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Veterinary
Parasitology
j our na l h o 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
Environmental
factors
influencing
the
transmission
of
Haemonchus
contortus
Michelle
C.
Santos,
Bruna
F.
Silva,
Alessandro
F.T.
Amarante
∗UNESP-UniversidadeEstadualPaulista,DepartamentodeParasitologia,InstitutodeBiociências,CaixaPostal510,18618-970Botucatu,SP,Brazil
a
r
t
i
c
l
e
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n
f
o
Articlehistory:
Received27January2012
Receivedinrevisedform20March2012 Accepted29March2012
Keywords:
Epidemiology Herbage Nematoda Sheep
Parasiticgastroenteritis
a
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s
t
r
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c
t
InfectionwiththegastrointestinalnematodeHaemonchuscontortuscausesconsiderable lossesinthesheepindustry.Inthisstudy,weevaluatedtheeffectthatclimatehason third-stagelarvae(L3)ofH.contortusintermsoftheirmigrationfromsheepfecestoBrachiaria decumbensgrass,aswellastheirdistributionamongtheforageplants.Fecalsamples con-tainingH.contortusL3wasdepositedonthesoilamongtheherbageataninitialheight of30cm.Samplecollectionbegan24haftercontaminationandwasperformedon alter-natedaysover13days.TheL3wererecoveredandquantifiedinthreestrata(heights)of grass(0–10cm,10–20cmand>20cm)aswellasintheremainingfecesandasuperficial layerofsoil,collectedfrombeneaththefeces.Inordertoobtainresultsunderdifferent environmentalconditions,fecalsamplescontainingH.contortusL3weredepositedon pas-tureinJanuary(summer),inApril(autumn),andJuly(winter).Inalloftheperiods,the L3wereabletomigratefromthefecestotheherbage.However,rains,accompaniedby highrelativehumidityandhightemperatures,apparentlyfavoredmigration.Thehighest L3recoveryrateinthepasturewasinthesummerobservationperiod,whichhadthe high-estnumberofdayswithmeasurableprecipitation,highrelativehumidity(>68.2%),and thehighesttemperaturesatthesoillevel(minimumandmaximummeansof19◦Cand
42◦C,respectively).Underthoseconditions,larvaebegantoreachtheupperstratumofthe
grass(>20cm)by24hafterthedepositionoffecalmatter,thenumberoflarvaehaving reachedthatstratumpeakingatsevendaysafterdeposition.Intheautumnobservation period,therewasnorainfallinthefirstfivedayspost-contamination.Duringthatperiod, highnumbersoflarvaewerefoundinthefecalsamplesdemonstratingthatfecescanactas areservoiroflarvaeintheabsenceofrain.Exceptfortwodaysinthesummerobservation period,whenmostoftheL3wererecoveredfromthetopsofbladesofgrass,L3where locatedpredominantlyatthebaseoftheherbage.Inconclusion,rainfallfavorsthe migra-tionofL3fromfecestoherbage.Inaddition,larvalmigrationupandalongbladesofgrass canoccurrelativelyrapidlywhenthetemperatureishigh.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Infectionwithgastrointestinalnematodescauses con-siderablelossesinthesheepindustry.Insmallruminants raisedintropicalandsubtropicalregions,themainparasite
∗ Correspondingauthor.Tel.:+55143816239;fax:+551438153744.
E-mailaddress:amarante@ibb.unesp.br(A.F.T.Amarante).
speciesisthegastrointestinalnematodeHaemonchus con-tortus. Anthelmintics have been used routinely in the prophylaxisofparasiticgastroenteritis.However,the fre-quent use of such drugs has resulted in the selection ofparasiteswithmultipleanthelminticresistance,which jeopardizesthetreatmentandcontrolofgastrointestinal nematodeinfections(Almeidaetal.,2010).
There is a need to develop new approaches to control parasitic gastrointestinal infection, such as
grazing management. Detailed knowledge regarding the biology of the free-living stages of nematodes is essential for understanding the epidemiology of parasitic gastroenteritis and can facilitate the develop-mentof husbandry strategiesthat seektolimit contact betweentheruminantsandtheirparasitesintheinfective stage.
