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Food
Microbiology
Fusarium
and
Aspergillus
mycotoxins
contaminating
wheat
silage
for
dairy
cattle
feeding
in
Uruguay
Agustina
del
Palacio,
Lina
Bettucci,
Dinorah
Pan
∗UniversidaddelaRepública,FacultaddeCiencias-FacultaddeIngeniería,Montevideo,Uruguay
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Articlehistory:
Received28September2014 Accepted25February2016 Availableonline4July2016 AssociateEditor:CarlosPelleschi Taborda Keywords: Wheatsilage Mycotoxins Fungi
a
b
s
t
r
a
c
t
WheatisoneofthemostimportantcultivatedcerealsinUruguayforhumanconsumption; however,whenharvestyieldsarelow,wheatisusuallyusedinensilingforanimalfeeding. Ensilingisaforagepreservationmethodthatallowsforstorageduringextendedperiodsof timewhilemaintainingnutritionalvaluescomparabletofreshpastures.Silageis vulner-abletocontaminationbyspoilagemoldsandmycotoxinsbecauseensilagematerialsare excellentsubstratesforfungalgrowth.Theaimofthestudywastoidentifythemycobiota compositionandoccurrenceofaflatoxinsandDONfromwheatsilage.Atotalof220 sam-plesofwheatwerecollectedfromfourfarmsinthesouthwestregionofUruguayweresilage practicesaredeveloped.ThemainfungiisolatedwereFusarium(43%)andAspergillus(36%), withFusariumgraminearumsensulatoandAspergillussectionFlavibeingthemostprevalent species.Aflatoxinconcentrationsinsilobagsrangedfrom6.1to23.3g/kg,whereasDON levelsrangedbetween3000g/kgand12,400g/kg.Whenevaluatingaflatoxigeniccapacity, 27.5%ofAspergillussectionFlavistrainsproducedAFB1,5%AFB2,10%AFG1and17.5%AFG2. AllisolatesofF.graminearumsensulatoproducedDONand15-AcDON.Theresultsfromthis studycontributetotheknowledgeofmycobiotaandmycotoxinspresentinwheatsilage.
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Introduction
Dairyproductsare one ofthe maincommoditiesexported byUruguay.Althoughpasturesconstitute themajorsource fordairycattlefeeding,silagegrainsarealsoused,supplying 125gperliterofmilkproduced.1Ensilingisaforage
preser-vationmethodthatallowsforgrainstorageduringextended
∗ Correspondingauthor.
E-mail:dpan@fing.edu.uy(D.Pan).
periodsoftimewhilemaintaininganutritionalvalue com-parable with that offreshpastures. Grain silagestorageis basedonthechemicalprocessesthatoccurinvegetable tis-sues whentheyare underanaerobic conditionsand inthe presenceoflacticacidbacteria,whichleadstoadecreaseinpH value(4–4.5),thusinhibitingseveralspoilagemicroorganisms. However, silageisvulnerable tocontamination byspoilage moldsbecauseensiledmaterialsareexcellentsubstratesfor
http://dx.doi.org/10.1016/j.bjm.2016.06.004
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
fungalgrowth,whicharepresentassporesinthefield, espe-ciallyinsoil.2Moreover,factorssuchasexcessivemoistureor
dryness,condensation,heat,poorcompressionofthe mate-rials,leakageofrainwaterandinsectinfestationcanleadto undesirablemoldcontamination,mycotoxinproductionand reductionofnutritionalvalue.3–5
Mycotoxins are low molecular weight products of fun-gal secondary metabolism, produced mainly by species of
Aspergillus,PenicilliumandFusarium.Theycancausehuman andanimaldisorderswhenconsumedorinhaled.6,7Exposure
tomycotoxinsthroughcontaminatedfeedisoneofthemajor riskfactorstoruminanthealth.7,8
AspergillusflavusandA.parasiticusarespeciesthatproduce aflatoxins (AFs), the most important carcinogenic myco-toxins. Acute aflatoxicosis in cattle has been thoroughly described.ConsumptionoffeedcontainingAFs mayreduce growth rate, decrease milkproduction,cause liver damage and depress immune function.9 Among AFs, aflatoxin B1
(AFB1) isthe most carcinogenic,and it is classified bythe International Agency for Research on Cancer (IARC) as a group1humancarcinogen.10 When animals eatfoodstuffs
containing AFB1, this toxin is metabolized and accumu-lated inmilkas aflatoxin M1(AFM1).11 Many studies have
demonstrated that AFM1 also displays toxic and carcino-genic effects; therefore it too has been included in IARC group1.12
Fusarium spp. are the most frequent wheat contami-nantfungiinUruguayand are particularlyprevalentwhen spring rains occur.13 Trichothecenes produced by Fusarium
speciesalterimmune-mediatedactivitiesinbovines.14 This
toxin isapotentinhibitor ofeukaryoticprotein biosynthe-sis,inducingvomiting,diarrhea,anemiaandfoodrefusalin animals.15–17
Theaimsofthisstudyweretoevaluatethefollowing:(1)the mycobiotacompositionofwheatsilagescollectedduringa4 monthperiod;(2)thetoxigenicabilityofisolatesofAspergillus
sectionFlaviandFusariumgraminearumsensulato(F. gramin-earums.l.)and(3) themycotoxins producedinwheatgrain silage.
