Campylobacter in broiler slaughter samples assessed by direct count on mCCDA and Campy-Cefex agar

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

Veterinary

Microbiology

Campylobacter

in

broiler

slaughter

samples

assessed

by

direct

count

on

mCCDA

and

Campy-Cefex

agar

Camila

Cristina

Gonsalves

_,

_Anderlise

_Borsoi

_,

_Gustavo

_Perdoncini

_,

Laura

Beatriz

Rodrigues

_,

_Vladimir

_Pinheiro

_do

_Nascimento

a_{CentrodeDiagnósticoePesquisaemPatologiaAviária(CDPA)–FAVET/UFRGS–Lab.Central–PortoAlegre,RS,Brazil}

b_{DepartamentodePatologiaExperimentaleComparada–FaculdadedeMedicinaVeterináriaeZootecnia(USP),Brazil}

c_{CursodeMedicinaVeterináriadaFaculdadedeAgronomiaeMedicinaVeterináriadaUniversidadedePassoFundo(UPF),Brazil}

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Articlehistory:

Received25February2015 Accepted13January2016 Availableonline20April2016 AssociateEditor:ElaineCristina PereiraDeMartinis

Keywords: Campylobacter

Agarplatecount mCCDA Campy-Cefex Broiler

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Campylobacterspp.causefoodborneillnessesinhumansprimarilythroughthe

consump-tionofcontaminatedchicken.TheaimofthisstudywastoevaluatetheUnitedStates DepartmentofAgriculture’s(USDA)recommendedmethodology,protocolMLG41.02,forthe isolation,identificationanddirectplatecountingofCampylobacterjejuniandC.colisamples fromthebroilerslaughteringprocess.AplatingmethodusingbothmCCDAand Campy-CefexagarsisrecommendedtorecoverCampylobactercells.Itisalsopossibletousethis methodindifferentmatrices(cloacalswabsandwatersamples).Cloacalswabs,samples frompre-chillerandpost-chillercarcassesandsamplesofpre-chiller,chilleranddirect supplywaterwerecollectedeachweekforfourweeksfromthesameflockata slaugh-terhouselocatedinanabattoirinsouthernBrazil.Sampleswereanalyzedtodirectlycount

Campylobacterspp.,andtheresultsshowedahighfrequencyofCampylobacterspp.on

Campy-Cefexagar.Fortheisolatedspecies,72%wereidentifiedasCampylobacterjejuniand38%as

Campylobactercoli.ItwaspossibletocountCampylobacterjejuniandCampylobactercolifrom

differentsamples,includingthewatersupplysamples,usingthetwo-agarmethod.These resultssuggestthatslaughterhousescanusedirectcountingmethodswithbothagarsand differentmatricesasamonitoringtooltoassessthepresenceofCampylobacterbacteriain theirproducts.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction

Campylobacterbacteriaareamajorcauseoffoodborneillness

inhumansandarethemostcommongastroenteritis-causing

∗ _{Correspondingauthor.}

E-mail:[email protected](C.C.Gonsalves).

bacteria in the world. In both developed and developing countries,theycausemorecasesofgastroenteritisthandoes foodborne Salmonella. The high incidence of Campylobacter

diarrheaanditsdurationandpossiblesequelaemakeithighly importantfromasocio-economicperspective.Indeveloping

http://dx.doi.org/10.1016/j.bjm.2016.04.025

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

countries,Campylobacterinfectionsinchildrenundertheage oftwo are particularly frequentand occasionally result in death.1

In the USA, Campylobacter is the second most isolated

agent of foodborne illness,2 _{and in the European Union}

(EU),Campylobacteristhemainpathogenthatcauseshuman gastroenteritis,3 _{with approximately 198,252 cases in 2009}

alone.4

Campylobacterbacteriacanbespreadbycontaminatedfood

and water, and chicken was implicated as the main con-taminationsource.5_{Duringthepoultryslaughteringprocess,}

carcasscontaminationcanoccur notonlybyhighbacterial loadspresentinthepoultry’sgastrointestinaltractbutalsoby thebacterialloadspresentinskinandfeathers.6_Inadditionto

initialflockcontamination,hygienicandsanitaryconditions duringboththeslaughteringprocessandcarcass conserva-tioncaninfluencethepresenceandlevelofCampylobacterin thefinalproduct.7