Development from the egg stage through infective third-stage larvae (L3)comprises a series of individual pre-infectivestages and processes. The migration of L3 fromfecestotheherbageisinfluencedbyclimatic con-ditionsandthemicroclimateoftheenvironmentprovided bytheforage(reviewedbyO’Connoretal.,2006). Para-sitesbehavein awaytomaximizeinfectionsuccess. In thecaseofH.contortus,theprobabilityofbeingingested bythehostisincreasediftheL3migratefromthefeces, wheretheinitialdevelopmenttookplacetothe vegeta-tionforruminantconsumption.FentonandRands(2004)
described a model that predicted three types of infec-tionbehavior formacroparasites:alwaysactivelyseeka host (a pure cruising strategy); always passivelyawait hostcontact(apureambushingstrategy); andaninitial cruisingphasefollowedbyambushing(amixedstrategy). According to those authors, H. contortus is an exam-pleofa parasitethatadoptsthemixedstrategy.In this case,inordertomaximizetheirchancesofhostcontact, infectivestages mustexit thedung and position them-selveswhere theyare more likely to be ingested by a host(e.g.,atthetips ofvegetation).Therefore,aninitial cruisingphasegreatlyincreasestheirchancesof encoun-tering a host. However, once at the tip of a blade of grassoraleaf,anysubsequentactivityisunlikelyto fur-therincreasetheirchanceofcontactingahost,atwhich pointtheyadoptanambush-onlystrategy,awaitinghost contact onthevegetation,also allowingthe L3 tosave energy.
Intheenvironment,theinitialcruisingappearstobe greatlyinfluencedbyclimaticconditions.For fecal mat-tercontaining L3 duringa dry season,greaternumbers of L3 tend to remain in the dung and those able to migrate tothe vegetation are most often foundat the base of the herbage. Conversely, during times of high humidityand hightemperatures, greaternumbersofL3 tend to befound higher up on vegetation(Silva et al., 2008).
This study was motivated by the need for more detailed information about the influence of climate on the dynamics of H. contortus L3 migration from sheep dung to the herbage. In previous studies car-ried out in the state of São Paulo, the collection of fecalsamples frompasture was initiated at sevendays aftercontamination (Oliveiraet al., 2008; Carneiro and Amarante, 2008; Silva et al., 2008). In all of those experiments, L3 numbers were markedly lower than were those recorded in control samples maintained in the laboratory. In the present study, we intended to determine when this loss begins to occur and whether climatic variables, such as temperature, rain-fall and luminosity, affect the numbers or behavior of
H.contortusL3.
2. Materialsandmethods
2.1. Studysiteandclimaticmeasurements
TheexperimentswerecarriedoutinapaddockatSão PauloStateUniversity,inthecityofBotucatu,Brazil.The dailyrainfallwasmeasuredatthestudysite,usingarain gauge(Multitec, SãoLeopoldo, Brazil)aswasthe maxi-mumandminimumdailytemperaturesatthesoillevel, withathermometer(JProlab,Curitiba,Brazil).The rela-tivehumidityandsolarradiationdatawereobtainedfrom theMeteorologicalStationoftheSãoPauloStateUniversity School ofAgricultural Sciences, Department of Environ-mentalScience,located8kmfromtheexperimentalsite.
2.2. CollectionandmaintenanceofH.contortusL3
DetailsabouttheisolationofH.contortuswere previ-ouslydescribedbySilvaetal.(2008).Inthepresentstudy, theH. contortusisolatewasmaintainedin lambdonors untilJuly2008.Subsequently,feceswerecollectedand cul-turedtorecoverL3thatwereusedtoinfectlambs fora test ofanthelminticefficacy,which demonstrated resis-tanceoftheH.contortusisolatetolevamisole,albendazole, ivermectin,moxidectin,closantelandtrichlorfon(Almeida etal.,2010).Fecescollectedfromalambtreatedwith mox-idectin(0.2mg/kgBW;Cydectin®,FortDodge)inOctober 2008,wereculturedtorecoverL3whichwerestoredand usedtoinfecttwoworm-freemalelambs,onasingle occa-sioninDecember 2008.Thesemonospecificallyinfected animalswerekepthousedandprovidedfecalsamplesfor thisstudy.