Materials
and
methods
Samples
Wheat was harvested from cultivated lands of four farms located in the southwest region of Uruguay, mechanically choppedandenclosedwithinpolystyrenebags.Compaction wasconductedinNovemberof2009,andthesilobagwasfirst openedinJanuary2010.Atotalof220samplesofwheat(4from freshlyharvestedgrainsand216fromstoredgrain)were ana-lyzedfromNovemberof2009atharvesttimeandat60,90and 120daysofensiling.Theremovalofmaterialwasperformed usingacuttingmachine.Samplingwasperformedmanually throughthe silosintransectsatthreelevels.From the cut edge,threepointsfromeachsectionofthesilo(upper,middle andlower)weresampledat50cmhorizontaldepth.Ateach time,1-kgsamplesfromeachsectionwerecollected.Samples belongingtoeach silowerehomogenizedand quarteredto obtain1-kgsub-samplesforanalysis.
Physicalpropertiesofsamples
Twenty-fivegramsofeachsampleweredriedinanSC1oven (SiacS.C.,Montevideo,Uruguay)toconstantweightinaPetri dish at 105◦C, and the moisture content of samples was expressed as percentage values (MC%). Water activity was determined byautomated analysis using anAqualab CX-2 (Decagon Devices,Inc.).pH measurementswere performed byadding90mLofdistilledwaterto10gofsilagematerials andhomogenizingthemixturefor15min.Thehomogenized mixturewasfiltered,andthepHofthefiltratewasmeasured withanOrionStarA211glasselectrodepHmeter(Thermo Scientific).
Analysisofmycobiota
One-hundredwheatgrainparticles fromeach samplewere placedinPetridishescontainingpotatodextroseagar(PDA) andincubatedat25◦Cundera12hwhite/12hblack fluores-centlightphotoperiod.18,19Theemergingcoloniesfromeach
particleofwheatweretransferredtoallowidentificationby meansofmacro-andmicromorphologicalcharacteristics, fol-lowingconventionalmycologicalmethods.20–26
ToconfirmmorphologicalidentificationsoftheisolatesofF. graminearums.l.specificPCRwasperformedusingtheprimers Fg16FandFg16R.27TheITSregionandpartsofthe-tubulin
andcalmodulingeneswereamplifiedtoconfirmthe identifi-cationofAspergillussectionFlaviisolates.28
Determinationofmycotoxinsinsilagessamples
The quantitative analyses of DON and aflatoxins in sam-ples were performed using competitive enzyme-linked immunosorbent assays (ELISA) using the Ridascreen® Fast
DONandRidascreen®FastAflatoxinSC(R-BiofarmAG)
com-mercialkits.Theprotocolsforextractionandquantification were performed according to manufacturer recommenda-tions.Thedetectionlimitsofthe techniquewere200g/kg forDONand2g/kgforaflatoxins.
Toxigeniccapacityoffungalisolates
The ability of potentially toxigenic isolates to produce mycotoxinsinvitrowastested.Aflatoxinanalyseswere per-formedusingthemethodologydescribedbyBragulatetal.29
Aspergillus section Flavi strains isolated from silage sam-pleswereincubatedinPetridishesofCoconutExtractAgar (CEA) (20% desiccated coconut; 1.5% agar) and incubated at 25◦C for 14 days in the dark. Then, 3 plugs were cut from each Petri dish and 1mL of methanol was added. After 1h in methanol, an aliquot (200L) was derivatized with 700L trifluoroacetic acid:acetic acid:water (20:10:70, v/v).Thederivatizedsolutionwasanalyzedusingareversed phase HPLC, consisting of a Shimadzu LC-10ADvp pump, aRF-10Axlfluorescencedetector(Shimadzu;excitationand emission wave lengthof360nm and 440nm, respectively), andaC18reversed-phasecolumn(150mm×4.6mmi.d.,5m
particlesize;Nucleodur®,Macherey-Nagel,Düren,Germany)
connected to a precolumn Security Guard (8mm×4mm i.d.,5mparticlesize;Nucleodur®,Macherey-Nagel,Düren,
Germany).Themobilephasewaswater:methanol:acetonitrile (4:1:1,v/v/v)ataflowrateof1.5mLmin−1.Theinjection vol-umewas20L.Aflatoxinproductionwasmeasuredinngg−1 ofculturemedium.Thelimitofdetectionwas1ngg−1ofB1 andG1and0.8ngg−1ofB2andG2(TrilogyAnalytical Labora-toryInc.,Washington,MO).