Broilercarcasses contaminated with Campylobacter have beendetectedinmanycountries.AstudyconductedinBrazil8

showedthat95ofthe96broiler carcassesexaminedtested positivefor Campylobacterat the end ofthe slaughter line. Accordingtothe World Health Organization,9 _{reducing the}

prevalenceorconcentrationataspecifiedpointinthe pro-ductionchainhasthepotentialtoreducetheriskofhuman incidencesifinterventionistaken.

Quantitative microbial risk assessment is a well-recognized component of modern risk analysis and is usedtoestimatetheimpactofaparticularhazard/product combinationand/orchangesinprocessingonpublichealth. Inthisregard,twomethodsforCampylobacterquantification were publishedby leadingauthorities, onebythe Interna-tionalStandardOrganization,ISO-TS10272-2,10_andanother

bytheUnitedStatesDepartmentofAgriculture,MLG41.02.11

Thelatterdescribesamethodfordirect platingand quali-tativeandquantitativeevaluationsusingCampy-Cefexagar fortheisolation,identificationandcountingofCampylobacter

spp. present in rinsed poultrycarcasses, sponges and raw productsamples.

ThepresentstudyaimedtotesttheMLG41.0211

method-ologyoncarcasssamplesandinalternativematrices,suchas cloacalswabsandwatersamples,fromthebroiler slaughter-ingprocesstocomparethenumberofrecoveredCampylobacter

cellsonCampy-CefexandmCCDAagarplates.

Materials

and

methods

Samples were taken once a week from a slaughterhouse locatedinsouthernBrazilforfourweeksinApril2013.Each time,threecloacalswabsandthreesamplesfrompre-chiller carcasses, post-chiller carcasses, pre chiller water, chiller wateranddirectsupplywaterwerecollected,with12samples ofeachtypeand72intotal.Thesampleswereplaced imme-diatelyintothermicboxeswithiceandsenttothelaboratory formicrobiologicalanalysis.

Quantitativeandqualitativeanalysis Controls

CampylobacterjejuniATCC33291andCampylobactercoliATCC

43578wereusedaspositivecontrolsforun-inoculatedagar platesandbrothsamplesets.Onecolonyfromeachpositive controlsamplewasconfirmed.

Carcassrinseprocedure

Carcasssampleswerecollectedpriortoplacementinthe pre-chillertankontheeviscerationline(pre-chillersamples)and immediatelyafterthepost-chiller(post-chillersamples).

Toperformquantitativeanalysisonthecarcassrinse sam-ples,carcasseswereputintosterileplasticbagswith400mL of1%BPW(1%BufferedPeptoneWater,Oxoid®_{,Basingstoke,}

Hampshire,UK)andmixedthoroughlybygentlyshakingfor 3min.Fromtherinsesolution,250␮Lwasstreakedontofour Campy-Cefexagarplateswithantimicrobialselective supple-ment(SR0155,Oxoid®_{,Basingstoke,Hampshire,UK)and5%}

sterilelakedequineblood(Ebefarma®_{,CachoeiradeMacacu,}

RJ,Brazil)usingsterileglassDrigalskiloops.Another100␮L fromtherinsesolutionwasstreakedontotwoCampy-Cefex agarplates.Theprocedurewasperformedinduplicatewith streakingontomCCDAagarplatescontaininganantimicrobial selective supplement (SR0155 Oxoid®_{, Basingstoke,}

Hamp-shire, UK).Theagarplateswere incubatedat42±1.0◦Cfor 48±2hundermicroaerobicconditions(Microerobac,Probac®_,

SãoPaulo,SP,Brazil).

