Evaluation of dot-blot test for serological diagnosis of bovine brucellosis

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h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

Evaluation

of

dot-blot

test

for

serological

diagnosis

of

bovine

brucellosis

Carla

Resende

Bastos

a,∗

_,

_Luis

_Antonio

_Mathias

a

_,

_Márcia

_Mariza

_Gomes

_Jusi

b

_,

Renata

Ferreira

dos

Santos

a

_,

_Glaucenyra

_Cecília

_Pinheiro

_da

_Silva

a

_,

Marcos

Rogério

André

b

_,

_Rosangela

_Zacarias

_Machado

b

_,

_Karina

_Paes

_Bürger

a

a_São_Paulo_State_University_(UNESP),_School_of_Agricultural_and_Veterinarian_Sciences,_Jaboticabal,_Department_of_Preventive_Veterinary

MedicineandAnimalReproduction,Jaboticabal,SP,Brazil

b_São_Paulo_State_University_(UNESP),_School_of_Agricultural_and_Veterinarian_Sciences,_Jaboticabal,_Department_of_Veterinary_Pathology,

Jaboticabal,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3March2017

Accepted30October2017

Availableonline13February2018

AssociateEditor:MilianeSouza

Keywords: Brucellaabortus

Animalhealth

Zoonoses

2-Mercaptoethanol

Complementﬁxationtest

a

b

s

t

r

a

c

t

The objective of this study was to standardize and validate the dot-blottest for the

serologicaldiagnosisofbovinebrucellosis,comparetheresultswiththosefoundinthe

2-mercaptoethanol(2-ME)andcomplementﬁxationtest(CF),andestimatetherelative

sen-sitivityandspeciﬁcityofthedot-blotcomparedtothesetests.Fiftybovinebloodserum

sampleswereusedfortheteststandardization,and1315sampleswereusedforevaluation

andcomparisonbetweenthetests;theresultswerecomparedusingtheKappaindicator.At

theendofstandardization,itwasestablishedasoptimalfortheantigenobtainedfrom

Bru-cellaabortusB19afterpassingthroughamicroorganismruptureprocess,thebloodserum

samplesdilutedat1:100,andtheconjugateat1:30,000.Thecomparisonofthedot-blot

resultswith2-MEshowedKappaindexof0.9939,sensitivityof99.48%,andspeciﬁcity99.91%,

withCF,Kappaindexof0.8226,sensitivity100%andspeciﬁcity95.32%.Usingthecombination

ofthetestresults2-MEandCFtoestablishthetrueconditionoftheanimal,thedot-blot

showedrelativesensitivityof100%,andrelativespeciﬁcityof99.91%.Theevaluatedtest

provedtobeeffectiveandreliable,besidesbeingeasytohandleandinterprettheresults.

anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

∗ _{Corresponding}_author_at:_São_Paulo_State_University_(UNESP),_School_of_Agricultural_and_Veterinarian_Sciences,_Jaboticabal,_Department

ofPreventiveVeterinaryMedicineandAnimalReproduction,AccessrouteProf.PauloDonatoCastellane,14884-900–Jaboticabal,SP–

Brazil.

E-mail:carlarbvet@gmail.com(C.R.Bastos).

