Evidence of cross-transfer of maternal antibodies through allosuckling in a mammal: potential importance for behavioral ecology

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through allosuckling in a mammal: potential importance

for behavioral ecology

Romain Garnier, Sylvain Gandon, Yannick Chaval, Nathalie Charbonnel,

Thierry Boulinier

To cite this version:

Romain Garnier, Sylvain Gandon, Yannick Chaval, Nathalie Charbonnel, Thierry Boulinier.

Ev-idence of cross-transfer of maternal antibodies through allosuckling in a mammal:

potential

importance for behavioral ecology.

Mammalian Biology, Elsevier, 2013, 78 (5), pp.361-364.

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ContentslistsavailableatSciVerseScienceDirect

Mammalian

Biology

j o ur n a l ho m e p ag e : w w w . e l s e v i e r . c o m / l o c a t e / m a m b i o

Short

Communication

Evidence

of

cross-transfer

of

maternal

antibodies

through

allosuckling

in

a

mammal:

Potential

importance

for

behavioral

ecology

Romain

Garnier

a,b,∗

_,

_Sylvain

_Gandon

a

_,

_Yannick

_Chaval

c

_,

_Nathalie

_Charbonnel

c

_,

_Thierry

_Boulinier

a

a_Centre_d’Ecologie_{Fonctionnelle}_et_Evolutive,_UMR_CNRS_5175,_Montpellier,_France

b_Department_of_Ecology_and_Evolutionary_Biology,_Princeton_University,_NJ_08544,_USA

c_INRA,_UMR_CBGP_{(INRA/IRD/Cirad/Montpellier}_SupAgro),_Campus_{international}_de_Baillarguet,_CS_30016,_F-34988_{Montferrier-sur-Lez}_Cedex,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received24September2012 Accepted23November2012 Keywords: Merionesunguiculatus Mongoliangerbil Ecologicalimmunology Host–parasiteinteractions

Transferofmaternalantibodies

Transgenerationalvaccinationdesign

a

b

s

t

r

a

c

t

Thetransferofmaternalantibodiesisacriticalmechanismfortheearlylifesurvivalofvertebrate new-borns.Inmammals,passivetransferofimmunecompoundscanoccurprenatallythroughtheplacenta andpostnatallythroughtheconsumptionofcolostrumandmilk.Insocialmammals,ithasbeen hypothe-sizedthatallosucklingmaybeawayforpupstobroadenandstrengthentheirpassiveaccesstoantibodies afterbirth,butempiricalevidenceforthismechanismisstilllacking.Inordertoinvestigatethepotential fortheoccurrenceofacross-transferofantibodiesbetweenpupsexposedtoseveralfemales,webredina commonenvironmentgroupsoftwofemalesMongoliangerbils(Merionesunguiculatus),eachpreviously injectedwithadifferentvaccine.Herewereportthedynamicsofpassivelyacquiredspeciﬁcantibodies intheserumofnewborns,showingthatpupsacquiredantibodiesfrombothfemalesofagroup.Our resultprovidestheﬁrstexperimentalevidenceofacross-transferbetweenlittersofpassivelyacquired antibodies.Wediscusshowsuchevidenceopensperspectivesforexploringthepotentialimportanceof horizontaltransferofimmunityinnaturalhost–parasitesystemsandhowthiscouldbeusedasatoolto answerimportantbehavioralecologyquestions.

Environmentalconditions encounteredby anewbornduring earlylifeareimportantforitsﬁtnesslaterinlife(Lindström1999). Parasites,inparticular,representamajorpressureonthe popu-lationdynamicsandevolutionoftheirhostspecies(Grenfelland Dobson1995;Tompkinsetal.2002).Theeffectofparasitesmay potentiallybeevenstrongerinyoungvertebrateswhoseimmune response is not completelyfunctional at birth (Frank 2002).In vertebrates, mothers have the ability to provide the newborn with a protection critical for early life survival (Boulinier and Staszewski2008;Grindstaffetal.2003)bytransferringsome spe-ciﬁcmoleculesproducedaspartoftheacquiredimmuneresponse, theantibodies(alsotermedimmunoglobulins[Ig];Brambell1970). In mammals, this transfer occurs through the placenta before birthand/orviathecolostrumandthemilkafterbirth(Baintner 2007).TheamountofIgstransferredthroughtheplacentais deter-minedbytheanatomical structureoftheplacenta(Chucrietal. 2010),andinparticularbyvariationsinthenumberoftissue lay-ersbetweenmaternalandfetalbloods.DifferencesinIgsubtypes arealsoimportant toconsiderasonlyimmunoglobulin G(IgG) canreachthenewborns bloodstream(Pastoret 1998).Similarly,

