h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Veterinary
Microbiology
Development
of
an
indirect
ELISA
based
on
recombinant
capsid
protein
to
detect
antibodies
to
bovine
leukemia
virus
Ana
Paula
Andreolla,
Luana
Marina
Scheer
Erpen,
Rafael
Frandoloso,
Luiz
Carlos
Kreutz
∗UniversidadedePassoFundo(UPF),FaculdadedeAgronomiaeMedicinaVeterinária(FAMV),LaboratóriodeMicrobiologiaeImunologia Avanc¸ada,PassoFundo,RS,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received15January2018
Accepted3May2018
Availableonline22May2018
AssociateEditor:RoxanePiazza
Keywords:
Bovineleukemiavirus
Retrovirus Diagnosis ELISA
a
b
s
t
r
a
c
t
Serologicaltestingandcullinginfectedanimalsarekeymanagementpracticesaiming
erad-icationofbovineleukemiavirusinfection.Here,wereportthedevelopmentofanindirect
ELISAbasedonBLVrecombinantcapsidprotein(BLVp24r)todetectanti-BLVantibodiesin
cattleserum.TheBLVp24rwasexpressedinEscherichiacoliandpurifiedbyaffinity
chro-matography,andthenusedtosetuptheELISAparameters.ThePolysorp®platecoatedwith
50ngofantigen/wellandbovineserumdiluted1:100gavethebestresultsduring
standard-ization.Usingserafrominfectedandnon-infectedcattlewesetupthecutoffpointat0.320
(OD450nm)withasensitivityof98.5%andspecificityof100.0%.Then,wetested1.187serum
samplesfromdairy(736samples)andbeefcattle(451samples)withunknownstatustoBLV.
Wefoundthat31.1%(229/736)and9.5%(43/451)ofsamplesamongstdairyandbeefcattle,
respectively,hadIgGstoBLV.TherateofagreementwithacommercialcompetitiveELISA
was84.3%withavalueof0.68.Thus,ourBLVp24riELISAissuitabletodetectBLVinfected
animalsandshouldbeausefultooltocontrolBLVinfectionincattle.
©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis
anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Bovine leukemia virus (BLV) is the etiological agent of
a neglected, silent lifelong infection commonly found in
dairycattle1 namedenzooticbovine leukosis(EBL).A large
∗ Correspondingauthor.
E-mail:[email protected](L.C.Kreutz).
percentile ofanimal infected bythe BLV mightbe
asymp-tomatic or aleukemic (AL), at least at the initial stagesof
infection, whileup to 30% developa persistent
lymphocy-tosis (PL)2 that in some cases, depending on the animal’s
Bolagenotype,3progressestoBcelllymphoma.4,5 The
infec-tion isconsiderederadicatedinseveralparts ofthe world6
but iswidelyspreadinNorth7–9 andmostSouthAmerican
countries10inwhichdairyfarmingisconsidered an
impor-tanteconomicactivity.Thereisnoofficialdataontherateof
infectioninBraziliandairyfarmandmostlimitedandbiased
https://doi.org/10.1016/j.bjm.2018.05.001
1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC
epidemiologicalstudiesindicatethattherangeof
seroposi-tiveanimalvaries from12.5% to100%.11,12 InSouthBrazil,
inwhichmosthigh productivity dairyfarmsare located, a
within-herdinfectionrateusuallyexceeds30%.13,14BLV
trans-missionmightoccurduringpregnancy,calvingorbyingesting
colostrumfrominfectedcows.15Intheherd,BLVspreading
occursmostlybymanagementpracticessuchasvaccination,
dehorning, ear tagging, artificial insemination and uterine
palpation8,16,17that, whenperformedwiththesame
equip-ment, might transfer BLV-infected cells from infected to
non-infectedanimals.
Historically,BLVwasconsideredaquitebenigninfection
ofcattlemainlybecauselymphomas weredetectedmostly
inolderanimalsthatwere stillkeptintheherd.1 However,
inthe lastdecade,several studiesindicatedthatBLV
nega-tivelyaffectsthe productivityofdairycows18–20 and thatB
andTlymphocytesfromBLV-infectedanimalshaveimpaired
immunefunctions,21whichcouldaffecttheoverallimmune
statusbyreducingtheabilitytorespondtovaccineantigens22
and bypredisposing toother infectious diseases including
mastitis.Furthermore,atremendousturnoveronBLVresearch
isonthewaydrivenbydataindicatingthatBLVmightbe
asso-ciatedwithcertain typesofhumancancer.23–25 Altogether,
thesedatashouldencourageofficialmeasurestointroduce
compulsorydiagnosisaimingtolimitthespreadofBLVand
initiatecontrolanderadicationprograms.
