Effects of Housing Management on Vaccination Success ...thesis.honors.olemiss.edu/1217/3/Benton...
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Effects of Housing Management on Vaccination Success Against Eimeria Infection in Chickens by Wilson Benton A thesis submitted to the faculty of The University of Mississippi in partial fulfillment of the requirements of the Sally McDonnell Barksdale Honors College. Oxford May 2018 Approved by ____________________________________ Advisor: Dr. Richard Buchholz ____________________________________ Reader: Dr. Stephen Brewer ____________________________________ Reader: Dr. Gregg Roman
Transcript of Effects of Housing Management on Vaccination Success ...thesis.honors.olemiss.edu/1217/3/Benton...
EffectsofHousingManagementonVaccinationSuccessAgainstEimeriaInfectionin
Chickens
by
WilsonBenton
AthesissubmittedtothefacultyofTheUniversityofMississippiinpartialfulfillmentoftherequirementsoftheSallyMcDonnellBarksdaleHonorsCollege.
OxfordMay2018
Approvedby
____________________________________Advisor:Dr.RichardBuchholz
____________________________________Reader:Dr.StephenBrewer
____________________________________Reader:Dr.GreggRoman
ii
©2018LouisWilsonBenton
ALLRIGHTSRESERVED
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ACKNOWLEDGEMENTS
IwouldliketofirstthankDr.RichardBuchholzforhiseffort,guidance,andpatienceasmyadvisor.IwouldalsoliketothankDr.StephenBrewerandDr.GreggRomanforactingasmysecondandthirdreaders.IamincrediblygratefultoDr.PhilStayer,
Dr.ErinRiley,andDr.DavidFrenchforgivingmetheopportunitytoperformresearchunderthem.Theyofferedinvaluableinsight,constantencouragement,andInowconsiderthemmyclosefriends.IamthankfulforDr.EmilyKimminauforherwillingnesstoadvisemewhilesimultaneouslycompletingherowndoctorate.I
wishherthebestasshefurtherstheresearchonEimeria.ThankstoSumnerStayerforthelonghoursshespentinthelabhelpingmakemyworkloadalittlelighter.Thankyoutothegrowers,servicetechs,labtechs,farmhands,andeveryoneelsewithwhomIbecamefriendsduringmyinternship.Iwishyouallthebest.Lastly,Iwanttothankmyfamily,specificallymyparents,LouisandDameaBenton,fortheir
constantloveandsupport.
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ABSTRACTLOUISWILSONBENTON:EffectsofHousingManagementonVaccinationSuccess
AgainstEimeriaInfectioninBroilerChickens(UnderthedirectionofDr.RichardBuchholz)
Coccidiosisisadiseaseofthegastrointestinalsystemofcommerciallyraised
chickens(Gallusgallus).Theinfectioniscausedbymultipleparasiticspeciesofthe
coccidiangenusEimeria.Eimeriaproliferatewithinlargepoultryhousesleadingto
decreasednutrientabsorption,anorexia,anddeathofthehost.Scheduled
vaccinationwithlivecoccidianoocystsshouldminimizeproductionlosses,but
variousmanagementfactorsmaycontributetovariationintheoutcome.I
investigatedwhetherbytrackinginfectioncyclesindetailitmightbepossibleto
improvepoultryproduction.Feceswerecollecteddailyfromfourfarmsacrossa
two-monthperiodtounderstandhowpeaksinoocystsheddingbythehosts
affectedmortalityandbirdweight.“2ndpeak”shedvalues(maximumOPGcounts
followingsecondaryinfection)showedsignificantcorrelationtoearlyweightloss
butnotmortality.Additionally,Ifoundnodifferencein2ndpeakvaluesbetween
malesandfemales;however,differencesindensitymayhavecausedtheeffect.
