10 the evaluation of candidate biomarkers of

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ContentslistsavailableatScienceDirect

Veterinary

Immunology

and

Immunopathology

jo u rn al h om ep a ge : w w w . e l s e v i e r . c o m / l o c at e / v e t i m m

Short

communication

The

evaluation

of

candidate

biomarkers

of

cell-mediated

immunity

for

the

diagnosis

of

Mycobacterium

bovis

infection

in

African

buffaloes

(

Syncerus

caffer

)

Wynand

J. Goosen

a_DST/NRF_Centre_of_Excellence_for_Biomedical_TB_Research/MRC_Centre_for_Tuberculosis_{Research/Division}_of_Molecular_Biology_and

HumanGenetics,FacultyofMedicineandHealthSciences,StellenboschUniversity,POBox19063,Tygerberg7505,SouthAfrica

b_Ezemvelo_KZN_Wildlife,_PO_Box_25,_Mtubatuba_3935,_South_Africa

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3September2014

Receivedinrevisedform20October2014 Accepted21October2014

Keywords: Africanbuffalo Bovinetuberculosis

Enzymelinkedimmunosorbentassay Interferongamma

Interferongamma-inducedprotein10

a

b

s

t

r

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Weevaluatedcommerciallyavailablebovineenzymelinkedimmunosorbentassays(ELISA) andahumanIP-10ELISAtomeasureIP-10,MIG,MCP-1,MCP-2,MCP-3andIL1-RAinbuffalo plasmainordertoidentifysensitivemarkersoftheimmuneresponsetoMycobacterium bovis-specificpeptides.Additionally,wefoundthatallcodingmRNAsequencesofthese cytokinesshowedveryhighhomologywiththeirhomologuesindomesticcattle(97–99%) asdidthederivedaminoacidsequences(97–99%).Thishighsequencehomologybetween cattleandbuffaloessupportstheuseofbovineELISAsforthedetectionthesecytokinesin buffaloes.MCP-1concentrationshowedapositivecorrelationwiththatofIFN-␥(p=0.0077) andappearstooccurinfargreaterabundanceinbuffaloeswhencomparedtohumans. UsingabovineIP-10ELISA,levelsofthiscytokinewerefoundtobesignificantlyincreased inantigen-stimulatedbloodsamplesfromM.bovistestpositivebuffaloes(p<0.0001)and IP-10wasdetectedinfargreaterabundancethanIFN-␥.MeasurementofIP-10withthis ELISAmayprovetobeasensitivemarkerofM.bovisinfectioninAfricanbuffaloes.

1. Introduction

InAfrica,buffaloes(Synceruscaffer)areoneofthemost important maintenance hosts of a variety of pathogens whichcausediseaseindomesticcattle(MichelandBengis, 2012).Theseincludeindigenousdiseasessuchas foot-and-mouthdisease,Corridordiseaseandaliendiseasessuchas bovinebrucellosisandbovinetuberculosis(BTB)(Michel andBengis,2012).Inordertobettercontrolsuchdiseases, thedevelopmentofimprovedtestsfortheirdiagnosisand anunderstandingoftheirpathogenesisandepidemiology isessential.

∗ _{Corresponding}_author._Tel.:₊₂₇₂₁_9389073;_fax:₊₂₇₈₆₅₄₂_6274.

E-mailaddress:[email protected](S.D.C.Parsons).

In the case of BTB, the immunological diagnosis of infectionreliesprimarilyonthedetectionofacell medi-ated immune responsetoMycobacteriumbovis antigens (Vordermeieretal.,2000).Examplesofsuchassaysinclude thesingleintradermaltuberculinskintest(SICTT)andthe

invitrointerferongammareleaseassays(IGRA),thelatterof whichquantifyantigen-stimulatedreleaseofthiscytokine. Recently, the specificity of IGRAs have been improved with theavailability of theBovigam PC-EC assay (BEC) utilizing peptidessimulatingtheM.bovisproteins6kDa earlysecretoryantigenictarget(ESAT-6)andthe10kDa culturefiltrateprotein(CFP-10)and theBovigamPC-HP assay (BHP) utilizing ESAT-6,CFP-10, peptides simulat-ingRv3615and3additionalmycobacterialantigens(Bass etal.,2013;Goosenetal.,2014).However,thesensitivityof BTBdiagnosisinbovidsremainssuboptimal(Vordermeier

http://dx.doi.org/10.1016/j.vetimm.2014.10.008

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Table1

Enzymelinkedimmunosorbentassaykitsandcomponents,andbuffaloplasmadilutions,usedtomeasureselectedcytokinesinQFT-processedwholeblood fromAfricanbuffaloes.

