Diversity of gastrointestinal helminths in Dall's sheep and the negative association of the abomasal

21 Pages·2017·3.48 MB·English

RESEARCHARTICLE
Diversity of gastrointestinal helminths in Dall’s
sheep and the negative association of the
abomasal nematode, Marshallagia marshalli,
with fitness indicators
O.AlejadroAleuy1*,KathreenRuckstuhl1,EricP.Hoberg2,AlasdairVeitch3,
NormanSimmons4†,SusanJ.Kutz5
a1111111111 1 DepartmentofBiologicalSciences,UniversityofCalgary,Calgary,Canada,2 MuseumofSouthwestern
BiologyandDepartmentofBiology,UniversityofNewMexico,Alburquerque,NM,UnitedStatesofAmerica,
a1111111111
3 124YorkCourt,NewGlasgow,NovaScotia,Canada,4 ProducersofDiamondWillow,PincherCreek,
a1111111111
Alberta,Canada,5 DepartmentofEcosystemandPublicHealth,FacultyofVeterinaryMedicine,Universityof
a1111111111
Calgary,Calgary,Canada
a1111111111
†Deceased.
*[email protected]
Abstract
OPENACCESS
Citation:AleuyOA,RuckstuhlK,HobergEP,Veitch
Gastrointestinalhelminthscanhaveadetrimentaleffectonthefitnessofwildungulates.
A,SimmonsN,KutzSJ(2018)Diversityof
gastrointestinalhelminthsinDall’ssheepandthe ArcticandSubarcticecosystemsareidealforthestudyofhost-parasiteinteractionsdueto
negativeassociationoftheabomasalnematode, thecomparativelysimpleecologicalinteractionsandlimitedconfoundingfactors.Weuseda
Marshallagiamarshalli,withfitnessindicators.
uniquedatasetassembledintheearlyseventiestostudythediversityofgastrointestinalhel-
PLoSONE13(3):e0192825.https://doi.org/
minthsandtheireffectonfitnessindicatorsofDall’ssheep,Ovisdallidalli,intheMackenzie
10.1371/journal.pone.0192825
Mountains,NorthwestTerritories,Canada.Parasitediversityincludedninespecies,among
Editor:EmmanuelSerranoFerron,Universitat
whichtheabomasalnematodeMarshallagiamarshallioccurredwiththehighestprevalence
AutonomadeBarcelona,SPAIN
andinfectionintensity.TheintensityofM.marshalliincreasedwithageandwasnegatively
Received:September11,2017
associatedwithbodyconditionandpregnancystatusinDall’ssheepacrossalltheanalyses
Accepted:January31,2018
performed.Theintensityoftheintestinalwhipworm,Trichurisschumakovitschi,decreased
Published:March14,2018 withage.Nootherparasitesweresignificantlyassociatedwithage,bodycondition,orpreg-
Copyright:Thisisanopenaccessarticle,freeofall nancy.OurstudysuggeststhatM.marshallimightnegativelyinfluencefitnessofadult
copyright,andmaybefreelyreproduced, femaleDall’ssheep.
distributed,transmitted,modified,builtupon,or
otherwiseusedbyanyoneforanylawfulpurpose.
TheworkismadeavailableundertheCreative
CommonsCC0publicdomaindedication.