Becauseoocystspergram(OPG)numberswerehomogenousacrossthehouseby
day21,“turnout”ofbirdsfrombroodendtooffenddidnotsignificantlyaffect
spatialdistributionofoocysts.Iconcludebydescribinghowcoccidianburden
variesamongchickenproducersnationallyandinternationallyandproposing
improvedmethodsforoocystmonitoring.
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TABLEOFCONTENTSLISTOFTABLESANDFIGURESCHAPTERONE Introduction................................................................1 WhatareCoccidia?.........................................................1 LifecycleofEimeriaSpp....................................................4 EimeriaofDomesticChickens(Gallusgallusdomesticus)....................5CHAPTERTWO Coccivac®-D2............................................................13
ImprovementsinProductivity............................................13
Application...............................................................16
TurnOut.................................................................17
CHAPTERTHREE
Problem..................................................................19
PreviousResearch........................................................22
Objectives.................................................................22
CHAPTERFOUR
MaterialsandMethods....................................................25
Sampling.................................................................28
OocystCounts............................................................31
InternationalData.........................................................32
StatisticalAnalysis........................................................32
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CHAPTERFIVE
EffectofLagTimeon3rdPeakOPGValues.................................34
Effectof2ndPeakOPGValuesonBodyWeightat4thWeek.................36
Effectof2ndPeakOPGValuesonMortality.................................37
InfluenceofSexon2ndPeakOPGValues...................................38
OPGCountsinBroodEndvs.OffEnd......................................39
Poolingvs.IndividualSamples............................................40
AssessmentofInternationalData..........................................42
CHAPTERSIX
EffectofLagTimeon3rdPeakOPGValues................................43
Effectof2ndPeakOPGValuesonBodyWeightat4thWeek.................44
Effectof2ndPeakOPGValuesonMortality.................................44
InfluenceofSexon2ndPeakOPGValues...................................45
OPGCountsinBroodEndvs.OffEnd......................................45
Poolingvs.IndividualSamples............................................46
AssessmentofInternationalData..........................................47
Conclusion................................................................48
LISTOFREFERENCES
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LISTOFTABLESANDFIGURES
Table1 TaxonomyofEimeria..............................................3
Figure1 TheLifecycleofEimeria.............................................7
Figure2 IntestinesofaPulletKilledbyEimerianecatrix......................8
Figure3 LesionScoredIntestinesInfectedwithEimeriamaxima..............9
Figure4 BirdsKilledbyEimeriatenella.....................................10
Table2 SpeciesSpecificCharacteristicsWithintheGenusEimeria..........12
Figure5 VaryingInfectionProgressions.....................................15
Figure6 FloorPlanofaTypicalChickenHouse..............................18
Figure7 TheDevelopmentalofAdaptiveImmunityinVaccinatedChicks.....20
Table3 TemperatureProgramUsedatAlloftheStudiedFarms.............27
Table4 DaysofFecalSamplingatFourFarmsAcrossChickAges7-28Days.30
Figure8 EffectsofLagTime...............................................35
Figure9 ComparisonofThirdPeakOPGCountsWithLagTime..............35
Figure10 Effectsof2ndPeakOPGon4thWeekBodyMass.....................36
Figure11 Effectsof2ndPeakOPGon4thWeekMortality......................37
Figure12 EffectsofSexonMeanOPG........................................38
Figure13 ComparisonofMeanOPGCountsBetweenBroodEndandOffEnd..39
Figure14 ComparisonofPoolingvs.IndividualSampling.....................40
Figure15 ComparisonofPollingvs.IndividualSampling(Day15).............41
Figure16 RandomResampling...............................................41
Figure17 MeanOPGCountsComparedAcrossRegions.......................42
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ChapterOne:IntroductionandBackground
Introduction
CoccidiosisisthemostcommondiseasethataffectstheUSpoultryindustry.
