Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

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Medical

Microbiology

Production

of

recombinant

flagellin

to

develop

ELISA-based

detection

of

Salmonella

Enteritidis

Seyed

Ali

Mirhosseini,

Abbas

Ali

Imani

Fooladi,

Jafar

Amani

,

Hamid

Sedighian

AppliedMicrobiologyResearchCenter,BaqiyatallahUniversityofMedicalSciences,Tehran,Iran

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Articlehistory:

Received4October2015 Accepted4April2016 Availableonline7July2017 AssociateEditor:RoxaneMaria FontesPiazza Keywords: SalmonellaEnteritidis FlagellinC IndirectELISA Bacterialdetection Foodcontaminated

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Food-bornediseases,causedbythepathogenicbacteria,arehighlyprevalentintheworld.

Salmonellaisoneofthemostimportantbacterialgeneraresponsibleforthis.Salmonella

Enteritidis(SE)isoneofthenon-typhoidSalmonellaethatcanbetransmittedtohumanfrom poultryproducts,water,andcontaminatedfood.Inrecentyears,newandrapiddetection methodssuchasenzyme-linkedimmunosorbentassay(ELISA)andpolymerasechain reac-tion(PCR)havebeendeveloped.Inthisstudy,recombinantFliC(rFliC)wasproducedtobe usedasanantigen.Theimmunizationwasconductedinmicewiththepurifiedrecombinant FliC(rFliC).ThemiceweresubcutaneouslyimmunizedwithrFliCandelicitedsignificant rFliCspecificserumIgGantibodies.AnindirectELISAsystemwasestablishedforthe detec-tionofSalmonellaEnteritidis.Ourresultsconfirmedthattherecombinantflagellincanbe oneoftheexcellentindicatorsforthedetectionofSalmonellaEnteritidis.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Salmonella represents a group of important gram-negative bacterialpathogens thatcauseintestinal andsystemic dis-eases in human and animal hosts after the ingestion of contaminatedwaterandfoodsuchaspoultrymeatandeggs.1

ApproximatelyonemillioncasesofSalmonellainfectionsare reportedeveryyearintheUnitedStates.2_Inprevious

descrip-tivestudies from differentplacesand samplesin Iran,the prevalence of Salmonella was found to be as 9.2% in 272 stool samples and 8% in 369 stool. In a study, 610 sam-pleswereobtainedfromchildrenunder12yearsofagewith

∗ _{Correspondingauthorat:AppliedMicrobiologyResearchCenter,BaqiyatallahUniversityofMedicalSciences,VanakSq.MolasadraSt.,}

P.O.Box19395-5487,Tehran,Iran.

E-mail:Jafar.amani@gmail.com(J.Amani).

37.5% prevalence of gastroenteritis, which is also caused byan importantentericpathogen bacterium.3–6 _{More than}

2500serovarshavebeenidentifiedforSalmonellaEnteritidis, based onantigenicdifferences inO,H1, and H2antigens.7

Among the 30 Salmonella serovars that are responsible for 73% casesofsalmonellosisinthe UnitedStates,Salmonella entericasubsp.entericaserovarEnteritidisorSalmonella Enter-itidis isan importantand dominant bacterial pathogen.It wasaprevalentcauseofhumansalmonellosisandcausative agentsoffoodborneillnessesworldwideduringtheearly1980s to thelate 1990s.8–10 _{Differentmethods(e.g.,conventional,}

immunological, and molecular-based methods) have been developedforthedetectionofSalmonella.Althoughthecurrent

http://dx.doi.org/10.1016/j.bjm.2016.04.033

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

culture-basedmethodsforthedetectionofSalmonellaare sen-sitive and inexpensive but atthe same time theyare very timeand material-consumingandneed initialenrichment. For example,the conventional methodforthedetection of

Salmonellae,includingSalmonellaEnteritidis,fromeggstakes 5–7days,islabor-intensiveandinvolvestheisolationofthe organismusingpre-enrichmentaswellasselective enrich-ment procedures and serological confirmation tests. This method is useful for the detection of small numbers of

SalmonellaEnteritidis.

Molecularmethods(PCR)aregoodbuttheyalsohavefew limitations.For PCR-basedmethods,the pathogenneedsto begrownandahighconcentrationofnucleicacidisrequired to be extracted.11,12 _{Bacterial flagellin is one of the outer}

membraneproteinsthatservemanyfunctionslikemobility, pathogenicity,and adjuvanticity and shows toll-like recep-tor(TLR)-ligandactivity.Itiseffectiveatverylowdoses13,14

andbindstotoll-like-receptor5(TLR5),whichispresenton theimmune-systemcells(epithelialcells,dendriticcells,and macrophage).One of the outmost flagellin proteinsis FliC whichhasamolecularweightof50–60kDa.15,16_Thebinding

ofFliCwithTLR5leadstoacascadeofreactionsthatresults intheproductionofpro-inflammatorycytokineslikeTNF-␣, IL-6,andIL-12.17

In this study, we produced recombinant flagellin (r-FliC) for the detection of Salmonella Enteritidis (SE) using enzyme-linked immunosorbent assay (ELISA) and proposing its usefulness in ELISA for the detection of

Salmonella.