2.3. Experimentalprocedures
Inordertoobtainresultsunderdifferent environmen-tal conditions, fecal samples containing H. contortus L3 wasdepositedonpasturein January(summer),in April (autumn),andJuly(winter)of2009.Ineachofthose obser-vationperiods,fecalsamplesweredepositedat70points, approximately1.5mapart.
Theexperimentalarea(504m2)waslocatedinside a
0.6-hapaddockcultivatedwithBrachiariadecumbensgrass, whichwasmaintainedungrazedduringthestudyperiod. Foreachobservationperiod,70sitesinthepasturewere chosenandmarkedwithstakes.Thecriterionforsite selec-tionwasaminimumherbageheightof30cm.Inorderto standardizetheheightofthepasture,theupperpartofthe foragewascutdownto30cmjustbeforethedepositionof fecalmattercontainingtheL3.Thiswasdonebyhand,with scissors,inordertopreventthecuttingsfromfallingonto thesoil.
Thesheephavingthehighestfecaleggcountwaschosen asthedonoranimal,andacollectionbagwasattachedtoits hindquarters.Thecollectionbagwaschangedtwicedaily andaftereachbagwasremoveditwasdatedandstoredin arefrigeratorat7◦C.Feceswerecollectedinthismanner
Foreach deposition,80fecalsampleswereprepared, eachwithatotalof30,000eggs.Theamountoffecesused ineachobservationperiodvarieddependingontheEPG valuepresented bythedonor animal. Themethodology forproducingtheL3andprocessingofsampleswasthat devisedbySilvaetal.(2008).Inbrief,fecesweregently pooledandthesamples,previouslyweighed,placedwith intact fecalpellets in 80 Petridishes that were keptin anincubator (at25◦C)where theyremainedfor7 days
toallowdevelopmenttoL3.Ofthe80 fecalsamples,70 weredepositedonthesoil,amongtheherbage,whilethe 10remainingsamplesservedascontrolculturesandwere immediatelyprocessedinthelaboratoryaccordingtothe techniqueofBaermann(UenoandGonc¸alves,1998),which allowedrecoveryandenumerationofL3ineachsample.
In the field, all L3-containing fecal samples were deposited in themorning(between 8:00and 9:00am). The dayof deposition wasconsideredday 0. The mea-surements were taken on alternate days, beginning on day1(24hafterdeposition).Therefore,sampleswere col-lected,between8:00and10:00am,ondays1,3,5,7,9, 11,and13,resultinginsevendaysofsampling.Foreach day,therewere10replicatesselectedrandomlyamongthe 70samples.Todelineatethecollectionareaandherbage height,a 10-cm diameter metalring wasattachedtoa rodgraduatedaccordingtostratifiedgrassheights(>20, 10–20,and0–10cm).Theringwasplacedoverthefecal matterdeposit,andbladesofgrassthatwereinsidethe circlewerecut,stratumbystratum.Eachcollected stra-tumwasstoredseparatelyinapre-labeledplasticbagfor subsequentprocessing in thelaboratory. Theremaining feceswerecollected,takingcaretorecoverallfecalpellets. Thesuperficiallayerofsoil,toadepthofapproximately 1cm,frombeneaththedungwasalsocollected.The sam-pleswerepackedseparatelyinpre-labeledplasticbagsfor subsequentprocessinginthelaboratory.ForL3recovery, material wasprocessedin accordance withthemethod describedbySilvaetal.(2008).
2.4. Statisticalanalysis
Datawereanalyzedbygenerallinearmodelwiththe programStatisticalAnalysisSystem,version9.2.(SAS Insti-tute,Inc.,Cary,NC,USA).Themodelincludedthreegrass heightstrata(0–10,10–20,and>20cm)andseven collec-tiontimepoints(days),withevaluationoftheinteraction between stratum and collectionday.Means were com-paredbyTukey’stestata5%significancelevel.Resultswere analyzedfollowinglogarithmictransformation(log(x+1)) andarepresentedasarithmeticmeans(±standarderror).