To perform qualitative analysis, 30mL of the rinsing solution was addedto 30mLofdouble strength blood-free Bolton enrichment broth (2X BF-BEB, Oxoid®, Basingstoke, Hampshire,UK) withselectivesupplement (SRE183,Oxoid®

Basingstoke,Hampshire,UK)andhomogenizedasdescribed above.Afterincubation,10␮LfromtheBF-BEBwasstreaked onto Campy-Cefex and mCCDA agar plates and incubated undermicroaerobicconditionsat42±1.0◦Cfor48±2h.

Cloacalswabs

Swabs werecollected duringbroilerhanging, oneswabper bird, and stored in a 10mL flask with 1% BPW (Oxoid®

Basingstoke,Hampshire,UK)at4◦C.Thetubeswere homog-enized, and the quantitative analysis followed the same methodologyasdescribedaboveforthecarcasses.Toperform the qualitative analysis, 3mL from the BPW homogenized solution was addedto 30mLofdouble strength blood-free Bolton enrichment broth (2X BF-BEB, Oxoid®_{, Basingstoke,}

Hampshire,UK) supplementedwithaselectivesupplement (SRE183,Oxoid®_{,Basingstoke,Hampshire,UK)andanalyzed}

asdescribedaboveforthecarcasses.

Watersamples

Watersamplesfrom theprechiller,chilleranddirectwater supply were collected in50mLsterileflasks and storedat 4◦Cinthelaboratory.Thewatersampleswerehomogenized andquantitativeandqualitativeanalysiswasperformedas describedaboveforthecarcasses.

Table1–FrequencyofCampylobacterspp.coloniesdirectlyisolatedfrommCCDAandCampy-Cefexagarplateswithor withoutbrothenrichmentindifferentsamplesfromthebroilerslaughteringprocess.

Sample Directisolation

AgarCampy-Cefex(%)

Directisolation AgarmCCDA(%)

Enrichmentbrothand agarisolation*_(%) Totalofanalyzed samples Swabs 100 100 100 12 Carcassespre-chiller 100 25 100 12 Pre-chillerwater 100 75 100 12 Chillerwater 100 66 100 12 Carcassespost-chiller 83 67 100 12 Watersupply 100 50 100 12

∗ _{Boltonbroth,Campy-CefexandmCCDAagars.}

Plateanalysisandresults

After incubation, all typical Campylobacter colonies were countedifthetotalnumberofcellsfellwithinarangeof15–300 colonies.Theinterpretationoftypicalcoloniesfollowedthe instructionsintheMLG 41.02protocol.11 _{Briefly,if thefour}

plateswith250␮Lwerecountable,thesumofthecountsfrom thefourplateswasdetermined.Iftheonlycountableplates were thetwo plates with100␮Lofsample,the total num-berofcellsonbothplates wasaveraged and multipliedby ten.Ifbothdilutionswerewithinthecountableinterval,the finalcountwasdeterminedbyaveragingtheresultscalculated above.AllcellcountresultsarerepresentedasCFU/mL.Ifthe finalcountwas>300CFUineachofthesixplates,“TNTC”(to numeroustocount)wasrecorded,oranestimatedcounting of>2100CFU/mLwasused.11

Examinationandconfirmationofcolonies

A typicalcolony from each samplewas picked to confirm and test motility, morphology, oxidase and catalase activ-ity (Probac®, São Paulo, SP, Brazil) and latex agglutination (Dryspot, DR0150,Oxoid®_{, Basingstoke, Hampshire,UK), as}

describedinMLG41.02.11_{Thesecolonieswerealsousedfor}

thepolymerasechainreaction(PCR)technique(seebelow). PCRmethodology

Multiplex PCR was performed as described by Perdoncini

etal.12_{toidentifyC.jejuniandC.coli.Briefly,30␮Lreactions}

containing10×buffer,1.5mMMgCl2,5mMdNTPs,twounits

ofTaqpolymerase,thermoDNAextract,4pmol/␮Lofeach16S rRNAprimer,2pmol/␮Lspecificprimersandultra-purewater upto30␮Lweremade.Thespecificprimersusedamplifythe followinggenes:mapA(Fa_{–CTATTTTATTTTTGAGTGCTTGTG,}