BLVinfectedanimal producearobustanti-BLVhumoral
immuneresponsethatmightbedetectedbyagargel
immun-odiffusion(AGID) or byimmune-enzymatic assays such as
ELISA.26,27 In Brazil, antigen to AGID is produced on fetal
lambkidney (FLK)cellspersistentlyinfectedwithBLVsbut,
unfortunately,itisscarceandnotreliable,andtheuseof
over-seasmadeELISAkitsfordiagnosisisrestrictedbyregulatory
agency,discouragingevenmorevoluntarydiagnosis.Because
several countries, mostly at the European Economic
Com-munityalreadyeradicatedEBL,thepresenceofBLV-infected
animalsintheherdmightsoonaffectinternationaltradeof
dairyproducts.Thus,controllingBLVspreadingamongstdairy
cattlebycontinuous diagnosis and culling infectedanimal
should becomemandatory toassure trading and safety of
dairyproducts.Withthisinmind,inthisstudyourmajorgoal
wastoevaluatearecombinantBLVcapsidproteinasantigen
todevelopanindirectELISA(iELISA)todiagnoseBLV-infected
cattle.
Material
and
methods
DNAextraction,cloningandsequencingofBLVcapsid proteingene
BloodsamplesfromacowinfectedbyBLV(positivebyAGID)
wascollectedwithEDTAandcentrifuged(1300×g/10min)to
obtainperipheralbloodmononuclearcells(PBMC)fromwhich
totalDNAwasextractedusingtheWizard®GenomicDNA
Purifi-cationKit(Promega,USA).TheamountofDNAwasmeasured
byspectrophotometryandstoredat−20◦Cuntiluse.
PCR primers were designed to amplify a 666bp DNA
fragment covering the complete nucleotide sequence of
the BLV capsid protein. Two restriction sites were added
in the primers to facilitate the directional cloning of the
amplifiedfragmentintothecloningvector(Forward(BamHI):
5-ATTAGGGGATCCCCAATCATATCTGAAGGGAATCGCAA-3;
Reverse(HindIII):5-TGGCAGAAGCTTTTAGAGAAGTGCAGGCT
GTTTCA-3).Theamplificationwasperformedusing100ngof
DNA,0.4Mofeachprimer,1.5UofPfuDNApolymerase,2.5L
of10× buffer supplementedwithMgSO4,100M ofdNTPs
(Promega, USA)and DNA/RNA-free water(Sigma, Brazil)to
afinalvolumeof50L.DNA wasdenaturedat95◦C/5min,
andthen amplifiedby35cycles ofdenaturing(94◦C/1min),
annealing(52◦C/40sec)andextension(72◦C/2min)followed
by a final amplification at 72◦C for 10min. The resulting
DNA fragment was analyzed by electrophoresis in agar
gel (1%), purified and cloned into the pGEM-T-Easy vector
(Promega, USA), and transformed into calcium
compe-tent TOP10 Escherichia coli (ThermoFisher Scientific®, USA).
Penicillin-resistantcloneswereobtainedandthepresenceof
the capsidproteingeneintothe vector(pGEM-BLVp24)was
confirmedbyPCRandbysequencingusingthevectorforward
primer (pUC/M13, Promega, USA). The sequence obtained
wascomparedwithreferencegenesavailableatGeneBank.
ExpressionofBLVcapsidprotein
ThepGEM-BLVp24vectorwasdigestedwithBamHIandHindIII
(Promega, USA) restriction enzymes and analyzed by low
meltingpointagargelelectrophoresis.TheDNAfragment
cor-responding to the complete sequence that codifies de p24
capsid protein was cut out of the gel, purified (Wizard SV
Gel, Promega,USA) and cloned into the pET-20 vector
pre-viously digested with the same restriction enzymes. The
resultingplasmid(pET-20-BLVp24)wastransformedinto
com-petentER2566E.coli(NewEnglandBiolabs,USA)forexpression
as a fusion protein containing histidine, a maltose
bind-ingprotein(MBP)and arestrictionsiteforthetobaccoetch
virus (TEV) protease at the N-terminus. The expressionof
therecombinantHis-Mbp-TEV-BLVp24(BLVp24r)wasinduced
overnightwith0.1Misopropyl-ˇ-D-thiogalactopyranoside(IPTG,
Sigma,USA).Thebacteriacellswerepelleted(4000×g,30min,
4◦C), suspended in lysis buffer (20mM NaH2PO4, 500mM
NaCl,10mMImidazol,pH8.0)andsonicatedthrice(70watts)
(Ultronic,Brazil).Then,thesonicatedbacteriawascentrifuged
(13,000×g, 1h, 4◦C) and the supernatant filtered (0.22M)
and purifiedusingtheÄkta PureChromatographySystem(GE
Healthcare, Germany) connected to a HisTrap (GE
Health-care,Germany)column.Thepurifiedproteinswerequantified,
aliquotedandstoredat−80◦untiluse.