∗ Correspondingauthorat:DepartmentofEcologyandEvolutionaryBiology,

PrincetonUniversity,NJ08544,USA.Tel.:+16092589830.

E-mailaddress:romaing@princeton.edu(R.Garnier).

postnatalabsorptionofantibodiesbynewbornsalsoreveals varia-tionsacrossmammalianspecies.Forinstance,inartiodactyls,no placental transferofIgsis possibleandcolostrumabsorptionis criticalforearlylifesurvival(Halliday1978).Incontrast,placental transferseemstobethedominantrouteforIgstransferinprimates whileIgsinthecolostrumandthemilkmaymostlyplayarolein thelocalprotectionofthegut(Sadeharjuetal.2007).Inrodents, bothprenatalandpostnatalabsorptionofantibodiesappeartobe importantfactorstoensurethesurvivalofthenewborn(Gustafsson etal.1994).

Hownewbornsgainaccesstocolostrumandmilkmaythusbe importantintermsofpassivetransferofimmunityinmany mam-malian species.Aninteresting wayfor newborns togetmilkis throughallosuckling,whenyoungindividualsfeedfromadifferent femalethantheirbiologicalmother.Allonursingisindeedwidely reportedinmammals(Packeretal.1992;Roulin2002)and off-springcouldgainimportantimmunologicalbenefitsbyacquiring antibodiesfromvariouslactatingfemales(immunologicalfunction ofallosucklinghypothesis;RoulinandHeeb1999).Thepotential foranimmunologicalfunctionofallosucklinghasbeensuggested throughtheefficienttransferofantibodiesfromafostermotherto hernon-biologicaloffspring(e.g.Gustafssonetal.1994;Halliday 1955).However,adoptioninthelaboratorydoesnotreflect condi-tionsencounterednormallyinsocialgroups.Inthewild,mothers andnewbornsareexposedtobiologicalandfostermothers/pups

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atthesametime.Mothersmaythuschoosewhichpupstosuckle andpupsmayinturnchoosefromwhichfemalestogetmilk.

In orderto investigatethepotential for theoccurrence of a cross-transferofantibodies,wefocusedonasocialmammal,the Mongoliangerbil(Merionesunguiculatus).Inthewild,thisspecies usuallyformsgroupsofonebreedingpairassociatedwitha num-berofsubordinates.Inthelabhowever,theycanbreedsuccessfully ingroups,withseveralfemalesgivingbirthtogether(French1994). Twogroupsof12“specificpathogenfree”females(obtainedfrom Janvier,LeGenet-St-Isle,France)werevaccinatedtwiceat8and12 weeksofagerespectivelyagainstinfluenza(0.1mL,intramuscular injection;Gripovac,Merial,France)orBorreliaburgdorferi(0.1mL, subcutaneousinjection;Merilym,Merial,France).Antibodies spe-cificofBorrelia(femalesvaccinatedagainstinfluenza)orspecificof influenza(femalesvaccinatedagainstBorrelia)remainedat unde-tectablelevelsthroughoutthestudy.

Aftertheboostervaccination,eachfemalewashousedduring 2weekswithamale.Topreventdirectinitialcontactand limit aggression,we split thecages with transparent Plexiglas walls (drilledwithholesofapproximately1cm).Wethenswitched indi-vidualsbetweeneachsideoftheseparationeverydayduringaweek togetthemusedtoeachother.Attheendoftheweek,the sepa-rationwasopenedandcontactbetweenthemaleandthefemale wasrenderedpossible.Whenaggressiveinteractionsamongadults wereneverthelessrecorded,individualswereimmediately sepa-rated.Thesameprocedurewasrepeatedtomatchfemalesbypairs basedontheirvaccinationstatus(onefemalevaccinatedagainst inﬂuenzaandtheotheragainstBorrelia).Thefemaleswere subse-quentlyhousedtogetherwithoutthemales.Pupswerethusborn inanenvironmentmimickinganaturalsocialsystemandexpected togainaccesstothemilkofbothfemalesandtodisplaydetectable levelsforbothantigens.