https://doi.org/10.1016/j.bjm.2017.10.002

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Introduction

Brucellosisisaninfectiousdiseaseofchroniccharacterthat

affectsanimals, causing greatlosses to livestock.Itis also

a public health problem, for being a zoonosis of

occupa-tionalcharacter,transmittedbycontactwithcontaminated

fetal membranes withthe causative agentBrucella abortus,

andfoodbornebyunpasteurizedmilkintake,freshcheeseand

undercookedmeatfromanimalswithbrucellosis.1,2

Due tothe socialand economicimportanceofthis

dis-ease,theMinistryofAgriculture,LivestockandSupplyofBrazil

(MAPA)hassetupacontrolanderadicationprogram,which

deﬁnedasofﬁcialtestsforthediagnosisofbovineandbuffalo

brucellosis:Milkringtest(MRT)usedformonitoringthehealth

conditionandasadiagnostictoolinepidemiological

surveil-lancesystems,RoseBengaltest(RB)asascreeningtestand

2-mercaptoethanol(2-ME) and complementﬁxation (CF) as

conﬁrmatorytestsinadditiontotheﬂuorescencepolarization

assay(FP).3

However,therearesomedifﬁcultiesconcerningthesetests,

suchasthe need forhighlytrained staff, the use oflabile

reagentsthat needtobeconstantly prepared,andtitrated,

toxicreagents.Thesefactshighlighttheneedtodevelopnew

diagnostictechniquesinordertocollaborateforthecontrol

anderadicationofbrucellosis.4

Theaimofthisstudywastostandardizeandvalidationthe

dot-blottechniquefortheserologicaldiagnosisofbovine

bru-cellosis,andcomparetheresultsobtainedbythistechnique

withtheonesfoundintheofﬁcialtests:complementﬁxation

and2-mercaptoethanol,andalsotoestimatetherelative

sen-sitivityandrelativespeciﬁcityofdot-blotincomparisonwith

ofﬁcialdiagnostictestsusedinthestudy.

Materials

and

methods

Fiftybloodserasamplesfromcattleofvariousbreeds,male

andfemale,fromdifferentpropertiesintheNorth,Northeast

andSoutheastregionsofBrazilwereusedfortest

standardiza-tion,samplespreviouslytestedandwiththesameresultsinall

ofﬁcialtestsandmore1315sampleswereusedforvalidation

ofthetests2-ME,CFanddot-blot.

Standardizationofdot-blot

Fromanimmunologicalpointofview,Brucellaantigenscan

bedividedintotwomajorgroups:proteinsandtheLPS.5_With

the intention of evaluate whatis the best antigenfor the

presentstudyweretestedthetwoantigens,theBrucella

abor-tussampleB19obtainedfromthecommercialvaccinesoldin

Brazilafterundergoingamicroorganismruptureprocess,by

themethodoffreezingfor5mininliquidnitrogenand

thaw-ingfor5mininwaterbathat37◦C,twentytimesinarow,

andquantiﬁedat3.1␮g/␮Lofprotein,thereferenceantigen

fordot-blottests.Thesecondantigentestedwas

lipopolysac-charideextractobtainedfromtheBrucellaabortusstrainS996

quantiﬁedat0.52␮g/␮L,theLPSwastestedforbeingoneof

themostimportantantigencombatedbyimmunoglobulins,

itisextremelyimmunogenicandcommonlydetectedin

sero-logicaltests.3

Inordertostandardizeandestablishtheidealamountof

antigenused,tests wereperformedwith0.5␮g,1␮g,1.5␮g

and2␮goftheantigenstested.Thequantitythatshowedthe

bestresultsforthetwoantigenstestedwas2␮g,concentration

whichallowedthevisibilityofthecolorreactionwithexcellent

sharpness,andwasthevalueestablishedforthemembrane

sensitization.

Forthetechniquestandardization,50controlsamplesof

bovinebloodserumwereused,17positivewithdifferent

titra-tions (weak,mediumandstrong), and 33negativeinthe3

tests:RoseBengaltest,2-MEandCF.

Thetechniquewasdevelopedaccordingtothepreviously

describedmethodology.7_The_test_started_by_cutting_the

nitro-cellulosemembrane(codeN-9888, Sigma-Aldrich,St.Louis,

MO,USA)intwoformats,squareandcircle,withthe

inter-esttoestablishtheshapethatwouldenablebettersolution

homogenization,andeconomyofmaterialwhenmakingthe

cuts.Toperformthemembranecutting,scissorswereusedfor

thesquareformat,andaholepunchforthecircleformat,both

properlysanitizedandhandledwithgloves.Tomakethecuts,

materialsofeasyacquisitionandmanipulationwereused,for

thepurposeoffacilitatingthetechnique.

Eachnitrocellulosemembranewassensitizedwith2␮gof

antigen,manipulatedwithforcepsandglovestoprevent

con-tamination,andplacedonasurfaceofhydrophobicmaterial.

Asforthesupportonwhichtocarryoutthereactions,two

typesofplatewereevaluated,thepolyacrylamideplatewith

96 wells,and thecellcultureplatewith24wellsand aﬂat

bottom.