Sodiumdodecylsulfatepolyacrylamidegelelectrophoresis (SDS-PAGE)
AliquotscontainingtheBLVp24rproteinelutedfromthe
His-TrapaffinitycolumnswereanalyzedbystandardSDS-PAGE
underreducingconditions(5%-mercaptoethanol)using5%
acrylamidestackinggeloverlaidon10%acrylamideresolving
gel.Samplescollectedpriortoandafterinductionwerealso
analyzedsimultaneously.TheSDS-PAGEgelwasstainedwith
CoomasieBlueR-250(Bio-RadLaboratories,USA)orusedfor
ProductionofpolyclonalantibodiestotheBLVp24r
TwoWistarratswereimmunizedtwice(subcutaneousroute,
21daysapart)with50g/doseofBLVp24rmixedtoMontanide
Gel01(15%v/v,Seppic,France).Bloodsampleswerecollected
priortoandduringimmunizationbypuncturingthecaudal
vein,andfinalbleedingwasperformedbycardiacpuncture.
All procedures withrats were performed underanesthesia
withIsoflurane(Cristalia,Brazil).Theexperimentalprotocol
wasapprovedbythelocalCommitteeonAnimalEthicsand
Usage(CEUA,protocol12/2014).
Bovineserumsamples
TheinhouseiELISAparameterswerestandardizedusing100
bovineserumsamplesthatwerepreviouslytestedinour
lab-oratorybyAGID(Tecpar,PR).Amongstthesesamples70were
negativeand30positivetoBLVantibodies.Outofthe70
nega-tivesamples,16werefromcowsthatwerealsonegativewhen
testedbyPCRtargeting theTax(nested PCR)and GAGBLV
genes.25ApositiveBLVreferenceserum(E05)kindlyprovided
bythe OIEreferencelaboratoryforEBLatthe Universityof
Leipzig(Germany)wasalsoincludedonthestudy.
Fortheepidemiologicalstudy,weused1.187bovineserum
samplesfromourVirologyDiagnosisLaboratoryandfromthe
VirologyLaboratoryattheFederalUniversityofSantaMaria
(UFSM).Sampleswereobtainedfromdairy(n=736)andbeef
(n=451)cattlefarmsatthecentralregionofRioGrandedoSul,
Brazil.Outofthese,werandomlyselected255serumsamples
andtestedthemalsobyacommercialanti-gp51competitive
BLVELISAkit(IngezimBLVCompac2.0,Spain)toestimatethe
rateofagreementandKappa()values.
Westernblotting
AfterSDS-PAGE, BLVp24r were transferredtonitrocellulose
membranes (Bio-Rad Laboratories, USA) using a semi-dry
apparatus(Electrosystems,Brazil). Thenitrocellulose
mem-branecontainingtheproteinswasblockedovernightat4◦C
withphosphatebufferedsaline(PBS,pH7.4)containing0.05%
Tween-20(PBS-T)and3%skimmilk(PBS-TSK)underconstant
shakingandthencutintostrip(4mmeach).Serumcollected
fromratspreviouslyandafterimmunizationwiththeBLVp24r,
andserumfromnon-infectedcows(IDGA/PCRnegative;n=8)
andcowsnaturallyinfectedwithBLV(n=8)wereanalyzedby
westernblotting.Ratorbovineserumsampleswerediluted
1:100in1%PBS-TSKandincubatedwiththemembranestrips
for1hat22◦Cunderconstantshaking.Then,themembranes
were washedthree times, 10mineach,with PBS-T.
Peroxi-daseconjugatedanti-rat,oranti-bovineIgGwholemolecule
wasdiluted1:1.000andincubatedwiththemembranesunder
thesameconditionsastheprimaryantibody.Afterwashing
thrice,themembraneswereincubatedwithperoxidase
sub-strate(4-Cloro-1-Naphthol+0.06% H2O2)for10minand then
transferredtodistilledwatertostopthereaction.
