An optimised protocol for molecular identification of Eimeria from chickens

ContentslistsavailableatScienceDirect

Veterinary

Parasitology

j o ur na l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / v e t p a r

An

optimised

protocol

for

molecular

identification

of

Eimeria

from

chickens

夽

Saroj

Kumar

,

Rajat

Garg

,

Abdalgader

Moftah

,

Emily

L.

Clark

,

Sarah

E.

Macdonald

,

Abdul

S.

Chaudhry

,

Olivier

Sparagano

,

Partha

S.

Banerjee

,

Krishnendu

Kundu

,

Fiona

M.

Tomley

,

Damer

P.

Blake

b_{SchoolofAgriculture,FoodandRuralDevelopment,AgricultureBuilding,NewcastleUniversity,NewcastleuponTyneNE17RU,United}

Kingdom

c_{PathologyandPathogenBiology,RoyalVeterinaryCollege,HawksheadLane,NorthMymmsAL97TA,UnitedKingdom} d_{FacultyofHealthandLifeSciences,NorthumbriaUniversity,NewcastleuponTyneNE18ST,UnitedKingdom}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3July2013 Receivedinrevisedform 13September2013 Accepted20September2013

Keywords:

Eimeriaspeciesidentification Chicken

COCCIMORPH MultiplexPCR NestedPCR Protocol

a

b

s

t

r

a

c

t

Molecularapproachessupporting identificationofEimeriaparasitesinfecting chickens havebeenavailableformorethan20years,althoughtheyhavelargelyfailedtoreplace traditionalmeasuressuchasmicroscopyandpathology.Limitationsofmicroscopy-led diagnostics,includingarequirementforspecialistparasitologicalexpertiseandlowsample throughput,areyettobeoutweighedbythedifficultiesassociatedwithaccessinggenomic DNAfromenvironmentalEimeriasamples.AkeysteptowardstheuseofEimeria species-specificPCRasasensitiveandreproduciblediscriminatorytoolforuseinthefieldisthe productionofastandardisedprotocolthatincludessamplecollectionandDNAtemplate preparation,aswellasprimerselectionfromthenumerousPCRassaysnowpublished.Such aprotocolwillfacilitatedevelopmentofvaluableepidemiologicaldatasetswhichmaybe easilycomparedbetweenstudiesandlaboratories.Theoutcomeofanoptimisationprocess undertakeninlaboratoriesinIndiaandtheUKisdescribedhere,identifyingfoursteps.First, sampleswerecollectedintoa2%(w/v)potassiumdichromatesolution.Second,oocysts wereenrichedbyflotationinsaturatedsaline.Third,genomicDNAwasextractedusinga QIAampDNAStoolminikitprotocolincludingamechanicalhomogenisationstep.Finally, nestedPCRwascarriedoutusingpreviouslypublishedprimerstargetingtheinternal tran-scribedspacerregion1(ITS-1).Alternativemethodstestedincludedsampleprocessingin thepresenceoffaecalmaterial,DNAextractionusingatraditionalphenol/chloroform pro-tocol,theuseofSCARmultiplexPCR(onetubeandtwotubeversions)andspeciationusing themorphometrictoolCOCCIMORPHforthefirsttimewithfieldsamples.

© 2013 Dirk Vulpius The Authors. Published by Elsevier B.V. All rights reserved.

夽 _{Thisisanopen-accessarticledistributedunderthetermsofthe} Creative Commons Attribution-NonCommercial-No Derivative Works License,whichpermitsnon-commercialuse,distribution,and reproduc-tioninanymedium,providedtheoriginalauthorandsourcearecredited.

∗ Correspondingauthor.

∗∗ Correspondingauthor.Tel.:+441707666041.

E-mailaddresses:[email protected](R.Garg), [email protected](D.P.Blake).