Materials

and

methods

Kits,enzymes,andreagents

Theplasmid extraction and gel purification kits were pro-curedfromiNtRON(Seongnam,Korea).Nickel-nitrilotriacetic acid(Ni-NTA)agarosewasobtainedfromQiagen(Maryland, State,USA).Primers weresynthesized bySinaclon(Tehran, Iran). Restriction endonucleases were obtained from Sina-clon(Tehran,Iran).T4DNAligasewassuppliedbyFermentas (Vilnius,Lithuania).Allotherreagentswere ofatleast ana-lytical grade and obtained from Sigma–Aldrich or Merck, Germany,includingkanamycin(40␮g/mL,Sigma), nitrocellu-losemembrane(PROTRAN),anti-polyhistidineantibodiesand anti-mouseIgGconjugatedwithhorseradishperoxidase(HRP) (RAYBiotech), and an ELISA reader (Bio-Rad, Berkeley, CA, USA).

Bacterialstrainsandcultureconditions

ThestandardstrainofSalmonellaentericaserovarEnteritidis (SE)(ATCC–13076,InstitutePasteurofIran)wasusedasthe sourceofflicgene.ItwasgrowninLuria-Bertani(LB)brothor LBagarat37◦C.Bacterialgenomewasextractedbythe CTAB-NaClmethod,andtheDNAconcentrationwasmeasuredbya spectrophotometer(Cecil,UK,OD260and280nm).Thequality oftheisolated DNAwasassessedbyelectrophoresison1% agarosegel.

Amplificationofflicgene

A colony of Salmonella Enteritidis was grown in Luria-Bertani broth (LB broth)overnight at 37◦C under constant agitation at 150rpm. The genomic DNA was extracted

from the Salmonella strain and flic gene was amplified

by polymerase chain reaction (PCR) using the following twospecificprimers(flicF: 5-tatagaattcatggcacaagtcattaatac-3 containing an EcoRI-engineered restriction site and flic

R: 5-tatataagcttttaacgcagtaaagagagg-3 containing a

HindIII-engineeredrestrictionsite).Theprimersweredesignedtoover the completesequence offlicgene (1518bp)locatedon the chromosomalDNA ofSalmonellaenteritidis asmentioned in thedatabaseavailableattheNationalCenterfor Biotechnol-ogyInformation(NCBI).Fortheamplificationoftheflicgene, thepolymerasechainreaction(PCR)wasstandardizedusing 10pMofeach gene specificprimers, 2␮L of25mM MgCl2,

10mMofeachdNTPs,2.5␮Lof10×enzymebufferand0.5U ofTaqDNApolymerase(Fermentas)ina-25␮Lfinalreaction volume. The amplificationwas carried out withthe initial denaturationofDNAat95◦Cfor5minfollowedby30cyclesat 95◦Cfor1min,annealingat55◦Cfor45s,extensionat72◦C for1min,andthenafinalextensionat72◦Cfor5min.The amplifiedproductwasanalyzedbytheelectrophoresisusing 1%agarosegelandethidiumbromideasatrackingdye.Forthe cloningofflicgene,theamplificationwascarriedoutwithpfu DNApolymerase(Fermentas,Lithuania)inareactionmixture (25␮L)containingDNAinthepresenceof4mMmagnesium sulfateand10pMofeachprimer.Thecyclingconditionswere initialdenaturationat95◦Cfor5min,followedby30cyclesat 95◦Cfor1min,55◦Cfor45s,72◦Cfor60sandthefinal exten-sionat72◦Cfor5min.ThePCRproductswere analyzedby electrophoresison1%agarosegelsandvisualizedbyethidium bromidestaining.

