The occurrence of aflatoxigenic Aspergillus spp. in dairy cattle feed in Southern Brazil

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

Food

Microbiology

The

occurrence

of

aﬂatoxigenic

Aspergillus

spp.

in

dairy

cattle

feed

in

Southern

Brazil

Augusto

Carlos

Favaretto

Variane

a,1

_,

_Fabiane

_Cristina

_dos

_Santos

b,1

_,

Fausto

Fernandes

de

Castro

b,1

_,

_Ione

_Parra

_{Barbosa-Tessmann}

b,∗

_,

Geraldo

Tadeu

dos

Santos

a

_,

_Magali

_Soares

_dos

_Santos

_Pozza

a a_Universidade_Estadual_de_Maringá,_Departamento_de_Zootecnia,_Maringá,_PR,_Brazil b_Universidade_Estadual_de_Maringá,_Departamento_de_Bioquímica,_Maringá,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received19October2017 Accepted17May2018

Availableonline14August2018 AssociateEditor:LuisAugustoNero

Keywords:

Feed Dairy

Aspergillus

a

b

s

t

r

a

c

t

Thepresenceofmycotoxinsorrelatedfungiinanimalfeedisamajorproblemforanimal andhumanhealth.Silageandconcentratedfeedsampleswerecollectedfrom21dairyfarms intheWesternpartofParanástateinSouthernBrazil.WateractivityandpHofallsamples weremeasured,andeachsamplewasanalyzedtocheckforthepresenceofaﬂatoxigenic

Aspergillus.Wateractivitywasobservedtobelowerintheconcentratedfeedsamples.ThepH waslowerinthesilagesamples,indicatingfermentationprocesses.Twosilagesamplesand fourconcentratedfeedsampleswerecontaminatedwithAspergillusspp.Sevenisolatesof

Aspergillusspp.wereobtainedandtheirpotentialtoproduceaflatoxinswasevaluated.Four oftheisolates,twofromthesilagesamplesandtwofromtheconcentratedfeedsamples, producedtheaflatoxinsB1,B2,G1,andG2inculturemedia.Theseisolateswereidentified asAspergillusparasiticusandAspergillusnomius.Thepresenceofaflatoxigenicisolatesof

Aspergillusspp.insilageandconcentratedfeedsamplesisamatterofconcern,becauseof theriskofaﬂatoxinproductionandcontaminationoftheanimalfeed.

Introduction

VariousspeciesofthegenusAspergillusarecommonlyisolated fromstoredfoods.1,2_Aﬂatoxins_are_mycotoxins_produced_by

thespeciesofthegenusAspergillus,subgenusCircumdati sec-tion Flavi (also referred to as the Aspergillus ﬂavus group)

∗ _{Corresponding}_author_at:_Universidade_Estadual_de_Maringá,_Departamento_de_Bioquímica,_Av._Colombo,_5790,_87020-900_Maringá,_PR,

Brazil.

E-mail:[email protected](I.P.Barbosa-Tessmann).

1 _The_three_ﬁrst_authors_have_contributed_equally_to_this_work_and_should_be_considered_as_ﬁrst_authors.

mainly bythe speciesAspergillus ﬂavus, Aspergillus parasiti-cus,andAspergillusnomius.3–6_About_50%_of_the_isolates_of_the

speciesA.flavuspredominantlyproduceaflatoxinsB1andB2. NearlyalltheisolatesofthespeciesA.parasiticusproduce afla-toxinsB1,B2,G1,andG2.3,4 _Aflatoxin_M1,_which_is_secreted

inmilkfromthemammaryglandsofbothhumansand lac-tatinganimals,isahydroxylatedmetaboliteofaﬂatoxinB1.

https://doi.org/10.1016/j.bjm.2018.05.005

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7_{Approximately} _0.5–6%_of_the_ingested_aﬂatoxin_B1_is

con-vertedtoaﬂatoxinM1andissecretedinmilk.7_According_to

theInternationalAgencyforResearchonCancer(IARC,2009), aflatoxinsareclassifiedasgroupIorcarcinogenictohumans, beingaflatoxinB1themosttoxic.8_Aflatoxin_M1_is_as_toxic_as

aflatoxinB1butistentimeslesscarcinogenic.The susceptibil-itytoaflatoxinsdependsonthespecies,age,dose,theextent ofexposure,nutrition,gender,andconcomitantexposureto othertoxins.Theliveristheprimarytargetorganinmammals, andaflatoxinscausehepatocellularcarcinoma.3,4

Milkhas high nutritivevalue because it contains many macro- and micronutrients, which are important for the growthofchildrenandmaintenanceofhumanhealth. Aﬂa-toxin M1 is thermostable and resistant to pasteurization.7

Humans can be exposed to aﬂatoxin M1 through endoge-nousproductionor byintakeofdairyproducts.Babiesand youngchildren,whomightconsumecontaminatedmilkorbe exposedbybreastfeeding,arethemostvulnerable.7

AccordingtotheUnitedStatesDepartmentofAgriculture, Brazilwasthesixthlargestmilkproducerintheworldin2018, onlybehindEuropeanUnion,UnitedStates,India,China,and Russia,achievingtheproductionof23billionliters.9

Accord-ingtotheBrazilianInstituteofGeographyandStatistics(2016), therehasbeenanincreaseinthe milkproductionbymore than50%duringthepastfewyears,ascomparedtothe begin-ningofthe21stcentury.10_The_highest_increase_has_occurred

intheSouthern region,which isthemajormilkproducing regioninBrazil,contributingto37%ofthenational produc-tionin2016.Inaddition,thisregionshowsaproductivityof 2966L/cow/year,whichis70%higherthantheBrazilian aver-ageof1709L/cow/year.10

Milk productivity is related to the animal’s productive potentialand breed genetics.11 _The _productive_potential _is

favored by environmental factors such as climatic condi-tionsandadequatenutrition.Tocompensateforpoorgrowth ofpastures, several dairy farms use forages, concentrates, and preserved feeds (hay or silage). As the production is intensiﬁed,thesesupplementsbecomethesolesourceof ani-malfeed.Thisiswherethe aﬂatoxinsbecomea matterof concern.12–14

Becauseofastrongcorrelationbetweenthepresenceof mold and the occurrence of mycotoxin, it is important to searchforthepresenceoffungiinanimalfeed.15–17_This

infor-mationcanindicatetheHazardAnalysisCriticalControlPoint (HACCP) withinthe food productionchain.18 _The_objective

ofthisstudywastoanalyzeanimalfeedforthepresenceof aﬂatoxigenicAspergillusindairyfarmslocatedinthewestern regionoftheParanáStateinSouthernBrazil.