3. Results
3.1. ClimaticconditionsandL3recovery
TheH.contortusL3containingfecalpelletsdeposited onthesoilamongherbageremainedexposedtoclimatic variationsfor13days.Asexpected,themaximumand min-imumdailytemperaturesmeasuredatgroundlevelwere differentforeachtimeoftheyear(Tables1–3).The heav-iestrainfalloccurredinJanuary/February(rainon7ofthe
13days,atotalof96.0mm)followedbyApril/May(rain on3ofthe13days,atotalof70.5mm)andJuly/August (rainon3ofthe13days,atotalof28.0mm).Themean rel-ativehumiditywasalsohigherinJanuary/Februarythan inApril/MayandJuly/August(80.9%vs.71.9%and73.0%, respectively).Asaconsequenceoftheheavyprecipitation andhightemperaturesinJanuary/February,therewas pro-gressive degradation of thefecalpellets, which didnot occurattheothertimesoftheyear(Table4).Herbage pre-sentedexternalmoisture(deworrainwater)atthetime ofsample collectiononalldays,exceptonfourdaysin July/August(Tables1–3).
Atalltimesoftheyear,thepercentagerecoveryofL3 fromthefieldsamples(ofsoil,feces,andtotal herbage) wasconsiderablylowerthanthatachievedfromthecontrol fecalsamples(Table4).
3.2. January/February2009
IntheJanuary/February(summer)observationperiod, larvaewererecoveredfromfecalsamplesinhighnumbers only on the first sampling day(i.e., 24h) after deposi-tion(Fig.1A).Thereafter,rainfallapparentlyfavoredthe degradationoffecalpelletsandlarvaldispersal intothe environment,resultingingreaterrecoveryofL3fromthe totalherbagesamplesthanduringeitheroftheothertwo observationperiods.Onthefirstdayofcollection,the low-estgrass height stratum (0–10cm) showed thehighest meannumberofL3(16.1±3.7).Overthesubsequent sam-plingdays, themean number ofL3 recovered fromthe uppermoststratum(>20cm)graduallyincreased,peaking onday7(at34.3±10.7),whereasthatrecoveredfromthe loweststratumgraduallydecreased(Fig.2A).However,on day13,thenumberofL3recoveredwassimilaramongthe threestrata(Fig.2A).
During this observation period, there was signifi-cantinteractionbetweencollectiondayandgrassheight stratum(p<0.05).However,therewerenostatistical dif-ferencesamongthedifferentgrassheightstrata,interms ofthemeannumberofL3recovered.
3.3. April/May2009
Table1
Maximumandminimumtemperatures(measuredatthesoillevel),dailyrainfall,relativehumidity(RH),andsolarradiation(SR),togetherwiththesample conditions,duringthesummerobservationperiod(January/February2009).
Date Temperature(◦C) Rainfall(mm) RHa(%) SRa(cal/cm2) Conditionofsamples
Maximum Minimum Herbage Feces Soil
(0)January28b 32 18 35 93.8 146 – – –
(1)January29c 25 22 3 89.8 215 Humid Humid/intact Humid
(2)January30 38 19 20 89.9 273 – – –
(3)January31c 34 20 0 78.5 420 Humid Humid/intact Humid
(4)February1 42 23 0 68.2 511 – – –
(5)February2c 41 19 4 78.9 249 Humid Humid/degraded Humid
(6)February3 38 20 0.5 80.4 409 – – –
(7)February4c 40 21 26.5 88.1 252 Humid Humid/degraded Humid
(8)February5 34 19 0 74.7 481 – – –
(9)February6c 40 19 0 70.4 533 Humid Humid/degraded Humid
(10)February7 39 20 7 74.2 0 – – –
(11)February8c 42 21 0 78.9 434 Humid Humid/degraded Humid
(12)February9 39 20 0 84.3 271 – – –
(13)February10c 38 20 0 82.3 308 Humid Humid/degraded Humid
aRHandSRrecordedattheMeteorologicalStationlocated8kmfromthestudysite. b Dayonwhichfecalsamplesweredepositedonthesoil.
c Samplecollectionday.
3.4. July/August2009
IntheJuly/August(winter)observationperiod,itrained immediatelyafterthedepositionof fecalmatter,which possiblyfavoredthepresenceoflarvaeinthetotalherbage samplesonthefirst dayofevaluation (Fig.1C). Similar towhatoccurredintheApril/Mayobservationperiod,L3 recoverywasgreatestinthelowestgrassheightstratum (0–10cm) on six of theseven samplingdays (Fig. 2C). Althoughinsmallnumbers,theL3werefoundinthe upper-mostgrassheightstratum(>20cm)onthefirstsampling day(24hafterthedepositionoffecalsamples).The over-allmeannumberofL3recoveredfromthe0–10cmstratum wassignificantlyhigherthanwasthatofL3recoveredfrom the10–20cmand>20cmstrata.