Rb_{–GCTTTATTTGCCATTTGTTTTATTA with 589pb, 50N)}

(Invitrogen®_{,SãoPaulo,SP,Brazil)andceuE(F}a

–AATTGAAAAT-TGCTCCAACTATG, Rb_{–TGATTTTATTATTTGTAGCAGCG with}

462pband a common region between species (16S rRNA), 50N (Invitrogen®_{, São Paulo, SP, Brazil) and F}a

–ATCTAAT-GGCTTAACCATTAAAC, Rb_{–GGACGGTAACTAGTTTAGTATT}

with857pb,50N(Invitrogen®_{,SãoPaulo,SP,Brazil)).}

Ampli-ficationreactionswerecarriedoutinathermalcycler(Swift MaxPro®_{,Esco,Hatboro,PA,USA)underthefollowing}

condi-tions:denaturationfor10minat95◦C,35cyclesat95◦Cfor 30s,annealingat59◦Cfor1minand30sandafinalextension

at 72◦C for 10min. Arcobacter spp. were used as negative

controls and C. jejuni ATCC 33291 and C. coli ATCC 43578 as positivecontrols.Tenmicroliter aliquotsofthe reaction mixtures were electrophoresed through 1.5% agarose gels (withtheadditionof20%ethidiumbromide)witha100-bp DNAladder(Invitrogen®_{,SãoPaulo,SP,Brazil)todetermine}

themolecularweight.Fragmentsweretransilluminatedwith UVlight.

Datastatisticalanalysis

DataweresubmittedforANOVAanalysisusingBioStatVersion 2009(AnalystSoft.Inc.,Alexandria,VA,USA).

Results

TheCampylobacterdirectplatecountfrequencywashigheron

the Campy-Cefexagar than on the mCCDAagar for differ-entsamplesfrombroilerslaughteringprocessasdescribedin

Table1.

Table 2 presents the results of quantitative analysis of

Campylobacter spp. directly counted on Campy-Cefex and

mCCDAagarplates.

TherewasahighfrequencyofC.jejuniinallPCR-analyzed samples(Table3).Inaddition,18%containedbothC.jejuniand

C.coliinthesamesample.

WithrespecttoallPCRsamples,2%containednoneofthe specificgenesusedtoidentifyC.jejuniorC.coli,althoughthey wereidentifiedasaCampylobacterspecies.

Discussion

Campylobacter spp. are recognized as a main cause of

humanenteritis outbreaksinboth developedand develop-ing countries.AlthoughBrazilisthe world’slargestpoultry meatexporter,dataregardingthispathogenarelimited,and atpresent,thereisnolegislationpertainingtoCampylobacter

riskanalysesorcontrolmethods.

TheCampylobacterisolationmethodologiesarelaborious,

andtherearemanybrothsandagarsavailable.Somestudies haveevaluatedtheeffectivenessofdifferentbrothsandagar plates fortheirabilitytoisolateCampylobacterfrom several matricestodevelopmoreefficientandlowercostmethods.13

Oyarzabal et al.14 _{evaluated 240samples ofbroiler carcass}

rinse samplesbyrecoveringCampylobacteronCampy-Cefex, mCCDAandCLA(Campy-Lineagar)agarplates.Theauthors concludedthatwithregardstotime,preparation,performance

Table2–DirectcountaveragesofCampylobacterspp.fromCampy-CefexandmCCDAagarplatesplatedwithdifferent samplesfromthebroilerslaughteringprocess.

Samples AgarCampy-Cefex

Average(CFU/mL) AgarmCCDA Average(CFU/mL) p* Swabs 1.3×103 _9.5×102 _0.307 Carcassespre-chiller 9.8×102 _8.3×101 _0.005 Pre-chillerwater 1.7×102 _5.4×102 _0.502 Chillerwater 8.0×102 _3.0×101 _0.139

Carcassespostchiller 1.5×102 _3.8×101 _0.194

Watersupply 7.3×101 _4.7×100 _0.318

∗ _{p<0.05,statisticallysignificant.}

Table3–PercentageofisolatedCampylobacterjejuniandCampylobactercolionCampy-CefexandmCCDAagarplatesas identifiedbyPCRanalysis.