Materials

and

methods

Samplecollection

Twenty-onerandomlychosendairyfarmswere visited dur-ingJanuaryof2015.Thedairyfarmswerelocatedinthecityof MarechalCândidoRondon,intheWesternregionoftheParaná StateinSouthernBrazil.Beforethesamplecollection,a ques-tionnairewasansweredbythefarmerstoidentifywhattypeof feedwasprovidedfortheanimals,howtheywerefed,andhow

thefeedwasstored.Thesilageswerewellcompactedandhad thecharacteristiccolorandodorofoptimumlacticacid fer-mentation.Theouterlayersofsilagethatwereincontactwith air(withoutthepolystyrenecover)appeareddry,andsome partsshowedslightfungalcontamination(spot).Eachsilage sampleconsistedofthreesub-samples,categorized as Sur-face–compositesampleobtainedfromthesilofront;Depth –compositesampleobtainedfromthesilointeriorat25cm depth;andSpot–compositesampleobtainednear contami-natedpoints,withinaradiusofupto20cm,withoutcollecting visiblydegradedorcontaminatedmaterial.19

Thesamplesoftheconcentratedfeedwerealsocollected frommostofthefarms.Severalstorageformsofthe concen-tratedfeedwereidentiﬁedinthevisitedfarms.Forinstance, bulksilos,feedbagspurchaseddirectlyfromagricultural hold-ings,andingredientspurchasedinbulkandstoredinreused bagsorcompartmentswithinthefarmitself.Whenthe con-centratedfeedwasstoredasasilo,asamplewascollected fromtheexitpointofthesilo.Whentheconcentratedfeed was keptinbags,sub-samples were collectedfrom various pointsofthebagincaseofasinglebagbeingused,orfrom severalbagsifmorethanonebagwerebeingused.Fromeach farm,the concentrated feed sub-sampleswere individually homogenizedandcombinedtomakeasample.

MeasurementofwateractivityandpHofsilageand concentratedfeedsamples

The measurement ofwateractivity ofthe silage and con-centrated feed samples was performed using a LabSwift water activity instrument (Novasina, Lachen, Switzerland). Thesamplepreparationandoperationoftheapparatuswere performedaccordingtotheinstructionsdescribedinthe oper-atingmanual.Afterhomogenizingthesample,aportionwas transferredtoandpackedinatestdishintriplicate.

ThepHmeasurementsofthesilageandconcentratedfeed sampleswereperformedbyadding9goftheeachsampleto 60mLofwaterina250-mLbeaker.Aftermixingfor5min,the sampleswerelefttorestfor30minandanaliquotfromthe supernatantwasusedtomeasurethepHusingapHmeter calibratedwithstandardbuffersolutionsofpH4.0and7.0.20

Isolationofthemicroorganisms

Twentygrams ofeach silageor concentrated feedsamples wereaddedto80mLofsterile0.1%peptonewaterin250mL Erlenmeyer ﬂasks. Thissuspension was incubated at25◦C for1hwithagitation at100rpm.Aliquots of100␮Lofthis suspension were spread on the surface of a Petri dish of diameter 10cm(in triplicate)containingA.ﬂavusand para-siticusagar(AFPA)(20g/Lyeastextract;10g/Lbacteriological peptone; 0.5g/Lferric ammoniumcitrate;and 15g/Lagar).2

Rapidly growing molds such as Rhizopus and Mucor were inhibitedbytheadditionofmalachitegreenataconcentration of2.5␮g/mLtothemedium,beforeautoclaving.Toprevent bacterialgrowth,641IU/mLofpenicillinand256.4␮g/mLof streptomycinwereasepticallysupplementedtothemedium, afterautoclavingandcoolingto60◦C.Theculturewas incu-batedat25◦Cwithaphotoperiodof12hforﬁvedays.

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A. ﬂavus and A. parasiticus and related species grow as orangecolored reverse coloniesintheAFPA,a characteris-ticwhichfacilitatestheirisolation.2_A_fragment_of_the_orange

coloredreversecolonygrownontheselectiveAFPAmedium was transferred toa tube containing potato dextrose agar (PDA)slant(for 1L:15gdextrose,20gagar,ﬁltrate of200g peeledpotatocookedin400mLofdistilled water)and cul-turedat25◦Cforﬁvedaystopromotesporeformation.Then, a1cm3_piece_of_the_PDA_culture_was_fragmented_by_agitation_in 20mLofsterilewater.Analiquotof100␮Lofthissuspension containing4.8×105 _spores _was_spread _on_2.5%_agar–water mediumin10cmdiameterPetridishes.Aftergrowingthe cul-tureat25◦Cforapproximately16to20h,afragmentofthe hyphaorasinglegerminatingsporewastransferredtofresh PDAslants.21_The_isolates_were_maintained_in_PDA_slants_at

4◦C,withpassagesdoneeverysixmonths.

Morphologyandcultureidentiﬁcationoftheisolates

Forthe taxonomicidentiﬁcation ofthe isolates,the follow-ingfeatureswereobserved:macroscopiccolonysurfaceand reverse characteristics inPDA and CzapekDox agar (30g/L sucrose,2g/LNaNO3,1g/LK2HPO4,0.5g/LMgSO4·7H2O,0.5g/L KCl,0.01g/LFeSO4·7H2O,15g/Lagar,pH7.3),andmicroscopic characteristics such as conidia, vesicle, conidiophore, and phialide.Theobservedcharacteristicswereanalyzed accord-ingtodichotomousidentiﬁcationkeysdescribedbyPittand Hocking.2

DNAextraction

To obtain mycelia without spores for the DNA extraction, thefollowingwasperformed. Afragmentofapproximately 1cm3 _of_a_monosporic_culture_in_a_PDA _slant_was _cut_into smallerpiecesandagitatedin5mLofsteriledistilledwater. Twomillilitersoftheobtainedsporesuspension(3.84×107₎ wereculturedin125mLErlenmeyerflaskscontaining25mL ofliquidAFP broth(withoutmalachite greenorantibiotics) orpotatodextrosebroth,forfiveandthreedays,respectively. Themicroorganismsweregrownwithoutagitation,at25◦C, withaphotoperiodof12hours.Aftercollectingthemycelial massbyfiltrationinsterilegauze,theobtainedmaterialwas maceratedwithliquidnitrogeninaporcelainrecipientuntil transformationinafinepowder.

Approximately300␮L ofthispowder wastransferred to a microtube and the DNA was extracted according to the methoddescribedbyKoenigetal.22 _with_the_{modiﬁcations}

reportedbyFariaetal.23_The_DNA_was_quantiﬁed_using_a

spec-trophotometerand theﬁnal concentrationwas adjustedto 100ng/␮L.

