brazilian journal of microbiology49(2018)347–350
h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Veterinary
Microbiology
Efficacy
of
leptospiral
commercial
vaccines
on
the
protection
against
an
autochtonous
strain
recovered
in
Brazil
Rafael
Bazaglia
Sonada
a,
Sérgio
Santos
de
Azevedo
b,
Francisco
Rafael
Martins
Soto
c,
Diego
Figueiredo
da
Costa
b,
Zenaide
Maria
de
Morais
a,
Gisele
Oliveira
de
Souza
a,
Amane
Paldês
Gonc¸ales
d,
Fabiana
Miraglia
a,
Sílvio
Arruda
Vasconcellos
a,∗aUniversidadedeSãoPaulo(USP),FaculdadedeMedicinaVeterináriaeZootecnia(FMVZ),SãoPaulo,SP,Brazil bUniversidadeFederaldeCampinaGrande(UFCG),CentrodeSaúdeeTecnologiaRural(CSTR),Patos,PB,Brazil cInstitutoFederaldeEducac¸ão,CiênciaeTecnologiadeSãoPaulo(IFSP),RãoRoque,SP,Brazil
dUniversidadedeSantoAmaro(UNISA),SãoPaulo,SP,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received16March2017
Accepted9June2017
Availableonline12October2017
AssociateEditor:MilianeSouza
Keywords: Leptospirosis Vaccination Productionanimals Control
a
b
s
t
r
a
c
t
Inswineandbovines,leptospirosispreventionandcontroliscarriedoutviavaccination
ofsusceptibleanimalsusingbacterins.However,theefficiencyofleptospirosisvaccines
hasbeenquestioned.Thisworkaimedtoinvestigatethepotencyoffiveleptospirosis
vac-cinessoldcommerciallyinBrazil,challengingtheanimalswithoneautochthonousstrain
ofLeptospira,Canicolaserovar,denotedLO4,isolatedfromswine.Thestandardprotocolwas
followed,andrenalcarriersofLeptospirawereidentifiedamongthesurvivinganimalsby
cul-tureandPCR.Ofthefivevaccinestested,onlytwoprovedeffective.Noneofthesurviving
animalswaspositivebyculture;however,oneanimalwaspositivebyPCR.Threeofthefive
vaccinessoldcommerciallyinBrazilfortheimmunizationofswineorbovinesfailedthetest
oftheefficacytoprotectthevaccinatedanimalsfollowingchallengewithanautochthonous
Leptospirastrain,Canicolaserovar.Thetwovaccinesprovidedprotectionagainsttherenal
carrierstateinthesurvivinganimals.Thecriteriausedtoproduceleptospirosisbacterins
soldcommerciallyinBrazilmustbereviewed.Theindustryshouldsupportresearcheson
leptospiralvaccinologytoimprovethequalityofthepresentvaccinesanddiscovernew
immunogenicstrains,becauseitisknownthatvaccinationisoneofthemostimportant
toolstoincreasethereproductionratesinlivestock.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mail:savasco@usp.br(S.A.Vasconcellos).
https://doi.org/10.1016/j.bjm.2017.06.008
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC
348
brazilian journal of microbiology49(2018)347–350Introduction
Vaccinationisoneofthemainmeansofcontrolling
leptospi-rosisinproductionanimals.1Vaccinationisperformedwith
bacterins,suspensionsofcompletepolyvalentbacterialcells
composedofthemostfrequentserovarsinaparticularregion
or country.2 Many commercial leptospirosisvaccines
avail-able in Brazil for the immunization of bovines and swine
are polyvalent, containing fiveor more serovars, including
Pomona,Icterohaemorrahagiae,Hardjo,Canicola,Wolffi,
Grip-pothyphosa,BratislavaandTarassovi.3
Theefficacyoftheswineorbovidaeleptospirosisvaccines
ishighlyquestionablebecausetheyinducelowproductionof
protectiveantibodiesandarerarelyproducedwiththestrains
that affect the herds.4 Despite the existence of promising
studiesrelatedtotheproductionofauniversalvaccinethat
canprovidecross-protectionbetweendifferentserovarsand
reducerenalcolonization,5thevaccinesonthemarketinduce
ashort-durationimmunityandgenerallydonotprevent
dis-easetransmission.6
SeveralleptospirosisvaccinessoldcommerciallyinBrazil
areproducedabroad,importedandonlypackagedinBrazil.
EventhoseproducedinthecountryusereferenceLeptospira
strains, which are antigenically distinct from those in the
field7andthusareincapableofpromotingeffectiveprotection
againstdisease,theinfectionortheestablishmentoftherenal
carrierstatewhentheanimalsareexposedtolocalstrains.8
Butit must beconsideredthat the mainobjectiveof
com-mercialvaccinesforlivestockisthereductionofreproductive
problems.9Inthetestoftheefficacyperformedinhamsters
ofnineleptospirosisbacterinssoldcommerciallyinBrazilfor
theimmunizationofdogs,onlytwowereeffective.10
Bearinginmind thequestions raisedabout the efficacy
oftheleptospirosisvaccinesforswineandbovidaecurrently
commercializedinBrazil,thisworktestedtheeffectiveness
offiveleptospirosisvaccines followingachallenge withan
autochthonousstrainofLeptospiraisolatedfromswine liver
inBraziland typedbymonoclonalantibodies asserogroup
CanicolaandserovarCanicola,strainLO4.