Thediseaseitselfcoststheglobalmarket$300millionannuallyduetodecreased
performanceandmortality,whiletheworldwidecostofpreventativemeasuresis
roughly$3billion(HighCostofCoccidiosisinBroilers,2013).Historically
producerslookedprimarilytoanticoccidialdrugsasananswertotheproblemwith
coccidiosis.However,heavyrelianceonsuchmeasuresallowedsomeparasitesto
escapesuppressionandreproduceresultinginresistantEimeriastrains.Theneed
forefficacyinmanagementwithoutthecostofresistancedevelopmentledtothe
creationoflivecoccidiosisvaccines.Livevaccinespromotethedevelopmentof
protectiveimmunitytoEimeriabyinducingauniforminfectionthroughoutflocks
(Chapman,2002).However,managementpracticesinfluencetheefficacyofthe
vaccine(Dalloul,2006).Myobjectivewastotrackoocystshedpatternsinan
attempttodeterminetheeffectcurrentgrowingprogramshaveonvaccinesuccess.
Thisresearchwasconductedduringasummerinternshipwithapoultryproducer
(ProducerX),whowishesnottobenamedinmythesis.
WhatareCoccidia?
CoccidiaareasubclassofthephylumApicomplexa(Table1)andare
membersoftheclassConoidasida.Thesesporeforming,singlecelled,obligate
intracellularparasitesareknownforthediseasetheycause:coccidiosis.Coccidia
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lifecyclesrequirethattheyfindahostinwhichtheycanliveandreproduce.Oncea
hostisinfectedwithcoccidiaitrunstheriskofdevelopingcoccidiosis,adiseaseof
theintestinaltractinbirdsandmammals(Brands,2000).
CoccidiasharethephylumApicomplexawithnumerouswell-known
parasites.Plasmodiumbelongstococcidia’ssisterclassAconoidasidaandcausesthe
diseasemalaria(Snowetal.,1997).Withincoccidia’sownclassistheparasite
Toxoplasmagondii,whichcausesthediseasetoxoplasmosis.Thisdiseasediffers
fromcoccidiosisinitsabilitytosetupinfectioninallanimals;however,itonly
reproducesincats(Dubey,1995).
TheorderEucoccidioridaisoneoffourwithinthesubclassCoccidia.This
ordercontainsthefamilyEimeriidae,whichincludesthegeneraEimeria,Isospora,
CystoisosporaandCyclospora.Thelattertwogeneraareassociatedwithinfections
foundinhumansthatareusuallytransmittedbyimported,improperlywashedfresh
berries,leafygreens,andherbs(CamaandMathison,2015).
Eimeriaisbyfarthemostspeciosegenuswithgreaterthan1,700described
species(Bartaetal.,1997).SpecieswithinEimeriathataffectcommercially
producedchickenswerethefocusofthisresearch.
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Kingdom EukaryaSuper-Phylum Alveolata
Phylum Apicomplexa
Class Conoidasida
Order Eucoccidiorida
Family Eimeriidae
Genus Eimeria
Species acervulina, praecox, maxima, brunetti,mitis,mivati,necatrix,tenella
Table1:ThetaxonomicclassificationEimeriaspeciesaffectingcommercialpoultryproduction.
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LifeCycleofEimeriaspp. ThelifecycleofEimeriaisinterestinginthatitinvolvesbothanexogenous
andanendogenousstage(Figure1).Theexogenousstagebeginsonceoocystsare
excretedbythechickenintotheenvironment.Itistherethatoocystsundergo
sporulationrenderingtheminfective.Withinthechicken,theendogenousstage
containsthreetofourseparateroundsofasexualreproductionfollowedbysexual
differentiation,afertilizationevent,andsheddingofunsporulatedoocysts(Shirley
etal.,2005).
Eimeriaaretransmittedtonewhostsviathefecal-oralroute.Following
ingestion,asporulatedoocysttravelsdowntheesophagusintothegizzardwhere
theoocystwalliscrushed.Therupturedwallsallowfortheliberationofsporocyts.