InhouseindirectELISA
Two commercially available polystyrene microplates
(Maxisorp® and Polysorp®, Nunc, USA) were evaluated
regardingtheabilitytoadsorbtheBLVp24r.Theplateswere
coated withBLVp24r(2g/well)dilutedin carbonate buffer
(pH9.6) at4◦Cfor12h and thenwashed threetimeswith
PBS-T. Platewells were blocked with PBS-TSM at 37◦C for
2h.Bovineserum positive(n=4) andnegative(n=4)toBLV
antibodies were diluted1:100inPBS-TSK 1%,added tothe
wellsandallowedtoreactwiththeantigenfor1hat37◦C,
in duplicates. After washing three times, peroxidase
con-jugated goat anti-bovine IgG (Sigma, USA) diluted1:10.000
in PBS-TSK 1% was added and the plates were incubated
as indicatedabove, followedbythree washesand addition
of substrate (3,3,5,5-tetrametilbenzidina+0.06% H2O2). The
plates were then incubated in thedarkat 22◦C for10min
andthereactionwasstoppedbyadding3NHCl.Theplates
werereadat450nmusingaSynergyHIplatereader(BioTek®,
USA). We used the optical density (OD) from three plates
to calculatethe mean positive/negative(P/N) ratio and the
mean within-platepercentcoefficientofvariation(CV%)as
previouslydescribed.28Theplateswerethencomparedusing
anindexobtainedbydividingtheP/NratiobytheCV%.
Afterselectingthebestplate(Polysorp®),theoptimal
anti-genconcentrationwasevaluatedinduplicatesusingdifferent
concentrationsoftheBLVp24r(2.0000,1.000,500,250,125,100
and50ng/well)inafinalvolumeof100L,andserafrom
nat-urallyinfected(n=16)ornon-infectedcows (n=16).For this
assay, the samplesnegativetoBLVantibodies bythe AGID
werefromcowsthatwerealsonegativetoBLVbyPCR.25The
optimalantigenconcentrationwasdefinedasthelowest
anti-genconcentrationthatcausednosignificantchangesinthe
absorbanceobtainedwiththepositiveandnegativesera.
The ideal serum dilution was determined by diluting
bovineserum(negativeandpositive,n=16each)from1:25to
1:200,andplatessensitizedwiththebestantigen
concentra-tion determinedintheprevious step(50ng/well). Theideal
dilution wasdetermined takinginto considerationthe best
specificityandsensitivityobtainingbythereceiveroperating
characteristics(ROC)curveperformedinallserainduplicates.
Thecut-offpointwassetbyanalyzingBLVnegative(n=70)
and positive (n=30) serum samples.Polysorp® plates were
coated with BLVp24r (50ng/well) and serum samples were
diluted1:100.Withthesamplesabsorbancevalues,we
deter-minedthethresholdfollowingtheROCcurveanalysis.
Statisticalanalysis
TheKolmogorov–Smirnovtestwasusedtodeterminethe
nor-maldistribution ofthe data. Theresultswere analyzedby
Kruskal–WallisorOneWayAnovafollowedbyTukeypost-test
according to the data. Significant differences were
consid-eredwhenp<0.05.Allthestatisticswereperformedusingthe
GraphPadPrismsoftware(GraphPad,USA).
Results
Cloning,expressionandcharacterizationofBLVp24r
A666bpDNAfragment(Fig.1A)wasobtainedandclonedinto
thepGEM-T-Easyvectorandthensub-clonedintothepET20
2 1 37 25 Rat serum Optical Density (450 nm) M 41 0 150 25 0.121 0.132 0.137 0.1490.1510.1640.1670.190 0.885 0.997 1.032 1.044 1.099 1.1871.488 1.491 0.116 1.032 37 50 75 100 250
Positive serum samples Negative serum samples
50 75 100 M 1 2 3 M 0.5 0.75 1.0 1.5 2.0 3.0
A
E
D
C
B
Fig.1–(A)Polymerasechainreaction(PCR)amplificationofthenucleotidesequencecorrespondingtotheBLVcapsid proteingene.LymphocytesfromBLV-infectedcows(lanes1)andfromanon-infectedcow(lane2)wereusedforDNA extractionandanalyzedbyPCR.Thefragmentamplifiedwas666nucleotidesinlength.M:1Kbpmolecularweightmarkers. (B)AnalysesoftherecombinantBLVcapsidprotein(BLVp24r).SampleswerecollectedpriortoIPTGinduction(lane1),after overnightinduction(lane2),andafterpurificationbytheHisTrapcolumn(lane3),analyzedbySDS-PAGEandstainedby Coomassieblue.Thesizeofthemolecularweightmarkersisindicatedontheleftside.(C)Westernblotanalysisofthe BLVp24rprotein.ThepurifiedBLVp24rproteinresolvedbySDS-PAGEandtransferredtonitrocellulosemembrane. MembranestripscontainingtheBLVp24rwereincubatedwithserumfromnon-infected(panelC;n=8)andnaturally BLV-infected(panelD;n=8)cows,andwithratserum(panelE)collectedpriorto(day0)orafterimmunization(day41)with theBLVp24r.AllserawerealsoevaluatedbyourinhouseiELISAandtheopticaldensity(OD450nm)ofeachserumis
indicatedbelowthestrips.M:molecularweightmarkers.