1. Introduction

Coccidiosis, caused by protozoan parasites belong-ingto thegenusEimeria, is oneof thecommonest and mosteconomically important enteric diseases of chick-ens’worldwide(Shirleyetal.,2005).SevenEimeriaspecies can infect the chicken (viz., Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria neca-trix, Eimeria praecox and Eimeria tenella) and all can

0304-4017/$–seefrontmatter© 2013 Dirk Vulpius The Authors. Published by Elsevier B.V. All rights reserved.

compromise economic production and animal welfare, resultinginpoorfeedconversionratios,failuretothrive andelevatedmortality(Longetal.,1976;Williamsetal., 2009). Conventionally, identification of Eimeria spp. is basedonmorphologicalfeaturesofthesporulatedoocyst, sporulation time and location/scoring of pathological lesionsintheintestinebuttheproceduresinvolvedrequire specialist expertiseand have seriouslimitations due to their subjective nature and overlapping characteristics amongdifferentspecies(Longand Joyner,1984).Mixed infectionsalsoposeaproblemfortheprecise discrimina-tionofspeciesusingmorphologicalmethods.Alternative species-specificdiagnosticsarerequiredtoinformroutine animalhusbandry,veterinaryinterventionand epidemio-logicalinvestigation.

OnesuchalternativeisEimeria species-specific poly-merasechainreaction(PCR).Overthelast20yearsseveral PCR assays have been developed that target genomic regionsof one ormore Eimeria speciesincluding theE. tenella 5S or small subunit rRNAs (Stucki et al., 1993; Tsujietal.,1999),thefirstandsecondinternaltranscribed spacer regions (ITS-1 and -2) (Gasser et al., 2001; Lew etal.,2003;Schnitzleretal.,1998;Suetal.,2003;Woods etal.,2000)andgene-specifictargetsincludingsporozoite antigengeneEASZ240/160(Molloyetal.,1998).Inoneof themostcomprehensivestudiesFernandezetal.(2003) designedspecies-specificprimersforEimeriaspp.froma groupofSCAR(Sequence-CharacterizedAmplifiedRegion) markersand usedthem todevelop a multiplexPCRfor thesimultaneousdiscriminationofdifferentEimeriaspp. inasinglereaction.Importantly,manyoftheseassayshave beenshowntobecapableofdetectinggenomicDNA rep-resentingasfewas0.4–8oocyst-equivalents(Fernandez etal.,2003;Haugetal.,2007),orasfewas10–20oocysts (Carvalhoetal.,2011a;Frölichetal.,2013).Nonetheless, routine application withfield samplesremains compli-cated by factors including DNA extraction from within the tough oocyst wall and faecal PCR inhibition (Raj etal.,2013).Broader uptakeofPCR-based Eimeria diag-nostics can be significantly enhanced by establishment ofanoptimisedprotocol. Similarly,identificationof the mostsensitiveandrobust primersfromthelarge num-berofEimeria-specificPCRassaysthatareavailableisan essentialsteptowardsstandardisedepidemiological anal-ysesappropriateforinternationalcomparison.Validation of collection, purification and PCR amplification proto-colsacrossdifferentlabs,in multiplecountries,is akey stepintheestablishmentofoptimalsamplingstrategies as we seek to improve understanding of parasite field biology.

Beyond PCR other approaches to species-specific identificationofEimeriaincludequantitativePCR(qPCR) (Morgan et al., 2009; Vrba et al., 2010), although cost is currentlylimiting for routineapplications, and Loop-mediatedIsothermalAmplification(LAMP;Barkwayetal., 2011).Importantly,accessingDNAfromwithintherobust oocyst wallis a challenge for all of these technologies whenworkingwithfaecalorlittersamples.Analternative computationalapproachistheuseofsoftwaretool COC-CIMORPH (http://www.coccidia.icb.usp.br/coccimorph), which is based on identification of sporulated oocysts

of Eimeria spp. of poultry by morphological analysis (Casta ˜nónetal.,2007).

In the present study three different parasite purifi-cation/DNA extraction procedures (QIAamp Stool Mini kit with and without faecal contamination, and phe-nol/chloroform)andthreedifferentPCRprotocols(nested PCRITS-1amplificationandmultiplexSCARPCRinaoneor twotubeformat)havebeentestedinIndiaandtheUKand comparedtothesoftwaretoolCOCCIMORPHfor diagnos-ticefficacyoncoccidiapositivefaecaldroppingscollected fromcommerciallyraisedpoultry.