4. Discussion
TwoaspectsofH.contortusL3ecologywereevaluatedin thepresentstudy:survivalandmigration.Regarding sur-vival,therewasnoquestionthattherecoveryoflarvaefrom thefecalpellets,vegetation,andsoilsurfacewaspoor.Even at24hafterthedepositionoffecalpelletsonpasture,the numberrecoveredwasonlyasmallfractionofthetotal numberpresentinthecontrolsamples.Similarresultshave beenreportedinotherfield studies(Levineetal.,1974; CarneiroandAmarante,2008;Silvaetal.,2008;Amaradasa etal.,2010).Althoughthefateofthelarvaeisunknown, itis thoughtthatthegreatmajoritydiewithinadayor two after presentationto the environment. Among the manylikelycausesofL3deathisexposuretotheelements
Table2
Maximumandminimumtemperatures(measuredatthesoillevel),dailyrainfall,relativehumidity(RH),andsolarradiation(SR),togetherwiththesample conditions,duringtheautumnobservationperiod(April/May2009).
Date Temperature(◦C) Rainfall(mm) RHa(%) SRa(cal/cm2) Conditionofsamples
Maximum Minimum Herbage Feces Soil
(0)April27b 29 17 0 74.8 373 – – –
(1)April28c 29 15 0 71.0 430 Humid Humid/intact Dry
(2)April29 25 12 0 66.8 409 – – –
(3)April30c 19 11 0 59.6 418 Humid Humid/intact Dry
(4)May01 30 12 0 71.7 423 – – –
(5)May02c 30 15 0 74.6 410 Humid Humid/intact Dry
(6)May03 30 16 46 76.0 372 – – –
(7)May04c 27 16 22 85.8 171 Humid Humid/intact Humid
(8)May05 19 11 0 70.9 385 – – –
(9)May06c 29 10 0 63.7 368 Humid Humid/intact Humid
(10)May07 35 15 0 68.7 338 – – –
(11)May08c 30 15 0 73.5 327 Humid Humid/intact Humid
(12)May09 30 18 2.5 70.3 314 – – –
(13)May10c 29 15 0 79.6 301 Humid Humid/intact Humid
aRHandSRrecordedattheMeteorologicalStationlocated8kmfromthestudysite. b Dayonwhichfecalsamplesweredepositedonthesoil.
Table3
Maximumandminimumtemperatures(measuredatthesoillevel),dailyrainfall,relativehumidity(RH),andsolarradiation(SR),togetherwiththesample conditions,duringthewinterobservationperiod(July/August2009).
Date Temperature(◦C) Rainfall(mm) RHa(%) SRa Conditionofsamples
Maximum Minimum Herbage Feces Soil
(0)July28b 21 14 17.5 90.5 205 – – –
(1)July29c 29 16 8.5 83.6 206 Humid Humid/intact Humid
(2)July30 28 15 0 83.7 229 – – –
(3)July31c 22 12 0 89.8 305 Humid Humid/intact Humid
(4)August01 25 13 0 83.5 287 – – –
(5)August02c 25 15 0 81.1 226 Dry Dry/intact Humid
(6)August03 30 14 2 64.8 258 – – –
(7)August04c 27 12 0 77.7 325 Dry Humid/intact Humid
(8)August05 28 13 0 83.1 291 – – –
(9)August06c 29 15 0 75.9 376 Humid Humid/intact Humid
(10)August07 28 14 0 49.4 406 – – –
(11)August08c 31 15 0 67.5 387 Dry Humid/intact Humid
(12)August09 33 12 0 48.8 407 – – –
(13)August10c 33 11 0 42.3 349 Dry Dry/intact Humid
aRHandSRrecordedattheMeteorologicalStationlocated8kmfromthestudysite. bDayonwhichfecalsamplesweredepositedonthesoil.
cSamplecollectionday.