(BLVp24r).Theinsertedfragmentwassequencedandaligned
toreferenceBLVgenome (GenBank accessnumberK02120,
AP018032, LC080653, HE967302 and KT122858) resulting in
96–99%identity(datanotshown).
TheexpressionofthepET-20-BLVp24constructioninE.coli
ER2566wasanalyzedbySDS-PAGEandtheresultingfusion
proteinHis-Mbp-TEV-BLVp24(BLVp24r)hadamolecularmass
of 67kDa (BLVp24r+MBP 43kDa) (Fig. 1B). The expression
yielded14mg/Lofultra-pureBLVp24r.
Theimmunogenicityand reactivityofthe BLVp24rwere
demonstratedbywesternblotusingserafromimmunizedrats
andserafromnon-infectedorfromcowsnaturallyinfected
withBLV. Sera fromnon-infectedcows (Fig.1C)and serum
collected from rats prior to immunization failed to
recog-nized BLVp24(Fig. 1E-0) whilesera from naturally infected
cowsandpost-inoculationratserumrecognizedthesame
pro-tein (Fig. 1D and1E-41).Thereactivity ofeachserum used
Table1–PerformanceofMaxisorp®andPolysorp®ELISA microplates.Thewellsofbothplateswerecoatedwith BLVp24r(2g/well)andevaluatedintriplicateswith negative(n=4)andBLV-positive(n=4)serasamplesto determinetheP/Nratio(P/N)andthepercentileofthe coefficientofvariation(%CV).Theindexvaluewasthen obtainedbydividingtheP/NratiobytheCV(%)from positivesamples.
Microplate P/N CV Index
Negative Positive
Maxisorp 2.04 7.1% 8.8% 0.23
Polysorp 4.50 2.2% 4.1% 1.1
iELISAandtheabsorbanceobtainedisindicatedinthelower panel.
Microplates
ThemeanODobtainedwithnegativeandpositiveserum sam-pleswassignificantlylower(p<0.01andp<0.05,respectively) whentestedwithantigenadsorbedtoPolysorp® microplate comparedto the Maxisorp® microplate (not shown). How-ever,the overall,analysisindicatedabetterefficiency with the Polysorp® microplates in that the P/N ratio obtained (4.5) was 2.2 times higher than the P/N ratio obtained with the Maxisorp® microplates (2.04). A lower CV was observed amongst negative and amongst positive samples withPolysorp®microplateswhichyieldedahighermicroplate index (Table 1). Considering that Polysorp® microplates
alloweda betterdistinction between positive and negative
samples(P/Nratio)comparedtoMaxisorp®microplates,they
werethenusedontheremainingexperiments.
Optimalantigenconcentrationandserumdilution
The ideal concentration of BLVp24r antigento be used in
thePolysorp®microplateswasevaluatedusingawiderange
ofantigensdilutionsand BLVnegative(n=16) andpositive
(n=16)bovineserum.Themean serumabsorbancereading
obtainedineachantigendilutionwasnotsignificantly
differ-ent(p>0.05)fromthenextlowerdilutionandthuswechoose
thelowerantigenconcentrationevaluated(50ng/well)to
per-formtheremainingassay(Fig.2).
Tosetupthebestserumdilutionwetestedsamplesdiluted
1:25,1:50,1:100and1:200withPolysorp®microplatescoated
with50ngofBLVp24r/well.Whenalldilutionswerecompared,
weobservedareduced sensitivityonlywithserum diluted
1:200(83.33%)comparedto91.67%ofsensitivityobtainedwith
theotherserumdilutions(notshown).Thus,weusethe
high-estpossibleserumdilution(1:100)fortheremainingstudies.