2. Materialsandmethods

2.1. Faecalsamplecollection

DuringNovember2011toApril,2012,atotalof45 com-mercialpoultryfarmsweresampledfromUttarPradesh and UttarakhandstatesofNorth India.Duringthesame period139commercialpoultryfarmsinEgypt,Libyaand theUKweresampled.Forcollectionofpoultrydroppings 50mlpolypropyleneconicaltubeswereused,eachwitha screwtopandcontaining5mlpotassiumdichromate(2% w/v).Theweightofeachtubewasrecordedandpooled fae-caldroppingswerecollectedstartingfromonecornerofa unitandfollowinga‘W’pathwayacrosstheunit,collecting onefreshdroppingeverytwotofivepacesdependingon thesizeoftheunituntilthetubewasfilledtothe10ml mark.Threetofivetubeswerefilledperunit.Eachtubewas thenproperlycappedandthecontentswerethoroughly mixed by vigorous shaking. The samples thuscollected weretransportedtothelaboratoryandrefrigeratedat4◦C untilfurtherprocessed.

2.2. Processingoffaecalsamples

Thetubeswithfaecalmaterialwereagainweighedand 1.6gsodiumchloridewasaddedtoeachtube.Then sat-uratedsaltsolutionwasaddeduptothe25mlmark.The tubeswerecappedtightlyandvigorouslyshakenuntilthe faecal material wascompletelybroken and mixedwell. Finally,thetubeswerefilledupto50mlmarkwith sat-uratedsaltsolutionandmixedthoroughly.Onthisfaecal suspension, 1–2ml of single distilled water was gently overlaid.Thesamplewaslefttostandfortenminutesand thencentrifugedat∼750×gfor8min.Usingadisposable Pasteurpipette,thelayerfromtheinterfacebetweenthe saturatedsaltandthewaterwastransferredtoanew50ml polypropyleneconicaltube.Thiswascontinuedforthree moretimestillnomaterialwasvisibleattheinterface.The newtubewasfilledupto50mlmarkwithsingledistilled waterandcentrifugedat∼750×gfor8–10min.The super-natantwascarefullyremovedwithoutdisturbingthepellet usingadisposablePasteurpipette,leaving3–5mlfluid.The supernatant wascheckedmicroscopicallyforunpelleted oocystsbeforediscarding.

screeningforunpelletedoocysts.Thepelletedoocystswere suspended in 1.0ml distilled ormolecular grade water. Afterthroughmixing,10␮lofthissamplewasdrawnfrom themicrofuge tubeandmixedwithsaturated salt solu-tionuptothe1mlmarkforestimating thefinal oocyst concentration(oocystspergramoffaeces,OPG)inthe sam-pleusingMcMasterchambers.Theeimerianoocystswere thenallowedtosporulatein2%w/vpotassiumdichromate solutionat27±2◦Cforthreedays.Followingsporulation, theoocystswerethoroughlywashedthriceinautoclaved distilledormoleculargradewaterfortaking photomicro-graphsandpelletedforDNAisolation.

2.3. IdentificationofEimeriaspp.byCOCCIMORPH

For the identification of eimerian oocysts, photomi-crographs of at least 50 individual sporulated oocysts wererandomlytakenfromeachsampleat10×/40×using a dry high power objective with a photomicrographic camera(Moticam5,HongKong)attachedtoatrinocular research microscope (Motic Trinocular Research Micro-scope BA210,Hong Kong). Theidentification of Eimeria spp. of chickens was done using COCCIMORPH soft-ware (http://www.coccidia.icb.usp.br/coccimorph/). The softwarewasdownloadedfromtheInternetandtheoocyst images(400× magnification)wereuploaded for species identificationasdescribedonline.TheEimeriaspp. iden-tifiedbythesoftwareineachsamplewasrecorded.

2.4. IsolationofgenomicDNA

For isolationofgenomicDNA,onlysamplesfoundto containmorethan500(India)or200(Egypt,LibyaandUK) OPGwereselectedforprocessing.

