Menezes-Souzab, Bruno Mendes Roatta, Rodrigo Dian de Oliveira Aguiar-Soaresa, Fernando Augusto Siqueira-Mathiasa, Jamille Mirelle de Oliveira Cardosoa, Rodolfo Cordeiro Giunchettia,b, Renata Guerra de Sác, Rodrigo Corrêa-Oliveirab, Cláudia Martins Carneir

(1)

ContentslistsavailableatScienceDirect

Molecular

Immunology

j ourna l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / m o l i m m

Dogs

immunized

with

LBSap

vaccine

displayed

high

levels

of

IL-12

and

IL-10

cytokines

and

CCL4,

CCL5

and

CXCL8

chemokines

in

the

dermis

Juliana

Vitoriano-Souza

a

_,

_Nádia

_das

_Dores

_Moreira

a

_,

_Daniel

_{Menezes-Souza}

b

_,

Bruno

Mendes

Roatt

a

_,

_Rodrigo

_Dian

_de

_Oliveira

_{Aguiar-Soares}

a

_,

Fernando

Augusto

Siqueira-Mathias

a

_,

_Jamille

_Mirelle

_de

_Oliveira

_Cardoso

a

_,

Rodolfo

Cordeiro

Giunchetti

a,b

_,

_Renata

_Guerra

_de

_Sá

c

_,

_Rodrigo

_{Corrêa-Oliveira}

b

_,

Cláudia

Martins

Carneiro

a,d

_,

_Alexandre

_Barbosa

_Reis

a,d,e,∗

a_Laboratório_de_{Imunopatologia,}_Núcleo_de_Pesquisas_em_Ciências_{Biológicas/NUPEB,}_Universidade_Federal_de_Ouro_Preto,_Ouro_Preto,_Minas_Gerais,_Brazil b_Laboratório_de_Imunologia_Celular_e_Molecular,_Instituto_de_Pesquisas_René_Rachou,_Fundac¸_ão_Oswaldo_Cruz,_Belo_Horizonte,_Minas_Gerais,_Brazil c_Laboratório_de_Bioquímica_e_Biologia_Molecular,_Núcleo_de_Pesquisas_em_Ciências_{Biológicas/NUPEB,}_Universidade_Federal_de_Ouro_Preto,_Ouro_Preto,

MinasGerais,Brazil

d_Departamento_de_Análises_Clínicas,_Escola_de_Farmácia,_Universidade_Federal_de_Ouro_Preto,_Ouro_Preto,_Minas_Gerais,_Brazil e_Instituto_Nacional_de_Ciência_e_Tecnologia_em_Doenc¸_as_Tropicais_-_INCT-DT,_CNPq,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received27March2013

Receivedinrevisedform22May2013

Accepted23May2013

Available online 1 August 2013

Keywords:

Caninevisceralleishmaniasis

Cytokines Chemokines Vaccine Saponin Adjuvants

Real-timePCR

a

b

s

t

r

a

c

t

Thecomplex interplaybetweencytokinesand chemokinesregulates innateand adaptive immune responses againstpathogens; specifically, cytokine and chemokine expressiondrivesactivation of immuneeffectorcellsandtheirrecruitmenttotissueinfectionsites.Herein,weinoculateddogswith Leishmaniabraziliensisantigensplussaponin(theLBSapvaccine),aswellaswiththevaccinecomponents, andthenusedreal-timePCRtoevaluatethekineticsofdermalexpressionofmRNAsofcytokines(IL-12, IFN-␥,TNF-␣,IL-4,IL-13,TGF-␤andIL-10)andchemokines(CCL2,CCL4,CCL5,CCL21andCXCL8)1,12,24 and48hafterinoculation.Wealsoevaluatedthecorrelationbetweencytokineandchemokine expres-sionanddermalcellularity.TheLBSapvaccineinducedhighlevelsofIL-12andIL-10expressionat12and 24h,respectively.Furthermore,weobservedpositivecorrelationsbetweenIL-12andIL-13expression, IFN-␥andIL-13expression,andIL-13andTGF-␤expression,suggestingthatamixedcytokine microen-vironmentdevelopedafterimmunizationwiththevaccine.Inoculationwiththesaponinadjuvantalone inducedachemokineandcytokineexpressionprofilesimilartothatobservedintheLBSapgroup.CCL4 andCXCL8chemokineexpressionwasupregulatedbytheLBSapvaccine.CCL5expressionwasinitially highestintheLBSapgroup,butat48h,expressionwashighestintheLBgroup.Informationaboutthe kineticsoftheimmuneresponsetothisvaccinegainedusingthisdogmodelwillhelptoelucidatethe mechanismsofandfactorsinvolvedinaprotectiveresponseagainstLeishmaniainfectionandwillaidin establishingrationalapproachesforthedevelopmentofvaccinesagainstcaninevisceralleishmaniasis.

1. Introduction

Human visceral leishmaniasis are heterogeneous diseases caused by obligate intracellular protozoan parasites of the genusLeishmaniaandaretransmittedbythebiteofthefemale

∗_{Corresponding}_author_at:_Laboratório_de_{Imunopatologia,}_Núcleo_de_Pesquisas

emCiênciasBiológicas,ICEBII,MorrodoCruzeiro,UniversidadeFederaldeOuro

Preto,35400-000OuroPreto,MinasGerais,Brazil.Tel.:+553135591694;

fax:+553135591680.

E-mailaddresses:alexreis@nupeb.ufop.br,alexreisufop@gmail.com(A.B.Reis).

phlebotominesandfly(Desjeux,2004).Dogsinfectedwithcanine visceralleishmaniasis(CVL)constitutethemaindomesticreservoir andplaya centralroleinthetransmissioncycleoftheparasite tohumans(Deane,1995).Therefore,acaninevaccinemaybethe mostpractical and effectivemethodfor reducing theincidence ofhumanvisceralleishmaniasis,andmayalsoprovideabasisfor thedevelopmentofasimilarvaccineforhumans(Gradoni,2001; Hommeletal.,1995;Mauel,2002).