Specificity,sensitivityandcut-offpointdetermination
SeventyAGIDnegativeserasamplesand30AGIDpositive
sam-pleswereusedtodeterminetheinhouseiELISAsensitivity
andspecificityfordifferentcut-offpointvalues.TheROCcurve
analysisoftheinhouseiELISAdataproducedpairedestimates
ofrelativesensitivityandrelativespecificityatdifferent
cut-offvalues.Acut-offofOD≥0.320wasrecommended.Atthis
0.0 50ng 50ng 100ng 125ng 250ng 500ng 100ng 125ng 250ng 500ng Concentration of BLVp24r/well 0.5 1.0 Positive Negative OD 450nm
Fig.2–Idealantigenconcentrationdeterminationforthein houseiELISA.Differentantigenconcentrationswere adsorbedtothePolysorp®microplatesandthenincubated withserumfromnon-infected(n=16)andnaturally BLV-infected(n=16)cowsdiluted1:100.Theantigen concentrationsevaluatedareindicated.Theresults representtheODmean±SEMateachantigen concentration.Thearrowindicatestheidealantigen concentration. 0
100% - Specificity%
Sensitivity%
0 25 25 50 50 75 75 100 100Fig.3–Receiveroperatingcharacteristic(ROC)analysis. TheROCcurvewascreatedusingtheresultsobtainedfrom analyzing70BLVnegativeand30BLVpositivebovine serumsamples(diluted1:100)bytheinhouseiELISA(50ng antigen/well)performedwiththePolysorp®microplate. TheareaundertheROCcurvewas0.9989.
cut-offvalueweobtainedarelativesensitivityof98.5%(95%
confidence interval=91.96–99.96%) and specificity of 100%
(95%confidenceinterval=95.01–100%)withalikelihoodratio
of75.TheROCcurvehadanareaunderthecurve(AUC)value
of 0.9989(95% confidence interval=0.9961–1.002; p<0.0001)
demonstratingahighlevelofaccuracyforthisiELISA(Fig.3).
Thus,sampleswithanabsorbancehigherthan0.320were
con-sidered positivewhenthenegativecontrolwas atleast1.5
Table2–Anti-BLVantibodiesprevalenceindairyand beefcattle.Serumsamplesfromdairycattle(n=736)and beefcattle(n=451)wereevaluatedinduplicatesbythe inhouseiELISA,using50ng/wellofBLVp24rin Polysorp®microplatesanda1:100seradilution. SampleswithaOD>0.320wereconsideredpositive.
Cattletype n Result
Positive Negative
Dairy 736 229(31.1%) 507(68.9%)
Beef 451 43(9.5%) 408(90.5%)
Total 1187 272(22.9%) 915(77.1%)
Table3–RateofagreementbetweentheiELISA(anti-p24 antibodies)andacommercialcompetitiveELISA (anti-gp51antibodies)evaluatedusing255samples selectedrandomlyamongstdairycattleserumsamples.
Competitivegp51ELISA Positive Negative Total
iELISABLVp24r
Positive 115 22 137
Negative 18 100 118
Total 133 122 255
EpidemiologystudyofBLVinfectionondairyandbeef cattleandcomparisonbetweeninhouseELISAand commercialassay
ByusingourinhouseiELISA,weanalyzed1.187serasamples fromdairy(736)andbeef(451)cattlefromtheNorthregion ofRioGrandedoSulState.Withindairycattle,wefound229 (31.1%)positivesamplesandwithinbeefcattleonly43(9.5%) sampleshadantibodiestoBLVp24r(OD>0.320)(Table2).
TheperformanceoftheinhouseiELISAtodetectanti-BLV
antibodieswascomparedwiththecommercialanti-gp51
com-petitiveELISAassay.Werandomlyselected255serapreviously
testedintheinhouseiELISA(118negativeand137positive)and
evaluatedtheminduplicatesusingthecommercialELISA.We
found122negativeand133positivesamples(Table3)
result-ingin84.3%ofagreementandaKappa(K)indexof0.68(good
agreement).
Discussion
Enzooticbovineleukosisusuallyoccursasasilent,
asymp-tomaticdisease in mostdairyfarms and forthis reasonit
hasbeenneglectedfordecades.Upto30%ofinfectedanimal
developapersistentlymphocytosis(PL)whichinsome
ani-malmightprogresstoBcelllymphoma.Therearenovaccines
andall infectedanimalsrespondtoinfection byproducing
antibodiesfirstlytothegp51viralenvelopeglycoproteinand
thentothep24viralcapsidprotein.Theseviralantigenshave
beengloballyexploredforserologicaldiagnosisofBLV
infec-tion; the gp51 is used mostly, but no exclusively, in AGID
assaysandthep24inimmunoassayssuchasiELISA.26,29–33
InfectedanimalsmightalsobedetectedbyPCRtargeting
con-servedviralgenes33;althoughhighlysensitive,PCRusagefor
epidemiologicalstudies is notpractical and convenient.In
Brazil,theAGIDassayistheonlyofficialdiagnosticmethod
acceptedbut,unfortunately,antigenproductionislimitedand
theassayyieldsunreliableresultsthatfrustrates
diagnosti-ciansanddifficultsarationalapproachtodiseasecontrol.In
addition,becausethepossibleinvolvementofBLVwith
cer-tain typesofhumancancer,23,24 EBLcontrolindairycattle
becomesamatterofpublichealthandshouldbecome
manda-tory.