Cloningandexpressionofflagellin

TheamplifiedPCR productwasdouble-digested withEcoRI

andHindIII,andclonedintotheprokaryoticexpression vec-tor pET-28a (+)at EcoRI/HindIIIsite to formthe expression plasmid pET-fliCwith kanamycinresistanceas aselectable marker.EscherichiacoliDH5␣transformantsgrownovernight onLBplatescontainingkanamycin(20␮g/mL)werescreened. For ligation, the flic gene PCR product and vector plasmid wereusedintheratioof2:1.Theligatedproductwasinitially propagated in E. coli DH5␣ competent cells.15 _The

trans-formed colonies were screened by colony PCR, restriction enzymeanalysis,andsequencing.TherecombinantpET-fliC plasmid extracted from E. coliDH5␣ cells waspurified and transformedintoE.coliBL21(DE3)strainfortheexpression of flagellin. The expression was induced by adding 1mM isopropyl-␤-d-1-thiogalactopyranoside(IPTG)togrowing cul-tureofthetransformedE.coliBL21(DE3)whenOD600reached 0.6,at37◦C,underconstantshakingat150rpm.Azero-time aliquot(uninducedculture)wasusedasthecontrol.The sam-ples were collected after24hand analyzedfor theprotein expressionin12%sodiumdodecylsulfate-polyacrylamidegel electrophoresis(SDS-PAGE).Therecombinantproteinwas fur-therconfirmedbyWesternblotanalysis.

Westernblotanalysis

WesternblottingwasperformedtoexaminetheFliC secre-tion from E. coli BL21 (DE3). In order to prepare samples,

E.coliBL21 (DE3)strain wascultured overnightinLB broth at37◦C. Thecultured bacteriawere harvested by centrifu-gationat6000rpmfor15min.Thesecretedproteinsample wasseparatedon12%SDS-PAGE.Theseparatedproteinswere transferredelectrophoreticallyusing transferbuffer(39mM glycine, 48mM Tris-base, 0.037% SDS, and 20% methanol, Bio-Rad) onto a polyvinylidene fluoride membrane (PVDF, Immobilon, Millipore) for immunoblotting. The membrane wasblockedunderconstant shakingfor24hwith5%(w/v) skimmed milk in phosphate-buffered saline (PBS), 0.05% Tween-20(PBS/T),pH7.4at4◦C.Themembrane was incu-bated with a 1:3000 dilution of mice anti-His-tag specific antibodyinthePBScontaining0.05%Tween-20(PBS/T),with gentleshakingatroomtemperaturefor1h.Afterwashingthe membranethricewithPBS/T,it wasfurtherincubated with goatanti-mouseIgGhorseradishperoxidaseconjugate,1:1000 dilutedinPBS/T,atroomtemperaturefor1handthenwashed thricewithPBS/T.3,3,5,5-Tetramethylbenzidine(TMB) sub-stratewasusedtovisualizethemembrane.

Solubilityandpurificationoftherecombinantflagellin proteinwithHis-tag

Thesolubility ofthe expressedproteinwas determined by resuspendingthebacterialpellet(24hpostIPTGinduction)in PBS.Aftersonication,thelysatewascentrifugedat12,000×g

(15min)andthesupernatantrepresentingthesolublefraction wascollected.Thepelletcontainingtheinsolublematterwas againresuspendedin8Murea.Bothextractswereanalyzed byresolvingon12%SDS-PAGE.Thepoly-His-tagcontaining recombinantflagellinwaspurifiedusingnickeldivalentions (Ni-NTA chromatography) (Maryland, USA) under denatur-ationconditionsasperthemanufacturer’sinstructions.The proteinconcentrationwasestimatedbyBradford’smethod18 andthepositiveelutesconfirmedbySDS-PAGEwerepooled andstoredat−20◦_{Cuntilfutureuse.}

Animalimmunization

FemaleBALB/cmice(18–22g)usedinthisstudywereobtained from Pasteur Institute of Iran, Tehran. The animals were housedandtreatedinaccordance withtheResearchEthics CommitteeaccordingtotheDeclarationofHelsinki.Themice were divided into test (n=5) and control groups (n=3). In thetestgroup,eachmousewasinjectedsubcutaneously,at the back ofthe neck, 10␮gof recombinant protein (rFliC) withcompleteFreund’sadjuvant.Subsequently,10␮gofrFliC proteinwasinjected asthefirst,second,and thirdbooster after0,14and28days,usingincompleteFreund’sadjuvant. The control group was injected with sterile PBS following the same protocol.Blood samples were collected from the mice one week after the second and third booster dose

(Table1).

Enzyme-linkedimmunosorbentassay(ELISA)forplasma IgG

Antigen-specificantibodyresponsesweredeterminedbythe enzyme-linkedimmunosorbentassay(ELISA).Polystyrene 8-well plates (MaxiSorp microtiter plates) were coated with 3␮gofrFliCdilutedin100␮Lcoatingbuffer(64mMNa2CO3,

136mMNaHCO3,pH9.8)andthenincubatedovernightat4◦C.