Analyte Plasmadilutiona _Manufacturer _Product_information

BovineIFN-␥ 1:2 MabtechAB,NackaStrand,Sweden Kitno.3115-1H-20 BovineIP-10 1:10 KingfisherBiotechInc.,StPaul,USA Primaryantibody:

PB0385B-100 Secondaryantibody: PBB0393B-050 Recombinantprotein: RP0079B-005 BovineMIG 1:2 BethylLaboratoriesInc.,Texas,USA Kitno.E11-803 BovineMCP-1 1:1000 BethylLaboratoriesInc. Kitno.E11-800 BovineMCP-3 1:10 MyBioSource,California,USA Kitno.MBS739771 BovineMCP-2 1:10 MyBioSource Kitno.MBS913274 BovineIL1-RA 1:2 MyBioSource Kitno.MBS740784 HumanIP-10 1:2 PeproTech®_,_Rocky_Hill,_NJ _Kit_no._900-K39 a_Final_plasma_assay_dilution_in_each_well.

et al.,2000)and itis possiblethatthequantification of biomarkersotherthanIFN-␥mayimprovethesensitivityof testssuchastheBECandBHPassays(Watersetal.,2003; Vordermeieretal.,2009;Jonesetal.,2010;Blancoetal., 2011).

Inhumans,candidatebiomarkersforthediagnosisof

Mycobacterium tuberculosis infection include monocyte-derived chemokine IFN-␥-induced protein 10 (IP-10) (Ruhwald et al., 2009), monokine induced by inter-ferongamma(MIG)(Abramoetal.,2006;Chakeraetal., 2011),monocytechemoattractantprotein(MCP)-1, MCP-2,MCP–3andinterleukin1receptorantagonist(IL1-RA) (Ruhwald et al., 2009; Chakera et al., 2011). Ruhwald etal.(2009)reportedsignificantlyhigherconcentrations ofIP-10,IL1–RA,MCP-1,MCP-2andMCP-3comparedto IFN-␥inantigen-stimulatedbloodsamplesfrompatients withactivetuberculosissuggestingthatall5biomarkers holdpromise asimmunological markersof tuberculosis in humans, with IP-10 and MCP-2 showing the great-est potential.Similarly, Abramoet al. (2006) showeda correlationbetweenMIGconcentrationandIFN-␥ produc-tion in 29 BCG-vaccinated controls and 24 TB patients, identifyingMIGasanovelbiomarkerforM.tuberculosis

infection.Incattle,Aranday-Cortesetal.(2012)reported thatIP-10,MIG,GranzymeAandIL-22mRNAexpression showpromiseasbiomarkersofantigen-inducedimmune responseswhichmightbeutilizedforthediagnosisofM. bovis infection. However,using a humanIP-10 ELISA,a studyincattlewhichwereexperimentallyinfectedwithM. bovisconcludedthatIP-10isapoordiagnosticbiomarker forthedetectionofthisinfectioninthisspecies(Waters etal.,2012).

Theaimofthisstudywastoevaluatetheuseof com-merciallyavailablebovineELISAsaswellasahumanIP-10 ELISA(Watersetal.,2012)forthemeasurementofselected candidatebiomarkersofimmuneactivationinresponseto

M.bovisantigensinwholebloodfromAfricanbuffaloes.

2. Materialandmethods

2.1. Animalsandsamplepreparation

In 2012, randomly selected buffaloes in the Hluh-luwe–iMfoloziParkwerecapturedandtestedforM.bovis

infectionusingtheSICTTaspreviouslydescribed(Parsons etal.,2011).AfterSICTTassessment,wholeblood (WB) wascollectedfromallSICTT-positivebuffaloesintosodium heparintubesbyjugularvenipuncture.Immediatelyafter collection,onemlofWBfromeachanimalwastransferred toaQFTTBAntigentube(containingpeptidessimulating ESAT-6,CFP-10andTB7.7)andaQFTNiltube(containing saline), respectively.The QFT tubes were shaken vigor-ously,andwithin4hofWBtransfertotheQFTtubes,these wereincubatedat37◦_C_for₂₀_h,_centrifuged_at₃₀₀₀_×_g

for6minandtheplasmafractionstoredat−80◦_C._To

iden-tifypositiveQFTreactors,plasmasamplesharvestedfrom theQFTtubeswereusedinamodifiedQFT(mQFT)assay usingacommerciallyavailablebovineIFN-␥ELISA(Table1) andacut-offof66pg/ml,aspreviouslydescribed(Parsons etal.,2011).Aspreviouslyreported,themQFTassayisa highlyspecifictestofM.bovisinfection(Parsonsetal.,2011, 2012;Goosenetal.,2014)andbuffaloesthattested posi-tiveonboththeSICTTandmQFTassayswereconsidered tobeinfectedforthepurposesofthisstudy.