Inadditiontothemechanicalbreakdowncausedbythegizzard,bothoocystand
sporocystaresubjectedtochemicaldegradationbytrypsinuponreachingthe
intestine.Thissecondaryliberationeventreleasestwosporozoitesfromeach
sporocyst.Sporozoitesarethemotile,infectivesubunitofasporulatedEimeria
oocyst(NortonandJoyner,1981).
Sporozoitesinvadeandinfectcellsoftheintestinalvilliknownas
enterocytes.Theyareabletorecognizeandattachtothesecellsthroughaprocess
knownas“glidingmotility.”Thismodeofcellularrecognitionisconservedacross
allApicomplexaspecies.Thesitesofdevelopmentwithineachenterocytevary
amongEimeriaspecies.Sporozoitesdevelopintoschizontsthroughanintermediate
stageinwhichtheyarecalledtrophozoites.Schizontshouseanasexual
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reproductionstageduringwhichnumeroushaploidnucleiareproduced.The
haploidcellsthatemergefromtheschizontsarecalledmerozoites.
Repetitionsoftheseasexualstagesarenumberedaccordingly(e.g.schizontI
andschizontII)andservetoamplifytheinfection(Shirleyetal.,2005).Firstround
merozoitesinallEimeriaspeciesimmediatelyinfectpreviouslyuninfected
enterocytesformingtrophozoitesandeventuallytheschizontIIgeneration
containingtheasexuallyreproducingmembersofmerozoiteII.Thereleaseofthe
secondroundofmerozoitesiswhereoneofthelargestdifferencesbetweenspecies
takesplace.Whilesomespeciesonlycarryouttworoundsofasexualreproduction,
othersdisplaythreeorevenfour.Forthispaper,onlythefirsttworoundswillbe
discussedasroundsthreeandfourareidentical.Afterbeingreleasedfromschizont
II,merozoiteIIwilleitherre-invadeenterocytesorundergoaprocessknownas
gametogenesiswherebythehaploidcellsdifferentiateintomale(microgametes)or
female(macrogametes)sexualcells.Microgametesinfectcellscontaining
macrogametesandservetofertilizethelatterformingazygote(Fantham,1910).
Zygoteswilldevelopinthecellandfinallybereleasedasunsporulated(non-
infective)oocysts.Immatureoocystsmovedowntheremainingportionofthe
digestivetractuntiltheyareexcretedwithfeces.Thecombinationofoxygen,
moisture,andtemperatureintheenvironmentwilldictatetheamountoftime
beforethesenewoocystswillsporulateandbereadytoinfectanewhost.
EimeriaofDomesticChickens(Gallusgallusdomesticus)
EightspeciesofEimeriaareknowntoinfectdomesticchickens.Thespecies
are:acervulina,praecox,maxima,brunetti,mitis,mivati,necatrix,andtenella.
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Aninthspecies,hagani,hasbeenproposedbysomeauthors(Levine,1938).
SuccessfulidentificationofEimeriaspeciesiscriticallyimportantinafield
setting.Thedifferentspeciesvaryinresponsetococcidiostaticdrugsandimpact
thebirdsdifferently.Forthatreason,researchershavedeterminedsixcriteriawith
whichitmightbepossibletoidentifydifferentspeciesofEimeria.Thesixtraits
observedare:(1)physicalpropertiesofoocysts,(2)hostandsitespecificity,(3)
morphologyoftheendogenousstages,(4)pathogeniceffects,(5)immunological
specificity,and(6)thetimingofthepre-patentandpatentperiodinexperimental
infections(JoynerandLong,1974).
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Figure1:ThelifecycleofEimeria.(A)indicatesthestartofthelifecycle-ingestionand(B)marksthereleaseofsporozoites.Numbers(1-11)ordertheprogressionofdifferenteventswithinthelifecyclestages.UsedwithpermissionfromGreif/Mattig/Weck-Heimann,“EimeriaSpp.Lifecycle.”Www.saxonet.de,www.saxonet.de/coccidia/coccid02.htm.Web.