The plates were washed threetimes withPBS/T (PBS con-taining0.05%,w/vTween-20).Nonspecificbindingsiteswere blockedwith100␮Lof3%(w/v)skimmedmilkinPBS/T(1h, 37◦C). Mouseserum sampleswere seriallydilutedto1:500 in PBS containing 0.03% Tween-20 and were added to the ELISA plates. Afterthreewashing steps, 100␮Lofthe goat anti-mouseIgGperoxidaseconjugate(Sigma)diluted1:5000 inPBS/Twasaddedtotheplates.Theplateswereincubated for1hat37◦CandthenwashedthriceinPBS/T.Toeachwell, 100␮LofTMBsubstrate(Sigma)wasaddedandincubatedat roomtemperaturefor15min.Thereactionwasstoppedwith 100␮Lof2MH2SO4andtheresultwasreadbyanELISAreader

atthe wavelengthof450nm(Bio-Rad, Berkeley,CA,United States).

PurificationofAnti-FliCIgGwithProteinGSepharose4B column

IgG antibodies were purifiedusing GSepharose4Bcolumn (Sigma).Atfirst,thecolumnwaswashedwith100mMTris–HCl buffer(pH=8)followedby10mMTris–HClbuffer(pH=8)until thecolumnwasreachedtoequilibrium.Theserumsamples wereloadedontothecolumnanditwaswashedwith100mM Tris–HCl buffer (pH=8) followed by 10mM Tris–HCl buffer (pH=8) untilallother proteinswereeluted. Theantibodies wereelutedfromthecolumnbypassing100mMglycinebuffer (pH=3).Attheend,thepurityofIgGwasconfirmedby SDS-PAGE(12%).

Thedeterminationofoptimumconcentrationsofthe antibodyandbacteria

ELISAwasperformedasdescribedaboveusingthedifferent concentrationsofthepurifiedanti-FliCIgGantibody(2,3,4, 5,10,20,30,40,50and60␮g)andaconstantconcentrationof theantigen(3␮g).Inanothersetofexperiment,ELISAwas per-formedwiththedifferentconcentrationsoftheantigen(0.625, 1.25,2.5,5,10,15and20␮g)andaconstantconcentrationof theantibody(10␮g).Subsequenttothis,whole-bacterial cell-ELISAwascarriedoutwiththestandardstrainofSalmonella enteritidisaccordingtoMcFarlandstandard(OD:0.257nm,cell density3×108_{CFU/mL).Inthisassay,theplateswerecoated}

withthedifferentconcentrationsofbacterialcells(108_,107_,

106_,105_,104_,103_,102_{,and10CFU/mL)andwereexposedtoa}

constantconcentration(10␮g)oftheantibody. Specificitytest

Thespecificityoftheindirect-ELISAwascheckedusinga vari-etyofotherbacteria,includingE.coli,Shigellasp.,Pseudomonas aeruginosa,Klebsiellasp.,StaphylococcusaureusandSalmonella

Table1–Summaryofimmunizationprotocols.

Groups Number InitialImmunization

componentsQuantity(␮l) Booster1components Quantity(␮l) Booster2components Quantity(␮l) Control 3 PBS 200␮1 PBS 200␮1 PBS 200␮1

rFliC 5 rFliC 10␮g rFliC 10␮g rFliC 10␮g

C.F.A 100␮1 I.F.A 100␮1 I.F.A 100␮1

C.F.A=CompleteFreund>sAdjuvant,I.F.A=IncompleteFreund>sAdjuvant.

againstSalmonellaEnteritidiswithadetectionsensitivityof 106_{CFU/mLandshowednocross-reactivitywithothertested}

bacterialspecies.Thecut-offwasOD0.7asdeterminedonthe basisoftheaveragecontrolsamplesplustwo-foldstandard deviations.

Clinicalsamples

TheinfectiousdoseofSalmonellais106_–107 _{cells.Therefore,}

theELISAwasperformedusingclinicalspecimenscollected fromthepatientswithSalmonellosis(20samples).Theassay protocol was based onthe previousELISA, optimized by a standardstrainofSalmonellaEnteritidiswith106_CFU/mLcells.

Statisticalanalysis

Allthedatapointswerethemean(±SD)ofthreeindependent setsofexperiments.SPSS andExcelsoftwarewere usedto determinemeansandstandarddeviations.Theresultswere subjected toStudent’st-test (Excel;Microsoft)for indepen-dentvariablesandthevalueswereconsideredsignificantat

p-values<0.05.

Results

Amplificationandcloningoffilcgene

SalmonellaEnteritidisgenomewasextracted(Fig.1A)andthe

flicgenewasamplifiedbyPCRusingthespecificprimers.The PCRproduct(1500bp)isshowninFig.1B.Thefragmentwas clonedinpET-28a(+)vectorandthentransformedintoE.coli

DH5␣. pET28a-flicplasmids were extractedfrom E. coli and digestedbyEcoRI/HindIIIand analyzed byagarosegel elec-trophoresis (Fig. 1C). Therecombinant pET28a-fliC plasmid extractedfromE.coliDH5␣cellswaspurifiedandtransformed toE.coliBL21(DE3)cellsfortheexpressionofFliCprotein.