Molecularidentiﬁcationoftheisolates

A 5.8S-ITS rDNA amplicon of approximately 600bp was obtained from the genomic DNA of the isolates by using the primers ITS4 5-CCTCCGCTTATTGATATGC and ITS5 5 -GAAGTAAAAGTCGTAACAAGG,previouslydescribedbyWhite et al.24 _The _{ampliﬁcation} _reactions _contained: ₅₀_mM _KCl;

10mMTris,pH7.5;1.5mMMgCl2;1.5UofPlatinumTaqDNA polymerase; 0.2mM ofeachdNTP; 25pmol ofeach primer;

and400ngoftheDNAsampleinafinalvolumeof25␮L.The cyclingconditionswere25cyclesof1minand30sat94◦C, 1minand30sat50◦C,and2minat72◦C,whichwere exe-cutedin aTechne TC-312thermocycler(England). Samples were heated for5minat94◦C,previousto thecycles, and wereincubatedfor10minat72◦C,afterthecycles.ThePCR productswerekeptfrozenat−20◦Cuntiluse.Negative con-trols (noDNAtemplate) wereusedtotestforthepresence ofDNAcontamination.TheamplificationofaDNAfragment wasconfirmedbyelectrophoresing10␮LofthePCRreactionin a1.5%agarosegelcontainingethidiumbromide(0.25␮g/mL) andvisualizingthegelunderUVlight.Theobtainedamplicons werepurifiedeitherbytheIllustraExoProStarTM_(GE Health-careLifeSciences,USA)orbythe Wizard® SVGelandPCR Clean-UpSystem(Promega,USA)andsequencedatthe Cen-terforHumanGenomeStudies(CEGH)fromtheUniversityof SãoPaulo(USP),SãoPaulo,Brazil.Theprimerusedin sequenc-ingwasoneofthoseusedintheamplification.Thesequencing wasdoneinonlyonedirection.Aftertrimmingthe5 and3 extremities,the5.8S-ITSrDNAobtainedsequenceswere com-paredwithsequencesdepositedindatabanks(GenBank)by usingMEGABLASTanalysis.

Detectionofaﬂatoxinbiosynthesispathwaygenes

Thepresenceoftheapa-2,omt-A,ver-1,andnor-1genes,which playaroleintheaﬂatoxinsbiosynthesispathway,was evalu-atedbyamultiplexPCRusingtheprimerspreviouslydescribed byShapiraetal.(APA-4505-TATCTCCCCCCGGGCATCTCCCGG and APA-1482 5-CCGTCAGACAGCCACTGGACACGG) and Geisen(omt15-GTGGACGGACCTAGTCCGACATCACandomt2 5-GTCGGCGCCACGCACTGGGTTGGGG; ver1 5 -CCGCAG-GCCGCGGAGAAAGTGGT and ver2 5 -GGGGATATACTCCCGC-GACACAGCC;andnor15-ACCGCTACGCCGGCACTCTCGGCAC andnor25-GTTGGCCGCCAGCTTCGACACTCCG).25,26

Theamplificationreactionscontained:50mMKCl;10mM Tris,pH7.5;1.5mMMgCl2;2.0UofPlatinumTaqDNA poly-merase;0.2mMofeachdNTP;25pmolofeachoneofthe8 primers;and400ngoftheDNAsampleinafinalvolumeof 25␮L.ThePCRreactionconsistedof30cyclesof1minat94◦C, 2minat65◦C,and2minat72◦C,alsoperformedintheTechne TC-312thermocycler(England).Previoustocycling,samples were heatedfor5minat94◦C.Aftercycling, sampleswere incubatedfor10minat72◦Candwerekeptfrozenat−20◦_C untiluse.ThenegativecontrolwasareactionwithoutanyDNA templatewhileA.parasiticusUEM443wasusedaspositive control.TheDNAamplificationwasevaluatedbyloading10␮L ofthePCRproductsina1.5%agarosegelcontainingethidium bromide(0.25␮g/mL).Afterthe electrophoresis,thegelwas visualizedandphotographedunderUVlight.

Analysisofaflatoxinproductioninspecificculturemedium Theevaluationofaflatoxinproductionpotentialoftheisolates wasperformedbyanalyzingtheproductionoffluorescence underultraviolet(UV)lightbythecultureincoconutmilkagar (CMA). Thismediumwaspreparedwith200mLofcoconut milk(SOCOCOS/A,Maceió,Alagoas,Brazil),600mLofdistilled waterhaving pH6.9,and 16gofagar.27 _Three _Petri_dishes

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thecenter bytouchingasterilewoodskewerstick covered withsporesatthe pointedend.Thedisheswereincubated at28◦Cwithaphotoperiodof12hforsevendays,and ﬂuores-cencewasobservedinaUVtransilluminatorwithemissionat 312nm.ThepurecultureofA.parasiticusUEM443,previously isolatedfrom peanut,and AspergillustamariiUEM15C were usedaspositiveandnegativecontrolforaﬂatoxinproduction, respectively.

The analysis of ammonium hydroxide vapor was con-ductedon7-day-oldculturesintheCMAmedium.Thedishes were inverted and drops of 28–30% ammonium hydroxide wereaddedtotheinnersideofthelid.Theproductionof aﬂa-toxinswasvisualizedbytheappearanceofpinkcoloraround andinthereverseofthecolonies.28

Thinlayerchromatography(TLC)analysisforaﬂatoxin production

AfragmentofamonosporiccultureinaPDAslant, measur-ingapproximately1cm3_,_was_cut_into_smaller_pieces_and_was agitatedin10mLofsteriledistilledwater.Oftheobtained sus-pension,a100␮Laliquot(containing9.6×105spores)without theagarfragmentswasspreadonPetridishescontainingmalt glucoseagar(MGA)[50g/Lmaltextract,50g/Lglucose,20g/L agar]orCMAmedium.23,29_The_dishes_were_incubated_at₂₈◦_C forsevendays.Onlyonedishwasculturedandanalyzedfor eachisolate.Withtheuseofasterilizedcorkborer,eightdisks of0.5cmdiameterwerecutfromeachculturedishandwere independentlytransferredtotwomicrocentrifugetubes(four diskseach),wheretheywerefragmentedand500␮Lof chlo-roformwasadded.29_This_mixture_was_agitated_at₁₀₀_rpm_for

60minatroomtemperatureandtheagarfragments contain-ingthemyceliawerediscarded.Theresultingextractswere combinedandpassedthroughasmallcolumnmadewitha 1.0mLpipettetip.Thiscolumn-tiphadglasswoolatthe bot-tom,ﬁlledwith1.5gofanhydroussodiumsulfate(Na2SO4), whichwascoveredwithsmallpiecesofﬁlterpaper.The elu-atewasdriedatroomtemperatureandtheobtainedresidue wasre-suspendedin20␮Lofchloroform.

Additionally, 100␮L aliquots from spore suspensions of eachisolate(9.6×105_of_spores)_were_used_to_inoculate₂₅_mL ofYeastExtractSucrose(YES)medium(20g/Lyeastextract and200g/Lsucrose)in125mLﬂasks.30_The_ﬂasks_were

incu-batedat28◦Cfor14days.Onlyoneflaskwasusedforculture andanalysisofeachisolate.Afterthisperiod,thecontentof eachflaskwasfilteredthroughplainfilterpaper.Tothefiltrate, 10mLofhexanewasaddedandthemixturewasagitatedina vortexmixerfor1min.Theorganiccomponentwasdiscarded and10mLofchloroformwasaddedtotheaqueous compo-nent.Thismixturewasagainagitatedinthevortexfor3min andwaslefttorestuntilthelayersseparated.Theorganiclayer ofchloroformwascollectedandfilteredbyafilterpapercoated withanhydroussodiumsulfate.Thesolventwasevaporated byincubationat50◦Covernight,andtheobtainedresiduewas re-suspendedin20␮Lofchloroform.