The capacity of F. graminearum s.l. isolates to pro-duce trichothecene was determined using multiplex PCR experiments30andasfollows:25goflonggrainricewasplaced
ina250mLErlenmeyerflaskand10mLofdistilledwaterwas added.Theflaskswereautoclavedtwiceduring2 consecu-tivedaysat121◦Cfor20min.Eachflaskwasinoculatedwith a7-day-culture-carnationleafagar(CLA)plugandincubated inthedarkfor28daysat25◦C.Attheendoftheincubation period,thecontentsoftheflaskweredriedat50◦Cfor24h andthenmilled.Fifteengramsofthedriedsamplewerethen extractedwith40mLacetonitrile:methanol(14:1,v/v),shaken for2hat150rpmandthenfilteredthroughWhatmanN◦1 fil-terpaper.Asyringe(3mLcapacity)pluggedwithglasswool anddry-packedwithalumina:carbon(20:1,w/w,500mg)was usedasamini-cleanupcolumn.A2mLaliquotofextractwas appliedtothecolumn, allowedtodrainundergravity, and the eluent collected. Thecolumn was washed with 500L acetonitrile:methanol:water(80:5:15,v/v),andthecombined eluents wereevaporated todrynessunderN2 at50◦C.The
cleanedresiduewasdissolvedin500Ltoluene:acetonitrile (95:5,v/v).Thestandardmycotoxinsandextractswereapplied toTLCplates(Merck5553,MerckKGaA,Darmstadt,Germany) andwereresolvedinasolventsystemcontaining8:1:1(v/v/v) chloroform:acetone:2-propanol.Theplatesweredevelopedby sprayingwith20%aluminumchlorideinethanolandheating to100◦Cfor7min.ThepresenceofDON,NIV,3-AcDONand 15-AcDON(TrilogyAnalyticalLaboratoryInc.,Washington,MO) productionwasdeterminedbyvisualcomparisonwithknown amountsofstandardsilluminatedwithUVlightat366nm.31
Dataanalyses
Correlationsbetweenvariableswereanalyzedusingthe Pear-son correlation coefficient. Fisher’s protected LSD testwas usedforcomparingthemeansoffungalpropagules, myco-toxin levels and sample storage time. All analyses were conductedusingSigmaSTATversion2.1(Chicago).
Results
and
discussion
Fungalcontaminationwasobservedinallofthewheat sam-ples.ThemainfungiisolatedwerefromthegeneraFusarium
(43%),Aspergillus(36%),Alternaria(33%),followedbySporothrix
(24%), Eppicoccum (12%) and Penicillium (6%). There was no significantdifferenceinthetotalfungalpropagules(p>0.05) amongthesamplingperiods,whichindicatesgoodprocessing practices.However,somegenera,suchasAlternaria, Eppicoc-cumandFusarium,occurred athigherfrequenciesinfreshly harvested wheat and were significantly different from the remainder ofthe sampling period (p<0.05) (Fig. 1).On the otherhand,Aspergillus, SporothrixandPenicillium, whichare knowntocausespoilageofstoredproducts,werepresentin highfrequenciesafter60daysofstorage(Fig.1).
0 10 20 30 40 50 60 70 80 90 100 Alternaria Penicillium Sporotrix Epicocum Aspergillus Fusarium Is o la ti on fr e qu en cy, %
Fig.1–Isolationfrequency(%)offungalgenerainsilage
samples; –harvest; –60daysofstorage; –90daysof
storage; –120daysofstorage.
Fusarium spp. and Aspergillus spp., the main toxigenic molds, were the prevalent genera in wheat silage. These resultsareinagreementwithseveralstudiesthatfound sim-ilarresultsincornandsorghumsilages.3,18,32–34Cornsilages
areheavilystudied,buttherearefewdataonmycofloraand mycotoxincontaminationinwheatsilages.