2.4.1. QIAampDNAStoolminikit

TotalgenomicDNAwasisolatedusingaQIAampDNA Stoolminikit(Qiagen,Germany)asperthemanufacturer’s protocolwithsomemodificationsfrom(i)oocystspurified asdescribedaboveor(ii)purifiedoocystssupplemented with100mgoocyst-negativefaecalmaterialcollectedfrom a specific pathogen free chicken to mimic the absence of a flotation step. Briefly, to the pelleted oocysts an equalvolumeofautoclavedglassballotinibeads measur-ing∼0.25–0.5mmindiameter(Sigma–Aldrich,USA)were added andcovered witha minimumvolumeASLbuffer (out of total 1.4ml tobe used for DNA isolation) sup-pliedwiththeDNAextractionkitorsterileTEbuffer.The oocystswerethendisrupted byvortexing(India;Spinix VortexShaker,Tarsons,India;maximumspeed)or bead-beating(Egypt,LibyaandUK,MiniBeadbeater-8,Biospec Products,Bartlesville,USA;settohomogenise)fortwo min-utes.Then,theremainingbufferASLwasaddedtothetube andthoroughlymixed.Thesuspensionwasthenheatedfor 5minat70◦CandprocessedaspertheQIAampDNAStool kitprotocol.TheDNAwaselutedtwicein100␮lTEbuffer asrecommendedbythemanufacturerandquantifiedusing absorbanceat260and280nm.

2.4.2. Phenol/chloroformDNAextraction

Total genomic DNA was isolated from purified oocysts using a standard phenol/chloroform extraction

protocolfollowingdisruptionusingaMiniBeadbeater-8 asdescribedpreviously(Blakeetal.,2003).

2.5. PCRamplification

AsummaryofthePCRassaystested,andtheprimers used,isprovidedinSupplementaryTable1.

2.5.1. IdentificationofEimeriagenusgenomicDNAby PCR

ThepresenceofEimeriagenusgenomicDNAwastested by PCR amplificationof the partial18S rDNA sequence usingtheprimersERIB1andERIB10asdescribedelsewhere (Schwarz et al., 2009). Briefly, each reaction contained 2_␮lgenomicDNAtemplate,25pmolforwardandreverse primer, 0.5U Taq polymerase (Invitrogen, Paisley, UK), 10mMTris–HCl,1.5mMMgCl2,50mMKCl and200␮M

dNTPs.Standardcycleparameterswere1×(5minat94◦C), 30× (30sat94◦C, 30sat57◦C, 2minat72◦C)and 1×

(10minat 72◦C). Post-amplification PCR productswere resolvedbyagarosegelelectrophoresis.

2.5.2. IdentificationofEimeriaspp.bynestedPCR

ThenestedPCRprotocolusingITS-1primerswas stan-dardisedfor identificationofEimeria species ofpoultry. PrimersamplifyingtheentireITS-1sequencewith flank-ingpartial18SrDNAand5.8SrDNAregionsofEimeriawere usedinthegenus-specificPCRphase,whilespecies-specific primerstargetingtheITS-1regionwereusedtoamplify theindividualEimeriaspeciesasdescribedelsewhere(Lew etal.,2003).

Briefly, each 25.0␮l PCR reaction included 2␮l of genomic DNA, 25pmol each of genus-specific primers, 1.25U of Taq polymerase, 200␮M each of dNTPs, and 2.5␮lofPCRbuffercontaining1.5mMMgCl2.Thethermal

cyclingwasdonewithaninitialdenaturingstepat94◦C for3minfollowedby30cyclesof94◦Cfor30s,55◦Cfor 30sand72◦Cfor 90sandafinal extensionat72◦Cfor 7min.TheproductoftheprimaryPCR (1.0␮lin25.0␮l reaction mixture) was used as template for thenested PCRwithspecies-specificprimersinindividualtubesusing thesameamplificationconditionsdescribedabove except-ingdifferentannealingtemperaturesfordifferentEimeria spp.(58◦CforE.mitis;61◦CforE.necatrixandE.praecox; 65◦CforE.tenella;71◦CforE.acervulina,E.maximaand E.brunetti).Negative,no-templatecontrolswereincluded witheachassayusingtripledistilledwaterinplaceof tem-plate.TheamplificationofspecificnestedPCRproductwas checkedbygelelectrophoresisin2%agarosegelsstained with0.5␮g/mlethidiumbromide.