In the search for a potential vaccine against CVL, various approachesinvolvingthedogmodelhavebeenemployed,andthe useofpurifiedfractionsfromparasiteextracts(e.g.,fucose man-noseligandantigen)(Borja-Cabreraetal.,2004;daSilvaetal.,2000)

(2)

56 (2013) 540–548 541

andfromparasitecultures(excreted/secretedantigens)(Lemesre etal.,2005,2007)have shownparticularpromise.Additionally, remarkableresultshave beenobtainedfollowing vaccinationof dogswithkilledparasitevaccines(Giunchettietal.,2007,2008a; Lasrietal.,1999;Mayrinketal.,1996;Moreiraetal.,2009;Panaro etal.,2001;Vitoriano-Souzaetal.,2008;Roattetal.,2012).These vaccinesrepresentanexcellenttoolforimmunoprophylaxisand controlofCVL,consideringtheirwide spectrumofantigenicity, aswellas theircost and safety(Giunchettietal., 2007,2008a; Lasrietal.,1999;Mayrinketal.,1996;Moreiraetal.,2009;Panaro etal.,2001; Vitoriano-Souzaetal., 2008).Moreover,inanimals thatreceivesaponinasanadjuvant,themajoradversereaction isminorlocalswelling,indicatingthatindogs,overalltolerance to the candidate vaccines appears to be adequate (Giunchetti etal.,2007,2008a,b;Moreiraetal.,2009;Vitoriano-Souzaetal., 2008).

Understanding the cellular immune responses of dogs to infectionwithLeishmaniaparasites iscrucial for vaccinedesign (Strauss-Ayalietal.,2007).InvolvementofIL-10intheimmune responseofLeishmania-infecteddogshasnotyetbeenestablished insomeorgans,becauseexpressionofIL-10mRNAisnotelevated inantigen-stimulatedperipheralbloodmononuclearcellsduring thecourseofanexperimentalinfectionorinthebonemarrowof naturallyinfecteddogs(Quinnelletal.,2001;Santos-Gomesetal., 2002).Lageetal.(2007)suggestedthatseverityCVLalsoismarked bythebalancedsplenicproductionoftype1and2cytokineswith thepredominantaccumulationofIL-10andIFN-␥asaconsequence ofincreasedparasiticloadandprogressionofthesymptoms. Fur-thermore,Menezes-Souzaetal.(2011)showedthatdogswithhigh skinparasitismexhibitedpredominantlyimmunoregulatory pat-ternofimmuneresponsecharacterizedbyincreasedexpressionof IL-10andTGF-␤.

IL-4mRNAisexpressedinthebone marrowofsome symp-tomaticdogs(Quinnelletal.,2001),andhighIL-4expressionwas recentlydetectedlocallyinskinlesionsofL.infantum–infected dogs(Brachelenteetal.,2005).Thus,thesecontradictoryfindings havebeenproventhattheroleofIL-4intheCVLprogressionstill unclearneedingmorestudiestoclarifytheirrealfunctioninthe naturalhistoryofthecaninedisease(Menezes-Souzaetal.,2011). Interferon-␥ expression in antigen-stimulated peripheral blood mononuclearcells,skinlesionsandbonemarrowofinfecteddogs hasbeenshowntobeelevated(Quinnelletal.,2001;Brachelente et al., 2005; Chamizo et al., 2005; Strauss-Ayali et al., 2005). Thus,thisIFN-␥increasedisresponsibleforthemainmechanism involvedinprotectiveimmuneresponseofdogsinfectedwithL. (L.)infantum,throughactivationofmacrophagestokillintracellular amastigotes(Vouldoukisetal.,1996).

The potential roles of chemokines in Leishmania infection include host defense functions such as leucocyte recruitment, participationincell-mediatedimmunity,cellactivationand anti-Leishmaniaactivity(RotandvonAndrian,2004).Chemokinesplaya vitalroleintheregulationofimmunitytovariousdiseasesaffecting theskin,includinghumancutaneousleishmaniasis(Guptaetal., 2011).However,theimmunomodulatoryprocesses triggeredby chemokines in assessments of the immunogenicity of vaccines againstCVLhavenotbeenwellstudied,andthereforeweaddressed thisissueinthepresentstudy.

SuccessfulimmunizationagainstinfectionbyLeishmaniaspp. requires theparticipationof both innate and adaptive immune responses.Thecytokineandchemokine microenvironment pro-motes immediate recruitment of cells that drive the immune response.Inthisprospectivestudy,weinvestigatedthekinetics oftheexpressionofcytokineandchemokinemRNAsintheskinof dogsafterimmunizationwithLeishmaniabraziliensisantigensplus saponinasanadjuvant(theLBSapvaccine),aswellasinoculation withtheseparatecomponentsofthevaccine.

2. Materialsandmethods

2.1. Animals

The details of the proposed study were presented to, and approvedby,theEthicalCommitteefortheUseofExperimental AnimalsoftheUniversidadeFederaldeOuroPreto, OuroPreto, MG, Brazil.As subjects, we used 12 mongrel dogs(males and females),8–12monthsold,whichwerebornandraisedinthe ken-nelsoftheCenterofAnimalScience,UniversidadeFederaldeOuro Preto.Thedogsweretreatedwithananthelminticandvaccinated againstrabies(Tecpar,Curitiba,PR,Brazil),caninedistemper,type2 adenovirus,coronavirus,parainfluenza,parvovirusandleptospira (VanguardHTLP5/CV-L;PfizerAnimalHealth,NewYork,NY,USA). Twodayspriortotheexperiments,thedorsalareaofallanimals wasshaved,andnolocalreactionswereobserved.

2.2. Immunizationprotocol

Thedogsweresubdividedintofourgroupsofthreedogsper group.TheSapgroupwasinoculatedwith1mgofsaponin(Sigma ChemicalCo., St.Louis, MO,USA), theLBgroupwasinoculated with600␮gofL.braziliensistotalproteins,theLBSapgroupwas inoculatedwith600␮gofL.braziliensistotalproteinsand1mgof saponin,andtheSgroup(control)wasinoculatedwithan equiva-lentvolumeofsterile0.9%salinesolution.Adjuvantsandantigens weredilutedwithsterile0.9%salinesolutiontoatotalvolumeof 250␮L.Followingapplicationofageneralanaesthetic(Thiopentax; Cristália,Itapira,SP,Brazil;7mg/kgofbodyweight),theinoculants wereintradermallyinjectedintotheshaveddorsalareaofthe ani-mals.Atintervals,skinbiopsieswerecollectedfromtheinoculation areasandstoredat−80◦_C_until_required_for_RNA_analysis.