Samples Campylobacterjejuni Campylobactercoli

Campy-Cefex mCCDA Campy-Cefex mCCDA

Swabs 12% 8% 10% 8% Carcassespre-chiller 8% 6% – 2% Pre-chillerwater 6% 6% – – Chillerwater 6% 2% 2% – Carcassespost-chiller 4% 6% – 6% Watersupply 4% – 4% –

and cost, Campy-Cefex and mCCDA agar obtained better Campylobactercountingresultsfromcarcassrinsesamples.In anotherpoultrystudy15_{wascomparedfiveagarplatesthat}

wereusedtoisolateCampylobacterasofcecalandfecal sam-plesobtainedfrom60 broilerchicken.ThemCCAagarwas moreefficientatisolatingthebacteriathanthemCCDA,CLA, CAP(Campylobacteragarplates)andCampylobacteragars.

Inthepresentstudy,thequantitativemethodsfromthe MLG41.0211 _{protocol showedreduced levelsof}

Campylobac-terinsamplescollectedalongtheslaughterline,fromcloacal swabs from live birds to post chiller carcasses, suggesting thattheprocessofslaughteringcanhaveabeneficialeffect onthemicrobiologicalstatusofcarcassesattheendofthe slaughteringline.Accordingly,Berrangetal.,16_reportedthat

improvedhygiene intheslaughteringprocessandconstant evaluationsofsuchhygienemeasuresallowedforareduction

inCampylobacterspp.numbersincarcassespriortoshipping

tomarkets.Thus,thenumberofbacteriafrominfectedflocks canbereducedduringprocessingintheslaughterhouse.

ThepresenceofCampylobacterinthe watersupply sam-ples(Table1)wasnotexpectedbecausechemicaltreatmentof thewatershouldeliminatebacteria;further,ifbacteriawere present,alowlevelshouldnothavebeendetectedbyadirect countmethod.ItisknownthatCampylobacterisableto com-posebiofilmsandthattheirformationhasbeenproposedas asurvivalmechanismoutsidethehostforprotectionagainst chemicalproducts,physicalcleaningprocessesand environ-mentalstress,amongothers.17_{Thus,thepresenceofbiofilms}

andthecontaminationofthewatersourcesarepossible expla-nationsforthesedata.

Ahigh(100%)prevalenceofCampylobacterincloacalswabs was also found in this study (Table 1) by direct counting on both types of agar. These data are in agreement with anotherstudy,18 _{who foundthat96.6% from30 samplesof}

cloacal swabs contained Campylobacter. Additionally, Evans andSayers19_{identifiedthesebacteriain91%ofchickencloacal}

swabs(20totalsamples)inGreatBritain,andFranchinetal.20

reportedthat75%oftheswabsfrombroilerflocksinsouthern Brazilwerepositiveforthesebacteria.

Regarding carcass contamination,we found Campylobac-tercontaminationin 83%ofpost-chiller carcasses,and the isolationfrequencybydirectplatecountingonCampy-Cefex agarwashigh(Table1).AfterenrichmentinBoltonbrothto boostlowcellnumbersinsomesamples,allpre-and post-chillercarcasssamplestestedpositive.However,thesedata werehigherthanthosereportedbytheEuropeans,whohad an average poultrycarcass contaminationlevel of 75.8%,21

and byKuana etal.,8 _{whoreportedthat 98.3%of60 broiler}

carcasseswerecontaminatedafterchillerprocessing.Inthe presentwork,asignificantdifferencewasfoundbetweenthe Campy-CefexandmCCDAplatesusedforcellrecoveryinthe analysisofpre-chillercarcasssamples,whereCampy-Cefex hadhigherCampylobactercellnumbers(Table2).Furthermore, itisimportanttonotethatdespitethehighisolation percent-agefromtheCampy-Cefexagar,noC.coliwasrecoveredin pre-andpost-chillercarcasssamples,afactthatisnotconsistent withothermatrices(Table3).