Analiquotofapproximately10␮Loftheextractsobtained fromthe MGA,CMA,and YEScultureswas addedto Thin-LayerChromatography(TLC)plates(Silicagel,Sigma–Aldrich, Germany).Theapplicationdotsweredriedatroom tempera-tureandthechromatogramwasdevelopedinasolventsystem

oftoluene–ethylacetate–chloroform–formicacid(7:5:5:2).The referenceisolateA.parasiticusUEM443wasanalyzedusingthe sameprocedure.Theaflatoxinstandardswereobtainedfrom Sigma-Aldrich(Germany).TheaflatoxinB1andB2standards were dissolvedintolueneataconcentrationof0.125␮g/␮L, and the aflatoxin G1 and G2 standards were dissolved in methanol:water(9:1,v/v)atthesameconcentration. Statisticalanalysis

Themeansandstandarddeviationsoftheresultswere com-paredusingDuncan’sNewMultipleRangeTest(MRT)ort-test

(˛<0.05),usingtheSAS-Mprogram.31

Results

Samplecollection

Theresultsofthedataaboutanimalfeedingandtheanimal feedsamplescollectedatdifferentdairyfarmsareshownin Table1.Itwasobservedthat95%ofthelactatinganddry ani-malswerefedusingacombinedsystemofgrazingandfeeding onthetroughduringthemilkingprocess(Table1).Inthecase ofconcentratedfeed,itwasfoundthat52%ofthefarmsused acommercialmixtureand43%formulateditwithinthefarm (soybeanmealandcorn,exceptatonefarmwhereonlywheat branwasused).In47.6%ofthosefarms,theconcentratedfeed wasstoredinbulkcarriers,whiletherestofthefarmsstored thefeedinbagsinthestable.Inthecaseofsilage,81%ofthe farmsusedhumidgrainand19%usedhumidgrainplusdry Tifton,hay,oroats.Thissilagewaskeptinsiloswithan aver-agesizeof1.6×3.9×2m,withstoragetimeofoneweektoone year.Thesilosweretrenchorsurfacestyleandwerecovered withcanvas.Onlyatfourfarms,inoculantswereaddedtothe silage.Roughly8–40kg(layersof15–40cm)ofthissilagewas useddailythroughouttheyear.Thefarmersdiscardedrotten partsofthesilagein86%ofthefarms.

Wateractivity,pH,andoccurrenceofAspergillusinsilage andconcentratedfeedsamples

Thewateractivityofthesilagesamplesrangedbetween0.95 and0.98(Table2)whilethevaluesobservedinconcentrated feed samplesrangedfrom 0.64to0.86 (Table3). Therewas a signiﬁcant differencein the pHvaluesofthe silage sub-samples(surface,depth,andspot)wheretheDepthsamples weremostacidic(Table2).

ThepHoftheSurfacesilagesamplesshowedlarge vari-ations(4.55–7.48)acrossdifferentfarms.ThepHoftheSpot silagesamplesrangedfrom4.46to7.42,whilethepHofthe Depthsilagesamplesrangedfrom4.37to6.98.Orangereverse coloniesinAFPAmediumoccurredonlyintwosilagesamples andinﬁveconcentratedfeedsamples(Tables2and3).Inthose samples,therewereonlyoneoracoupleofcoloniesthatwere allusedtoobtaintheisolates.Becauseofthat,theywerenot counted.

Eight monosporicfungiisolateswere obtainedandthey are listed in Table 4. The features of the isolates such as colony color on PDA; formation and size of conidia; and

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Table1–Animalfeedingandanimalfeedsamplescollectedatdifferentdairyfarms. Farm Lactating cows feedinga Drycows feedinga Concentrated feed Concentrated feedstorage

Silageb _Silage_silo_size

H(D)×L×Wc Silagesilo type Silage storage timee Silagesilo cover Silage additive

1 Combined Combined Formulated Silo H.G. 1.5×3×2 Trench 3W Canvas –

2 Combined Combined Formulated Silo H.G. NMd _Surface ₆_M _Canvas _–

3 Combined Combined Formulated Bag H.G. 1×3.5×2 Trench 1W Canvas –

4 Combined Combined Commercial Bag H.G. 1.8×5×2 Trench 1Y Canvas –

5 Combined Combined Commercial Bag H.G. 1.9×4.5×2 Trench 1Y Canvas –

6 Combined Combined Commercial Silo H.G. 2×4.5×2 Trench 1M Canvas –

7 Combined Combined Commercial Silo H.G. 1.6×5×2 Trench 3M Canvas –

8 Combined Combined Formulated Silo H.G.,Tifton 2×3.5×2 Surface 2M Canvas –

9 Combined Grazing Formulated Silo H.G.,Hay,or

Oat

2×4×2 Trench 1M Canvas –

10 Combined Combined Commercial Bag H.G.,Tifton 1.6×4.5×2 Trench 2M Canvas Inoculants

11 Combined Combined Formulated Silo H.G. 1×5×2 Trench 2M Canvas Inoculants

12 Combined Combined Commercial Bag H.G. 1.8×3×2 Surface 2W Canvas –

13 Combined Combined Commercial Bag H.G. 1.5×3.5×2 Trench 8M Canvas –

14 Combined Combined Commercial Silo H.G. 1.6×4×2 Surface 1M Canvas Inoculants

15 Combined Combined Wheatbrain - H.G. 1.2×3×2 Trench 2W Canvas –

16 Combined Combined Commercial Silo H.G. 1.5×3×2 Trench 2W Canvas –

17 Combined Combined Formulated Silo H.G. 2×4.5×2 Trench 6M Canvas Inoculants

18 Combined Combined Commercial Bag H.G. 1.9×4×2 Surface 3W Canvas –

19 Combined Combined Formulated Bag H.G. 1.5×4×2 Trench 2M Canvas –

20 Combined Combined Formulated Bag H.G. 1×3×2 Trench 1M Canvas –

21 Combined Combined Commercial Bag H.G.,Tifton NMd _Trench ₁_W _Canvas _–

a _Combined₌_grazing_and_feeding_on_the_trough_during_the_milking_process. b _H.G.₌_humid_grain.

c _H(D)_×_L_×_W₌_Height_(Depth)_×_Length_×_Width,_in_meters. d _NM₌_not_measured.

e _W,_M,_and_Y₌_weeks,_months,_and_years.

Table2–Thewateractivity,pH,andoccurrenceoforangereversecoloniesinthesilagesamples.