The sensitized membranes were blocked for 12h with

300␮LTBS(20mMTris,500mMNaCl,pH7.5)withaddition

0.05%Tween20and5%powdermilkintoeachwelloftheplate

with24wellsand200␮Lintoeachwelloftheplates,with96

wellstominimizetheoccurrenceofnon-speciﬁcreactions,8

leavingtheplatestirringat4◦C.

Afterincubation,theblockingsolutionwasremoved,and

500␮Loftheserumtobetested,waspipettedintoeachwellof

theplate,with24wellsand200␮Lintoeachwelloftheplate

with96wells,dilutedintheproportions1:25,1:50and1:100in

TBS0.05%Tween20withpowdermilkat5%.Thematerialwas

thenincubatedfor2hatroomtemperatureunderconstant

agitation.

Aftertheincubationwasﬁnished,thewellswerewashed

withTBS0.05%Tween20.Then,itwasevaluatedwhichwas

the optimalnumber of washes1,2 or 3times, each wash

lasting5min.Subsequently,300␮LofconjugateIgGofrabbit

anti-totalbovineIgGlinkedtoalkalinephosphatase(coden.

A0705,Sigma-Aldrich,St.Louis,MO,USA)waspipettedinto

eachwelloftheplatewith24wellsand200␮Lintoeachwell

oftheplatewith96wells,inthedilutions1:4000;1:10,000;and

1:30,000.

The material was incubated for 1h at room

tempera-ture.Conjugatewasremovedandthreewashes,5mineach,

wereperformedusingTBS0.05%Tween20.Thebandswere

visualizedbytheadditionoftheenzymesubstrate

5-bromo-4-chloro-3-indoyl phosphate/nitroblue tetrazolium chloride,

followingthemanufacture’srecommendations(coden.

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I II III IV V

VI VII VIII IX X

Fig.1–Resultsobservedinthedot-blottestforthe diagnosisofbrucellosisincattle.Themembranesinrow1 (samplesI,II,III,IV,andV)showedpositiveresultswith differentcolortints,andinrow2(samplesVI,VII,VIII,IX, andX)membraneswithnegativeresults,withoutreaction.

Thedevelopmentlasted 5min,sincethis isthe time in

whichthepositivemembranesdistinctlystain,andthe

neg-ativeonesremainwiththeoriginalcolor,followedbya5min

rinseindistilledwatertoremoveanynon-speciﬁccolor

reac-tionfromsubstrateresidue.

Whenreadingtheresults,theinterpretationwasbyvisual

observationof the membranes. Thepositive sampleswere

standardizedtocontainastrongorweakpurplishcircle,

sur-roundedbyacirclewithoutstaining,andthenegativeones

withoutanystaining,asshowninFig.1.

Serologicaltests

The1315sampleswereevaluatedbytheconﬁrmatorytests:

2-MEandCF.The2-MEtestwasperformedaccordingtothe

recommendationsofTechnicalManualPNCEB,3 _and_the _CF

test,wasperformedaccordingtothemethodologydescribed

in study techniques for brucellosis laboratories.9 _The

ani-malswithpositiveresultsinthesetwotestswereconsidered

infected,andtheanimalswithnegativeresultsinthesetwo

testswereconsidereduninfected.

Dataanalysis

Thesensitivityandspeciﬁcityvaluesandconﬁdenceintervals

(CI)wereobtainedfollowingrecommendedcriteria.10 _The_Z

testwasusedtoassessthesigniﬁcanceofthekappa

indica-torfollowinginterpretationcriteria,11_being_established_that

theone-tailednullhypothesiskappadoesnotdifferfromzero.

ThecalculationswereperformedusingthepackageepiR,

soft-wareR.

Results

and

discussion

Standardizationofdot-blottest

Theshapethatprovidedthebestresultsformembranewasa

circle,andamongthetestedantigens,theoneobtainedfrom

Brucellaabortusafterundergoingamicroorganismrupture

pro-cess,providedthebestresult,whichwastheoneusedinthe

testingof1315samplesinthisstudy.

Thestandardizeddot-blottestshowedimportant

charac-teristicsforthefeasibilityoftheiruseinroutinediagnostic

laboratories,suchastheuseofaneasilyproducedantigen,

high performance,and providingagreatdistinctionforthe

visualizationoftheresultsofpositiveandnegativesamples.4

Thesupportthatallowedthebestagitation,not

demonstrat-ingcompetitionwiththemembrane,wasthecellcultureplate

with24wellsandaﬂatbottom.