2.6. IdentificationofEimeriaspp.bymultiplexPCR

ThemultiplexPCRusingSCARprimersforidentification ofthesevenEimeriaspeciesthatinfectchickens(Fernandez etal.,2003)wasstandardisedusingpureDNAsamplesfrom theHoughtonstrainsofeachEimeriaspp.

dNTP,5.0mMMgCl2,3.5UTaqDNAPolymerase,and1.6×

amplificationbuffer(suppliedbythemanufacturer)ina finalvolumeof25␮lreactionmixture.Thermocycling con-ditionsweresetat96◦Cfor5minforinitialdenaturation, followed by 30 cycles of 1min at 94◦C, 2min at 65◦C and90sat72◦C,withafinalextensionat72◦Cfor7min. Oncetheaboveconditionswerestandardisedfor individ-ualprimerpairs,alltheprimerpairswereputtogetherina single50␮lreactionmixtureforsingle-tubemultiplexPCR withthesamecyclingconditionsasdescribedabove.For two-tubemultiplex PCR,amplificationswereconducted separatelyintwotubes;tube1containedtheprimersfor E.acervulina,E.brunettiandE.mitiswhiletube2contained primersforE.maxima,E.necatrix,E.praecoxandE.tenella. AlltheconditionsforPCRremainedasdescribedabove.The amplificationofspecificPCRproductswerecheckedbygel electrophoresisin2%agarosegelsstainedwith0.5␮g/ml ethidiumbromide.

2.7. Statisticalanalysis

TheresultsofEimeriaspeciesdetectionforeachassay werecomparedbyChi-squareanalysisusingSPSSversion 20 (IBM, US).Results wereconsidered significantwhen p<0.05.

3. Results

3.1. GenomicDNAextraction

3.1.1. Protocolselection

Triplicateenvironmentalfaecalsampleswerecollected from30farmsand examinedmicroscopicallytoconfirm thepresenceofEimeriaoocysts(10×/20×).Oocystswere purified,pooledperfarmtostandardiseandsplitfor par-allelprocessingby(i)QIAampDNAStoolkit,(ii)QIAamp DNA Stool kit plus faecal contamination and (iii) phe-nol/chloroform.UsingtheEimeriagenus18SrDNAassay 93%(28/30)ofthesamplesprocessedusingtheQIAamp DNAStoolkitwerePCRpositiveand100%ofthesamples containing≥5000OPGatthebeginningoftheprocesswere positive(Table1).Theadditionoffaecalmaterialreduced thePCRpositiverateto30%withonlyoneof17samples containingfewerthan20,000oocystsfoundtobepositive. Usingphenol/chloroformextraction77%(23/30)samples werePCRpositivewitha100%successrateonlyoccurring above20,000startingOPG.

3.1.2. Protocolsensitivity

Theprotocolfoundtobemosteffective(QIAampDNA Stoolkitafteroocystflotation)wassubsequentlytestedon alargernumber offieldsamplestoinvestigate diagnos-ticsensitivity.In total 139farmswerevisited, ofwhich 100werepositive (71.9%)for Eimeriaoocystsby micro-scopicexaminationwithOPG rangingfrom0.2×103 _to

191.3_×103_{.Alloocystpositivesampleswereprocessed.}

Using the Eimeria genus 18S rDNA assay 96% (96/100) ofthe sampleswerePCR positive and 100% of samples containing≥5000OPGwerepositive(Table2).Sensitivity droppedbelow80%onlywhensamplescontainingfewer

than 500OPGwere processed,although thenumber of samplestestedatthislevelwasverysmall.

3.2. OptimalidentificationofEimeriaspp.

Out of 45 poultryfarms screenedin North India, 37 (82.2%)werepositiveforEimeriaspp.bymicroscopic exam-inationwithOPGrangingfrom0.1×103_to242.5×103_.Out

ofthe37coccidiapositivefarms,30farmshadOPG lev-elsabove500andthuswereselectedforfurtherEimeria speciesidentificationstudies.

3.2.1. COCCIMORPH

COCCIMORPHisacomputationalapproachforparasite identificationincaseofEimeriaspp.fromthechicken. Digi-talimagesof50individualunidentifiedsporulatedoocysts of Eimeria spp. were uploaded on tothe software. The software then analysed the oocyst on the basis of dif-ferent features namely, curvature characterisation, size, symmetryandinternalstructurecharacterisationforthe identificationofeimerianspecies.IdentificationofEimeria spp.usingCOCCIMORPHsoftwarerevealedthepresence ofE.acervulina,E.maxima,E.mitis,E.praecox,E.necatrix andE.tenella,in96.7%,36.7%,90.0%,3.3%,23.3%and16.7% offarms,respectively(Fig.1,SupplementaryTable2).E. brunetti wasnot recordedin anyof thefarmsscreened usingCOCCIMORPH.