Material
and
methods
Bioethicspermission
Thestudy wasapprovedbythe bioethicscommitteeofthe
VeterinaryMedicineandZootechnyFacultyoftheUniversity
ofSãoPaulo,Protocol1430/2008.
Animals
Youngandhealthymalegoldenhamsters(Mesocricetus
aura-tus)weighing50–90gwereused.Theanimalswerehousedin
polypropyleneboxes,withtheirweightshomogeneously
dis-tributedbetweengroups.Theboxeswerelinedwithsawdust,
andtheanimalsreceivedtapwaterandpelletizedcommercial
feedadlibitum.
Vaccines
Five polyvalent bacterin vaccines produced forthe
immu-nizationofswineorbovidaeandsoldcommerciallyinBrazil
are here identifiedas vaccinesA, B,C,Dand E: VaccineA
–serovarsCanicola,Grippotyphosa,Hardjo,
Icterohaemorra-hagiae, Pomona and Wolffi; Vaccine B– serovars Canicola,
Hardjo, Icterohaemorrahagiae,Grippotyphosa and Pomona;
VaccineC–serovarsPomona,Grippotyphosa,Canicola,
Ictero-haemorrahagiae, Wolffi and Hardjo; Vaccine D – produced
forswineimmunizationagainstparvoviruses,erysipelaand
leptospirosis,andinthecaseoftheleptospirosis,includesthe
serovarsBratislava,Canicola,Grippotyphosa,Hardjo,
Ictero-haemorrahagiaeandPomona;VaccineE–producedforswine
immunizationagainstparvovirosis,erysipelasand
leptospi-rosis and, in the case of the leptospirosis, includes the
serovarsPomona,Grippotyphosa,Canicola,
Icterohaemorra-hagiae,BratislavaandHardjo.
Potencytest
The vaccines were evaluated in hamsters according to
the standard procedure, AmericanCode FederalRegulation
norm,11 but as the tested vaccines were produced for the
immunizationofbovineandswineandnotfordogstheywere
dilutedat1:800oftherecommendeddosebytheirrespective
manufactures.
Challengestrain
The challenge was performed with the LO4 strain of the
serogroupCanicola,serovarCanicolaisolatedfromtheliver
ofaslaughteredpig12andtypedbyacollectionofmonoclonal
antibodies(RoyalTropicalInstitute,Amsterdam,The
Nether-lands)asLeptospirainterrogansserovarCanicola.13
Experimentalgroups
Thehamstersweredistributedintosixcagesof10animals:
(1)agroupvaccinatedwithbacterinA;(2)agroupvaccinated
withbacterinB;(3)agroupvaccinatedwithbacterinC;(4)a
groupvaccinatedwithbacterinD;(5)agroupvaccinatedwith
bacterinE;and(6)acontrolgroupthatwasnotvaccinated.
All the animals were manually restrained forthe
subcuta-neousapplicationof0.25mLofthebacterin.After15days,the
animalswerechallengedwith0.2mLoflivecultureofthe
chal-lengestrainviatheintraperitonealroute.Theanimalswere
observedfor14consecutivedays,countingthosethatdiedof
leptospirosis.Attheendofthisperiod,totestforleptospiral
renalinfections,thesurvivinganimalswereeuthanizedina
CO2chamberandnecropsiedsothattheirkidneyscouldbe
harvestedforcultureandPCR.
Determinationoflethaldose(LD50)ofchallengestrain
Forthechallenge,suspensionsoflivertissuefromhamsters
experimentallyinfectedwithLO4strainsandeuthanizedin
theagonicphaseofthediseasewereprepared.Thesetissues
were macerated inthe proportion1.0goftissuefor9.0mL
ofmodifiedEMJHliquidmedium,thendilutedto10−1,from
whichserialdilutions(10−2,10−3,10−4,10−5,and10−6)were
carriedout.Thechallengeinoculaweretitratedinhamstersin
brazilian journal of microbiology49(2018)347–350
349
Table1–HamstersimmunizedagainstleptospirosisandchallengedwithstrainLO4,serovarCanicola,theproportionof animalskilledbyleptospirosisbyeachvaccine,theconclusionsforthevaccinesandthecontrol,andtheproportionsof animalsshowingpositiverenalcultureandPCRforleptospirosis.