2.2. SequencingofselectedbuffalocytokinemRNAs

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Table

of HotStarTaq DNA polymerase (reagents from Qiagen) and0.4mMdNTPs(PromegaCorporation,Fitchburg,WI, USA). ThePCRreactionswereinitiatedbyincubation at 95◦_C _for ₁₅_min_and _consisted _of₄₅ _cycles_of ₉₄◦_C _for

1min,56◦_C_for₁_min_and₇₂◦_C_for₁_min_after_which_PCR

products were incubated for a further10min at 72◦_C.

These amplification productswere sequenced withthe 3130xlGeneticAnalyzer(AppliedBiosystems,FosterCity, CA, USA) according to the manufacturer’s guidelines. Cytokine protein sequences were then inferred from cDNA sequences using the EMBOSS Transeq software (http://www.ebi.ac.uk/Tools/st/embosstranseq/). The degree of homology betweenall buffalo sequences and homologous sequences of cattle were calculated using ClustalW2software.

2.3. CytokineELISAs

TheconcentrationofIP-10,MIG,MCP-1,MCP-2, MCP-3and IL1-RAintheQFT-processedplasma sampleswas measuredusingcommerciallyavailableELISAs (Table1) accordingtothemanufacturer’sinstructions.Furthermore, theabsoluteconcentrationofIP-10inthesesampleswas calculatedwithreferencetoadilutionseriesof recombi-nanthumanandbovineIP-10forthehumanandbovine ELISAs,respectively(Table1).

2.4. Statisticalanalysis

Foreachanimal,theconcentrationofeachanalytewas characterizedas theELISAopticaldensity (OD)and the antigen-dependentsecretionofeachanalyte(ODTB-Nil₎_was

defined as theOD valueof theQFT TB Antigen plasma sample(ODTB₎_minus_that_of_the_QFT_Nil_plasma_sample

(ODNil_)._Using _these_OD_values, _a _Wilcoxon_signed-rank

testwasusedtodetermineifthemedianconcentrationof eachanalytewassignificantlydifferentinplasmafromthe QFTNilandTBAntigentubes.Furthermore,absolute con-centrationsofantigen-specificIFN-␥andIP-10werealso comparedusingaWilcoxonsigned-ranktest.The correla-tionbetweentheODTB_value_for_each_analyte_and_that_of

IFN-␥wascalculatedasSpearman’scorrelationcoefficient.

3. Resultsanddiscussion

The coding mRNA sequences of IP-10, MIG, MCP-1, MCP-2,MCP-3andIL1-RAwereobtainedfortwoAfrican buffaloes andsubmittedtotheNCBI sequencedatabase (http://www.ncbi.nlm.nih.gov/genbank/) (Table 2). The availability of these sequences should prove useful for the investigation of buffalo immunology by means of gene expressionassays aspreviouslydescribed(Parsons etal.,2012).Thesequencesshowedveryhighhomology withthoseof cattle(97–99%)asdidtheinferredamino acidsequences(97–99%)(Table2).Thisdegreeof homol-ogybetweenbuffaloandcattlesuggeststhatcommercial bovineELISAkitscouldbeusedtoquantifytheseproteins inplasmasamplesfrombuffaloes.

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Fig. 1.Optical densities (including medianand interquartile range) obtainedinELISAsofplasmasamplesharvestedfromQFTNiltubes() andQFTTBAntigentubes(•)followingovernight wholeblood incu-bationat37◦_C_for_(a)_IFN-_␥_,_IP-10,_and_MIG,_and_(b)_IL1-RA,_MCP-1,

MCP-2andMCP-3(n=18SICTT-positive/mQFT-positiveAfricanbuffaloes;

*_p_<_0.0001).