Descrip-tiveandkinectstudiesofthebiopsiedtissuewereperformedat1, 12,24and48h.Thehistologicalanalysiswasperformedaccording toVitoriano-Souzaetal.(2008).Briefly,ahistologicalexamination (morphometricanalysisandleucocytedifferentialcounting)of sec-tionsstainedwithhaematoxylinandeosin(HE).Thekineticsofcell migrationwasevaluatedwithinthreeskinlayers(outerdermis, innerdermisandhypodermis)andthepredominanceofcellsin theinflammatoryinfiltrateandtheirdistributionwithintheskin layerswereassessed.Aquantitative(morphometric)analysisof theinflammatorycellspresentintheskinlayerswasperformed by acquiring digital images of pre-marked areas. Images were capturedat40 and 100× magnificationusinga LeicaDM5000B micro-camera (Leica Microsystems-Switzerland Ltd., Heerbrugg, Switzerland)and LeicaApplicationSuitesoftware(version2.4.0 R1), andanalyzed withtheaidofLeica QWin(V3)software.In ordertoidentifywhichtypesofcellswererecruitedtothesites ofdifferentinoculationssites,theinflammatorycells(neutrophils, eosinophils,macrophagesandlymphocytes)werecountedandthe resultswereexpressedin percentages.Herein,correlation anal-ysisbetweenthe cellularityand thecytokines and chemokines mRNA expression in the skin of these same animals was per-formed.

2.3. ExtractionoftotalRNAandsynthesisoffirst-strandcDNAs

(3)

542 56 (2013) 540–548

Table1

Sequencesofforward(F)andreverse(R)primersusedforreal-timePCRquantificationofmRNAexpressioninskinbiopsiesofimmunizeddogs.

Gene Primersequence(5′_–3′₎ _Product_length_(bp) _Reaction_efficiency_(%) _R2 _GenBank_accession_no. GAPDH

F TTCCACGGCACAGTCAAG 115 99.1 0.996 AB038240

R ACTCAGCACCAGCATCAC IL-12p40

F CAGCAGAGAGGGTCAGAGTGG 109 96.5 0.989 U49100

R ACGACCTCGATGGGTAGGC IFN-␥

F TCAACCCCTTCTCGCCACT 113 95.4 0.967 AF126247

R GCTGCCTACTTGGTCCCTGA TNF-␣

F CGTCCATTCTTGCCCAAAC 94 97.2 0.983 DQ923808

R AGCCCTGAGCCCTTAATTC IL-4

F CACCTCCCAACTGATTCCAA 123 96.9 0.991 AF239917

R CTCGCTGTGAGGATGTTCAA IL-13

F CCTCCTCAGAGCAAAGTG 148 96.7 0.973 AF244915

R CCCAGCACAAACAAAGAC IL-10

F AGAACCACGACCCAGACATC 129 97.1 0.993 U33843

R CCACCGCCTTGCTCTTATTC TGF-␤1

F AGGATCTGGGCTGGAAGTG 134 95.1 0.981 L34956

R CGGGTTGTGCTGGTTGTA CCL2

F CCTGCTGCTATACACTCA 91 96.3 0.979 U29653

R GCTTCTTTGGGACACTTG CCL4

F TCCTACTGCCTGCTGCTT 76 95.9 0.981 AB183194

R GCTGGTCTCAAAGTAATCTGC CCL5

F TTCTACACCAGCAGCAAG 136 98.7 0.991 AB098562

R TTCTACACCAGCAGCAAG CCL21

F AGTCTGGCAAGAAGGGAAAG 60 96.7 0.972 AB164433

R GGGTCTGTGGCTGTTCAGT CXCL8

F ACACTCCACACCTTTCCAT 116 98.7 0.977 AF048717

R GGCACACCTCATTTCCATTG

bymeasuringtheOD ratioat 260/280nm usingNanovue® _(GE Healthcare, USA). To confirm the integrity of extracted RNA, electrophoresisofafractionofeachRNAsamplewasperformed onadenaturing agarosegeland verifiedboth the18Sand 28S ribosomal RNA(rRNA) bands. Theprocedureincluded a DNAse treatment step, and the efficiency of this step was evaluated byPCRamplificationofthecDNAreactionmixturewithoutthe additionoftheThermoScriptenzyme.EachquantitativePCRrun wasperformedwithtwointernalcontrolsassessingbothpotential contaminationbygenomicDNA(noreversetranscriptaseadded) and reagent purity (no cDNA added). First-strand cDNAs were synthesizedfrom1.0␮goftotalRNAusingtheThermoScript RT-PCRSystem(InvitrogenBrasil,SãoPaulo,SP,Brasil)witholigo-dT primersaccordingtothemanufacturer’sinstructions.

2.4. Designofprimersforgeneevaluation

PrimersweredesignedwiththeaidofGeneRunner(ver.3.05, HastingSoftwareInc.)usingspecificcaninesequencesobtained fromGenBank(http://www.genbank.nlm.nih.gov).Thesequences oftheprimersemployedarelistedinTable1.Theprimerswere synthesizedbyEurogentec(Southampton,UK)andreconstituted innuclease-freewater.

2.5. Real-timePCR

Real-time PCR was performed on an ABI Prism 7000 DNA SequenceDetectionSystemusing10␮LofSYBRGreenPCRMaster

Mix(PEAppliedBiosystems,FosterCity,CA,USA),4␮Lof100mM primersand8␮LofcDNAdilutedat1:5carriedoutinafinal vol-umeof20␮L.Thesampleswereincubatedat95◦_C_for₁₀_min_and

thensubmittedto40cyclesof95◦_C_for₁₅_s_and₆₀◦_C_for₁_min,

duringwhichtimefluorescencedatawerecollected.Theefficiency ofeachpairofprimerswasevaluatedbyserialdilutionofcDNA accordingtothe protocoldeveloped byPE Applied Biosystems. Toevaluategeneexpression,weperformedthreereplicate anal-yses,andtheamountoftargetRNAwasnormalizedwithrespect totheendogenous control (referencegenes) geneGAPDH. Data weregeneratedbymeansofthe2−Ct_method_using_the_mean valueoftheCtofthecontrolgroupasthecalibrator(Livakand Schmittgen,2001).PCRproductswereclonedwithpGEM-TEasy Vector(Promega)andsequencedtocheckspecificityusinganABI 3100AutomatedSequencer (PEApplied Biosystems)and a Dye TerminatorKit.