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Figure2:IntestinesofapulletkilledbyEimerianecatrix.
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Figure3:LesionscoredintestinesinfectedwithEimeriamaxima.Scoresfromlefttoright:+4,+3,+2,+1,+1.Thefirstfoursamplesaresplayedandtheinnerepitheliumisvisible.Thefifthsamplehasnotbeencut.
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Figure4:BirdskilledbyEimeriatenella.Bloodycecaareespeciallyevidentwithinbothindividuals.
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Oocystpropertiesareoneoftheeasiesttraitstoobserveinthefieldby
utilizingthefecalfloatationtechnique.UnlikeotherspeciesofEimeria,thespecies
thatinfectchickensdonotdisplaypolarcaps,differentiationinoocystwalls,
obviousmicropyles,ordifferentiationinsporocysts.Duetothelackofthese
properties,notingthesizeandshapeoftheoocystisconsideredtheonlyvalidway
toroughlydeterminewhichspeciesispresent.
Nosinglemethodofidentification(Table2)isadequatetoidentifyaspecies
ofEimeria.Withinthelast10years,themitochondrialgenomesofseveralspeciesof
Eimeriahavebeensequenced.Theobservedsequencevariabilityallowsforthe
differentiationofsomespeciesinpoultry.ThisisalargestepforwardinEimeria
research;however,muchworkislefttobedone.Morerecentlyresearchershave
aimedatsequencingEimeriagenomesintheirentiretyandhavebeensuccessfulin
thecaseofEimeriatenella(Ogedengbeetal.,2014).Asdatabasesgrow,sotoodoes
thehopeofnewmethodstotreatandpreventcoccidiosis.Nevertheless,atthistime
attemptsatusingcoccidianmoleculesasantigensinvaccinepreparationhavebeen
unsuccessful(LillehojandTrout,1993).Myresearchinvestigateshowmanagement
affectsthesuccessoflivecoccidiavaccines.
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Species OocystProperties
Host/SiteSpecificity MorphologyofEndogenousStages
PathogenicEffects
ImmunologicalSpecificity
Pre-PatentandPatentPeriod
acervulina Small-midsized Anterior1/3ofintestine
Developssuperficialtohostcellnucleus
300,000-1million:causesymptoms/notlethal
Speciesspecific
praecox Large/ovoid Anterior1/3ofintestine
Speciesspecific Firstshed:ninehoursbeforeothers
maxima Large/ovoid Middle1/3ofintestine
Developsbeneathhostcellnucleus
10,000oocysts-lethal
Speciesspecific
brunetti Large/ovoid Lower1/3ofintestine 10,000oocysts-lethal
Speciesspecific
mitis Small/spherical Anterior1/3ofintestine
Speciesspecific
mivati Small/spherical Anterior1/3ofintestine
300,000-1million:causesymptoms/notlethal
Speciesspecific
necatrix Medium/teardrop Middle1/3ofintestine
Large2ndgenerationschizonts
Speciesspecific
tenella Medium/teardrop Lower1/3ofintestineandcecum
Large2ndgenerationschizonts
Speciesspecific
Table2:SpeciesspecificcharacteristicswithinthegenusEimeria.