F.graminearums.l.,F.acuminatum,F.poae,F.sambucinum,F. sporotrichioides,F. oxysporum,F. nygamai,F. lateritium,F. equi-setiandF.subglutinanswereidentified;F.graminearums.l.was theprevalent species,witharelativedensityof93%,which has been reported by other authors forwheat atthe field stage.13,35–37Theseresultsdifferfromthosefoundforcornand
sorghumsilages,inwhichF.verticillioidesisthemostfrequent species.2,18,33,38
WithinthegenusAspergillus,onlyAspergillussectionFlavi, A. niger aggregate and A. clavatus were identified, with
AspergillussectionFlavishowingthehighestrelativedensity (98%)inaccordingwithitsoccurrenceincornandsorghum silagesinArgentina and Brazil.5,18 Onthe other hand,this
result differsfrom resultsreported byanother author who foundhighincidencesofA.fumigatusincornsilages.39The absenceofA.fumigatusinstudysamplesdecreasesthedual hazardsofingestionofpathogenicsporesandpotential myco-toxinproduction.40
TheisolatesofSporothrixschenckii-likemorphogroup41are
notcommonlyfoundinsilages,butinthiswork,theisolate frequencywas23.8%andwasonlyfoundinpost-fermentation silagesamples.ThiscouldbebecauseS.schenckiiisa dimor-phic fungusthatgrowsasayeast(35–37◦C)andasmycelia (25–30◦C)inpresenceofCO2.42,43 Thisspeciesisan
impor-tanthuman-pathogenicfungusandistheetiologicagentof sporotrichosis. The most common clinical presentation of thisdiseaseisthecoetaneousformwithorwithoutregional lymphaticinvolvement.Sporotrichosisisconsideredtobea hazard amongworkerswhoseoccupationsbringthem into frequentandsometimestraumaticcontactwithplant mate-rialorsoil.44
F.graminearums.l.wasthemostfrequentisolateonrecently harvested grains and grains storedup today 60.However,
AspergillussectionFlaviwasrecordedafter60daysofstorage asshowingasignificantnegativecorrelationwithF. gramin-earum s.l. (r=−0.97,p<0.05). F. graminearums.l. exhibited a negativecorrelationwithtimeofstorage(r=−0.97,p<0.05),
Table1–Physicalpropertiesandmycotoxinlevelsinwheatsilagesduringthestoragetime.
Time(days) aW±SD pH±SD MC%±SD Deoxynivalenol(g/kg) Aflatoxins(g/kg)
Mean±SD Range Mean±SD Range 60 0.693±0.057 6.39±0.5 11.7±2.3 5300±1691 3000–6800 3±5 nd-11.9 90 0.672±0.051 6.49±0.30 12.4±1.9 5525±1499 3400–6800 12±8 nd-18.1 120 0.709±0.043 6.69±0.30 14.6±3.4 6007±2122 3800–7900 17±9 6–23
aw,wateractivity;SD,standarddeviation;MC,grainmoisturecontent;nd,notdetected.
whileAspergillussection Flavishowedapositivecorrelation (r=0.99,p<0.05).
Thestudyofpre-existingmycobiotainagivensubstrate cansometimesbeusedasabasisforestimating contaminat-ingmycotoxins.Wheat grainsare frequentlycontaminated simultaneouslybyseveralfungi,whichareeachableto pro-duceseveraltoxins.
Due to a lack ofunified pre-existing methodologies for mycotoxin analysisinwheat silagesamples,we chosethe mostappropriateavailablemethodsforthesesamples. Mea-suredmycotoxinlevels inwheatsilagesamplesareshown
inTable1. Thepresenceofmycotoxinswas detectedinall
samplesanalyzed.Thelevels ofaflatoxinsrangedfrom 6.1 to23.3g/kg,whereas DONlevels werebetween3000g/kg and12,400g/kg,withrecentlyharvestedsamplesshowing higherandsignificantlydifferentlevels(p<0.05).Therewere nosignificantdifferencesinaflatoxinlevels(p>0.05)among thesamplingperiods.
Freshlyharvestedsamplesdidnotshowaflatoxin contam-ination,butahighpercentageofsilosampleswerepositive foraflatoxinscontamination,suggestingthatAspergillus sec-tionFlaviandaflatoxincontaminationwasenhancedduring storage.However,only16.7%oftheseshowedlevels exceed-ingtherecommendedlimitforfeeddestinedfordairycattle and the maximum permissible level for human food in Uruguay(20gkg−1).45,46 Similarresultswerefoundincorn
andsorghumsilagesfromArgentinaandBrazil,18,33while
afla-toxincontaminationwasabsentinwheatsilagesfromIsrael and the Netherlands,47,48 probably duetothe different
cli-maticconditionsinUruguay.