3.2.2. NestedITS-1PCR

NestedPCRusingITS-1primerwasstandardisedwith pureDNAofallsevenspeciesofEimeria.SpecificPCR ampli-consofE.acervulina(321bp),E.brunetti(311bp),E.maxima US strain(145bp), E.maximaAustralianstrain (145bp), E.mitis1(328bp),E.mitis5(193bp),E.necatrix (383bp), E.praecox(116bp)andE.tenella(278bp)werevisualised (datanotshown).Infieldsamples,ITS-1basednestedPCR identifiedE.acervulina,E.brunetti,E.maxima,E.mitis,E. praecox,E.necatrix andE.tenellain 93.3%,10.0%, 86.7%, 96.7%,66.7%,80.0%and 100%farms,respectively (Fig.1, SupplementaryTable2).In16farms,boththe Australian-andUS-typestrainsofE.maximawereidentified,whilein tenfarmsonlytheUS-typestrainofE.maximawaspresent. Similarly,E.mitiswasidentifiedbyprimersspecificforboth E.mitis1andE.mitis5inallthefarmsthatwerepositive forE.mitis.MixedinfectionsofEimeriaspp.wererecorded inallfarmswithaminimumofatleastthreespecies(in fourbroilerfarms).AllsevenEimeriaspp.wereidentified inthreefarms.

3.2.3. SCARmultiplexPCR

Table1

ComparisonofthreeDNAextractionprotocolsforthedetectionofeimeriangenomicDNAwithinchickenfaecalsamplesbyPCRtargetingtheEimeriagenus 18SrDNA.

OPG n Stoolkit+F Stoolkit−F Phenol/chloroform−F

<1000 6 0 5 3

1000–5000 5 1 4 3

5001–20,000 6 0 6 4

20,001–100,000 7 3 7 7

100,001–200,000 6 5 6 6

Total 30 9 28 23

OPG,oocystspergramstartingmaterial;Stoolkit,QIAampDNAStoolkit.+F,includingcontaminatingfaecalmaterial;−F,withoutcontaminatingfaecal material;n,numbersamplestestedperOPGgroup.

Table2

TheinfluenceoffaecalsampleoocystconcentrationonPCRsensitivityforeimeriangenomicDNA.Samplespreparedusingtheoptimaloocyst flota-tion/QIAampDNAStoolkitDNAextractionprotocolwithaPCRtargetingtheEimeriagenus18SrDNA.

OPG Numberfarms TheoreticaloocystsperPCRa _{Numberpositive Percentpositive}

<500 5 <25 3 60

500–1000 5 25–50 4 80

1001–2000 5 50–100 5 100

2001–5000 10 100–250 9 90

5001–10,000 20 250–500 20 100

10,001–50,000 36 500–2500 36 100

50,001–100,000 15 2500–5000 15 100

100,001–200,000 4 5000–10,000 4 100

Total 100 96

OPG,oocystspergramstartingmaterial.

a_{TheoreticaloocystsperPCRcalculatedbaseduponprocessing5gfaeceswithDNAelutionduringextractionin200␮landinclusionof2␮lperPCR.}

ofthefarmsscreenedbyone-tubemultiplexPCR.A maxi-mumoftwoEimeriaspp.wereidentifiedinsixfarms,while for11farmsnoEimeriaspp.wererecordedbyone-tube multiplexPCR.However,two-tubemultiplexPCR identi-fiedE.acervulina,E.maxima,E.mitis,E.praecox,E.necatrix andE.tenella,in36.7%,43.3%,53.3%,56.7%,6.7%and46.7% farms,respectively(Fig.1,SupplementaryTable2).A max-imumoffiveEimeriaspecieswereidentifiedinfivefarms, whileintwofarmsnoEimeriaspp.weredetectedby two-tubemultiplexPCR.E.brunettiwasneveridentifiedusing themultiplexPCRinone-ortwo-tubeformats.