Expressionandpurificationofrecombinantprotein

Therecombinant FliC proteinwith N-terminalHis-tag was expressed inE.coliBL21-DE3 cells(Fig. 2A)and purifiedby Ni-NTAaffinitychromatography.TheSDS-PAGEofthe puri-fiedproductisdepictedinFig.2B.TheWesternblottingwith

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M

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FliC

_FliC

1518bp

A

B

C

1518bp

1000bp

1000bp

Fig.1–GenomicDNAextraction,PCRproducts,andthedigestionanalysisonanagarosegel.(A)LaneM,DNAsizemarker; Lane1,Salmonellagenome.(B)LaneM,DNAsizemarker;Lane1,fliCgene.(C)LaneM,DNAsizemarker;Lanes1–5,digested constructsbyEcoRIandHindIIIrestrictionenzymes.

1

M

2

8 7 M

6 5 4 3

2

1

1

2

3

M

A

B

C

Fig.2–Expression,purification,andidentificationofrFliC,stainedwithCoomassieblueR-250.(A)ExpressionofrFliC.M, proteinsizemarker.Lane1non-inducedtransformed(pET28awithoutinsert)BL21DE3ascontrol;Lane2induced

transformed(pET28awithinsert).(B)PurificationstepsofrFliC.M,proteinsizemarker.Lane1,clearedlysatebeforeloading onthecolumn;Lane2,3,flowthrough;Lane4,5,washing;Lane6,7,8,elutionwith250mMimidazole.(C)Westernblotting ofrFliC.M,proteinsizemarker;Lane1,non-inducedtransformed(pET28awithoutinsert)BL21DE3ascontrol;Lane2,the supernatantofinducedtransformedBL21DE3;Lane3,purifiedrecombinantprotein.

3.5 3 2.5 2 1.5 1 0.5 0 100 200 400 800 Reciprocal dilution 1600 OD:450nm 3200 Without Without Ab Ag Control

rFlic after first injection rFlic after second injection rFlic after third injection

Fig.3–SerumantibodyresponseofBALB/cmice immunizedwithrecombinantFliCproteins.

theanti-His-tagantibodyconfirmedthepresenceofa55-kDa protein(Fig.2C).

ImmuneresponseelicitedwithrFliC

Thepatternsofantibodyproductionfollowedbythe immu-nization at three settings of the first, second and third injectionswere thesameexceptfortheincreasedantibody titeraftereachphaseoftheimmunization(Fig.3).This sug-geststhattheadjuvantedrecombinantflagellininjectedinto theBALB/cmicehasgoodimmunogenicproperties.Thesera obtainedfromthe controlmicethatwere injectedwiththe samepreparationwithoutflagellindidnotrevealany signifi-cantleveloftheanti-flagellinantibody.

Purificationofanti-FliCIgGwithProteinGSepharose4B column

Anti-FliC IgG was purified with Protein G Sepharose 4B column chromatography and achieved a concentration of 2.339mg/mL.Inthe12%SDS-PAGE,theproteinappearedas singlebandconfirmingitspurity(Fig.4A).

Theevaluationoftheoptimumconcentrationsofantibody andSalmonellaEnteritidisforspecificELISA

AspecificELISAwasdesignedwithaconstantconcentration ofantigen(3␮g)andvaryingantibodyconcentrations(2,3,4, 5,10,20,30,40,50 and60␮g)forthedeterminationofthe optimumconcentrationofantibody.Aconcentrationof10␮g antibodywasfoundtobetheoptimum(Fig.4B).AnotherELISA wasperformedforthedeterminationofantigenwitha seri-allydilutedconcentrationofrecombinantflagellin(0.625,1.25, 2.5,5,10,15and20␮g)andaconstantconcentrationof anti-body(10␮g).Onthebasisofthisresult,3␮gwasconsidered forantigencoating(Fig.4C). Forthe detectionofSalmonella

Enteritidisinfection,bacterial-ELISA wasdesignedwiththe standardstrainofSalmonella(106_{CFU/mLcells)(Fig.4D).}

Specificitytest

The specificity ofthe indirect-ELISA was establishedusing a variety of other microbial strains and showed no cross-reactivity with other tested bacteria. Based on the control samplesthecut-offwasdeterminedtobe0.7andtheELISA testassayedon106_{CFU/mLbacteria.TheresultsoftheELISA}

areshowninFig.5. Samplesresults

TheclinicalsamplesweretestedwithELISAforthedetection ofSalmonellaantigen.TheresultsofELISAareshowninFig.6.