(weather).Toevaluatethisfactor,wemeasuredanumber ofclimaticvariables.Thetemperatureatthesoillevelin theshadeofherbage >20cminheightvariedwidely.For instance, intheJanuary/February (summer)observation period,themaximumandminimumtemperatureswere 42◦Cand19◦C,respectively.Additionalmortalitycaused
by ultravioletlight exposure couldalsohelp to explain thehighlarvalmortalityratesonpasture(vanDijketal., 2009).Althoughwedidnotmeasurethevariationin rela-tivehumiditythroughoutthedayatthegroundlevel,itis likelythattherewasalsoconsiderablefluctuationbetween moistanddryconditionsonthesurfaceoftheherbage.It hasbeenreportedthat,inthelaboratoryandinthefield, infectivelarvaeareabletosurviveseveralcyclesof des-iccation/rehydrationinaprocesscalledanhydrobiosis,in whichthemetabolicactivityisdecreasedandthesurvival ofthelarvaeis prolonged(Lettiniand Sukhedeo,2006). However,accordingtoSiambaetal.(2011),repeatedcycles ofdesiccation-revivalcycles,duetodiurnalvariationsin pasturehumidity,canreducetheviabilityofH.contortus
larvae,whichsuffercontinuousdepletionofirreplaceable lipid reserves.Such a reduction in viability might have occurredinthepresentstudy.
Thedegradationoffecalpelletsoccurringinthesummer observationperiod wasattributedtohightemperatures
andhighhumidity.Theactivitiesofmicroorganismsmight alsohavehadsomeinfluenceonfecaldegradationandin L3mortality.Forexample,nematophagousfungicantrap andkilltheL3ofH.contortus(Camposetal.,2008).
Despitethelow survivalofthefree-livingstages,the numberofH.contortuslarvaeproducedandremainingon pastureissufficienttocausesevereinfectionsin suscepti-blesheep.Thisispossibleduetomassiveeggproduction. Assuming that, in sheep, fecal output corresponds to approximately5%ofbodyweight(Amaranteetal.,2007), aneweweighing50kgandshedding1,000EPGcan elimi-nateapproximately2.5millioneggsperday.Ifonly0.01% ofthoseeggsgiverisetoL3thatareabletosurviveand migratetopasture,therewillbe25,000L3 reachingthe pastureeveryday.
ThereisevidencethatH.contortusL3reachvegetation merelybychance.AccordingtoSciaccaetal.(2002),the behaviorofH.contortusL3suggestsrandomcrawling,with frequentreversalsofdirection,whichisdifferentfromthat ofAncylostomacaninumandStrongyloidesstercoralis,two skin-penetratingspecies,whoseL3exhibitnegative geo-taxis,crawlingagainstthepullofgravity,presumablyin attemptstocontactapassinghost.
In all of the experimental periods analyzed in the presentstudy,L3wereabletomigratefromthefecalpellets
Table4
NumbersofHaemonchuscontortusthird-stagelarvae(L3)recoveredfromfieldsamples(ofsoil,feces,andherbage),aswellasfromcontrolsamples,by observationperiod(timeofyear).
Observationperioda NumberofH.contortusL3larvae(mean±standarderror)
Control Soilb Fecesb Herbageb Notrecoveredb
Summer 18,610
±141
2.24±0.30 9.74±3.55 8.23±0.91 18,589.79 (0.01%) (0.05%) (0.04%) (99.90%)
Autumn 12,160
±540
5.96±1.77 160.70±26.33 2.30±0.28 11,991.04 (0.05%) (1.32%) (0.02%) (98.61%)
Winter 11,102
±677
1.66±0.29 20.27±2.73 3.72±0.36 11,076.35 (0.02%) (0.18%) (0.03%) (99.77%)
aDatarepresenttheoverallmeanvaluesof13-dayobservationperiod.
bPercentagesinparenthesesrepresenttheproportionsoflarvaerecovered(ornotrecovered)fromthefieldsamplesinrelationtothenumberoflarvae
Fig.1.AveragenumbersofHaemonchuscontortusthird-stagelarvae(L3)recoveredfromfieldsamples(ofsoil,feces,andherbage)duringeachofthethree 13-dayobservationperiods:January/February(A);April/May(B);andJuly/August(C).ThemeannumberofL3infecalsamplesdepositedonpasturein thosethreeobservationperiodswas18,610,12,160,and11,102,respectively.Barsrepresentstandarderrorofthemean.
totheherbage.However,rainfallandhighrelative humid-ityaccompaniedbyhightemperaturesapparentlyfavored migration,withL3recoveryfrompasturebeinggreatestin thesummerobservationperiod,duringwhichtherewas measurablerainfallon7ofthe13days,highmeanrelative humidity(>68.2%),andthehighesttemperaturesatthesoil level(minimumandmaximummeansof19◦Cand42◦C,
respectively).