4. Discussion

AccurateidentificationofEimeriaspp.isimportantnot only for the diagnosisof disease but also for manage-mentofsubclinicalinfection,developmentandapplication of effective control strategies, and biological and epi-demiological study (Lee et al., 2010; Sun et al., 2009). Traditionally,identificationofEimeriaspp.hasbeenbased onthemorphologicalcharacteristicsofoocysts,parasite biology,clinicalsignsoftheaffectedanimals,andthe typ-icalmacroscopiclesionsassessedduringnecropsy(Long and Joyner,1984).However,in a natural setting mixed infectionsofdifferentEimeriaspp.arecommonly encoun-teredandmorphologicalcharacteristicsandpathological changes mayoverlap, hindering accuratediagnosis and underminingdetection of subclinical disease (Longand Joyner,1984;RiceandReid,1973).Thus,ithasbeen sug-gestedthatthesemethodsshouldnotbeusedinisolation for differentiation of Eimeria species (Long and Joyner, 1984;Lopezetal.,2007).Alternativesincludemolecularor computationalapproachessuchasPCR,qPCRandthe soft-wareCOCCIMORPH.PCRassayscapableofidentifyingand differentiatingEimeriaspp.havebeenavailableformore than20yearsbut,despiterecognitionasthe‘goldstandard’ ofdetection formany pathogens, this technologyisyet toreplacetraditional coccidialdiagnostics (Brooketal., 2008;OlanoandWalker,2011;Stuckietal.,1993).Features ofeimerianbiologyincludingtheresistanceoftheoocyst walltoanythingotherthanmechanicaldisruption,limiting accesstotemplateDNA(formostavian-infectingspecies), andPCRinhibitionbythesurroundingfaecalmaterialhave discourageduseofPCR.WhileseveralPCRassayshavebeen describedtoidentifyspecificEimeriaspeciesveryfew stud-ieshavefocusedontheapplicabilityofthesetechniques foridentifyingEimeriaspp.incommerciallyraised poul-trythroughouttheworld(Carvalhoetal.,2011a,b;Frölich etal.,2013;Haugetal.,2008).Developmentofa standard-isedprotocolsupportingmediumthroughputdiagnostic samplingforEimeriawillenhancethevalueofsuchdata whilepromotingtheapplicationofPCRandcomparison betweenstudies.

Followingcollectionoffreshenvironmentalfaecal sam-plesweexploredtwoDNAextractionproceduresandthe influenceofresidualfaecalcontamination.Theinclusionof faecalmaterialdramaticallyreducedPCRsensitivitywith genomicDNApurified usingtheQIAamp DNAStoolkit, supportingthevalueofevenarudimentarypre-extraction parasite purification step. The cause of this inhibition remainsunclearatpresent.TheInhibitExstepoftheStool

kit protocol is designed to adsorb substances that can degradeDNAandinhibitdownstreamenzymaticreactions andshouldminimisePCRinhibition.Whileitispossible that the faecal PCR inhibitor concentrationover loaded the InhibitEx matrix it is more likely that the residual faecaldebrisreducedtheefficiencyofthecolumn purifica-tionstep.Insupportofthishypothesiscomparablestudies using sieved faecal samples withand withoutflotation were not similarly affected, although this protocol was notadoptedowingtoqualitycontrolissuesavoiding con-taminationbetweensamplesduringprocessing(datanot shown). Using Eimeria oocysts enriched by flotation in saturated salineconsiderably improvedPCR sensitivity, where theStool kitperformedconsiderably betterthan thephenol/chloroformextraction(93%comparedto77%). Extensionofthesestudiestoincludealargersamplepanel withtheStoolkit revealedanoverall sensitivityof96%, with100%accuracywhenstartingwithanOPGinexcess of5000(theequivalentof250oocystsperPCRfromthe beginningoftheprotocol).DNAprecipitationcouldbe con-sideredtoconcentratetheDNAtemplateandimprovePCR sensitivity,althoughtheadditionalcomplexityislikelyto belimitinginamediumthroughputsurveillancesystem. Thus,thelowfalsenegativerateandtheimprovedhealth and safetyassociatedwitha non-phenolbasedprotocol supportedadoptionoftheparasiteflotation/QIAampDNA Stoolkitprotocol.