Atbirth,newbornsweremarkedsubcutaneouslywithan indi-vidualcombinationoftattooinkdots.At3weeksofage,beforethe ﬁrstmarkingfaded,anadditionalnumberedeartag(Monel 1005-1,NationalBand&TagCo.,Newport,KY,USA)wasusedtoensure alonglastingindividualmarkingofindividuals.Thesameeartags werealsousedforthemarkingoffemalesthroughoutthe experi-ment.Toassesspassiveantibodyacquisition,bloodwasregularly obtainedfromthenewbornsduringtherearingperiod.Atday1 (i.e.,within24hafterbirth,ascageswerecheckedeveryday)and day8,abloodsample(20–30␮L)wasobtainedbycardiacpuncture usinganinsulinsyringewith30Gneedle.Aftereyeshadopened, bloodsamplingconsistedofapunctureeachweek(startingday 19,anduptoday48)intheretro-orbitalvenalsinuswitha hep-arinizedcapillarytube(75␮L),alternativelyfromtheleftandright eye.Bloodwasthenstoredindrytubesandcentrifugatedwithinan hour.Theplasmawascollectedandkeptfrozenat−20◦_C_pending

analyses.Thissamplingprotocolwaschosentofollowthe new-bornsforasufﬁcientlylongperiodafterweaning(between21and 30day;NorrisandAdams1972).

Antibody levels in newborns were measured using specific commercialEnzymeLinkedImmuno-SorbentAssays(ELISA).An indirect ELISA kit was used for influenza (ID Screen Antibody InfluenzaACompetition,IDVet,Montpellier,France).The percent-ageofinhibition(PI)relativetoanegativecontrolwasusedasa measureofantibodylevel.HighPIvaluesindicatehighplasma con-centrationsofspecificantibodiesagainstinfluenza.AdirectELISA kitwasusedfor Borreliaanalyses(BorreliaIgG+VlsEELISA,IBL InternationalGMBH,Hamburg,Germany).Asthiskitisdesigned forhumans,wereplacedthesecondaryantibodybyaperoxidase conjugatedrabbitanti-gerbil IgG(ImmunologyConsultants Lab-oratory,Portland,OR, USA).Opticaldensity(OD)wasusedas a measureofantibodylevelsinthenewbornsandhighODvalues reflectedhighserumlevelsofBorreliaspecificantibodies.Analyses wererunonceforeachsampleduetothelimitedamountofserum

availableandwerelimitedtothe4pupsthatreachedadulthoodas completedynamicsovertherearingperiodwererequiredtoassess thedynamicsofthepassivetransferofantibodies.

ThepresentstudycomplieswiththeguidelinessetintheGuide for the care and use of laboratory animals (National Research Council2011)andtheprotocolhasbeenapprovedbytheAnimal CareandUseCommittee–LanguedocRoussillon(projectnumber CEEA-LR-1003).Inaddition,becauseoftherepeatedsamplingof newbornsrequiredbytheprotocol,wechosenottoincludeany controlgroupinordertominimizethenumberofanimalsused.