Introduction
DataAvailabilityStatement:Theunderlyingdata
hasbeenuploadedto(https://osf.io/ey7kx/). Itiswidelyacceptedthatnematodeparasitescanhaveasubtlebutsignificantdetrimental
Funding:WethanktheKillamProgramandNSERC effectsonhostfitness[1–3].Inwildungulates,forinstance,experimentalandobservational
CREATEHostParasiteInteractionProgramfor workhasshownthatgastrointestinalhelminthscannegativelyinfluencepregnancyrates[4,5],
fundingO.AlejandroAleuy.Someinitial bodycondition[5–8],offspringsurvival[9]andmortality[9–11].Thestudyoftheseeffectsin
assessmentsoftheSimmonsCollectionswere
wildungulatesfromArcticandSubarcticregionsisimportantforavarietyofreasons:i)
supportedthroughtheBeringianCoevolution
humancommunitiesintheseregionsdependonwildungulatesasasourceoffood,aseco-
Project(BCP),fundedbytheNationalScience
nomicdriversthroughsporthuntingandtourism,andasafocusforculturalactivities[12],ii)
Foundation(DEB0196095and0415668),an
explorationofparasitediversityandhistoryacross ArcticandSubarcticregionsareamongtheareaswithleasthumandisturbanceglobally,
PLOSONE|https://doi.org/10.1371/journal.pone.0192825 March14,2018 1/21
MarshallagiamarshalliisnegativelyassociatedwithfitnessindicatorsinDall’ssheep
thenorthernroofoftheworld,plannedand however,thecurrentandpredictedclimatechangeismoreprominentinhighlatitudescaus-
coordinatedbyJosephA.Cook(UniversityofNew ingrapidanddeepecologicalchangesandalteringhost-parasitedynamics[13–15],iii)wild
Mexico)andEricP.Hoberg(formerUnitedStates
animalandparasitecommunitiesarecomparativelymoresimplethanintemperateandtropi-
NationalParasiteCollection).Thefundershadno
calsystemsdecreasingthenumberandcomplexityofpotentialconfoundingfactors,andthus,
roleinstudydesign,datacollectionandanalysis,
servingasanidealmodelfortestingecologicalmechanisms[4,16]
decisiontopublish,orpreparationofthe
manuscript. TheDall’ssheep(Ovisdallidalli)isawildungulateendemictothearcticandsubarctic
regionsofNorthAmerica,includingAlaska(AK,UnitedStates),Yukon(YT,Canada),and
Competinginterests:Theauthorshavedeclared
NorthwestTerritories(NT,Canada)[17].ItbreedsinlateNovemberandearlyDecemberwith
thatnocompetinginterestsexist.
birthsoccurringthefollowingApril-May.Duringtheshortsummer,ewesseeksolitudeand
protectioninruggedterrainforgrowthanddevelopmentoflambsandtorecovertheirbody
reserves[18,19].Dall’ssheepareanimportantsubsistencespeciesfornorthernindigenouspeo-
pleaswellasavaluablesporthuntingprize,appreciatedfortheirhigh-qualitymeatandlarge
hornsize[20,21].
TheparasitologicalinformationonDall’ssheepislimitedtoserologicalsurveysofmicro-
parasiteslikeToxoplasma gondii[22],descriptionofEimeriaspp.[23],detailedtaxonomicand
ecologicalstudiesontheprotostrongylidnematodesParelaphostrongylus odocoileiandProto-
strongylusstilesi[24–26],andbroad-basedfecalandgastrointestinalparasitesurveys[27–30].
ThenematodeMarshallagiamarshallihasbeendocumentedasthegastrointestinalmacropara-
sitewiththehighestinfectionintensitiesandprevalencethroughoutDall’ssheepdistribution
range[28–30].ThepathophysiologicalchangescausedbyM.marshalliincludeamarkedloss
offunctionalparietalcellsintheabomasumofthehostresultinginanincreaseofabomasal
pH,andanincreaseofbothserumpepsinogenandserumgastrinconcentrations[31,32].The
clinicalpresentationissimilartoothergastrointestinalparasitesincludingappetitedepression,
weightloss,constipationanddiarrhea[31,33].BasedonfieldobservationsinAlaska,Neilsen
andNeiland[29]suggestedthatM.marshallimightchronicallyweakenadultsheepandalter
thedigestioncapacityofcrudeproteinanddrymatterinlambsduringtheirfirstwinter.
DespitetheimportanceofDall’ssheepforthelocaleconomyandsubsistence,plusthemount-
ingevidenceonthedetrimentaleffectofparasitesonwildungulatesinarcticandsubarctic
regions[4,5,34],littleisknownabouttheimpactsofgastrointestinalhelminthsandparticularly
M.marshallionDall’ssheep.
Theobjectivesofthisstudyweretodescribethediversityofgastrointestinalparasitesin
Dall’ssheepandtodeterminetherelationshipbetweenparasiteintensityandfitnessindica-
tors.Todothis,weusedanunprecedentedhistoricalcollectionofparasitesandassociated
datainDall’ssheepcollectedfromtheMackenzieMountains,Canadain1971–1972.Wehad
threespecificobjectives;to(i)describethediversityandintensityofgastrointestinalhelminths,
andtheirrelationshipwithageclassesandsex,(ii)determinetheassociationbetweenM.mar-
shalliintensityandbodyconditionindicators,andfinally,(iii)determinetheassociation
betweeninfectionintensityofM.marshalliandpregnancystatusinadultfemalesDall’ssheep.