Discussion

Non-typhoidalSalmonellaentericaserovarscausemostofthe casesofbacterialfood-bornediseasesworldwide.Salmonella

Enteritidis,whichisoneofthemostcommoncausesoffood poisoninginhumans,cancausediseasessuchas gastroen-teritis,bacteremia,fever,intestinal,andfecalinfection.19_Over

1 M 2

Control Log 10 CFU/mL Purified Anti-Flic antibody

Control

10µg Anti-Flic antibody

Antigen concentration (µg) Bacterial count log 10

60 50 40 20 10 5 4 3 2 Without Ag Without Ab Without Ag 1 2 3 4 5 6 7 8 Without Ab Concentration (µg) OD:450nm OD:450nm OD:450nm

A

C

B

Fig.4–ThepurificationofIgGantibodyandestablishingELISAusingtherecombinantFliCantigen.(A)PurificationofIgG antibodybyGSepharose4Bcolumn.Lane1,SDS-PAGEofpurifiedIgGwith2ME;M,proteinsizemarker.Lane2,SDS-PAGE ofpurifiedIgGwithout2ME.(B)DevelopmentofELISAafterIgGpurification.(C)Developmentofantigenwiththe

determinationofpurifiedantibody.(D)BacterialELISA.

increasingincidenceofnon-typhoidSalmonellabacteremia.20

Therefore,arapidandproperdetectionofthisbacteriumis importantforfoodsafety.Thebacterialculturebasedmethods forthe identificationofSalmonellaaretime-consuming(5–7 days)andrequireenrichmentoftheculture.21_Assuch,

sev-eralscreeningmethodssuchasnucleicacid-basedassaysand immunology-basedassayshave been examinedfora rapid detectionofSalmonellaintherecent decades.20,22–24 In ear-lierstudies,lipopolysaccharides(LPS)presentonthebacterial surface were used in the ELISA-assay for the detection of

Salmonella,but theproblem withlipopolysaccharidesisthe cross-reactivitywithothergram-negativebacteria.25–27

There-fore, flagellin (FliC) was sought as an alternative for rapid diagnosisofSalmonellaand,duringthe lastdecade,several studieshaveuseditforthedetectionofSalmonella.Forthe productionofrecombinantFliC(rFlic),inthisstudy,astandard strainofSalmonellaentericaserovarEnteritidiswasusedthat producesanantigenwithhighimmunogenicity.28_ADNA

frag-ment ofthe expectedsize (near 1518bp) wasamplified by PCRandexpressedusingchemicallyinducible T7promoter (pET28a).ThebestconditionfortherecombinantrFliC expres-sionwasachievedat1mMIPTG concentration,the culture temperatureof37◦Candwithovernightinduction.However,

theproteinexpressionshowedanincreasewiththe incuba-tiontimeupto18h.rFliCwasseparatedonSDS-PAGEandits biochemicalnaturewasconfirmedbyimmunoblottingusing miceanti-His-tagspecificantibody.rFliCwaspurifiedbased on6XHis-tagusingtheNi-NTAcolumn.ThepurifiedrFliCwas used forthe immunizationofmice. TheELISA testresults showed that all immunized miceproduced a high titerof

2.5 2 1.5 1 0.5 0 –0.5 Salmonella Shigella Pseudo mo nas E.coli Staph Klebsiella Witho ut Ag Witho ut Ab

Bacterial cells:106 _CFU/mL

OD:450nm

Fig.5–Cross-reactivityofmouseanti-S.enteritidisIgGwith othermicrobialstrains.Assayswereconductedintriplicate.

2.5 2 1.5 1 0.5 0

Bacterial cells: 106 CFU/mL

s1 s2 s3 s4 s5 s6 s7 s8 s9 _{s10 s11 s12 s13 s14 s15 s16 s17 s18 s19 s20} Control

OD:450nm

Fig.6–ELISAofclinicalsamplesforthedetectionofSalmonellaEnteritidis.Negativecontrolsarealsoindicated.Assay cut-offwascalculatedbyreplicateanalysisofnegativesamples.

anti-FliCantibodyuponinjectingrFliCthreetimes.Thenan ELISA system was designedfor the detectionof Salmonella

Enteritidis,which isanimportantpathogenofhumanand poultry.Accurateandquicktestsareessentialtoensurefood safety by the governments.29 _{The availability of sufficient}

quantitiesofrecombinant antigens forthe rapiddetection ofSalmonellacanbe achievedinserum samplesina short time.30_{Thus,newmethodsforquickdetectionofSalmonella}

havebeendevelopedinthepastdecade.Amongthe molec-ulartechniques,PCRisacommonandaccuratemethodand canalsobeusedforthedetectionoflowlevelsofpathogenic bacteria.31_{ELISAcanbeusedtoscreenavarietyofsamples}

andcomponentsinthepresenceofSalmonellaspp.