In theApril/May (autumn) observationperiod, there wasnorainfallinthefirstfivedaysafterdepositionofthe fecalmatter.Thehighnumbersoflarvaefoundinthefecal samplescollectedduringthisobservationperiodsuggest thatthedungactedasareservoiroflarvaeintheabsence ofrain,aspreviouslydescribed(Silvaetal.,2008;Carneiro andAmarante,2008;vanDijketal.,2009).Inexperiments carriedoutinalaboratoryatroomtemperature(20–24◦C)
andrelativehumiditybetween71%and83%,vanDijkand
Morgan(2011)notedthatfreewaterappearedtobe essen-tialforlarvalreleasefromdungbutwasnotessentialto ascend herbage.Therefore, rainis necessaryfor making fecalpelletshumidandsoft,whichallowslarvaetrapped insidethosepelletstoexitandmigratetoherbagethatisin closecontactwiththedung.Therefore,afilmofmoisture, throughwhichlarvaewould“swim”,appearstobe neces-saryfortheirmigrationalongthebladesofgrass.However, evenintheabsenceofrainintheinitial5days,some lar-vaewereabletomigratetotheherbageduringtheautumn observationperiod.Itislikelythattherewasaquantityof dewwatersufficienttoallowthismigration.
Fig.2.AveragenumbersofHaemonchuscontortusthird-stagelarvae(L3)recoveredfromherbagesamples,bygrassstrata(0–10cm,10–20cm,and>20cm) duringeachofthethree13-dayobservationperiods:January/February(A);April/May(B);andJuly/August(C).ThemeannumberofL3infecalsamples depositedonpastureinthosethreeobservationperiodswas18,610,12,160,and11,102,respectively.Barsrepresentstandarderrorofthemean.
inGhana,increasednumbersofL3duringtherainy sea-sonanddecreasednumbersorabsenceofL3 duringthe dry season.In a studyconducted in thestate of Texas,
Amaradasaetal.(2010)identifiedastrongpositive corre-lationbetweenrainfallandtotalaveragedailyH.contortus
larvalcountsonpasture.Theseresultsalsoshowthatrain facilitatestheliberationoflarvaefromsheepfeces. How-ever,whenheavyrainsoccurinashortperiodoftime,there isareductioninlarvalrecovery,asoccurredaftertwo con-secutivedaysofrain(totalof68mmondays6and7)in theautumnobservationperiodofourstudy.Itislikelythat theheavyrainswashedlarvaeawayfromthefecal mat-terdepositionsite,resultinginlow larvalrecoveryfrom pasture.
Ontwodaysduringthesummerobservationperiodof ourstudy,L3recoverywasgreatestfortheuppermostgrass
Inthepresentstudy,larvaewererecoveredonall sam-plingdaysduringeachofthe13-dayobservationperiods. Thesewererelativelyshortobservationperiods.However, in experiments of longer duration, it hasbeen demon-stratedthatH.contortuslarvaecansurviveonpasturefor severalweeks,especiallywhen thetemperatureismild, around17◦C(CarneiroandAmarante,2008).Thesurvival
ofL3forextendedperiodswasalsoobservedinastudy con-ductedinCameroon,wherelarvaeofH.contortusmigrated totheherbageandsurvivedforapproximately11weeks duringtherainyseason(NdamukongandNgone,1996).
Inconclusion,rainfallfavorsthemigrationofL3from dung to herbage. In addition, high temperatures can increasethespeedoflarvalmigrationupandalongblades ofgrass.
Acknowledgements
MichelleC.SantosandBrunaF.Silvareceivedfinancial supportfromFAPESP(Fundac¸ãodeAmparoàPesquisado EstadodeSãoPaulo)andAlessandroF.T.Amarante from CNPq(Conselho NacionaldeDesenvolvimento Científicoe Tecnológico,Brazil).
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