Materialandmethods
Thesimmonscollection
From1968to1972anextensivescientificcollectionofDall’ssheepoccurredaspartofademo-
graphicstudyofsheepintheMackenzieMountains,NorthwestTerritories(NWT),Canada
[35].Thetwolargestcollectionsoccurredinthewintersof1971and1972and,inadditionto
thephysiologicalandreproductivedata,gastrointestinalparasiteswerecollectedbyDr.Anne
Currier,aveterinarianfortheCanadianWildlifeService,FortSmith,NWT.Afterpreliminary
evaluationintheearly1970s,nofurtherworkwasdoneontheparasitecollectionandits
whereaboutswereunknownuntilthespringof2000,whenitwaslocatedasanorphaned
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MarshallagiamarshalliisnegativelyassociatedwithfitnessindicatorsinDall’ssheep
collectionattheCanadianMuseumofNature(CMN)inOttawa(Ontario,Canada).Thespeci-
mensandassociateddocumentationwererecoveredandevaluatedbySusanKutzalongwith
EricHobergandAlasdairVeitch,whodeterminedthatitwasanextremelyvaluable,unprece-
dentedintactdatasetandparasitespecimencollectionforDall’ssheep.Itisnowknownas
“TheSimmonsCollection”.
Thecollectionconsistedoffourboxescontainingapproximately100vialsofparasites
each,aswellasasmallcollectionofspecimensmountedonslides.Allthedocumentation
wasstoredinasinglebindercontainingindividualcardswiththeparasitologicalinforma-
tionfromeachsheepandanextensivescientificcorrespondenceamongtheprincipalinves-
tigatorsinvolvedinthisresearch,includingDr.NormanSimmons(projectleader,Fort
Norman),Dr.AnneCurrier(veterinarian,FortSmith,postmortemexaminations,isolation
andquantificationofhelminths),Dr.LaurelP.E.Choquette(Ottawa,identificationofhel-
minths),Dr.EricBroughton(veterinarypathologist,Ottawa,lungpathology),andDr.G.
G.Gibson(Ottawa,involvedinthelungwormstudies).Inaddition,Dr.NormanSimmons
providedtheoriginalpostmortemcards(Fig1)andsummarysheetswiththeinformation
onphysiologicalandreproductiveparametersfromeachsheepcollectedin1971and1972
(seebelow).
Fig1.Originalpostmortemcard.CardusedbyDr.NormanSimmonstorecordtheindividualphysiologicaland
reproductiveinformationofDall’ssheepcapturedintheMackenzieMountainsduringFebruary1971–1972.
https://doi.org/10.1371/journal.pone.0192825.g001
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MarshallagiamarshalliisnegativelyassociatedwithfitnessindicatorsinDall’ssheep
Dall’ssheepcapturesanddatacollection
TheDall’ssheepcollectionoccurredinFebruary1971and1972fromseveralindependentsub-
populationsinhabitingtheareasofMountainandMoosehornriversinthenorthcentralMac-
kenzieMountains(65degreesN;128degreesW),Canada.Thecaptureswereperformedwith
ahigh-poweredriflefiredfromahelicopter,includingallsheepinthefirstgroupencountered
exceptforthoseclearlyidentifiableasrams.Theinformationonphysiologicalandreproduc-
tiveparametersoneachsheepwasrecordedonindividualpost-mortemcardsandsummary
sheets[35].Thepostmortemcardsincludedinformationonsex(i.e.male,female),agein
yearsbasedonbothcountsofhornringsandtoothcementumannuli,reproductivestatus
determinedbythepresenceorabsenceofafoetus,bodyweight(Kg),severalbodymeasure-
ments(e.g.totalbodylength,chestcircumference)andabodyconditionscorebasedonthe
presence/absenceofabdominalandbackfat(Fig1).
ThegatheringofspecimensanddataongastrointestinalparasiteswasperformedbyDr.