TheELISAtest,designedinthisstudy,didnotshowany cross-reactionwithotherbacteria.Sensitivityanalysisofthis testshowedthatitcoulddetect103_{bacteriainclinical}

sam-ples,giventhattheinfectiousdoseofSalmonellaisabout106

approximately.

Besidesours,thestudiesofMinicozzi(2013),5_Jindal(2012)13

andBang(2012)21havealsoreportedtheproductionof recom-binantflagellarantigen(r-FliC)anddevelopmentofELISAfor thedetectionofSalmonella.Inpreviousworks,the investiga-tionswerecarriedoutmainlywithseraobtainedfrominfected flocksandbirdsbut,inthisstudy,weusedmiceforthe pro-ductionofantibodyandProteinGSepharose4Bcolumnfor the purification. Theflic gene ofSalmonella Enteritidiswas amplified, cloned successfully using pET-28a(+) expression vectorand wasintroduced intoE. coliBL21 (DE3)asa suit-ablehost.Therecombinantflagellinwaspurifiedsuccessfully anddemonstratedgoodimmunogenicpropertiesbasedonthe immunizationofBALB/cmiceanditsabilitytoinducea sig-nificantserumflagellin-specificIgGimmuneresponse,which wasmeasuredbyELISA.Weprovidefurtherevidencethatthe recombinantflagellincanbeoneoftheexcellentindicators forthedetectionofSalmonellaEnteritidis.

Conflicts

of

interest

Theauthorsdeclarethattheyhavenocompetinginterest.

Acknowledgments

This article is a part a Ph.D. thesis being prepared by Ali Mirhosseini. The study was supported by the grants from AppliedMicrobiologyResearchCenterBaqiyatallahUniversity ofMedicalSciences.

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s

1.HyeonJ-Y,ChonJ-W,ChoiI-S,ParkC,KimD-E,SeoK-H.

DevelopmentofRNAaptamersfordetectionofSalmonella

Enteritidis.JMicrobiolMethods.2012;89(1):79–82.

2.KilicA,BedirO,KocakN,etal.Analysisofanoutbreakof

Salmonellaenteritidisbyrepetitive-sequence-basedPCRand

pulsed-fieldgelelectrophoresis.InternMed.2009;49(1):31–36.

3.RanjbarR,GiammancoGM,FarshadS,OwliaP,AleoA,

MamminaC.Serotypes,antibioticresistance,andclass1

integronsinSalmonellaisolatesfrompediatriccasesof

enteritisinTehran,Iran.FoodbornePathogDis.

2011;8(4):547–553.

4.ChashniS,HassanzadehM,FardM,MirzaieS.

CharacterizationoftheSalmonellaisolatesfrombackyard

chickensinnorthofIran,byserotyping,multiplexPCRand

antibioticresistanceanalysis.ArchRaziInst.2009;64(2):

77–83.

5.NosratyS,FallahF,SabokbarA,DezfoolyanM,AdabianS,

EsmaeilnejadN.DetectionofSalmonellaenteritidis,typhi

andtyphimuriuminfoodsbymultiplexPCRinchildren

hospital.BMCInfectDis.2012;12(suppl1):P95.

6.DallalMMS.PrevalenceofSalmonellaspp.inpackedand

unpackedredmeatandchickeninsouthofTehran.

JundishapurJMicrobiol.2014;7(4).

7.FiererJ,GuineyDG.Diversevirulencetraitsunderlying

differentclinicaloutcomesofSalmonellainfection.JClin

Invest.2001;107(7):775–780.

8.MinicozziJ,SanchezS,LeeMD,HoltPS,HofacreCL,Maurer

JJ.Developmentofrecombinantflagellarantigensfor

serologicaldetectionofSalmonellaentericaserotypes

Enteritidis,Hadar,Heidelberg,andTyphimuriuminpoultry.

Agriculture.2013;3(3):381–397.

9.DesinTS,KösterW,PotterAA.Salmonellavaccinesinpoultry:

10.OraczG,FeleszkoW,GolickaD,MaksymiukJ,KlonowskaA,

SzajewskaH.RapiddiagnosisofacuteSalmonella

gastrointestinalinfection.ClinInfectDis.2003;36(1):112–115.

11.SiregarTH,EllimanJ,OwensL.Developmentofrealtime

polymerasechainreactionfordetectionofSalmonella

typhimuriumandSalmonellaenteritidisinfish.Squalen.

2013;7(2):51–58.

12.AmaniJ,AhmadpourA,FooladiAAI,NazarianS.Detectionof

E.coliO157:H7andShigelladysenteriaetoxinsinclinical

samplesbyPCR-ELISA.BrazJInfectDis.2015;19(3):278–284.

13.JindalG,TewariR,GautamA,PandeySK,RishiP.

