavailable.

Atpresent,thereisstillasubstantialriskofmisidentificationgiventhatthescreeningofticksfor C.burnetiifrequentlyreliesonthedetectionofasinglegene,basedondiagnosticPCRassays,

Coxiella burnei (Nine Mile strain from Dermacentor andersonii)

Coxiella burnei

Coxiella

A

Coxiella burnei (Dugway strain) Coxiella burnei (CbuK strain) Coxiella burnei (CbuG strain) Coxiella burnei (CbC2 strain) Coxiella burnei (CbB18 strain) Coxiella burnei (CbB1 strain)

Coxiella burnei from Amblyomma trigrinum Coxiella burnei (Cb109 strain)

Coxiella burnei (RSA331 strain) Coxiella burnei (Z3055 strain)

CLB of Ornithodoros denmarki CLB of Ornithodoros capensis CLB of Ornithodoros amblus CLB of Ornithodoros sphenicus CLB of Ornithodoros peruvianus CLB of Ornithodoros marimus CLB of Argas monachus CLB of Ornithodoros rostratus CLB of Ornithodoros brasiliensis CLB of Amblyomma variegatum CLB of Ornithodoros kairouanensis CLB of Ornithodoros rupestris

CLB of Ornithodoros marocanus CLB of Ornithodoros sonrai CLB of Ornithodoros erracus CLB of Ornithodoros occidentalis CLB of Rhipicephalus sanguineus CLB of Rhipicephalus turanicus CLB of Rhipicephalus pusillus CLB of Rhipicephalus geigy CLB of Rhipicephalus decoloratus CLB of Rhipicephalus annulatus CLB of Rhipicephalus australis CLB of Rhipicephalus microplus Rickesiella grylli

Rickesiella pulae Legionella pneumophila Legionella longbeachae

CLB of lxodes sp.

CLB of lxodes ricinus CLB of lxodes hexagonus CLB of lxodes uriae

CLB of Dermacentor marginatus CLB of Dermacentor silvarum CLB of Amblyomma americanum CLB of Amblyomma cajennense CLB of Haemaphysalis punctata

B

D

C

Figure1.CladogramoftheCoxiellaGenusBasedonRepresentativeDNASequencesAvailableintheGenBankDatabase (adaptedfrom[49]and[117]).Membersofthetwosister-generaofCoxiella(RickettsiellaandLegionella)havebeenaddedto delineatetheCoxiellagenus.ThefourCoxiellacladesarelabeledA–D.CLB,Coxiella-likebacteria;circles,Coxiellastrainsprimarily characterizedinticks;blue,Coxiella-likebacteria;red,C.burnetii;Black, bacteriabelongingto bacterialgeneraotherthanCoxiella.

546 TrendsinParasitology,November2015,Vol.31,No.11

withoutconfirmationbysequencingthattheobtainedPCRproductsarespecificforC.burnetii.

Indeed, while sequencing has revealed the presence of mutations specific to Coxiella-like bacteria,ithasalsohighlightedgeneticsimilaritiesbetweenCoxiella-likebacteriaandC.burnetii.

AsdetailedinTableS1,themostroutinelytargetedgenesareIS1111(atransposaseinsertion element for which PCR kits are commercially available), sod (superoxide dismutase), icd (isocitratedehydrogenase)andcom1(encodinga27kDaoutermembraneprotein).Interest- ingly, instudiesinwhich severalofthese genesareamplifiedfromthe sametick samples, amplification may take place for a specific gene whereas another is not amplified (e.g., [27,42,65,66]),andthismaysuggestthatthedetectedbacteriaisnotC.burnetii.Accordingly, aCoxiella-likebacteriumfoundinBothriocrotonauruginanswasshowntoharboranIS1111-like element 90%identical tothe IS1111insertionsequenceof C.burnetii[67];as aresult,the detectionofIS1111mayrevealinfectionbythisCoxiella-likebacteriumratherthanbyC.burnetii.

Similarly,Reevesetal.[68]showedthataC.burnetiisodBgeneamplifiedfromtheCoxiella-like bacteriafoundinCarioscapensisdisplayed>92%identitywiththesodBgenefromC.burnetii;

however,theydidnotdetectIS1111norcom1.Conversely,highdifferencesexistbetweensod gene sequences from C. burnetii and from the Coxiella endosymbiont of A. americanum (Genbank accession number CP007541). Taken together, these results suggestthat Cox- iella-likebacteriasharegeneticfeatureswithC.burnetii,butthatthesequencesincommonare variable.

ThesemethodologicalproblemsmaybeencounteredwithothersupposedlyC.burnetii-specific geneticmarkersbecauseseveralgenesusedtodetectC.burnetiihavenowbeenfoundintick- borneCoxiella-likebacteria[53,65,69,70].Inrecentyears,remarkableprogresshasbeenmade indesigningnewPCR-basedtechniquestodetectC.burnetii.Thesepromisingmethodsinclude multiple-locusvariablenumbertandemrepeat(MLVA)analysis,multispacersequencetyping (MST),andSNP genotyping[71–75].Theycanbeusedto rapidlyandsensitively detectC.

burnetiiinavarietyofclinicalandenvironmentalsamples.Thesemethodsweredevelopedusing abroadpanelofC.burnetiistrains,butCoxiella-likebacteria,whosegenotypeprofilesremain entirelyuncharacterized,werenotincluded.Consequently,theabilityofthesetechniques to distinguishbetweenC.burnetiiandCoxiella-likebacterianeedstobefurthertestedbeforethey canbeappliedtoticksamples.

Overall,PCR-basedscreeningthatdoesnotusecomplementaryPCR-productsequencingmay notbespecificenoughtounambiguouslyidentifyC.burnetii,andmaythusoverestimatethe prevalenceofthepathogeninticks.

Box2.InsightsonMaternallyInheritedBacteriainArthropods

Symbiosis,inwhichdifferentspeciesengageinlong-termandintimateassociations,isaubiquitousfeatureoflife.

Arthropods,inparticular,areknowntoengageinexceptionallydiverseassociationswithspecificbacterialendo- symbiontsthatliveexclusivelywithintheircellsandundergomaternal(transovarial)transmissiontotheiroffspring [118,119].Theseheritablebacteriausespecificadaptivestrategiestospreadandpersistwithinarthropodpopula- tions,eitherprovidingfitnessbenefitstofemalehostsorsubtlymanipulatinghostreproduction.Twocategoriesof endosymbiosesareusuallyrecognized, althoughintermediates andtransitionsarefrequent.Thefirstcategory consistsofobligate(primary)mutualisticsymbiontsthatarenecessarytosupportnormalhostdevelopmentand assisttheirhostinvariousfunctionssuchascomplementationofthediet.Forexample,mostblood-feedinginsects(e.

g.,bedbugs,kissingbugs,tsetseflies...)harborobligatesymbiontsthatprovideBvitamins,whicharenecessaryto completetheirlifecycle[118].ManyCoxiella-likebacteriaofticksseemtobelongtothiscategory.Thesecondcategory consistsoffacultative(secondary)symbiontsthatarenotrequiredforhostsurvival.Someprotectagainstcertain environmentalstresses,suchasheatorattackbyparasitoidsandpathogens[80,120].Othersarereproductive parasitesthatspreadbyincreasinghostreproductionthroughdaughters(thetransmittingsex)attheexpenseof reproductionthroughsons[121,122].

Overall,heritableendosymbioticbacteriaareofecologicalandevolutionaryimportancetotheparticulararthropod speciesthatareinfectedbecausetheypotentiallymediatetheacquisitionofimportantecologicaltraitsordrivechangesin reproductivetraits[118,122,123].

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