Studies using direct plate counting methods have indi-catedthatselectiveenrichmentdoesnotincreasetherecovery

of Campylobacter from fecal or cecal samples or chicken

carcasses.22,23_{Thesedatadifferfromthepresentstudywhere}

100% of enriched samples were positive for Campylobacter

spp.Kiessetal.24_{demonstratedthatdirectplatingservesan}

advantageinisolatingCampylobacterfrompoultrylitter sam-ples; 37%ofthe sampleswere positiveforCampylobacteras testedbydirectplatingand2%werepositivefollowing enrich-ment.DespiteourhigherCampylobacterfrequencyinenriched post-chiller samples, the directcount method was able to

recoverandquantifyCampylobacterinagreementwith Oyarz-abal et al.,14 _{who demonstrated the valueofdirectplating}

instudyingCampylobacterspp.contaminationofpoultry car-casses.

MultiplexPCRanalysisidentified72%ofsamplesas

posi-tiveforC.jejuniand38%aspositiveforC.coli(Table3).Similar

results were demonstrated in the European Union, where 60.8%ofcecalsamplestestedpositiveforC.jejuniand41.5% testedpositiveforC.coli.21_{InsouthernBrazil,astudy}

con-ductedbyPerdonciniet al.12_{sampledeightdifferentpoints}

fromabroilerslaughterhouseline.TheyidentifiedC.jejuniin 75%ofthesamplesandC.coliin10%ofthesamples.In addi-tion,bothC.jejuniandCcoliwerepresenttogetherin15%ofall samples.Incontrast,200samplesofbroilercecalcontentfrom asouthernBrazilslaughterhousewereevaluated,anditwas foundthat44%werepositiveforC.coliand2%werepositive

forC.jejuni.25

Studieshavealsobeenperformedwithregardstothe

differ-entCampylobacterserotypescolonizingbirds.Shibiny-Eletal.26

reportedthatitisnotcommontoisolatemorethanonetype orsubtypeofCampylobacterfromthesamebird.Theauthors suggestthatC.jejuniandC.colicompeteequallyandshowed adeclineinC.jejuniandC.colidominanceinisolatesfrom 35-day-oldbirds.Incontrast,thepresentstudy,aswellasthe studybyPerdoncinietal.,12 _{isolatedtwodifferentserotypes}

fromthesamesample;18%ofthesamplescontainedbothC.

jejuniandC.coli.Infact,bothserotypesappearedinthesame

frequencyincloacalswabs.

In this work, the abilityto isolate C. colifrom different matricesbyCampy-CefexandmCCDAagarplateswasvariable (Table3).AccordingtoWHO,1_{manydifferentformsofmedia}

canbeusedintherecoveryofCampylobacterspp.,andalthough mCCDAagaristherecommendedmedium,alternativesmay beused.Themaindifferencebetweenmediaisthe degree towhicheachinhibitscontaminatingflora,but allselective agentsallowforthegrowthofbothC.jejuniandC.coli.

Conclusions

Thedirectplatingmethodsappliedinthisstudy wereable to recover Campylobacter from different poultry matrices. Onlyfrompre-chillerwaterdidtheCampy-Cefexagardirect countingmethodrecoverstatisticallyhighCampylobactercells numbers.Basedon theresultsofthis study,it isplausible tosuggest that bothCampy-Cefexand mCCDAagar plates can increase the chancesofrecovering C. colifrom swabs, carcassesandwatersamples.Thepresentworkalso demon-strated thatdirect countingofCampylobacter from samples atdifferent sitesinthebroiler slaughterhouseisuseful for identifyingcontaminationpointsandlevelsandisapossible toolforcontrollingCampylobactercontaminationatBrazilian slaughterhouses.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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