Farmsamples Wateractivitya _pHa _Orange_reverse

coloniesin

AFPAmedium

Surface Depth Spot Surface Depth Spot

1 0.98 0.97 0.98 7.48 4.87 5.00 – 2 0.98 0.99 0.97 5.23 4.37 5.40 – 3 0.98 0.98 0.98 6.69 5.36 6.92 – 4 0.99 0.98 0.97 5.32 5.28 5.33 – 5 0.98 0.98 0.98 4.84 5.04 5.50 – 6 0.97 0.97 0.97 4.49 5.20 4.46 – 7 0.99 0.98 0.98 6.15 6.08 5.46 – 8 0.97 0.97 0.97 4.70 4.40 5.09 X(Spot) 9 0.97 0.97 0.98 5.10 5.44 6.16 – 10 0.97 0.97 0.98 4.67 4.51 4.98 – 11 0.98 0.98 0.98 5.16 4.81 6.02 – 12 0.98 0.98 0.97 5.43 6.23 5.85 – 13 0.98 0.98 0.97 5.64 6.21 5.41 – 14 0.98 0.98 0.98 5.35 6.98 7.42 – 15 0.98 0.97 0.98 5.51 6.74 6.11 – 16 0.97 0.97 0.97 4.94 6.96 7.26 X(Spot) 17 0.98 0.98 0.98 5.31 4.95 7.34 – 18 0.97 0.98 0.98 6.27 5.25 5.42 – 19 0.98 0.97 0.98 4.55 4.68 6.02 – 20 0.97 0.97 0.98 6.67 4.45 6.80 – 21 0.97 0.95 0.98 5.20 5.20 6.70 – Averageb _0.98_±_0.001a _0.98_±_0.001a _0.98_±_0.001a _5.46_±_0.12ab _5.38_±_0.01b _5.90_±_0.02a

a _Data_represent_the_average_of_duplicate.

b _Averages,_inside_an_analysis,_followed_by_different_letters_are_{statistically}_different_according_to_t-test_and_Duncan’s_test_(˛₌_0.05)._The

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Table3–Thewateractivity,pH,andoccurrenceoforangereversecoloniesintheconcentratedfeedsamples.

Farm Concentratedfeedtype Wateractivitya _pHa _Orange_reverse

coloniesin AFPAmedium 2 Formulated 0.83 5.24 – 3 Formulated 0.69 6.32 X 4 Commercial 0.67 6.84 – 5 Commercial 0.64 6.33 – 6 Commercial 0.69 6.27 – 7 Commercial 0.68 6.83 – 8 Formulated 0.70 6.88 X 10 Commercial 0.69 7.00 – 11 Formulated 0.69 6.36 X 12 Commercial 0.65 7.39 – 13 Commercial 0.67 6.66 – 14 Commercial 0.71 6.27 – 15 Wheatbran 0.72 6.42 X 16 Commercial 0.68 6.70 – 17 Formulated 0.71 6.82 – 18 Commercial 0.67 6.33 – 19 Formulated 0.68 5.86 – 20 Formulated 0.86 6.02 – 21 Commercial 0.67 6.47 X Average 0.72±0.06 6.27±0.51

a _Data_represent_the_average_of_duplicate.

characteristics of the vesicle, conidiophore, and phialide enabledustoclassifysevenoftheminthegenusAspergillus

andoneoftheminthegenusTrichoderma.2

The molecular identification, which was carried out by sequencingtheamplified5.8S-ITSrDNAgenefragmentand comparingwithsequencesdepositedinGenBank,identified theisolatesasshowninTable4.The5.8S-ITSrDNAsequences of the A. flavusand A. nomius isolates could not separate them.However,theiraflatoxinsproductionprofilehelpedin theiridentification.Theobtainedsequencesweredeposited in GenBank and their accession numbers are listed in Table4. 1Kb mar ker Negativ e control UEM 443 − A. parasiticus UEM 3C − A. nomius UEM 8C2 − A. nomius UEM 8CI − A. flavus UEM 11C − A. flavus UEM 8S − A. parasiticus UEM 16S − A. par asiticus UEM 15C − A. tam arii apa-2 (1,032 bp) omt-A (797 bp) ver-1 (537 bp) nor-1 (400 bp) bp 3054 2036 1636 1018 506/517 396 344 298

Fig.1–ThemultiplexPCRreactions.Agarosegel(1.5%) stainedwithethidiumbromide.1KBmarker(Thermo FisherScientiﬁc,USA)isthemolecularmarker.

Detectionofaﬂatoxinbiosynthesispathwaygenes

ThemultiplexPCRreactionsresultsareshowninFig.1and Table4.Theonlygenethathadafragmentampliﬁedfrom all isolates’ genomicDNA wasthe nor-1gene. Theprimers directedtotheapa-2andver-1geneshaveproducedmostof thenegativereactions.

Aﬂatoxinproductionanalysis

Theresultsoftheevaluationofaflatoxinproductioninspecific culturemediumandbyTLCarelistedinTable4.A represen-tativeresultofthefluorescenceinCMAandcolorchangeof coloniesbyammoniumhydroxidevaporisshowninFig.2.A representativeTLCchromatogramoftheYESmediumextracts ispresentedinFig.3.

Discussion

There wereno signiﬁcantdifferences(˛<0.05)inthewater activitiesbetweenthesilagesub-samples:Surface,Depth,and Spot(Table2).Inaddition,therewasinsigniﬁcantvariationin wateractivitiesbetweenthesilagesamplesofdifferentdairy farms.delPalacioetal.reportedlowervaluesforwater activ-ity inwheatsilage(around0.70).32 _This_could _be_attributed

eithertothedifferentsilagesubstratesorthemethodology, whereintheyevaluatedthewateractivitybycalculatingthe lossofmassafterdrying.32

According totheliterature,the minimumlevel ofwater activityforthegrowthofA.parasiticusspeciesandthe produc-tionofaﬂatoxinsis0.83(17%).4,33_The_water_activity_results_of

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Table4–Identiﬁcationandaﬂatoxinproductionanalysisoftheisolates.

Isolatesb _Species _GenBank

acession number (AN) GenBankAN ofsimilar sequences(% ofidentity) PCR Fluorescence inCMAa Ammonium hydroxide vapora TLC

apa-2omt-Aver-1nor-1 MGA CMA YES

UEM443c_A._parasiticus _MG964332 _JQ316518.1 (100%) HQ340110.1 (100%) + + + + + + B1,B2, G1,G2 B1,B2, G1,G2 B1,B2, G1,G2 UEM3C A.nomius MG964333 MG857640.1 (100%) MG857637.1 (100%) + + − + − − − − B1,B2, G1,G2 UEM8C1 A.ﬂavus MG964334 MG734749.1 (100%) KY319338.1 (100%) + + + + − − − − −

UEM8S A.parasiticus MG964335 KY937934.1

(100%) HQ340110.1 (100%) − − − + − − − − B1,B2, G1,G2 UEM8C2 A.nomius MG964336 MG857640.1 (100%) MG857637.1 (100%) + + + + − − − − B1,B2, G1,G2 UEM11C A.ﬂavus MG964337 MG734749.1 (100%) KY319338.1 (100%) + + + + − − − − − UEM15C A.tamarii MG964338 MG857652.1 (100%) LC127424.1 (100%) − − − − − − − − −

UEM16S A.parasiticus MG964339 KY937934.1

(100%) HQ340110.1 (100%) − + + + + + B1,B2, G1,G2 B1,B2, G1,G2 B1,B2, G1,G2 UEM21C Trichoderma longibrachiatum MG964340 MF144551.1 (100%) KY225659.1 (100%) NT NT NT NT NT NT NT NT NT

a _The_analysis_was_done_in_triplicate.

b _The_samples_number_indicates_their_origin_regarding_the_number_of_silage_or_concentrated_samples._The_letter_after_the_number_indicates

theiroriginregardingConcentratedfeed(C)orSilage(S)samples.

c _A._parasiticus_UEM_443,_positive_control.