With this in mind, here we describe the productionof
arecombinantBLVcapsidprotein(BLVp24r)andits usefor
developmentofanindirectELISAassaytodetectanti-BLV
anti-bodiesinblood samples.Thevectorandexpressionsystem
waschosenaimingtoimprovetherecombinantproteinyield,
solubilityandpurificationwithoutlosingimmunogenicityand
recognition by antibodies from BLVnaturally infected
ani-mals.Indeed,therecombinantproteinwaseasilypurifiedby
standardprocedures andits immunogenicitydemonstrated
inrats.Furthermore,theproteinwasrecognizedby
antibod-iesfromBLVnaturallyinfectedcows,bothbywesternblotand
ELISA,andbytheOIEreferenceserumE05,kindlyprovided
bytheOIEreferencelaboratoryforEBLattheUniversityof
Leipzig(Germany),thatwasalsousedintheiELISAsettings.
Inaddition,theMBPtailisnotimmunogenicinrats,oreven
otherspecies,whichallowstherecombinantfusionproteinto
beusedindevelopingdiagnosticassays.34
Wefoundthatmicroplatecharacteristicsaffectedthe
out-comeoftheassay.Overall,negativeandpositiveserasamples
testedwiththePolysorp®microplatehadalowerabsorbance
readingbuttheP/Nratioandthe%CVfoundwithbothtypes
ofplatesindicatedthatthePolysorp® microplatesshouldbe
usedtobetterdifferentiatenegativefrom positivesamples.
Interestingly,inapreviousstudyinwhichweexpressedthe
capsidproteinofHepatitisEVirus(HEV)inthesame
expres-sion system we found that Maxisorp® microplates had a
higherantigenbindingefficiencyandalowerCVcomparedto
Polysorp® microplates.28Weareawarethatantigen
adsorp-tiononthesolidphasecanbeaffectedbyhydrophobicand
hydrophilic domains present on the molecules and by the
typeofplate,underlyingthenecessitytotestdifferentplates
during the initialstep of an ELISA optimizationfor a
tar-getantigen.Consideringthemaximalbindingcapacity/cm2
of the well (0.5g protein) and the final volume used in
the assay (100L), we estimated that saturation would be
achievedwith0.37gofantigen.However,thedifferenceson
P/Nratioand%CVwerenotsignificantwhenwecompared
differentantigenconcentrationand,therefore,wechoosethe
lowestantigenconcentrationtested(50ng/well)tocarryout
the study. Eventhough antigenconcentration might affect
ELISA parameters, similar recombinant antigen
concentra-tionswereusedinpreviousstudytodetectBLV30andother
viral infections.35 Furthermore,our BLV p24r was fused to
MBPandresultedinaproteinwithahighermolecularmass
(67kDa) when compared to the BLV capsid protein per se
(24kDa).Whenlargerproteinsareused,lowerconcentrations
(g/well) might be required to optimize ELISA
parame-ters.Wehypothesizethat MBP wouldcontributetoprotein
adsorptiontothemicroplatewellleavingrelevantantigenic
epitopescompletelyuncoveredandavailableimprovingthe
efficiency of antibody binding to the recombinant protein
and reducing the amount ofantigensrequiredtooptimize
Theperformanceofour inhouse iELISA was compared
tothe AGIDandacommercialcompetitiveELISA thatused
aperoxidase-labeledmonoclonalantibodyasdetecting
anti-body.Therewasa72%agreementbetweentheinhouseiELISA
andAGIDandthekappaindexwaslow(0.42;datanotshown).