2.6. Statisticalanalysis

(4)

56 (2013) 540–548 543

Fig.1.RelativeexpressionofmRNAsoftypeI(IL-12,IFN-␥andTNF-␣),typeII(IL-4andIL-13)andimmunoregulatory(IL-10andTGF-␤)cytokinesinthedermisofdogs

1,12,24and48hafterinoculationwithL.braziliensisantigens(LBgroup; ),saponin(Sapgroup; )orL.braziliensisantigensplussaponin(LBSapgroup;).Significant

(5)

544 56 (2013) 540–548

3. Results

3.1. CytokinemRNAexpressionprofilesintheskinofdogsafter immunization

EvaluationofcytokinemRNAexpressionisessentialfor improv-ingourunderstandingoftheprotectivecellularimmuneresponse inducedbyvaccines againstcanineleishmaniasis. Therefore,we evaluatedtheexpressionofIL-12,IFN-␥,TNF-␣,IL-4,IL-13,

TGF-␤andIL-10mRNAsinthedermisofdogs1,12,24and48hafter inoculationwiththeLBSapvaccineoritscomponents(Fig.1).At 12h,expressionofIL-12intheLBSapgroupwashigherthanthat intheSapgroup,and,interestingly,theexpressionofIL-10was alsohigherintheLBSapgroupthanintheSapgroup.Nosignificant differencesintheexpressionofIFN-␥,TNF-␣,IL-4,IL-13orTGF-␤

wereobserved(Fig.1).

Wecarriedoutadetailedanalysisofthecorrelationsbetween type1,type2andimmunoregulatorycytokines(Fig.2)andfound thatintheLBgroup,therewasapositivecorrelationbetweenIL-13

andTNF-␣(p<0.0001,r=0.8927).IntheSapgroup,therewere pos-itivecorrelationsbetweenIL-13andTNF-␣(p=0.0002,r=0.8749), betweenIL-13andIL-12(p=0.0040,r=0.7622) andbetween IL-13andIFN-␥(p=0.0170,r=0.6708).IntheLBSapgroup,positive correlationswereobservedbetweenIL-13andIL-12(p<0.0001, r=0.9455)andbetweenIL-13andIFN-␥(p=0.0194,r=0.6606).In theLBgroup,apositivecorrelationwasobservedbetweenIL-10 and IL-4(p=0.0014, r=0.8344), and intheLBSap groupa posi-tivecorrelationwasobservedbetweenTGF-␤andIL-13(p=0.0020, r=0.7948).

3.2. ChemokinemRNAexpressionprofilesintheskinofdogsafter immunization

We evaluated the expression of the mRNAs of CCL2, CCL4, CCL5,CCL21andCXCL8(IL-8)chemokines1,12,24,and48hafter inoculationtheLBSapvaccineoritsindividualcomponents(Fig.3). At24hafterinoculation,expressionofCCL4mRNAintheLBSap groupwas11.6timesthatintheLBgroup(p<0.05).At1hafter

IL-10

IL-4

IL-13

0 10 20

p<0.0001

r=0.8927

0 20 40

0 10 20

p=0.0020

r=0.7948

TNF-

α

0 10 20

p=0.0002

r=0.8749

IL-13

0 20 40

0 10 20

p=0.0170

r=0.6708

0 30 60

0 20 40

p=0.0194

r=0.6606

0 10 20

p=0.0040

r=0.7622

0 20 40

p<0.0001

r=0.9455

IFN-

γ

IL-12

p=0.0014

r=0.8344

(A) Type I versus Type II Cytokines

0 10 20

0 15 30

(B) Type II versus Immunoregulatory Cytokines

TGF-β

IL-13

Fig.2.Correlationsbetweenrelativeexpressionof(A)typeIandIIcytokinemRNAsand(B)typeIIandimmunoregulatorycytokinemRNAsinthedermisofdogsinoculated

withL.braziliensisantigens(LBgroup; ),saponin(Sapgroup; )orL.braziliensisantigensplussaponin(LBSapgroup;䊉).Pearson’scorrelationindexes(r)andp-values

(6)

56 (2013) 540–548 545

Fig.3. RelativeexpressionofmRNAofchemokinesCCL2,CCL4,CCL5,CCL21andCXCL8inthedermisofdogs1,12,24and48hafterinoculationwithL.braziliensisantigens

(LBgroup; ),saponin(Sapgroup; )orL.braziliensisantigensplussaponin(LBSapgroup;).Significantdifferences(p<0.05)comparedwithLB,SapandLBSapare

indicatedbytheletters“a,”“b”and“c,”respectively.

inoculation,CCL5expressionwassignificantlyupregulatedinthe LBSap groupin comparison withexpression in theLB and Sap groups (4.0-fold and 6.8-fold,respectively; p<0.05). At12 and 24hafterinoculation,CCL5expressionintheLBgroupwashigher thanthatintheSapgroup(28.2-foldand72.1-fold,respectively; p<0.05). Also, 48h after inoculation, we observed increases in expressionof these chemokine (CCL5)in the LBgroup relative toexpressionintheSapandLBSapgroups(6.9-foldand5.6-fold, respectively;p<0.05).Inaddition,CXCL8expressionwas signif-icantlyhigherintheLBSapgroupthanintheLBandSapgroups

24h(13.9-foldand6.4-fold,respectively;p<0.05)and48hafter inoculation(13.6-foldand4.7-fold,respectively;p<0.05).There werenosignificantdifferencesinexpressionofCCL2andCCL21 betweenthegroups.

3.3. Correlationofinflammatorycellsintheouterandinner dermiswithcytokineandchemokineexpression

(7)

546 56 (2013) 540–548

Neutrophils

Outer dermis

Inner dermis

0 50 100

0 2 4

p=0.0005

r=0.8917

CCL5

0 40 80

0 2 4

CCL5

0 40 80

0 30 60

p=0.0240

r=0.6434

IL

-4

Macrophages

Neutrophils

mRNA Relative expression 2

-ΔΔCT

Number of cells (%)

0 40 80

0 10 20

p=0.0479

r=0.5803

IL-13

0 40 80

0 2 4

p=0.0030

r=-0.8020

CCL5

0 40 80

0 20 40

p=0.0138

r=0.6859

IL-12

0 40 80

0 20 40

p=0.0125

r=0.6930

IL-13

0 40 80

0 30 60

p=0.0015

r=0.8085

IFN

-γ

0 40 80

0 20 40

p=0.0040

r=0.7615

TNF

-α

Lymphocytes

0 40 80

0 10 20

CCL5

p=0.0345

r=-0.6117

0 40 80

0 30 60

p=0.0351

r=0.6103

IL-12

p=0.0083

r=-0.7464

Fig.4.CorrelationsbetweenrelativeexpressionofcytokineandchemokinemRNAsandpercentagesofmacrophagesandneutrophilsintheouterandinnerdermisofdogs

afterinoculationwithL.braziliensisantigens(LBgroup; ),saponin(Sapgroup; )orL.braziliensisantigensplussaponin(LBSapgroup;䊉).Pearson’scorrelationindexes

(r)andp-valuesareshownonthegraphs.