AnneCurrier.Adultswormsfromtheabomasum,smallintestine,largeintestineandcaecaof
eachsheepwereisolatedfromeachorgan,provisionallyidentified,counted,andstoredin10%
formalin.Between2003–2005,S.Kutz,togetherwithEP.Hoberg,retrievedthesevials,rehy-
dratedthespecimenswhenneeded,andthenmountedthemonmicroscopeslidesandcleared
themin5%glycerolforsubsequentexaminationandidentificationatthespecieslevelaccord-
ingto[36–42].
Dataanalyses
Sheepbodycondition. Weusedtwodifferentapproachestoassessbodyconditionofthe
sheep:i)acategoricalbodyconditionscore(BCS)basedonthepresenceorabsenceofsubcu-
taneousfatreserves,andii)anon-destructiveresidualindexbasedonmassandmorphometric
measurements(scalemassindex,SMI)[43].
TheBCSwasdeterminedinthefieldandrecordedinthepostmortemcards.Sheepwithno
abdominalorbackfatwereclassifiedas‘Lean’;sheepwithabdominalbutnotbackfatwere
classifiedas‘Fair’;sheepwithabdominalandsomebackfatwereclassifiedas‘Good’,and
sheepwithmuchorgoodbackfatwereclassifiedas‘VeryGood’.
TheSMIisarecentinnovationthatstandardizesbodymassatafixedvalueofalinearbody
measurementbasedonthescalingrelationshipbetweenmassandlength[43,44].Thisisaver-
satileindexasitisindependentfrombodysize,sex,andphysiologicalstatus,andunlikemost
residualindices,itcanbeusedtocompareacrosspopulations[45].TheSMIwascalculated
accordingto[43]asshowninthefollowingformula:
(cid:20) (cid:21)
L bSMA
SMI ¼M 0 ð1Þ
i L
i
whereM isthebodymassandL isthebodylengthofindividuali,theb istheregression
i i SMA
coefficientintheStandardizedMajorAxisregressionofln-transformedMonln-transformed
L,andL isanarbitraryvalueofL.Normallythearbitraryvalueusedisthearithmeticmeanof
0
Lforthesampleunderstudy.Finally,SMIisthepredictedscalemassindexforindividuali.In
thecaseofpregnantfemales,wecorrectedtheirweightbysubtractingtheweightoftheirfetus
(meanfetusesweight:0.236kg,SE=0.013)beforecalculatingtheirSMI.
Parasiteintensity,prevalence,andspeciesdominance. Wedeterminedtheintensityof
infection(i.e.mediannumberofworms)andprevalence(i.e.percentageofpositivecases+
95%confidenceintervals)foreachspeciesofgastrointestinalhelminthbysex,pregnancystatus
andyearofcollection.Inthecaseofinfectionintensityandprevalencebysheepage,weused
twodifferentapproaches:i)tocomparejuvenileswithadultsweusedtwoagecategories;
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MarshallagiamarshalliisnegativelyassociatedwithfitnessindicatorsinDall’ssheep
juveniles(<24monthsofage)andadults(>24monthsofage),andii)todeterminetheage
patternofinfectionforthesixmostabundantparasitespeciesweusedfiveagecategories:
lambs(<1year),yearlings(1yearold),youngadults(2to<6years),matureadults(6to<11
years)andoldadults(11yearsandolder).Inalltheanalyses,thedifferencesininfection
intensitybetweencategorieswereexploredusingat-testwithWelchCorrectiontoaccountfor
differencesinvariance.Inthecaseswheretheassumptionofnormalitywasnotmetapermu-
tationt-testwith10,000permutationswasused.Thedifferencesinparasiteprevalenceamong
categoriesweredeterminedusingchi-squaredtestwithMonte-Carlosignificancetestproce-
dures(2000replicates)toaccountforlowcellcounts(<5)inthecontingencytables.Totest
differencesinparasiteinfectionintensitybyBCSweusedaone-wayANOVAandlog+1trans-
formedparasiteintensitytoaccountfornormallydistributedresiduals.TheTukeyHDStest
wasusedforpairwisecomparisons.Intheunivariateanalysesdescribedabove,alltheobserva-
tionsavailableforeachspecieswereused.