NT=non-tested.

parasiticusgrowthandaflatoxinproduction.Infact,theonly twostrainsofA.parasiticusobtainedinthisworkwereisolated fromsilagesamples.A.flavuscausesmaximumgrowthand aflatoxinproductionathighervaluesofwateractivity,ranging from0.9upto0.99.4,33–35_The_strains_of_A._flavus_and_A._nomius

(anaﬂatoxigenicspeciesphenotypicallysimilartoA.ﬂavus)

obtainedinthisworkwereisolatedfromconcentratedfeed samples.36_Although_the _low_water_activity _of_concentrated

feedsamplesshouldnotsupportaﬂatoxinproductionbythe aﬂatoxigenic Aspergillusspecies, the presenceofAspergillus

indicatesthattheycouldgrowandproduceaﬂatoxinifthe samplesmoistureincreased.

Silagesamplesweremoreacidicthanconcentratedfeed samples.AhighernumberofSilageDepthsampleshad pH valueslower than5.0,indicatingtheoccurrenceof fermen-tationprocesses,which arecharacteristicofwell-preserved

silage.Alonsoetal.alsoreportedmoreacidicpHinthe cen-tralsamplesofmaizesilage.37_The_optimum_initial_pH_levels

foraﬂatoxinproductionbyA.parasiticusrangedfrom5.0to7.0, withlowerpHlevelsyieldingmoreB1toxin.34_The_average_pH

oftheobtainedsamplesindicatesthatthisfunguscouldgrow inthesamplesandproduceaﬂatoxins,providedthatsufﬁcient moistureispresent.

AftersearchingsequencedatabanksforDNAhomologous totheobtained5.8S-ITSrDNAsequences,arangeofidentity of100%wasdetermined(Table4).Twomonosporicisolates of aﬂatoxigenic A. parasiticus(UEM 8S and UEM 16S) were obtainedfromtwosilageSpotsub-samples(Table2).Fungal developmentisnotcommoninwell-madeandstoredsilages due to the biochemical processes that occur in the silo. However,fungidevelopinconditionssuchaspoorstorage, whichfavorstheirgrowth.Oneconditionwhichtraditionally

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Fig.2–FluorescenceinCMAandcolorchangeinducedbyammoniumhydroxidevapor.(A)Thebackofadishcontaining CMAmediumwithacolonyofA.parasiticusUEM16SphotographedunderUVlight(312nm)inatransilluminator.(B)The frontofadishcontainingCMAmediumwithacolonyofA.parasiticus16Sexposedtoammoniumhydroxidevapor.

leads to the “spots” caused bythe growth offungi is the inadequatesealingofasiloorthepresenceofholes inthe coveringcanvas.TwomonosporicisolatesofaflatoxigenicA. nomius(UEM3CandUEM8C2)andtwomonosporicisolates ofnon-aflatoxigenicA.flavus(UEM8C1 andUEM11C)were alsoobtainedfromthreeconcentratedanimalfeedsamples (Table 3). The higher incidence of aflatoxigenic Aspergillus

intheconcentratedfeedsamplescomparedwiththesilage samplesmayreﬂectthedifferenceinthesubstratematerial usedintheirpreparation.Inaddition,theconcentratedfeed couldalsobecontaminatedduringitspreparationordueto inadequatestoragefacilitiesatthefarms.

Fungal toxins generally are multi-ring structures and hence require a sequence of structural genes for their biological synthesis. Therefore, there is no speciﬁc PCR to detectaﬂatoxigenicAspergillusspecies.38_Multiplex_and_single

PCRsystems havebeen used fordetection ofaﬂatoxigenic

Aspergillusisolates,performedbothasconventionaland

real-timePCR.17,23,25,26,29,39_The_multiplex_PCR_system_used_in_this

workwasalreadyusedtodiscriminateamongaﬂatoxigenic

B1 B2 G1 G2 UEM 443 − A_{. par} asiticus UEM 8S − A_{. par} asitic us UEM 3C − A_{. nomius} UEM 8C2 − A_{. nomius} UEM 8CI − A . flavus UEM 11C − A_{. fla} vu s UEM 15C − A_{. tamar} ii UEM 16S − A_{. par} asiti cus

Fig.3–ATLCplatewiththeextractsoftheisolatesgrown inYESmedium.TheplatewasphotographedunderUV light(312nm)inatransilluminator.B1,B2,G1,andG2are

thestandards(1.5␮g)ofaﬂatoxins(Sigma–Aldrich, Germany).TheextractofA.parasiticus(UEM443)reference isolatewasalsoanalyzed.

and non-aﬂatoxigenic Aspergillus isolates.29,39 _This_reaction

usedthepairofprimersAPA-450andAPA-1482designedby Shapiraetal.,whichampliﬁesaDNAfragmentof1032bpof theA.parasiticusapa-2gene,whichcodesforatranscription factor containing a zinc cluster DNA binding motif that activatesaﬂatoxinB1andB2biosynthesisbycontrollingthe expressionofthenor-1andver-1genes.25_Three_other _pairs

of primers described by Geisen were also used: omt1 and omt2,whichamplifyaDNAfragmentof797bpfromtheA. parasiticusomt-Agene,thatcodes fortheenzyme sterigma-tocystinO-methyltransferase;ver1andver2, whichamplify aDNAfragmentof537bpfromtheA.parasiticusver-1gene, which codesforthe enzymeversicolorin Adehydrogenase; and nor1andnor2, thatamplifyaDNA fragmentof400bp oftheA.parasiticusnor-1gene,whichcodesfortheenzyme norsolorinicacidreductase.26

IthasbeenproposedthatthelackofamplificationofDNA fragments ofoneof the testedgenes may signify that the isolatehaslostormutatedthisgene.Threeoftheobtained isolates(A.flavusUEM8C1,A.nomius8C2,andA.flavus11C) andthepositivecontrol(A.parasiticusUEM443)hadDNA frag-ments amplifiedfromall four testedgenes, indicatingthat theywerepotentiallyaflatoxigenic.Otherthreeisolateshad atleastoneDNAfragmentamplified(A.nomiusUEM3C,A. parasiticus UEM 8S, and A. parasiticus UEM 16S), indicating thattheycouldbeaflatoxigenic.TheisolateUEM15Chadno DNAfragmentamplifiedfromitsgenomicDNAwiththeused primers,whatisinagreementwithitsmolecular identifica-tion asA.tamarii,aspeciesalsobelongingtothesubgenus