(Fig.4).Weobservedimportantcorrelationsbetweenchemokine and cytokineexpression and cellularityin the outerdermis. In the Sap group, macrophages and CCL5 expression were posi-tivelycorrelated(p=0.0005,r=0.8917),andneutrophilsandCCL5 expression were negatively correlated (p=0.0083, r=−0.7464). IntheLBSapgroup,neutrophilswerepositivelycorrelatedwith IFN-␥(p=0.0015,r=0.8085),IL-12(p=0.0351,r=0.6103)andIL-4 (p=0.0240,r=0.6434)expression.

Intheinnerdermis,weobservedapositivecorrelationbetween neutrophilsandIL-13(p=0.0479,r=0.5803)intheLBgroup.As in theouter dermis, there wasa negative correlationbetween CCL5and neutrophils(p=0.0030,r=−0.8020)in theSapgroup. Inthe LBSapgroup,we observed positivecorrelations between neutrophilsandIL-12(p=0.0138,r=0.6859)andbetweenTNF-␣

(p=0.0040,r=0.7615)andIL-13(p=0.0125,r=0.6930),aswellas anegativecorrelationbetweenlymphocytesandCCL5(p=0.0345, r=−0.6117).

4. Discussion

ThesearchfornewimmunoprophylacticsagainstCVLshould focusonprospectiveantigenscapableofinterveningintheimmune systeminsuchawayastoinduceaspecificimmunologicalevents related to parasite elimination. The development of immunity againstinfectiousagentssuchasLeishmaniaspp.requiresthe con-certedactionofcomponentsofboththeinnateandtheadaptive immunesystems(Teixeiraetal.,2005).Thedifferentialregulation oftype1andtype2responsesandtheassociationofThcytokines andimmunefunctionareimportanttostudytheadaptiveimmune responsetoLeishmaniainfections(Menezes-Souzaetal.,2011).

(8)

56 (2013) 540–548 547

of neutrophilsto theskinof animalsimmunized withSap and LBSaparesimilartothereactionsobservedinanimalsthatexhibit resistancetoLeishmaniainfection(Vitoriano-Souzaetal.,2008). Basedontheseresultsitispossibletohypothesizethatneutrophils functionatthefrontlineoftheimmunesystem,responding imme-diatelyupon request, anddirect themigration ofother cellsto theinoculation area somehours afterwards (macrophages and lymphocytes).It appears,therefore, thatneutrophils participate effectivelyintheadaptiveimmuneresponsefromthevery begin-ningoftheprocess(Vitoriano-Souzaetal.,2008).Inthissense,the investigationofthekineticsofcellmigrationtotheinoculationarea isextremelyrelevantsincethenumberandtypesofcellsrecruited immediatelyafterinoculationwillstimulatetheinnateimmune systemandwillinfluencethedevelopmentofacquiredimmunity. Toexpandourinvestigationoftheimmuneresponsetriggered bythecomponentsoftheLBSapvaccine,weevaluatedexpression ofcytokine andchemokine mRNAsin theskinof dogs,seeking biomarkersofimmunogenicityinthecontextoftheinnateimmune response.Wecarriedoutakineticsstudyoftherelativeexpression ofthemRNAsoftype I,IIandimmunoregulatorycytokinesand chemokinesinducedbythevaccineanditsseparatecomponents atthesiteofinoculationafter1,12,24and48h.Additionally,we evaluatedthecorrelationsbetweencytokineandchemokinemRNA expressionandcellularityintheouterandinnerdermis.

WeanalyzedexpressionofIL-12,IFN-␥,TNF-␣,IL-4,IL-13,

TGF-␤andIL-10mRNAsinthedermisofdogsimmunizedwiththeLBSap vaccineandfoundthatexpressionofIL-12andIL-10mRNAsat12 and24h,respectively,washigherintheLBSapgroupthaninthe Sapgroup.Moreover,weobservedpositivecorrelationsbetween expressionofthemRNAsoftypeI(IL-12,IFN-␥,TNF-␣)andtype IIcytokines(especiallyIL-13)in thegroupsinoculated withthe separatevaccinecomponentsand withtheLBSapvaccineitself. Theseresultssuggestthatafteradministrationofeachinocula,a microenvironmentofmixedtypeIandIIcytokinesdevelopsinthe skinofanimals.Cytokinesplaya decisiverolein theregulation oftheimmuneresponsedirectedagainstL.infantum, determin-ingtheprevention orprogression of theinfection(Carrillo and Moreno,2009).Severalstudieshavedemonstratedthatamixed cytokinepatterncanbeassociatedwithresistanceor susceptibil-ityin both vaccineand Leishmania-infection models(Raziuddin andAbdalla,1994;Peruhype-Magalhaesetal.,2006).Inmurine leishmaniasis,severalresearchershaveobservedthatIL-13 synthe-sispromotesinitialIFN-␥productionandinfluencestheassembly andmaturationoftissuegranuloma.However,suchexperiments havenot addressedthemechanism(s)bywhich IL-13regulates theexpressionofanti-leishmanialtype1response(Murrayetal., 2006).Menezes-Souzaetal.(Menezes-Souzaetal.,2011) demon-strated that asymptomatic Leishmania-infected animals present highexpressionofIL-13withconcomitantIFN-␥production.The authorssuggestthatinflammatorycytokineprofiles,particularly thosedrivenbyIFN-␥,TNF-␣andIL-13,couldbeusedas biomark-ersforasymptomaticclinicalformsinCVL.

InstudiesofHIVinfectionswasdemonstratedeffectsofboth IL-13andIFN-␥onHIV-1macrophageinfections(Meylanetal.,1993; Kornbluthetal.,1989),toenhanceAgpresentingfunction(deWaal Malefytetal.,1993;Marshalletal.,1997),andtobeassociatedwith improveddiseasestatus(increasedCD4ordecreasedHIVviralload) suggestacontributionbythesecytokinesinmaintainingimmune functionfollowingHIV-1infection(Robertetal.,1999).