Thespeciesdominanceamonggastrointestinalparasiteswasdeterminedbyusingtwo
approaches:aDominanceIndex(DI)andaDominanceValue(DV).TheDI,originallypro-
posedby[46],considersspeciesprevalenceandiscalculatedbymultiplyingthetotalnumber
ofspeciesibythenumberofhostsinfectedwithspeciesianddividedbythetotalnumberof
hostsexaminedsquared.Inthisindex,adominantspecieswasdefinedasoneinwhichtheDI
isgreaterthan1,andaco-dominantspeciestheonewhichtheDIvaluewasbetween0.01and
0.99.TheDV,originallyproposedby[47],considersthetotalnumberofparasitesintheentire
hostsample.DViscalculatedsimplybydividingthetotalnumberofspeciesiintheentire
hostsamplebythetotalnumberofgastrointestinalparasitesincludingallthespeciesinthe
hostsample,andmultiplyingtheresultbyonehundred.Onlythesheepwithcompleteinfor-
mationforalltheparasitespecieswereincludedinthecalculationofbothapproachesinorder
toobtaincomparableresultsamongspecies.
Fitnessindicatorsandgastrointestinalparasites. Therelationshipbetweenfitnessindi-
catorsinthehost(i.e.BCS,SMIandpregnancy),andtheinfectionintensityofgastrointestinal
helminthswasexploredusingtwoapproaches:i)GeneralizedLinearModels(GLM),this
approachprovideaflexibleframeworktostudytheassociationofcontinuous(i.e.SMI)and
discrete(i.e.BCSandpregnancystatus)outcomesandasetofindependentvariables.How-
ever,thenumberofpredictorvariablesinthecandidatemodelisdirectlylimitedby,among
otherfactors,samplesize(e.g.>10outcomeeventsperpredictorvariable)[48].Themodels
withtoofewoutcomeeventsrelativetothenumberofpredictorvariablesmightproduce
biasedestimatesofregressioncoefficients[49],andii)PartialLeastSquaresRegression
(PLS-R)providesaflexibleframeworktostudytheassociationofcontinuousvariables(i.e.
SMI)andasetofindependentvariables.Thisapproachisparticularlyusefulwhenthenumber
ofsampleunitsislowwithrespecttothenumberofmeasuredindependentvariables[50].Spe-
cifically,thePLS-Rwasusedtoinvestigatethesimultaneouseffectofhelminthsco-infectionin
hostfitness.
TheGLMswerefittedasfollows:inthecaseofBCSweusedordinallogisticregressionto
accountfortheorderonthecategoriesintheoutcomevariable.Proportionaloddsratioswere
obtainedbytheexponentiationofthecoefficientsfromtheindependentvariablesinthefinal
model.Inordertomeetthesamplesizerequirementsperpredictorvariable,theparameters
includedwerereducedto:infectionintensityofonlythehelminthspeciesnegativelyassociated
withfitnessindicatorsintheunivariateanalyses(i.e.M.marshalliandT.schumakovitschi),
infectionintensityofalltheintestinalparasitespeciescombined,ageinyearsandpregnancy
status.TheSMIwasanalyzedusingaGLMwithaGaussianerrorstructureandaloglinkfunc-
tionandtheparametersincludedinthemodelswerethesameasintheanalysesofBCS.
Finally,aGLMwithbinomialerrorsstructureandaloglinkfunctionwasusedtoanalyze
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MarshallagiamarshalliisnegativelyassociatedwithfitnessindicatorsinDall’ssheep
pregnancystatusinadultfemalesheepusingthesameparametersincludedintheanalysesof
BCSplusbodycondition(i.e.SMI).Inordertofacilitatetheinterpretationofcoefficientsand
proportionaloddsratiosinallthemodelsfitted,parasiteintensitywasdividedbyonehundred
beforebeingincludedinthemodels.Bydoingthiswere-scaledtheindependentvariabletoa
moremeaningfulunitfromoneparasitetoonehundredparasites.Inordertoincludepreg-
nancystatusasanindependentvariable,onlyadultfemaleswereincludedintheanalyses.In
allthecaseswhenGLMswereusedthemodelcomparisonwasdoneusingAkaikeInformation
Criterion(AIC)[51].