CircumdatisectionFlavi,butconsideredasanon-aﬂatoxigenic species.36

WhentheAspergillusisolatesweretestedforthe produc-tionofaflatoxinsbyevaluatingthepresenceoffluorescence inCMAanddevelopmentofpinkcolorbyammonium hydrox-idevapor,onlytheisolatesA.parasiticusUEM443(control)and UEM16Swereobservedtoproduceaflatoxins(Table4,Fig.2). TLCanalysisoftheextractsobtainedafterculturingthese iso-latesinMGA,CMA,andYESindicatedthattheycouldproduce allfouraflatoxins(Table4,Fig.3).However,theotherisolates eitherdidnotproduceaflatoxinsoronlyhaveproducedinthe

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YESmedium.Thereareseveralfactorsthataffectaﬂatoxins productionsuchastemperature,pH,moisture,medium com-position,timeofculture,radiationandinoculumsize.34_It_is

possiblethattheYESmediumprovidedthebestconditions foraflatoxinsproductionbytheisolatesundertheemployed conditionsoftemperature,photoperiod,andtimeofculture. Inagreementwiththe literature,the isolatesofA. parasiti-cusUEM8SandUEM16S,andofA.nomiusUEM3CandUEM 8C2,produced all four aflatoxins in YESmedium (Table4, Fig.3).4,36_The_{non-production}_of_any_aflatoxin_by_the_isolates

ofA.ﬂavusUEM8C1andUEM11Calsoagreeswiththe litera-ture,becauseitisknownthat50%oftheisolatesofA.ﬂavus

arenon-producers.3,4

Amongtheaflatoxinproducerisolatesfoundinthiswork, onlythe isolate A. parasiticusUEM 8S had solely oneDNA fragmentamplifiedinthePCRreaction.Theother producer isolateshadatleastthreeifnotallfourtestedgenes ampli-fied.Theresultssuggestthatthenon-amplifiedgenesmaybe mutated,disallowingPCRamplificationwiththeusedprimers, but still coding functional proteins, consideringthat these isolates were able to produce the aflatoxins. These differ-ences may reflectgenetic variationsamongthe isolates of

A.parasiticusandA.nomiusandindicatethattheusedpairs ofprimersdonothaveenoughresolutiontoseparate aflatox-igenicfromnon-aflatoxigenicstrains.Thefactthataflatoxin negativeA.flavusstrainspresentedamplificationofalltested genesisintriguing,butthispatternwasalreadyreportedin theliterature.23,26

Thepresenceof aﬂatoxigenic Aspergillus inanimal feed hasalreadybeenreportedfromotherpartsofBrazilandthe world.32,40–43 _The_presence_of _{aﬂatoxigenic} _A._parasiticus _in

silageandofaflatoxinogenicA.nomiusinconcentratedfeed revealedbythepresentstudyrepresentsapotentialhealthrisk totheanimalsandhumans,becauseoftheriskofsecretionof M1aflatoxinsinmilkbythebioconversionthatoccursinthe rumen.Inaddition,in14.3%ofthefarms,thespottedsilage wasnotdiscarded.Nevertheless,itisimportanttopointout thatthepresenceofaflatoxigenicA.parasiticusandA.nomius

inanimalfeed,evenunderoptimumconditionsof develop-ment,doesnotimplyactualproliferationandthepresenceof aﬂatoxins.

Conclusion

Inthisstudy,samplesofsilageandconcentratedfeedwere collectedfrom21dairyfarmsinSouthernBrazilandanalyzed formoisture,pH,andthepresenceofaﬂatoxigenicAspergillus.

ThepresenceofaﬂatoxigenicA.parasiticusintwosilage sam-plesandofaﬂatoxigenicA.nomiusintwoconcentratedfeed samplesisapotentialcauseofconcernforanimalandhuman health.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgment

The authors are thankful to CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) of the EducationMinistryandCNPq(ConselhoNacionalde Desen-volvimento Científico e Tecnológico) of the Science and TechnologyMinistryofBrazilforthescholarshipgiventoA.C.F. Variane, F.C.dos Santos,and F.F. deCastro. Wealsothank AraucáriaFoundation,theParanáStateScience,Technology andSuperiorStudiesSecretariatforfundingthiswork(Grant N◦12247/2013).

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1.GouramaH,BullermanLB.AspergillusﬂavusandAspergillus parasiticus:aﬂatoxigenicfungiofconcerninfoodsandfeeds: areview.JFoodProtect.1995;58:1395–1404.

2.PittJI,HockingAD.FungiandFoodSpoilage.Sydney:Academic Press;1997.

3.RichardJL.Somemajormycotoxinsandtheirmycotoxicoses –anoverview.IntJFoodMicrobiol.2007;119:3–10.

4.KumarP,MahatoDK,KamleM,MohantaTK,KangSG. Aﬂatoxins:aglobalconcernforfoodsafety,humanhealth andtheirmanagement.FrontMicrobiol.2017;7:1–10.Article 2170.

5.OlsenM,JohnssonP,MöllerT,PaladinoR,LindbladM. Aspergillusnomius,animportantaﬂatoxinproducerinBrazil nuts?WorldMycotoxinJ.2008;1(2):123–126.

6.EhrlichKC,KobbemanK,MontalbanoBG,CottyPJ. Aﬂatoxin-producingAspergillusspeciesfromThailand.IntJ FoodMicrobiol.2007;114(2):153–159.

7.IqbalSZ,JinapS,PirouzAA,FaizalARA.AﬂatoxinM1inmilk anddairyproducts,occurrenceandrecentchallenges:a review.TrendsFoodSciTechnol.2015;46:110–119.

8.IARC–InternationalAgencyforResearchonCancer. Chemicalagentsandrelatedoccupations.Areviewofhuman carcinogens.100F.France:IARC–WorldHealthOrganization; 2009:225–248.Accessed15.08.2018.

9.USDA–UnitedStatesDepartmentofAgriculture.Foreign AgriculturalServices.Dairy:WorldMarketandTrade.USA: USDA;2018.Accessed15.08.18.

10.IBGE–InstitutoBrasileirodeGeograﬁaeEstatística. Produc¸ãodapecuáriamunicipal.ProdPecMunic.2016:44. 11.OltenacuPA,BroomDM.Theimpactofgeneticselectionfor

increasedmilkyieldonthewelfareofdairycows.Anim Welfare.2010:9–49,19(S).

12.MaganN,OlsenM.MycotoxinsinFood:DetectionandControl. Abington,UK:WoodheadPublishing;2004.