Thedilutionthatdemonstratedthebestresultsforserum,

was1:100andforconjugate1:30,000,anditwasestablished

thenumberofthreewashesperstep.Animportantfeatureof

thetestisthefactthatitdoesnotrequirespeciﬁcequipment

norgenerateanyrisktothehandlershealth.

Comparisonbetweenthetests

Duetothedifﬁcultiesrelatedtotheisolationoftheetiologic

agent,the serologicalmethodsarebest suitedforthe

diag-nosisoflargeherds.Foreffectivecontrolanderadicationof

brucellosis,itisnecessaryforthediagnosistobesafe,andof

viableexecution,andforthisreason,theserologictestsare

indicated.12_To_verify_the_feasibility_of_using_the_dot-blot,_the

resultswerecomparedtotheofﬁcialBrazilianconﬁrmatory

tests2MEandFC.

Thecomparisonoftheresultsofdot-blotwith2-MEshowed

analmostperfectagreement,11_with_Kappa_index_of_0.9939_(CI

95%:0.939–1.0484),sensitivityof99.48%(CI95%:97.11–99.91)

and speciﬁcityof99.91%(CI95%:99.49–99.98),Ztest(35.71;

P=1.35×10−279);withCFtheKappaindexof0.8226(CI95%:

0.7690–0.8761)meansanalmostperfectagreement11_;

sensi-tivityof100%(CI95%:97.42–100.0)andspeciﬁcityof95.32%

(CI95%:94.03–96.47),Ztest(30.1;P=2.29×10−199).

Using the combinationofresultsofthe 2-MEand CFto

establishthetrueconditionoftheanimal,thedot-blotshowed

relativesensitivityof100%(CI95%:97.25–100.00)andrelative

speciﬁcityof99.91%(CI95%:99.48–99.98).Thecombinations

ofresultsobtainedinthetestsaresummarizedinTable1.

The results of this study have show almost perfect

agreement between the ofﬁcial tests and the dot-blot

(Tables2and3),aswellasinotherstudies13,14_when

compar-ingthedot-blottotheRoseBengaltestandCF,andcomparing

Table1–Combinationsofresultsa_obtained_in_the_tests 2-mercaptoethanol(2-ME),complementﬁxation(CF)and dot-blotforserologicaldiagnosisofbovinebrucellosis.

Numberofserums 2-ME CF Dot-blot

1086 − − − 136 + + + 52 + − + 11 − A − 12 I − − 9 I + + 4 + A + 2 I A + 1 + − − 1 − − + 1 I − + Total 1315

a _+:_positive_test_result;_I:_{inconclusive;}_−:_negative_test_result;_and A:anti-complementary.

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Table2–Comparisonbetweenthetests 2-mercaptoethanol(2-ME)anddot-blotforthe

serologicaldiagnosisofbovinebrucellosisusing2-MEas thegoldstandardinthecomparison.

2-Mercaptoethanol Total

Dot-blota _Positive _Negative

Positive 192 1 193

Negative 1 1097 1098

Total 193 1098 1291

a _Sensitivity₌_99.48%_(CI_95%:_{97.11%–99.91%);}_speciﬁcity₌_99.91%

(CI95%:99.49%–99.989%);kappa=0.993(CI95%:0.9393–1.0484).

Table3–Comparisonbetweenthetestscomplement ﬁxation(CF)anddot-blotfortheserologicaldiagnosisof bovinebrucellosis,usingCFasthegoldstandardinthe comparison.

Complementﬁxation Total

Dot-blota _Positive _Negative

Positive 145 54 199

Total 145 1153 1298

a _Sensitivity₌_100%_(CI_95%:_{97.42%–100.0%);}_speciﬁcity₌_95.32%_(CI

95%:94.03%–96.47%);kappa=0.822(CI95%:0.7690–0.8761).

thetechniquetoagentisolationfordiagnosisofinfectionby

Brucellamelitensis.