InthecaseofthePLS-R,theindependentvariablesincludedinthemodelweretheintensity
ofeachgastrointestinalparasitespecies(ninespecies),thespeciesrichness(i.e.numberofpar-
asitespeciesperhost),ageinyears,andpregnancystatus,resultinginatotalof12independent
variables.ThesingledependentvariablewasSMI.Inordertoincludepregnancystatusasan
independentvariable,onlyadultfemaleswereincludedintheanalysis.BothGLMsandPLS-R
wereperformedwiththeRstatisticalsoftware(RDevelopmentCoreTeam)andonlysheep
withcompleteinformationinalltheindependentvariableswereincluded.Asignificancelevel
ofp<0.05wasusedinalltheanalyses.
Results
Generalresults
Atotalof112Dall’ssheepwerecollectedfromtheMackenzieMountainsduring1971and
1972[35].However,TheSimmonsCollectionattheCMNcontainedparasitologicalinforma-
tiononlyfrom104animals(23from1971and81from1972);88adultfemales(20from1971
and68from1972),6yearlingfemales(1from1971and5from1972),8yearlingmales(2from
1971and8from1972)andtwomalelambscapturedin1972.Itwasnotpossibletodetermine
thewhereaboutsoftheinformationonthe8remainingsheep.
Prevalenceofgastrointestinalhelminthswas100%.Eightdifferentnematodespecieswere
identified;MarshallagiamarshalliandOstertagiagruehnericollectedfromtheabomasum;
Nematodirusandersoni,Nematodirusdavtiani,Nematodirusoiratianus interruptus,Nemato-
dirusspathiger,andTrichostrongylus sp.collectedfromthesmallintestine;andTrichurisschu-
makovitschiandSkrjabinemaoviscollectedfromthelargeintestine(cecumandcolon).In
addition,thecestodeMonieziasp.wasidentifiedinthesmallintestine.Therewerenodiffer-
encesinparasiteprevalenceandintensitybetween1971and1972foranyoftheparasitespe-
ciesanalyzed,totalcountofintestinalparasitesortotalgastrointestinalparasites(S1
Table).Forthisreason,thedataintherestofmanuscriptwereanalyzedandpresented
groupingbothyears.Informationonabomasalhelminthsspecieswasavailableonlyfor79
sheep.TotalcountofNematodirusspp.wasavailablefor102sheepbutNematodirusspecies
identificationwereonlyavailablefor54sheep.Marshallagia marshalliwasthemostcom-
monparasitespeciesamongallsheepwiththehighestprevalence(100%,n=79,95%Con-
fidenceInterval[95%CI]=94.1–100)andhighestintensityofinfection(median=224,
range22–2457).Marshallagia marshalliisadimorphicspecieswithtwodistinctmale
forms[37].Theminormorphotype,designatedasMarshallagia occidentalis,wasnot
observedintheSimmonscollection.CountsofM.marshallireflectedinthecurrentpaper
wouldthusrefersolelytothemajormorphotype.Nematodirusspp.weredetectedin95%
(n=102,95%CI=88.2–98.1)ofthesheepalthoughwithsignificantlylowerintensityof
infection(median=98,range1–1427)thanM.marshalli(Permutationtest,Z=3.074,
p-value<0.01).AmongallNematodirusspp.,Nematodirusspathigerhadthehigherpreva-
lence(94.4%,n=54,95%CI=83.7–98.6)andinfectionintensity(median=78,range
2–485).FormoredetailsseeTable1.Marshallagia marshalliwasthespecieswiththe
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MarshallagiamarshalliisnegativelyassociatedwithfitnessindicatorsinDall’ssheep
Table1. PrevalenceandmedianinfectionintensityofgastrointestinalhelminthsinDall´ssheepcategorizedasJuveniles(sheepyoungerthan24months,including
twolambsyoungerthan12months)andadults(sheep24monthsandolder).