13.MurphyPA,HendrichS,LandgrenC,BryantCM.Food mycotoxins:anupdate.JFoodSci.2006;71(5):51–65. 14.Fink-GremmelsJ.Mycotoxinsincattlefeedsandcarry-over

todairymilk:areview.FoodAdditContamA.2008;25(2): 172–180.

15.GonzálezHHL,MartinezEJ,PacinA,ResnikSL.Relationship betweenFusariumgraminearumandAlternariaalternata contaminationanddeoxynivalenoloccurrenceonArgentina durumwheat.Mycopathology.1999;144:97–102.

16.LoriGA,SisternaMN,HaidukowskiM,RizzoI.Fusarium graminearumanddeoxynivalenolcontaminationinthe durumwheatareaofArgentina.MicrobiolRes.2003;158:29–35. 17.PassoneMA,RossoLC,CiancioA,EtcheverryM.Detection

andquantiﬁcationofAspergillussectionFlavispp.instored peanutsbyreal-timePCRofnor-1gene,andeffectsof

(10)

storageconditionsonaﬂatoxinproduction.IntJFood Microbiol.2010;138:276–281.

18.FAO–FoodandAgricultureOrganizationoftheUnited Nations.ManualontheapplicationoftheHACCPsystemin mycotoxinpreventionandcontrol.FAOFoodNutrPap. 2003:73.

19.ICMSF–InternationalCommissiononMicrobiological SpeciﬁcationforFoods.Samplingformicrobiological analysis:principlesandspeciﬁcapplications.In:

Micro-organismsinFood.UK:BlackwellScientiﬁcPublications; 1995.

20.SilvaDJ,QueirozAC.Análisedealimentos:métodosquímicose biológicos.3.ed.Vic¸osa:EditoradaUniversidadeFederalde Vic¸osa;2002.

21.NelsonPE,ToussounTA,MarasasWFO.FusariumSpecies:An IllustratedManualforIdentiﬁcation.UniversityPark:

PennsylvaniaStateUniversityPress;1983.

22.KoenigRL,PloetzRC,KistlerHC.Fusariumoxysporumf.sp. cubenseconsistsofasmallnumberofdivergentandglobally distributedclonallineages.Phytopathology.

1997;87(9):915–923.

23.FariaCB,SantosFC,CastroFF,etal.Occurrenceoftoxigenic AspergillusﬂavusincommercialBulgurwheat.FoodSci Technol.2017;37(1):103–111.

24.WhiteTJ,BrunsT,LeeS,TaylorJ.PCRprotocols:aguideto methodsandapplications.In:InnisMA,GelfandH,Sninsky JJ,WhiteTJ,eds.PCRProtocols:AGuidetoMethodsand Applications.SanDiego:AcademicPress;1990.

25.ShapiraR,PasterN,EyalO,MenasherovM,MettA,Salomon R.DetectionofaﬂatoxinogenicmoldsingrainsbyPCR.Appl EnvironMicrobiol.1996;62(9):3270–3273.

26.GeisenR.Multiplexpolymerasechainreactionforthe detectionofpotentialaﬂatoxinandsterigmatocystin producingfungi.SystApplMicrobiol.1996;19(3):388–392. 27.LinMT,DianeseJCA.Coconut-agarmediumrapiddetection

ofaﬂatoxinproductionbyAspergillusspp.Phytopathology. 1976;66:1466–1469.

28.SaitoM,MachidaS.Arapididentificationmethodfor aflatoxinproducingstrainsofA.flavusandA.parasiticusby ammoniavapour.Mycoscience.1999;40:205–211.

29.CriseoG,BagnaraA,BisignanoG.Differentiationof afatoxin-producingandnon-producingstrainsofAspergillus ﬂavusgroup.LettApplMicrobiol.2001;33:291–295.

30.DavisND,DienerUL,ElridgeDW.ProductionofaﬂatoxinB1 andG1byAspergillusﬂavusinsemisyntheticmedium.Appl Microbiol.1966;29:378–380.

31.CanteriMG,AlthausRA,VirgensFilhoJS,GigliotiEA,Godoy CV.SASM-Agri:Sistemaparaanáliseeseparac¸ãodemédias

emexperimentosagrícolaspelosmétodosScoft-Knott, TukeyeDuncan.RevBrasAgrocomp.2001;1(2):18–24. 32.delPalacioAD,BettucciL,PanD.FusariumandAspergillus

mycotoxinscontaminatingwheatsilagefordairycattle feedinginUruguay.BrazJMicrobiol.2016;47:1000–1005. 33.RomoMAM,CartagenaMACR,FerriEER,FernàndezGS.

Minimalmoisturecontentforgrowthandaﬂatoxin productionbyAspergillusparasiticusinmixedfeeds. Mycopathologia.1986;95:145–148.

34.KlichMA.Environmentalanddevelopmentalfactors influencingaflatoxinproductionbyAspergillusflavusand Aspergillusparasiticus.Mycoscience.2007;48:71–80.

35.MedinaA,RodriguezA,MaganN.Effectofclimatechangeon AspergillusﬂavusandaﬂatoxinB1production.FrontMicrobiol. 2014;5:1–7,article348.

36.KurtzmanCP,HornBW,HesseltineCW.Aspergillusnomius,a newaﬂatoxin-producingspeciesrelatedtoAspergillusﬂavus andAspergillusparasiticus.AntonieLeeuwenhoek.

1987;53:147–158.

37.AlonsoV,VergaraLD,AminahuelC,etal.Physiological behaviourofgliotoxigenicAspergillusfumigatussensustrictu isolatedfrommaizesilageundersimulatedenvironmental conditions.FoodAdditContamA.2015;32(2):236–244. 38.LevinRE.PCRdetectionofaﬂatoxinproducingfungiandits

limitations.IntJFoodMicrobiol.2012;156:1–6. 39.FärberP,GeisenR,HolzapfelWH.Detectionof

aﬂatoxinogenicfungiinﬁgsbyaPCRreaction.IntJFood Microbiol.1997;36(2–3):215–220.

40.daSilvaJ,PozziC,MallozziM,OrtegaE,CorreaB.Mycoﬂora andoccurrenceofaﬂatoxinB1andfumonisinB1during storageofBraziliansorghum.JAgricFoodChem. 2000;48:4352–4356,19.

41.KellerLAM,PereyraMLG,KellerKM,etal.Fungaland mycotoxinscontaminationincornsilage:monitoringrisk beforeandafterfermentation.JStoredProdRes.

2013;52:42–47.

42.DavariE,MohsenzadehM,MohammadiGh,Rezaeian-Doloei R.CharacterizationofaﬂatoxigenicAspergillusﬂavusandA. parasiticusstrainisolatesfromanimalfeedstuffsin northeasternIran.IranianJVetRes.2015;16(2): 150–155.

43.RahimiS,SohrabiN,EbrahimiMa,TebyanianM,ZadehMt, RahimiS.Studyingtheeffectofaflatoxingenesaflpandaflq onAspergillusflavusandAspergillusparasiticusinthecattle feedusedinindustrialanimalhusbandries.ActaMedica Mediterr.2016;32:2091–2100.