2007,2009).However,usingcommercialbovineELISAs, only IFN-␥ and IP-10 levels were found to be signifi-cantlyincreasedinantigen-stimulatedbloodsamplesfrom SICTT-/mQFT-positive buffaloes (Figs.1 and 2). Further-more,MCP-1wastheonlycytokinetoshowasignificant

Fig.2.ThedifferenceinplasmalevelsofIFN-␥,IP-10,MIG,IL1-RA, MCP-1,MCP-2andMCP-3harvestedfromQFTTBantigentubesandQFTNil tubesfollowingovernightwholebloodincubationat37◦_C_(n₌₁₈

SICTT-positive/mQFT-positiveAfricanbuffaloes).

correlation with IFN-␥ (p=0.0077). The results for the remaininganalyteswereunexpectedandweconcludethat theseareeitherpoorindicatorsofantigenstimulationin buffaloesundertheconditionsdescribedorthatthebovine ELISAsdidnotefficientlydetectthesebuffaloproteins.The latteris unexpectedgiventhehighdegreeofhomology observedbetweencattleandbuffalotranscriptsofthese analytes(Table2).However,wecannotruleoutthat tran-scriptional,translationalandpost-translationaldifferences betweencattleandbuffaloesmayalterproteinepitopes. ParticularlysurprisingweretheresultsforthehumanIP-10 ELISAwhichdetectednegligiblelevelsofIP-10inallbuffalo plasmasamples(0–137pg/ml;Fig.1).Thisisincontrastto findingsfromastudywhichusedthesameELISAreagents todetecthighlevelsofIP-10inplasmafromcattlefollowing experimentalinfectionwithM.bovis(Watersetal.,2012). Ourfindingsmayindicatethattheanti-humanIP-10 anti-bodiesusedinthisstudyhavepoorcross-reactivitytothis proteinofbuffaloesdespitetheirhighpredictedhomology withcattleIP-10(Table2).

For the MCP-1 ELISA, plasma samples were diluted 1:1000.Nonetheless,MCP-1ELISAsignalswerehighly cor-relatedwithIFNg(p=0.0077)suggestingthattheresults areatruereflectionofMCP-1abundance.Absolute con-centrations of MCP-1 were not calculated; however, in humans,atypicalplasmadilutionfactorforsuchanassay is1:8(Ruhwaldetal.,2009)andourfindingssuggestthat MCP-1isparticularlyabundantinantigen-stimulated buf-faloplasma.ThiscytokineisassociatedwithaTh2biasand enhancedproductionofIL-4(Deshmaneetal.,2009),both ofwhichareassociatedwithgreaterBTBpathologyin cat-tle(Thackeretal.,2007).Moreover,anincreaseinMCP-1 plasmaconcentrationduetoapolymorphisminthe MCP-1genepromoterregionhasbeenshowntobeassociated withanincreasedsusceptibilitytopulmonary tuberculo-sisinhumans(Flores-Villanuevaetal.,2005).Ourfindings, therefore,warrantfurtherinvestigationoftheroleof MCP-1inbuffaloBTB.

Using thebovineIP-10 ELISA,levels ofthis cytokine were shown to be elevated in antigen-stimulated samples in all SICTT-positive/mQFT-positive buffaloes (Figs. 1 and 2). In addition, antigen-specific IP-10 was released in significantly greater abundance than IFN-␥

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In conclusion, we have shown thatIP-10 is a useful markerofimmuneactivationbyM.bovisantigenswhen using thebovine IP-10 ELISA. Our results highlight the limitationsimposedonveterinaryresearchbythelackof optimalimmunologicalreagentsandtheneedtointerpret experimentalresultsinthislight.Our findingsalso sug-gestthatthediagnosticpotentialofIP-10forBTBincattle bere-evaluatedusingspecies-specificreagents.Studiesare currentlyunderwaytocomparethesensitivityofIP-10 andIFN-␥asdiagnosticbiomarkersofM.bovisinfectionin Africanbuffaloes.

Acknowledgments

WethanktheNationalResearchFoundation(NRF)and Medical Research Council (MRC) for financial support. Opinionsexpressedandconclusionsarrivedatarethoseof theauthorsandarenotnecessarilytheviewsoftheNRFor MRC.Furthermore,weacknowledgetheClaudeLeon Foun-dationandtheNRFSouthAfricanResearchChairInitiative inAnimalTuberculosisforpersonalfundingandsupport. Lastly,we thank theWildlifeCapture Unitof Ezemvelo KZN-Wildlifefortheirassistance.

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