Juveniles Adults
n No.ofinfected Prevalence% Median(Range) n No.ofinfected Prevalence%(95%CI) Median(Range)
(95%CI)
Abomasalparasites
Ostertagiagruehneri 13 0 0(0–28.3) NA 66 1 1.5%(0–9.3) 360(1)
Marshallagiamarshalli 13 11 100(67.8–100) 252(22–853) 66 66 100(93.1–100) 223.5(42–2457)
Smallintestineparasites
Monieziasp. 16 2 12.5(2.2–39.6) 1.5(1–2) 86 13 15.1(8.6–24.8) 1(1–3)
Trichostrongylusspp. 16 0 0(0–24.1) NA 86 2 2.3(0.4–8.9) 2(1–3)
Nematodirusandersoni 12 7 58.3(28.6–83.5) 13(3–66) 42 21 50(35.5–64.5) 27(2–189)
Nematodirusdavtiani 12 6 50(25.3–74.6) 29.5(5–176) 42 13 30.9(18.1–47.2) 19(3–490)
Nematodirusoiratianusinterrruptus 12 9 75(42.8–93.3) 119(20–431) 42 25 59.5(43.3–73.9) 67(4–469)
Nematodirusspathiger 12 11 91.6(59.8–99.6) 109(6–202) 42 40 95.2(82.6–99.2) 73(2–485)
Largeintestineparasites
Trichurisschumakovitschi 16 16 100(75.9–100) 29.5(5–210) 86 75 87.2(77.9–93.1) 14(1–131)(cid:3)
Skrjabinemaovis 16 6 37.5(16.3–64.1) 18.5(1–68) 86 55 63.9(52.8–73.8) 5(1–45)(cid:3)
Parasitesgrouped
TotalNematodirusspp. 16 16 100(75.9–100) 232.5(4–630) 86 81 94.1(86.3–97.8) 87(1–1427)
TotalIntestinalparasites 16 16 100(75.9–100) 312(45–643) 86 86 100(94.7–100) 122.5(2–1481)
TotalGastrointestinal 13 13 100(71.7–100) 516(173–1234) 66 66 100(93.1–100) 414.5(65–3715)
Significantdifferencesinintensitybetweenageclassesareindicatedby(cid:3)(p<0.05,Randomizationtest).
Nodifferencesinprevalencebetweenageclasseswerefoundinanyofthespeciesorgroupsofparasites(p<0.05,chi-squaredtest).
https://doi.org/10.1371/journal.pone.0192825.t001
highestdominanceindexanddominancevalue,followedinbothcases,byN.spathigerand
N.oiratianus interruptus.ThesedominanceindicatorswereseveraltimeshigherinM.mar-
shallicomparedtoanyotherspeciesinbothjuvenileandadultsheep(Table2).
Table2. SpeciesdominanceofgastrointestinalhelminthsinDall´ssheepcategorizedasJuveniles(sheepyoungerthan24months,includingtwolambsyounger
than12months)andadults(24monthsandolder).
Juveniles(n=12) Adults(n=39)
DominanceIndex DominanceValue DominanceIndex DominanceValue
Abomasalparasites
Ostertagiagruehneri 0 0 0.0002 0.01
Marshallagiamarshalli 280.1(cid:3) 47.9 478.5(cid:3) 61.9
Smallintestineparasites
Monieziasp. 0.03 0.04 0.023 0.02
Trichostrongylusspp. 0 0 0.005 0.01
Nematodirusandersoni 7.8(cid:3) 2.3 14.7(cid:3) 3.7
Nematodirusdavtiani 13.4(cid:3) 4.6 11.9(cid:3) 4.6
Nematodirusoiratianusinterruptus 89.4(cid:3) 20.4 40.9(cid:3) 9.4
Nematodirusspathiger 96.9(cid:3) 16.6 124.7(cid:3) 17.0
Largeintestineparasites
Trichurisschumakovitschi 47.7(cid:3) 8.2 18.2(cid:3) 2.6
Skrjabinemaovis 0.15(cid:3) 0.09 2.24(cid:3) 0.6
SpeciesclassifiedasDominant(DominanceIndex>1)areindicatedby(cid:3).