However,inourexperiencewiththeavailableAGIDassay,we
noticedthateventhepositivecontrolserumprovidedwith
thekitcommonlyfailstogiveareliableresult,whichis
diffi-culttoreadevenbyexperienceddiagnosticians.Furthermore,
becauseiELISAismoresensitivethanAGIDindetectingeven
smallamounts ofantibodytoagivenantigen,27,32 animals
recentlyinfectedmight notbedetectedbyAGID. However,
whencompared tothe commercial ELISA,we found84.3%
ofagreementbetweentheresultswithavalueof0.68and
the predictive positiveand negativevalues were 0.839and
0.847,respectively. Althoughtheagreement foundbetween
theassays isconsideredgood,it isworthnothing thatthe
commercial assay detects antibodies to the BLV gp51
gly-coproteinusingaperoxidase-labeledcompetingmonoclonal
antibody.Thus,inthis case,because gp51 isahighly
vari-ablesurfaceglycoprotein,animals infectedwithadifferent
genotype10mightnothaveantibodiestotheepitopetargeted
bythedetectingmonoclonalantibodyandwouldresult
neg-ativeinthecommercialbut positiveintheinhouseiELISA
thatusesthehighlyconservedBLVp24rprotein.Infact,one
animalpositivebyiELISAandWesternBlotassay(Fig.1C,
sec-ondstripOD450 0.997)endednegativewhenassayedbythe
commercialELISA,strengtheningthehypothesisthat
differ-entgenotypesofBLVarecirculatingondifferentgeographical
areasand wouldescapedetectionbyserologicalassaythat
wouldtargetuniqueepitopes.Intheotherhand,animals
neg-ativetotheinhouseiELISAbutpositiveinthecommercial
assaycouldhavebeeninfectedrecentlyanddeveloped
anti-bodiesmostlytotheviralsurfaceglycoprotein.Thus,inthis
situation,theidealiELISAshouldcontainbothp24andgp51
antigensnominimizethepossibilityoffalse-negativeresults.
Inanycase,controllingBLVinfectioninaherdrequires
period-icaltestingtoremovepositiveanimalsaslongasanypositive
animalisstilldetectedwithintheherd.
BLVcausesasilent,life-longinfectionofdairycattlewhich
hasbeen mostlyunderestimatedor evenovershadowedby
other diseases. However, as the economic impact of BLV
oncow’shealthandmilkproductionisappreciated18–20,36,37
and,mostrecently,withitspossiblelinkwithhumanbreast
cancer,23,25 a major switch on diagnosis and disease
con-trolshouldbeexpected.Thus,rapidandreliableserological
testshouldbepromptlyavailableatlowcosttomilk
farm-ersanderadicationpoliciesshouldbecomemandatory.Using
ourinhouse iELISA,wetested736 dairyand451 beef
cat-tleserasamplesfromtheNorthregionofRioGrandedoSul
andfoundaprevalenceof31.1%(229/736)and9.5%(43/451)
respectively.Fordairycattle,theprevalencetoBLVinfection
remainssimilartothatreportedinprevious studies.13,14 In
fact,wewereexpectingahigherprevalence.Thus,we
hypoth-esizethatcurrentinvasivemanagementprocedureslikeear
tagging,vaccination,artificialinseminationandtuberculosis
testing are performedtaking into consideration therisk of
transmittingBLVamongstcattle,mainlybecausemost
veteri-nariansareawareoftherelativelyhighprevalenceofwithin
herdinfectionalreadyreportedindairycattlefromthisregion.
Bydoingso,iatrogenictransmissionofBLViskeptat
mini-mum.Also,weareawarethatmostveterinariansrecommend
thatonceaninfectedanimalisdetected,itshouldberemoved
fromtheherdormanagedinsuchawaytoreducetheriskof
transmittingBLVtonon-infectedcows.
In conclusion, the in house iELISA we developed using
BLVp24rmightbeexploredasacommercialtesttodetectBLV
infected animals. The proteinexpressions and purification
systemweusedyieldsenoughproteintomakeitaffordable
andsimpletouse.BecauseBLViswidespreadindairycattle
andweshowedahighrateofinfectionalsoinbeefcattle,and
becauseoftherecentreportsofBLVinfectioninhumans,
con-trollingBLVinfectionincattlebecomesapublichealthissue
thatshouldbecomemandatory.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
AnaPaulaAndreollawasaMasterStudentsupportedbythe
Fundac¸ãoUniversidade dePassoFundoand thenbyCAPES
(01947247000).L.M.S.Erpeenhasanundergraduatefellowship
from the ConselhoNacionaldeDesenvolvimento Científico
e Tecnológico – CNPq. The Laboratório de Microbiologia e
Imunologia Avanc¸ada is equipped with funding from the
Secretaria de Desenvolvimento Econômico, Ciência e
Tec-nologia(SDECT–Convênio46/2014,processo328-25.00/14-0).
L.C.KreutzholdsafellowshipfromCNPq(Produtividadeem
Pesquisa–PQ307900/2016-9).
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