Vaccinesused Proportionofdeadanimals Conclusionforvaccinesandcontrol Positiverenalcultures PositiverenalPCRs
VaccineA 10/10a Ineffective — — VaccineB 4/20 Effective 0/16b 1/16c VaccineC 10/10 Ineffective — — VaccineD 1/10 Effective 0/9 0/9 VaccineE 10/10 Ineffective — — Control 10/10 Ineffective — —
a Numberofanimalskilledbyleptospirosis/totalnumberofimmunizedanimals.
b NumberofanimalswithpositiverenalcultureforLeptospira/totalnumberofanimalssurvivingthechallenge. c NumberofanimalspositiveforPCR/totalnumberofanimalssurvivingthechallenge;—,notcarriedout.
ofoneofthedilutions(10−1to10−10)oftheinfectious
inocu-lumbytheintraperitonealroute.Theanimalswereobserved
dailyfor14days,andthelethaldose(LD50)wascalculatedby
anestablishedmethod.14Theinfectiveinoculaappliedinthe
firsttrialwas1440andintheretest309.
Investigationoftherenalcarrierstateofleptospiresin survivalanimals
On the 14th post-infection day (p.i.d.), the surviving
ham-sterswereeuthanizedinaCO2chamberandnecropsied.Their
kidneys were aseptically harvested, and a fragment ofthe
organwastestedforLeptospirabypolymerasechainreaction
(PCR).15 Theremainingsegmentsofkidneyweremacerated
and dilutedto10−1 ina buffered salinesolution and then
dilutedtwicemore(10−2and10−3).Onehundredmicroliters
perdilutionwasplatedintubeswithBakelitelidscontaining
5.0mLFletcher’ssemisolidmedium,whichwereincubatedin
anovenat28–30◦Cforsixweeksandexaminedweekly16for
thegrowthring(Dingerzone)ofLeptospira.Thepresenceof
Leptospirawasconfirmedbydarkfieldmicroscopy.
Results
Basedonthefirstchallengeandtheexaminationofrenal
tis-sueofthesurvivinganimals,onlyvaccineDwasfoundtobe
effective,withonlyoneanimalkilledbyleptospirosis.All
ani-malsvaccinatedwithA,CandEdiedofleptospirosis,asdidall
oftheanimalsinthecontrolgroup(Table1).Inthefirst
chal-lengetheresultsforvaccineBwereinconclusive,withthree
deathsandnoneoftherenaltissuefromanysurviving
ani-malwaspositiveforLeptospirabycultureorPCR.Intheretest,
vaccineBappearedtobeeffective;however,PCRtestingofthe
renaltissuesofthe16survivinganimalsyieldedonepositive
result,butallcultureswerenegative.
Discussion
and
conclusion
Inspiteof theinvestigation ofin vitro potencytestof
Lep-tospiravaccines,9,17thevaccination-challengetestsperformed
inhamsterare still animportantprocedurefor controlling
thesebacterins.Ofthefivecommerciallyavailable
leptospi-rosisbacterinsproducedfortheimmunizationofbovidaeor
swineinBrazil,onlytwo,vaccinesBandDwerefoundtobe
effective.Theresultsagreewithotherfindings10of
unsatisfac-toryperformancebyleptospirosisbacterinssoldinBrazilfor
theimmunizationofdogs;sevenofthoseninevaccinesfailed.
VaccinesBandDalsopreventedtherenalcarrierstatein
thesurvivinganimals.WhenvaccineBwasretested,one
ani-malwaspositivebyPCR14daysfollowingthechallenge.This
findingisincontrasttotheresultsofthestudyofthecanine
vaccine,inwhichtheanimalsbecameculture-positiveafter
vaccinationwithtwooffivevaccines.10
Althoughthe maingoal oflivestockvaccinationagainst
leptospirosisisthedecreaseofreproductiondrawbacksand
notnecessarilythesterileimmunity,therenalcarriageof
Lep-tospireinvaccinatedanimalscanpromotetheenvironment
contaminationandthespreadofthebacteriaintotheherd
andalsotootheranimalhostsincludingwildones.8,17
ThepositivePCRfromtheanimalvaccinatedwithvaccine
Bdoesnotdefinitivelyindicatethatthevaccinewasnot
effec-tiveagainsttherenalcarrierstate.PCRismoresensitivethan
cultureandcandetectDNAbothofdeadandlive
microorgan-isms,whileculturerequiresviablebacteriaandisvulnerable
tosamplecontamination.18
Itisimportanttohighlightthelargenumberofvaccines
thatfailedfollowingchallengewiththeindigenousBrazilian
strain. Noleptospirosisvaccine producedinBrazilincludes
localstrains,insteadtheyincludereferencestrainsthatcould
beantigenicallydistinctfromfieldstrains.7Evenvaccinated
animals arenotprotectedagainstinfectionand renal
colo-nizationbyLeptospira.8
It isnecessary torevise the criteria appliedtothe
pro-duction oftheleptospirosisbacterins sold commerciallyin
Brazilfortheimmunizationofswineandbovines.The
indus-try should supportresearchesin leptospiralvaccinology to
improvethequalityofthepresentvaccinesanddiscoverynew
immunogenicstrains,becauseitisknownthatvaccinationis
oneofthemostimportanttoolstoincreasethereproduction
ratesinlivestock.9
Conflicts
of
interest
350
brazilian journal of microbiology49(2018)347–350Acknowledgements
WewouldliketothankFundac¸ãodeAmparoàPesquisado
Estadode SãoPaulo (FAPESP)forthefinancial support(no.
2010/17001-1).
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