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MarshallagiamarshalliisnegativelyassociatedwithfitnessindicatorsinDall’ssheep
Distributionofgastrointestinalparasitesbyageandsex
Weanalyzedtheagedistributionpatternofinfectionintensityforthesixmostabundantpara-
sitespecies(i.e.fiveageclassesofthehost):M.marshalli,N.andersoni,N.davtiani,N.oiratia-
nusinterruptus,N.spathigerandT.schumakovitschi(Fig2).Trichurisschumakovitschihada
significantlyhigherinfectionintensityinimmaturesheep(Permutationtest,Z=-2.265,p-
value=0.023),withyearlingshavingsignificantlyhigherinfectionintensitythanyoungadults
(Pairwisepermutationtest,W=-3.081,p-value=0.012)andmatureadults(Pairwisepermuta-
tiontest,W=-2.851,p-value=0.013).TheoppositetrendwasobservedforM.marshalli,with
higherinfectionintensityinmatureadultsthaninyoungadults(Pairwisepermutationtest,
W=2.681,p-value=0.0219).Theinfectionintensityamongtheremainingparasitespecies
didnotdifferamongageclasses.Comparisonofinfectionintensitybetweensexeswasdone
onlyinyearlingsasnooldermaleswerecaptured.TheinfectionintensityofT.schumakovitschi
inmalesheep(n=8,median=65,range11–210)wassignificantlyhigherthaninfemales
(n=8,median=16.5,range=5–62)(Permutationtest,Z=-1.84,p-value=0.021).Therewas
nodifferenceofinfectionintensitybetweensexesforotherparasitespecies.
Infectionintensityofparasitesandbodycondition
Thebodyconditionofadulteweswasnegativelyassociatedwiththeinfectionintensityofgas-
trointestinalparasites.InformationinBCSwasavailableonlyfor63adultsand12juveniles.
Fig2.InfectionintensityofgastrointestinalhelminthspeciesinadultDall’ssheepcategorizedbyageclass.Lambsandoldadultswereexcludedfrompairwise
comparisonbecauseofsmallsamplesize(n<5).Significantdifferenceamongcategories(p<0.05)arerepresentedwith“(cid:3)”.
https://doi.org/10.1371/journal.pone.0192825.g002
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MarshallagiamarshalliisnegativelyassociatedwithfitnessindicatorsinDall’ssheep
Fig3.InfectionintensityofgastrointestinalhelminthsinadultDall’ssheepcategorizedbybodycondition(BCS).A)TotalinfectionintensityofM.marshalli
(Pairwisecomparisons,n=42,Lean-VeryGood,W=-2.674,p=0.0249).B)InfectionintensityofT.schumakovitschi(Pairwisecomparisons,n=61,Lean-VeryGood,
W=-2.816,p=0.0291).Significantdifferencesamongcategories(p<0.05)arerepresentedwith“(cid:3)”.
https://doi.org/10.1371/journal.pone.0192825.g003
Thetotalcountofgastrointestinalhelminths(log -transformed)inadultfemaleswithboth
10
LeanandFairBCSwassignificantlyhigherthaninsheepinVeryGoodBCS(ANOVA,F =
3,38
6.965,p<0.001).Forindividualparasitesspecies,onlytheintensityofM.marshalliandT.
schumakovitschiwasnegativelyassociatedwithBCS(M.marshalli,n=42:Permutationtest,
Z=-2.966,p-value=0.0013,Fig3A;T.schumakovitschi,n=61:Permutationtest,Z=-2.756,
p=0.005,Fig3B).TheinfectionintensityofM.marshalli(median=223.5,range=42–2457)
wassignificantlyhigherthanT.schumakovitschi(median=14,range=1–131)(Permutation
test,p<0.001).TheBCSinjuvenilesheepwasnotassociatedwitheithertotalcountofgastro-
intestinalhelminths,M.marshallioranyparasitespecies.However,thesamplesizewaslow
andtherewasanunbalanceddistributionamongBCScategories.WithrespecttoSMI,adult
Dall’ssheepwithlowerSMIhadsignificantlyhigherinfectionintensitiesofMarshallagiamar-
shalli(Fig4).JuvenilesheepalsohadthesamepatternofhigherinfectionintensityofM.mar-
shalliwithlowerSMI,however,theassociationwasbarelynotsignificant.Trichuris
schumakovitschialsohadanegativeassociationwithSMIinjuvenilesheep(Fig4).Noother
singlespecies,nortotalintestinalparasitesasawhole,werenegativelyassociatedwithSMI.
Asimilarnegativeassociationbetweenbodyconditionindicatorsandtheintensityofgas-
trointestinalparasiteswasobservedwhendatawereanalyzedusingamultivariateapproach.In
thecaseofBCS,thebestmodelincludedM.marshalliinfectionintensity/100(logodds
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MarshallagiamarshalliisnegativelyassociatedwithfitnessindicatorsinDall’ssheep
PLOSONE|https://doi.org/10.1371/journal.pone.0192825 March14,2018 10/21