Despite preventive measures to limit agonistic interactions, females showedhighlevels ofaggressiveness toward males.In addition,highratesofcannibalismoffemalesonpupsoccurred rapidlyafterbirth.Thelattermadeitimpossibletokeeptrackofthe exactnumberofbirthsoverthe12femalepairssetup.Foronlyone pairoffemales,sevenpupswerefoundaliveatday1.Threeofthese newbornsdiedduringtheirfirstweek,butthetwofemaleswere abletoraisethefourremainingpupstoadulthood.Atbirth,three ofthosefourpupshadhighanti-Borreliaantibodylevelswhilethe remainingonehadamuchlowerlevel(Fig.1).AsIgGagainst Bor-reliacanbetransmittedthroughtheplacentainrodents(Morshed etal.1993),thisresultsuggeststhatthosethreepupswereborn totheBorreliavaccinatedmother.Thefemalevaccinatedagainst influenzalikelygavebirthtotheremainingone.Anti-Borrelia anti-bodylevelsshowedasteepincreaseduringthefirstweekofage whichcanbeexplainedbytheacquisitionofmaternalantibodies fromthemilk.Allindividualsreachedveryhighlevelsby8days ofage.Theantibodylevelsremainedrelativelystablebetween8 and19daysofage,which isexpectedasnewbornsfedonmilk duringthisperiod.Afterwards,antibodylevelsdecreasedrapidly andreachednon-detectablelevelsby40daysofage.The dynam-icswassimilarforanti-influenzaantibodiesthroughouttherearing periodofnewborns(Fig.2).However,atbirth,allindividualshadno detectableantibodiesindicatingthatantibodiesagainstthe nucleo-proteinofinfluenzavirusesmaynotbetransmittedthroughthe placenta.

Allnewbornsdisplayedatsomepointduringtherearingperiod detectableantibodylevelsspecificallydirectedagainstboth Borre-liaandinfluenza.Althoughtherewasnocontrolgroupofoffspring raisedbyunvaccinatedmothers,thedecayofantibodylevels fol-lowingweaningisnotconsistentwithastimulationoftheimmune systemofthenewborns.It ratherfitsthedynamicsexpectedif thenewbornspassivelyacquired antibodiesfromboth females. Thisresultdemonstratestheefficiencyofallosucklingasasource ofantibodiesinnewbornsandprovidesevidenceforapotential immunologicalfunctionofallosuckling.Thetransmissionof anti-bodiesfromamothertofosteroffspringhasbeendescribedbefore butthisisthefirsttimethatcross-transferofantibodieshasbeen reportedinasocialcontext,whenfemalescanchoosewhichyoung tosuckleandwhennewbornscanchoosefromwhichfemaleto suck.

Anumberofchallengesrelatedtothebreedingofgerbilshave reduced thesamplesizein thepresentstudy. Awaytoreduce aggressivenessproblemscouldbetorearnon-sexuallymature indi-vidualsingroupsofonemaleandtwofemales.Becausethesocial groupwouldnothavebeenmodiﬁedrecentlybeforethefemales givebirth,lowerlevelsofinfanticidecouldbeexpected(e.g.Elwood 1991).Anotherpossibilityistopairfemalesonlyafterthepupswere born.Inthislattercase,theidentityofthemotherofthepupswould beknownandonecouldthusavoidsamplingnewbornsinthevery fewdaysfollowingbirth.However,suchadesignwouldlimitthe accessofpupstothecolostrumofnon-biologicalmothersandin turnmaymodifythedynamicsofacquisitionofpassiveantibodies bythenewborns.

Therapiddecayofantibodylevelsstartingafter20daysofage isassociatedwithweaning,whichusuallyoccursbetween21and

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Fig.1.Borreliaspeciﬁcantibodylevelsintheserumofthepupsatdifferentage.Antibodylevelsareexpressedasopticaldensities(OD)measuredbyELISA:ahighvalue

ofODcorrespondstohighantibodylevels.Colorsrefertothevaccinationofthebiologicalmother(greyforBorrelia,blackforinﬂuenza)andsymbolsarespeciﬁcofthe

individual.Plaincurvesrepresentthedynamicsofantibodiesacquiredfromthebiologicalmotherwhiledashedcurvesrepresentthedynamicsofantibodiesacquired

throughallosuckling.

30days inMongolian gerbils(Norrisand Adams1972).Atthat time,newbornsstopfeedingonmilkanddonotacquirepassive immunityfromtheirmothers.Thetransferofantibodiesfromthe intestinallumentothebloodstreamofthenewbornindeedrelies onareceptor,theneonatalFcreceptor(FcRn)whichhasinmice beenshowntobeexpressedinthegutonlyduringtheneonatal period(RoopenianandAkilesh2007).Ifandhowthisreceptoris expressedintheintestineofnewborngerbilsisunknownbutit probablyinﬂuencestheacquisitionofpassiveantibodies.