In this study, we observed increased expression of some chemokinesintheskinofdogsinoculatedwiththevariousvaccine components.Isimportanttonotethatchemokinesproducedatthe siteofinfectionarecriticalindeterminingthecellsthatinfiltrate thesiteandthusindefiningtheeventualoutcomeofthedisease (Teixeiraetal.,2006).Inaddition,Leishmaniaparasitesalsopossess achemotacticfactorthatselectivelyattractsneutrophilswhichcan

serveashostcellsintheearlystagesofinfection(vanZandbergen etal.,2002).

Our datashowedincreasedexpressionofCCL4 mRNAin the LBSapgroup24hafterinoculation.Inaddition,significant upregu-lationofCCL5mRNAexpressionwasobserved1hafterinoculation intheLBSapgroup.Attheotherbiopsytimes,thelevelsofCCL4 andCCL5mRNAswerehigherintheLBgroupthanintheother groups.ThesedatademonstratetheabilityoftheLBSapvaccineto inducetheproductionofchemokines(CCL4andCCL5),whichare importantintherecruitmentofimmunecellstotheinoculation site.Thesechemokinespreferentiallyrecruitmonocytestotheskin, suggestingahoststrategyforcontrollingparasitismduringongoing CVLinfection(Menezes-Souzaetal.,2011).Inaddition,the expres-sionofCCL5mayberelatedtoresistancetoinfectionbyLeishmania followingthecascadeofeventsleadingtoparasitecontrolduring L.majorinfection(Santiagoetal.,2004).

Inthisstudy,weobservedhigherlevelsofCXCL8expressionin theskinofdogsvaccinatedwiththeLBSapvaccinethaninthedogs intheothergroups.InthefirstfewhoursofLeishmaniainfection, CXCL8productionamplifiestherecruitmentofneutrophilstothe infectionsite(vanZandbergenetal.,2002;Badolatoetal.,1996; Venuprasadetal.,2002).Neutrophilsrepresentthefirstgroupof leukocytestoarriveatthesiteofinfection,andtheyarethought toserveasTrojanhorsesforeventualentryofintomacrophages, theultimatecellularhostforLeishmania.However,theinitialinflux ofneutrophilsseemstobebeneficialforLeishmaniasurvivalinthe infectedtissue(vanZandbergenetal.,2004).

Theinteractionsofcytokinesandchemokineswithvariouscells in theskinlayers after immunizationare importantfor under-standingthe mechanisms bywhich adaptive immunityagainst Leishmaniainfectionisactivated.Ourcorrelationanalysisbetween celltypesandchemokineexpressionintheouterdermisshoweda positivecorrelationbetweenmacrophagesandCCL5andanegative correlationbetweenneutrophilsandCCL5intheSapgroup.These dataconfirmtheroleofCCL5intherecruitmentofmacrophages andothermonocytes.IntheLBSapgroup,neutrophilswere posi-tivelycorrelatedwithincreasedexpressionofIFN-␥,IL-12andIL-4 mRNAs,andamixedprofileofcytokineswasobservedintheskin.In theinnerdermis,neutrophilsandIL-13expressionwerepositively correlatedintheLBgroup.Aswasthecaseintheouterdermis,CCL5 expressionandneutrophilswerenegativelycorrelatedintheSap group.IntheLBSapgroup,neutrophilswerepositivelycorrelated withexpressionofIL-12,TNF-␣andIL-13mRNAs.

The developmentof immunitydepends on themigration of appropriate cell populations to infected sites, which in turn dependsontheexpressionofcytokinesandchemokines. Addition-ally,theknowledgeofthecellprofileofthelocalinflammatory infiltratecanprovidesupplementaryinformationconcerningthe immune response in the microenvironment of the inoculation (Stewartetal.,1984).Takentogether,ourcurrentresultsallowus toconcludethatintheskinmicroenvironmentofdogsimmunized withLBSapvaccine,therewasamixedcytokineprofileanda dis-tinctiveexpressionprofileforCCL4,CCL5andCXCL8mRNAs.Our datacanbeexpectedtocontributetotheestablishmentofa ratio-nalstrategyforthedevelopmentofvaccinesandimmunological therapiesagainstvisceralleishmaniasis.

Acknowledgements

(9)

548 56 (2013) 540–548

gratefulfortheuseoffacilitiesatCEBIO,UniversidadeFederalde MinasGerais,andRedeMineiradeBioterismo(FAPEMIG),andfor supportwiththeprovisionofexperimentalanimals.

References

Badolato,R.,Sacks,D.L.,Savoia,D.,Musso,T.,1996.Leishmaniamajor:infectionof

humanmonocytesinducesexpressionofIL-8andMCAF.Experimental Parasi-tology82,21–26.

Borja-Cabrera,G.P.,CruzMendes,A.,ParaguaideSouza,E.,HashimotoOkada,L.Y.,

de,A.T.F.A.,Kawasaki,J.K.,etal.,2004.Effectiveimmunotherapyagainstcanine

visceralleishmaniasiswiththeFML-vaccine.Vaccine22,2234–2243.

Brachelente,C.,Muller,N.,Doherr,M.G.,Sattler,U.,Welle,M.,2005.Cutaneous

leishmaniasisinnaturallyinfecteddogsisassociatedwithaThelper-2-biased immuneresponse.VeterinaryPathology42,166–175.

Carrillo,E.,Moreno,J.,2009.Cytokineprofilesincaninevisceralleishmaniasis.

Vet-erinaryImmunologyandImmunopathology128,67–70.

Chamizo,C.,Moreno,J.,Alvar, J.,2005.Semi-quantitativeanalysisofcytokine

expressioninasymptomaticcanineleishmaniasis.VeterinaryImmunologyand Immunopathology103,67–75.

daSilva,V.O.,Borja-Cabrera,G.P.,CorreiaPontes,N.N.,deSouza,E.P.,Luz,K.G.,

Palat-nik,M.,etal.,2000.AphaseIIItrialofefficacyoftheFML-vaccineagainstcanine

kala-azarinanendemicareaofBrazil(SaoGoncalodoAmaranto,RN).Vaccine 19,1082–1092.

deWaalMalefyt,R.,Figdor, C.G.,Huijbens,R.,Mohan-Peterson,S.,Bennett,B.,

Culpepper,J.,Dang,W.,Zurawski,G.,deVries,J.E.,1993.EffectsofIL-13on

phenotype,cytokineproduction,andcytotoxicfunctionofhumanmonocytes: comparisonwithIL-4andmodulationbyIFN-gorIL-10.JournalofImmunology 151,6370.