ImmunologicalcharacterizationofrecombinantSalmonella

entericaserovarTyphiFliCproteinexpressedinEscherichia

coli.AMBExpress.2012;2(1):1–9.

14.BergmanMA,CummingsLA,BarrettSLR,etal.CD4+Tcells

andtoll-likereceptorsrecognizeSalmonellaantigens

expressedinbacterialsurfaceorganelles.InfectImmun.

2005;73(3):1350–1356.

15.HajamIA,DarPA,SekarSC,etal.Expression,purification,

andfunctionalcharacterisationofflagellin,aTLR5-ligand.

VetItal.2013;49(2):181–186.

16.CummingsLA,WilkersonWD,BergsbakenT,CooksonBT.In

vivo,fliCexpressionbySalmonellaentericaserovar

Typhimuriumisheterogeneous,regulatedbyClpX,and

anatomicallyrestricted.MolMicrobiol.2006;61(3):795–809.

17.HaikoJ,Westerlund-WikströmB.Theroleofthebacterial

flagelluminadhesionandvirulence.Biology.

2013;2(4):1242–1267.

18.BollagD,MichaelD,StuartJ.ProteinMethods.2nded.New

York:JohnWileyandSons,IncPub;1996.

19.DehghaniB,RasooliI,GargariSLM,NadooshanMRJ,OwliaP,

NazarianS.ImmunogenicityofSalmonellaentericaserovar

Enteritidisvirulenceprotein,InvH,andcross-reactivityofits

antiserawithSalmonellastrains.MicrobiolRes.

2013;168(2):84–90.

20.KuhnKG,FalkenhorstG,CeperTH,etal.Detecting

non-typhoidSalmonellainhumansbyELISAs:aliterature

review.JMedMicrobiol.2012;61(1):1–7.

21.BangJ,ShuklaS,KimY,KimM.Developmentofindirect

competitiveELISAforthedetectionofSalmonella

Typhimurium.RomBiotecholLett.2012;17:7194–7204.

22.RodpaiaE,MoongkarndiaP,TungrugsasutbW,

PhisannoradejaR,KanaratcS.Comparisonofmultiplex

polymerasechainreactionandimmunoassaytodetect

Salmonellaspp.,S.Typhimurium,andS.EnteritidisinThai

chickenmeat.ScienceAsia.2013;39(2):150–159.

23.MajeedLJ,ZnadKH,ThamirMK,BaqirHI.StudyofELISAand

antibioticsensitivitytestforSalmonellaenteritidisas

experimentalinfectioninmice.Um-SalamaSciJ.

2009;6(1):114–122.

24.MandalJ,AkhterMZ,KabirMS,AhsanS.Developmentofa

multiplexpolymerasechainreactionprotocolforthe

simultaneousdetectionofSalmonellaentericaserovarTyphi

andClass1integron.AsianPacJTropDis.2014;4:S808–S812.

25.IsomakiO,VuentoR,GranforsK.Serologicaldiagnosisof

Salmonellainfectionsbyenzymeimmunoassay.Lancet.

1989;333(8652):1411–1414.

26.DalbyT,StridMA,BeyerNH,BlomJ,MølbakK,KrogfeltKA.

RapiddecayofSalmonellaflagellaantibodiesduringhuman

gastroenteritis:afollowupstudy.JMicrobiolMethods.

2005;62(2):233–243.

27.StridMA,DalbyT,MølbakK,KrogfeltKA.Kineticsofthe

humanantibodyresponseagainstSalmonellaenterica

serovarsEnteritidisandTyphimuriumdeterminedby

lipopolysaccharideenzyme-linkedimmunosorbentassay.

ClinVaccineImmunol.2007;14(6):741–747.

28.CrayfordG,CoombesJL,HumphreyTJ,WigleyP.Monophasic

expressionofFliCbySalmonella4,[5],12:i:-DT193doesnot

alteritspathogenicityduringinfectionofporcineintestinal

epithelialcells.Microbiology.2014;160(Pt11):2507–2516.

29.LeeK-M,RunyonM,HerrmanTJ,PhillipsR,HsiehJ.Reviewof

Salmonelladetectionandidentificationmethods:aspectsof

rapidemergencyresponseandfoodsafety.FoodControl.

2015;47:264–276.

30.KremerC,O’MearaK,LaytonS,HargisB,ColeK.Evaluation

ofrecombinantSalmonellaexpressingtheflagellarprotein

fliCforpersistenceandenhancedantibodyresponsein

commercialturkeys.PoultSci.2011;90(4):752–758.

31.RickeS,PillaiS,NortonR,MaciorowskiK,JonesF.

ApplicabilityofrapidmethodsfordetectionofSalmonella

spp.inpoultryfeeds:areview.JRapidMethodsAutom