Studies performed with infected cattle, naturally and

experimentally,indicatedthattheCFhavedrawbacks,such

astheneedforhighlytrainedpersonnel,andtheuseoflabile

reagents,thatneedtobeconstantlypreparedandtittered,4_is

highlyassociatedwiththeisolationoftheetiologicalagent,

with98%speciﬁcity,detecting93negativeanimalsof95that

werefreeoftheinfection15_and_also_showing_good_sensitivity

havingtheleastamountoffalsenegativereactions.16

In another study comparing the diagnostic tests for

brucellosis17_the_CF_was_the_most_efﬁcient_test,_detecting_the

diseasein17 of19infectedanimals inthepre-vaccination

testsand22of23infectedanimalsinpostvaccinationtests.

Theseresultsareduethehighantibodydetectionsensitivityof

thetest,requiringonly5␮g/mLofIgMand10␮g/mLofIgG1.18

Factorsthatattesttheuseofthistestasareferenceforthe

evaluationofanotherserologicaltest(Table3).19

Althoughthe conﬁrmatory serological tests individually

presentsatisfactoryresults(Tables2and3),onecannot

dis-regard the occurrence of divergent results between them

(Table1),indicatingthepossibilityoffalse-negativeresultsin

theconﬁrmatorytests.20_In_order_to_reduce_this_bias,_the

asso-ciationofofﬁcialconﬁrmatorytestswasusedtoestablishthe

trueconditionoftheanimalinrelationtobrucellosis.19,21

The data from Table 4 demonstrates that the results

obtainedwithdot-blotwereexcellentwhencomparedtothe

trueconditionoftheanimal,establishedbythesamplesthat

obtainedthesameresultsinCFand2-MEtests.

Someserologicaltestshavedrawbacks,suchastheuseof

largeamountsofreagents,theuseoftoxicsubstancessuch

as2-MEtest,4_and_problems_such_as_technical_{difﬁculties,}

pro-zoneeffectandanticomplementarysera,asCF.19_Thus,_the

Table4–Dot-blottestresultscomparedtothetrue conditionoftheparticularanimaldeterminedbythe combinationofresultsinthetests2-mercaptoethanol andcomplementﬁxationfortheserologicaldiagnosisof brucellosis.

2-ME+CF Total

Dot-blota _Infected _Not_infected

Positive 136 1 137

Total 136 1087 1223

a _Sensitivity₌_100%_(CI_95%:_{97.25%–100.00%);}_speciﬁcity₌_99.91%

(CI95%:99.48%–99.98%).

useofaneasilyhandledtestlikedot-blot,whichusesnotoxic

orlabilereagents,andgetsaprecisedistinctionbetweenthe

positiveandnegativeserum,generatesasecurityinexecution

andinterpretationofresults.14

New diagnostic techniques are emerging however, and

many have proved themselves impossible in routine use,

becauseoftheneedofexpensiveequipment,andrequiring

highlytrainedteams,factorsnotnecessaryinthetechnique

standardizedinthisstudy.

Thedot-blotpresentsadvantagesoverotherprimary

bind-ingassays.Inthistechnique,theantigenadherestothecenter

ofthenitrocellulosemembrane,whichensurestheuseofan

establishedantigenconcentration,and itssimplicity,

preci-sionandspeeddemonstratethattheassaycanbeusedinthe

ﬁeldforlargescalediagnosisineradicationprograms,suchas

thebovinebrucellosisone.12

Itisanattractivetestforroutinediagnosis,because the

nitrocellulosemembranehasahighafﬁnityforproteins,being

effective to correctly identifyreactive individuals (positive)

andnon-reactive(negative)toanetiologicalagent.22

Asreportedinrecentresearch,23 _the_study_of_brucellosis

servedandservesasthebasisforsomeofthegreatadvances

inepidemiology,andthedevelopmentandveriﬁcationofgood

resultsofadiagnostictestprovidesatechniquenotonlyfor

thediseaseinquestion,butitalsoopensspaceforevaluation

and standardizationofthe testforother speciesandother

infections.

The dot-blot standardized in this study showed high

relative sensitivity, high relative speciﬁcity, and had good

agreementwiththetestsusedforcomparison,managingto

detectantibodiesagainstBrucellaabortusinbovineserum.It

isaviabletest,easytoperformandinterpret,aswellassafe

forthehandler,provingsuitablefortheserologicaldiagnosis

ofbovinebrucellosis.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

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