Deane,L.M.,Deane,M.P.,1995.Leishmaniosevisceralurbana(nocãoenohomem)

emSobral,Ceará.Hospital,75–87.

Desjeux,P.,2004.Leishmaniasis:currentsituationandnewperspectives.

Compar-ativeImmunology,MicrobiologyandInfectiousDiseases27,305–318.

Giunchetti,R.C.,Correa-Oliveira,R.,Martins-Filho,O.A.,Teixeira-Carvalho,A.,Roatt,

B.M.,deOliveiraAguiar-Soares,R.D.,etal.,2007.Immunogenicityofakilled

Leishmaniavaccinewithsaponinadjuvantindogs.Vaccine25,7674–7686.

Giunchetti,R.C.,Reis,A.B.,daSilveira-Lemos,D.,Martins-Filho,O.A.,Correa-Oliveira,

R.,Bethony,J.,etal.,2008a.Antigenicityofawholeparasitevaccineas

promis-ingcandidateagainstcanineleishmaniasis.ResearchinVeterinaryScience85, 106–112.

Giunchetti,R.C.,Correa-Oliveira,R.,Martins-Filho,O.A.,Teixeira-Carvalho,A.,Roatt,

B.M.,deOliveiraAguiar-Soares,R.D.,etal.,2008b.AkilledLeishmaniavaccine

withsandflysalivaextractandsaponinadjuvantdisplaysimmunogenicityin dogs.Vaccine26,623–638.

Gradoni,L.,2001.Anupdateonantileishmanialvaccinecandidatesandprospects

foracanineLeishmaniavaccine.VeterinaryParasitology100,87–103.

Gupta,G., Dey, R.,Bhattacharyya, S., Majumdar, S.,2011. Role of exogenous

chemokinesasimmunotherapeutictoolagainstvisceralleishmaniasis.Science AgainstMicrobialPathogens:CommunicatingCurrentResearchand Technolog-icalAdvances,605–612.

Hommel,M.,Jaffe,C.L.,Travi,B.,Milon,G.,1995.Experimentalmodelsfor

leishman-iasisandfortestinganti-leishmanialvaccines.AnnalsofTropicalMedicineand Parasitology89(Suppl.1),55–73.

Kornbluth,R.S.,Oh,P.S.,Munis,J.R.,Cleveland,P.H.,Richman,D.D.,1989.

Inter-feronsandbacteriallipopolysaccharideprotectmacrophagesfromproductive infectionbyhumanimmunodeficiencyvirusinvitro.JournalofExperimental Medicine169,1137.

Lage,R.S.,Oliveira,G.C.,Busek,S.U.,Guerra,L.L.,Giunchetti,R.C.,Correa-Oliveira,R.,

etal.,2007.Analysisofthecytokineprofileinspleencellsfromdogsnaturally

infectedbyLeishmaniachagasi.VeterinaryImmunologyandImmunopathology 115,135–145.

Lasri,S.,Sahibi,H.,Sadak,A.,Jaffe,C.L.,Rhalem,A.,1999.Immuneresponsesin

vaccinateddogswithautoclavedLeishmaniamajorpromastigotes.Veterinary Research30,441–449.

Lemesre,J.L.,Holzmuller,P.,Cavaleyra,M.,Goncalves,R.B.,Hottin,G.,Papierok,G.,

2005.Protectionagainstexperimentalvisceralleishmaniasisinfectionindogs

immunizedwithpurifiedexcretedsecretedantigensofLeishmaniainfantum promastigotes.Vaccine23,2825–2840.

Lemesre,J.L.,Holzmuller,P.,Goncalves,R.B.,Bourdoiseau,G.,Hugnet,C.,Cavaleyra,

M.,etal.,2007.Long-lastingprotectionagainstcaninevisceralleishmaniasis

usingtheLiESAp-MDPvaccineinendemicareasofFrance:double-blind ran-domisedefficacyfieldtrial.Vaccine25,4223–4234.

Livak,K.J.,Schmittgen,T.D.,2001.Analysisofrelativegeneexpressiondatausing

real-timequantitativePCRandthe2(-DeltaDeltaC(T))Method.Methods25, 402–408.

Marshall,J.D.,Robertson,S.E.,Trinchieri,G.,Chehimi,J.,1997.PrimingwithIL-4and

IL-13duringHIV-1infectionrestoresinvitroIL-12productionbymononuclear cellsofHIV-infectedpatients.JournalofImmunology159,5705.

Mauel,J.,2002.VaccinationagainstLeishmaniainfections.CurrentDrugTargets:

Immune,EndocrineandMetabolicDisorders2,201–226.

Mayrink,W.,Genaro,O.,Silva,J.C.,daCosta,R.T.,Tafuri,W.L.,Toledo,V.P.,etal.,

1996.PhaseIandIIopenclinicaltrialsofavaccineagainstLeishmaniachagasi

infectionsindogs.MemoriasdoInstitutoOswaldoCruz91,695–697.

Menezes-Souza,D.,Correa-Oliveira,R.,Guerra-Sa,R.,Giunchetti,R.C.,

Teixeira-Carvalho,A.,Martins-Filho,O.A.,etal.,2011.Cytokineandtranscriptionfactor

profilesintheskinofdogsnaturallyinfectedbyLeishmania(Leishmania)chagasi presentingdistinctcutaneousparasitedensityandclinicalstatus.Veterinary Parasitology177,39–49.

Meylan,P.R.,Spina,C.A.,Richman,D.D.,Kornbluth,R.S.,1993.Invitrodifferentiation

ofmonocytoidTHP-1cellsaffectstheirpermissivenessforHIVstrains:amodel systemforstudyingthecellularbasisofHIVdifferentialtropism.Virology193, 256–267.

Moreira, N., Giunchetti, R.C., Carneiro, C.M., Vitoriano-Souza, J., Roatt, B.M.,

Malaquias,L.C.,etal.,2009.Histologicalstudyofcellmigrationinthedermis

ofhamstersafterimmunisationwithtwodifferentvaccinesagainstvisceral leishmaniasis.VeterinaryImmunologyandImmunopathology128,418–424.