AswheatgrainsarrivetosiloscontaminatedwithF. gramin-earums.l.,theycouldprovidetheinitialDONlevelspresented inthesilage.Duringthestoragetime(120days),areductionof 43%inDONcontaminationwasdetected.Conversely,incorn silages,DONlevelsincreasedduringstorage.33,49Inthisstudy,
thereductionintheoccurrenceofDONcouldbeconsidered anadvantageofusingsilobags.
Fewstudieshavehighlightedtheimportanceof character-izingthetoxicogenic profileoffungalspeciesisolatedfrom silages.AspergillussectionFlaviandF.graminearums.l.werethe dominantspecies,andvariousstrainswere abletoproduce aflatoxins and DON, respectively. The presence of toxico-genicstrainsincommoditiesconstitutesapotentialriskfor animal health because the toxins can be produced in the substrate.Thiswasconfirmedbythepresenceofaflatoxins and DON contaminationin silagesamples. When evaluat-ing aflatoxigenic capacity, 27.5% ofAspergillus section Flavi
strainsproducedAFB1,5%AFB2,10%AFG1and17.5%AFG2. Trichothecenemycotoxin genotypes and chemotypes were
determined forisolatesofF.graminearums.l.Themultiplex PCR assay demonstrated that all isolateswere ofthe DON genotype.Theseassaysdemonstratedthatallstrainswiththe DONgenotypewerealsoofthe15-AcDONgenotype.Allstrains withthe15-AcDONgenotypeproduced15-AcDONandDONas assessedbychemicalanalyses.
Thephysical propertiesofsamples didnotvary signifi-cantlyduringstoragetime(p>0.05).Table1showsthephysical propertiesofthesilages.Wateractivities,pHandMC%ranged from0.693aw to0.709aw,from6.39to6.69andfrom11.7to
14.6,respectively.However,somesampleshadaw,moisture
contentandpHlevelsthatallowedfungalgrowthand myco-toxin production.Fungalgrowth generallyoccurs whenthe siloisnotwellpacked,thewateractivityexceeded0.7–0.8and pHvaluesrangedover6.39,50,51
Itisknownthattheminimalwateractivitynecessaryfor fungal growth isbelow thatrequiredfor theproductionof aflatoxins. Assuch,52Laceyet al.suggests0.78and 0.83as
theminimumwateractivityvaluesthatfavorproliferationof
A.flavusandtheproductionofaflatoxins,respectively.Inour case,althoughvalueswerelowerthanthoserequired,there was alargenumberofAspergillussectionFlavi isolatesand highaflatoxinlevels,probablyduetooxygenpresenceorhigh temperatures.SomefarmersinUruguayremovesilagefor ani-malfeedingbyshoveling;thispracticebreakscompactionof thesilowithintheextractionareaandfavorsaeration,thereby changingtheredoxpotential.
Ontheotherhand,Fusariumisnotveryadaptabletothe anoxic and acidic environmentsof silobags,5 which could
bethereasonwhyF.graminearums.l.wasmainlypresentat harvestandcouldbeanadvantageofusingthistypeofsilo.
WheatsilagesshowedahighpresenceofAspergillus sec-tionFlaviaswellashighcontaminationofDONandaflatoxins, whicharehazardoustohumanandanimalhealth.Thehigh fungalandmycotoxinlevelspresentinsilagecouldaffectthe palatability offeed andcould causeanimal diseaseor pro-duction losses. For dairy cattle, the problem does not end here becausemycotoxinsinfeed canleadtoproductionof metabolicproducts(AFM1)indairyproductsthateventually affecthumanhealth,mainlythatofinfants.Theadoptionof regularscreeningforDONandaflatoxinlevelsinsilagesis rec-ommendedtoavoidexposureofanimalstocontaminatedfeed andintroductionofthesecompoundsintothefoodchain.To date,noinformationexistsaboutthepresenceof mycotox-insandtheir derivativesinmilkfrom Uruguay.Theresults inthepresent studycontributetothe sparseknowledgeof mycobiotaandcontaminationofmycotoxinsinwheatsilage. Furtherstudiesontheecophysiologicalaspectsof contami-nant specieswouldhelptoidentifyfactorsthat needtobe
controlledto diminishthe development and productionof mycotoxinsinsilostorage.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgement
This work was supported byAgencia Nacional de Investi-gacióneInovación(ANII),projectnumber:INI-X-2010-2-2903.
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