Ourresultsuggeststhatnewbornsmayreceiveantibodiesfrom severallactatingfemalesofasocialgroup.However,hownewborns willhaveaccesstoantibodiesislikelytovarywiththesynchrony ofbirthswithingroups.Offspringfromtheﬁrstlitter,usuallyborn tothedominantfemale,mayforinstancebeabletobroadenand

strengthentheirrepertoireofantibodieswiththecolostrumof fos-termothers.Conversely,pupsbornlatertosubordinatefemales wouldonlygainaccesstoalimitedamountofantibodiesfromthe dominantfemale’smilk.Theexistenceofanefﬁcientcross-transfer of antibodies may also contributeto the decision of dominant femalestoallowsubordinatebreeding.Insomesocialmammals, suchasbandedmongooses(Mungosmungo),subordinatefemales canalsobeforcedoutofthegroupduringthecourseofthe ges-tation (Cant et al. 2010; Gilchrist 2006). Excluded females can subsequentlybereintegratedinthegroupafteraborting.Should thisabortionoccurlateingestation,ithasthepotentialtoresult inspontaneouslactationwhich mayfurtherreducetheamount ofmaternalcarerequiredfromthedominantfemale.Inaddition, subordinatefemalesmayexperiencea ratherdifferentparasitic

Fig.2.Inﬂuenzaspeciﬁcantibodylevelsintheserumofthepupsatdifferentage.Antibodylevelsareexpressedaspercentageofinhibition(PI)bycomparisonwithacontrol

sample:ahighvalueofPIcorrespondstohighantibodylevels.Colorsrefertothevaccinationofthebiologicalmother(greyforBorrelia,blackforinﬂuenza)andsymbolsare

speciﬁcoftheindividual.Plaincurvesrepresentthedynamicsofantibodiesacquiredfromthebiologicalmotherwhiledashedcurvesrepresentthedynamicsofantibodies

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environmentduringthecourseofexclusionandbroadenthe reper-toireofthepupsofthedominantfemalethroughcross-transferof antibodies.

Theuseofvaccines (Staszewskiand Boulinier2004)as indi-vidual markers of females could allow the exploration of the cross-transferofantibodiesinthewild.Ifeachfemalefromasocial groupreceivesa differentvaccine,antibodiesagainst each vac-cinecouldbesearchedfor innewborns andused toassessthe transferofantibodiesthroughallosuckling.Thechoiceofthe vac-cinesshouldbemadecarefullynotonlytoensuresafetyforthe vaccinatedindividualsbutalsotheavailabilityofaccurateELISA techniquestomeasurespeciﬁcantibodylevelsinthepups.A sin-glebloodsampleofthenewbornatacarefullychosentimewould be sufﬁcient to obtain evidence for the cross-transfer of anti-bodies. Finer resolution antibodydynamics couldallowfor the explorationoftheeffectofbirthsynchronywithingroups.Social mammalssuchasmeerkats (Clutton-Brock etal.2001),banded mongooses(Cant2000)orrodents(e.g.theAlpinemarmot,Cohas andAllainé2009)forwhichlong-termmonitoringprogramsare alreadyimplemented mightproveparticularlysuitableforsuch studies.

Acknowledgements

TheauthorsthankAudreyArnal,CarineBrouat,Philippe Per-ret,AurorePonchonandAnneXuerebfordiscussionsandhelpat variousstagesoftheexperimentandtwoanonymousreviewers forcommentsonanearlierversionofthemanuscript.Technical helpfortheimmunologicalanalyseswasprovidedbythePACE.The membersofthe“Terraind’Expérience”platformattheCEFE-CNRS playedanimportantrolebydesigningandproducingthePlexiglas separations.Financialsupportforthisstudycamefromthe Cen-treNationaldelaRechercheScientiﬁque(CNRS)andtheAgence NationaledelaRecherche(ANR)grants07JCJC0128“EPICE”toSG and11BSV7003“EVEMATA”toTB.RGwassupportedbyaPhD fellowshipfromtheFrenchMinistryofResearch.

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