Murray,H.W.,Tsai,C.W.,Liu,J.,Ma,X.,2006.VisceralLeishmaniadonovaniinfection

ininterleukin-13−/− mice.InfectionandImmunity74,2487–2490.

Panaro,M.A.,Acquafredda,A.,Lisi,S.,Lofrumento,D.D.,Mitolo,V.,Sisto,M.,etal.,

2001.Nitricoxideproductionbymacrophagesofdogsvaccinatedwithkilled

Leishmaniainfantumpromastigotes.ComparativeImmunology,Microbiology andInfectiousDiseases24,187–195.

Peruhype-Magalhaes,V.,Martins-Filho,O.A.,Prata,A.,SilvaLde,A.,Rabello,A.,

Teixeira-Carvalho,A.,etal.,2006.Mixedinflammatory/regulatorycytokine

pro-filemarkedbysimultaneousraiseofinterferon-gammaandinterleukin-10and lowfrequencyoftumournecrosisfactor-alpha(+)monocytesarehallmarksof activehumanvisceralLeishmaniasisduetoLeishmaniachagasiinfection.Clinical andExperimentalImmunology146,124–132.

Quinnell,R.J.,Courtenay,O.,Shaw,M.A.,Day,M.J.,Garcez,L.M.,Dye,C.,etal.,2001.

Tissuecytokineresponsesincaninevisceralleishmaniasis.JournalofInfectious Diseases183,1421–1424.

Raziuddin,S.,Abdalla,R.E.,el-Awad,E.H.,al-Janadi,M.,1994.Immunoregulatoryand

proinflammatorycytokineproductioninvisceralandcutaneousleishmaniasis. JournalofInfectiousDiseases170,1037–1040.

Roatt,B.M.,Aguiar-Soares,R.D.,Vitoriano-Souza,J.,Coura-Vital,W.,Braga,S.L.,

Correa-Oliveira,R.,etal.,2012.PerformanceofLBSapvaccineafter

intrader-malchallengewithL.infantumandsalivaofLu.longipalpis:immunogenicity andparasitologicalevaluation.PLoSONE7,e49780.

Robert,T.,Bailer,A.H.,Sun,Junwei,Joseph,B.Margolick,Martin,Melissa,Kostman,

Jay,Montaner,LuisJ.,1999.IL-13andIFN-gsecretionbyactivatedTcellsin

HIV-1infectionassociatedwithviralsuppressionandalackofdiseaseprogression. TheJournalofImmunology,7534–7542.

Rot,A.,vonAndrian,U.H.,2004.Chemokinesininnateandadaptivehostdefense:

basicchemokinesegrammarforimmunecells.AnnualReviewofImmunology 22,891–928.

Santiago,H.C.,Oliveira,C.F.,Santiago,L.,Ferraz,F.O.,deSouza,D.G.,de-Freitas,

L.A.,etal.,2004.InvolvementofthechemokineRANTES(CCL5)inresistance

toexperimentalinfectionwithLeishmaniamajor.InfectionandImmunity72, 4918–4923.

Santos-Gomes,G.M.,Rosa,R.,Leandro,C.,Cortes,S.,Romao,P.,Silveira,H.,2002.

Cytokineexpressionduringtheoutcomeofcanineexperimentalinfectionby Leishmaniainfantum.VeterinaryImmunologyandImmunopathology88,21–30.

Stewart,R.J.,Holloway,L.J.,Isbister,W.H.,1984.Peritonealneutrophilia:apotential

indicatorofthesurgicalacuteabdomen.AustralianandNewZealandJournalof Surgery54,565–568.

Strauss-Ayali,D.,Baneth,G.,Shor,S.,Okano,F.,Jaffe,C.L.,2005.Interleukin-12

aug-mentsaTh1-typeimmuneresponsemanifestedaslymphocyteproliferation andinterferongammaproductioninLeishmaniainfantum-infecteddogs. Inter-nationalJournalforParasitology35,63–73.

Strauss-Ayali,D.,Baneth,G.,Jaffe,C.L.,2007.Splenicimmuneresponsesduring

caninevisceralleishmaniasis.VeterinaryResearch38,547–564.

Teixeira,C.R.,Teixeira,M.J.,Gomes,R.B.,Santos,C.S.,Andrade,B.B.,Raffaele-Netto,

I.,etal.,2005.SalivafromLutzomyialongipalpisinducesCCchemokineligand

2/monocytechemoattractantprotein-1expressionandmacrophage recruit-ment.JournalofImmunology175,8346–8353.

Teixeira, M.J., Teixeira, C.R., Andrade, B.B., Barral-Netto, M., Barral, A., 2006.

Chemokinesinhost–parasiteinteractionsinleishmaniasis.Trendsin Parasito-logy22,32–40.

vanZandbergen,G.,Hermann,N.,Laufs,H.,Solbach,W.,Laskay,T.,2002.Leishmania

promastigotesreleaseagranulocytechemotacticfactorandinduce interleukin-8releasebutinhibitgammainterferon-inducibleprotein10productionby neutrophilgranulocytes.InfectionandImmunity70,4177–4184.

vanZandbergen,G.,Klinger,M.,Mueller,A.,Dannenberg,S.,Gebert,A.,Solbach,

W.,etal.,2004.Cuttingedge:neutrophilgranulocyteservesasavectorfor

Leishmaniaentryintomacrophages.JournalofImmunology173,6521–6525.

Venuprasad,K.,Banerjee,P.P.,Chattopadhyay,S.,Sharma,S.,Pal,S.,Parab,P.B.,

etal.,2002.Humanneutrophil-expressedCD28interactswithmacrophageB7

toinducephosphatidylinositol3-kinase-dependentIFN-gammasecretionand restrictionofLeishmaniagrowth.JournalofImmunology169,920–928.

Vitoriano-Souza,J.,Reis,A.B.,Moreira,N.D.,Giunchetti,R.C.,Correa-Oliveira,R.,

Carneiro,C.M.,2008.Kineticsofcellmigrationtothedermisandhypodermis

indogsvaccinatedwithantigeniccompoundsofLeishmaniabraziliensisplus saponin.Vaccine26,3922–3931.

Vouldoukis,I.,Drapier,J.C.,Nussler,A.K.,Tselentis,Y.,DaSilva,O.A.,Gentilini,M.,

etal.,1996.Caninevisceralleishmaniasis:successfulchemotherapyinduces