MolecularidentificationofEimeriaspp.usingPCRwas supplemented duringthese studies by the online COC-CIMORPH tool, an innovative approach developed for identificationofeimerianoocystsofpoultryandrabbitsin whichdigitalimagesofunidentifiedsporulatedeimerian oocystsareuploadedforspeciesidentificationonthebasis ofsporulatedoocystmorphology(Casta ˜nónetal.,2007). COCCIMORPHwasmosteffectivewithE.acervulinaandE. mitis,demonstratinggoodagreementwiththenested ITS-1PCRassay,althoughitfaredlesswellwithE.brunetti,E. praecoxandE.tenella.Indeed E.brunettiwasnot identi-fiedinanysample,althoughtheoccurrenceofthisspecies wasfoundtobelowthroughoutthestudy.Perusalof avail-ableliteraturerevealedthatnodataexistsontheuseof thissoftwareforidentificationofEimeriaspp.infield sam-ples.Ithaslongbeenrecognisedthatthesizeandshape rangesofeimerianoocystsarewide,overlapsubstantially betweenspecies(Longetal.,1976)andmayvarydueto environmentalandphysicalfactors(Jones,1932;Joyner, 1982).Further,infrequentspeciescanremainundetected usingCOCCIMORPHgiventhatasmallsubsamplemaynot presentatruerepresentationofthetotalsample.Assuch, while COCCIMORPH canbe a valuable tool for prelimi-naryscreening/identificationpurposesorin theabsence ofalaboratoryitshouldbereinforcedwithmicroscopicor molecularvalidation.

Comparison of the identification technologies tested herepromoteuseofthenestedITS-1PCRassayasitwas abletoidentifyalloftheEimeriaspp. thatwere identi-fiedbySCARmultiplexPCRand/orCOCCIMORPHwithjust fourexceptions(oneE.acervulina,oneE.maximaandtwo E.necatrix;Fig.1andSupplementaryTable2).Thesegaps mayhavebeenduetovariationsintheITS-1sequence,as hasbeenreportedpreviouslyinthecaseofE.tenellafrom India(Bhaskaranetal.,2010).Whileitis clearthat PCR canfacilitatethedetectionofminorityEimeriaspecies sub-populationswhichmaybemissedbyroutinemicroscopy (Frölich et al.,2013), therelianceof PCR onvery small primerannealingsiteswithinatargetgenomealsorisks false negatives where geneticdiversity occurs.Relevant ITS-1diversityhasalreadybeendescribedforE.maxima andE.mitis,reflectedbytheinclusionofmultipleprimer pairsinthenestedPCR(Lewetal.,2003;Schnitzleretal., 1999).Indeeditshouldbenotedfromthepresentstudy thatboththeUSandAustralianITS-1E.maximasequence typeswereevidentinNorth Indianpoultry.Thus, while thenested ITS-1assayprovided thebestspecies cover-agewithalowfalsenegativerate,additionalassayswill beimportantifcomprehensivesurveillanceisrequired.

Identificationof chickenEimeriaspecies is ofutmost importancefor effectivecontrolofclinicaland subclini-calcoccidiosis.Conventionalparasitologicaltechniquesare timeconsumingandrequireexpertise,whichis increas-inglyexpensiveandscarce.Computationalidentification onthebasisofoocystmorphology(COCCIMORPH)provides a valuable diagnostic tool but failed to correctly iden-tifymanyspeciesinpracticalfieldapplication.Theuseof molecularbiologicaltechniquestodiscriminatebetween differentspeciesof poultrycoccidiahasbeenlimitedto datebuttheprovisionofprotocolssupportingtheir cost-effective,robustandstraightforwardapplicationwithan

easytointerpretoutputcanimproveuptakeindeveloped anddevelopingregions.AsthecostofPCRequipmentand reagentscontinuestodrop,itisfeasiblethatthe proto-colsdescribedherewillbedevelopedandintegratedinto routinepoultrymanagementandveterinarysurveillance.

Acknowledgements

Authors are thankful to the Indian Council of Agri-cultural Research, New Delhi and the Director, Indian VeterinaryResearchInstitute,Izatnagarforproviding nec-essaryfacilities.ThefinancialassistanceprovidedbyDFID andBBSRC,UKintheformofCIDLIDprojectBB/H009337 (Anticoccidial vaccine development: the importance of geneticdiversityanddeliverystrategy)andtheLibyan Gov-ernmentforthePhDstudentshipawardedtoA.Moftahis dulyacknowledged.Thismanuscripthasbeenassignedthe referencePPB00587bytheRVC.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbe found,intheonlineversion,athttp://dx.doi.org/10.1016/ j.vetpar.2013.09.026.

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