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ChapterTwo:CoccidiosisVaccinesinCommercialPoultryProductionCoccivac®-D2
Coccivac®-D2isavaccinemanufacturedbyMerck&Co(Kenilworth,New
Jersey)forthepreventionofcoccidiosisinchickens(Gallusgallusdomesticus).This
livevaccineincorporatesnon-attenuatedoocystsofseveralspeciesofEimeria
knowntoinducediseaseandmortalitywithincommerciallyrearedflocks.The
speciescontainedwithinthevaccineare:Eimeriaacervulina,E.brunetti,E.maxima,
E.mivati,E.necatrix,andE.tenella.Inoculatedbirdsprogressthrougha“controlled”
infectionwitheachofthesespecies,whichallowsforthedevelopmentofbothan
immunologicallyspecificandnonspecificresponseagainstdevelopmentalstagesof
Eimeria.Immunityacquisitionrequiressuccessivephasesofoocystsheddingand
re-ingestion(thus,reinfection)bythebirds.Bycontinuallycyclingtheoocysts,
birdsremainexposeduntiltheirimmunesystemhasafullydevelopedresponseto
theEimeriaspeciescapableofsuppressinginfection(Ahmadetal.,2016).Despite
theartificialoriginoftheinfection,heavyloadsofthevaccinestrainscanstillinduce
coccidiosis.However,thestrainsofeachspeciesinCoccivac®-D2arestilldrug
sensitiveandcanbeeliminatedwithanticoccidialdrugs(Chapman,2000;Tewari,
2011)towhichfieldstrainsinpoultryhousesarenowresistant.
ImprovementsinProductivity
Poultryproducersareattemptingtobalancethecostofcoccidiosistreatment
withthebenefitstomeatproduction.Thesecondlargestexpensethatcommercial
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producersface,behindthepurchasingofgeneticallytestedchicks,isfeed.Forthat
reasontheFCR,orfeedconversionratio,isofparamountimportance.Thisratio
measurestheefficiencywithwhichanindividualconvertsfeedintoadesired
product(usuallybodymass).ProducerXtypicallyobservesa2:1FCRintheir
uninfectedbroilerflocks.However,birdsladenwithcoccidiaexperience
degradationoftheintestinallining,whichreducesnutrientabsorption.Thisresults
inanFCRvalueof2.02-2.05:1(Stayer,2017).Whilehundredthsofapoundappear
insignificant,thatvaluemultipliedbyseveralmilliontoaccountforalarge-scale
operationquicklybecomesastaggeringloss.Coccidiosisvaccinesimprovegastro-
intestinalabsorptionviahostsuppressionofcoccidianreproductionthereby
increasingthegrowthefficiencyofeachbirdintheflock.
Bodyweightuniformityisofparticularinteresttoproducersasitaffects
carcass-processingefficiency.Inanunvaccinatedhouse,fieldstraincoccidiawill
infectandcyclelaterinthelivesofthebirdsthaninavaccinatedhouse(Figure5).
Thislateonsetofcoccidiosisresultsindrasticdifferencesinweightatthetimeof
slaughter.Discrepanciesinweightoccurduetobirdsexperiencingvaryinglevelsof
coccidiosisduringtheperiodofhighestdailyweightgain(30-40days).Useof
vaccineschallengesbroilerswithcoccidiaearlyintheirlives,whichallowsthebirds
ampletimetorecoverfromanyweightlossbeforetheyareslaughtered.While
weightuniformityisnotasimportantinbreeders,studieshaveshownhens
burdenedbydiseasesproducefewereggsthantheirhealthycounterparts(Klasing2007).Justasbefore,coccidiosisvaccinesexposethesebirdsearlyintheirlivesso
thattheirproductivephaseisnotimpeded.
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Figure5:Infectionwithinvaccinatedhousesusuallyprogressesandisresolvedbeforecriticalperiodsofgrowth.Naturalinfectionsoccurlaterinthelifespanandsignificantlyimpactdevelopment.
1stPeak
2ndPeak
3rdPeak
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
OocystBurden
Age(Days)
VaccinatedHouse
UnvaccinatedHouse
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Application
Themostimportantaspectofanycoccidiosisvaccineapplicationis
homogeneity.Ifachickismissedbyinitialvaccinationatthehatcheryitrunsthe
riskofgettingcoccidiosis.Unvaccinatedchicksmaybeinfectedbyexistingoocysts
inthepoultryhouseorbylargenumbersofoocystsshedbyvaccinatedbirdsona
roughlysevendaycycleuntilimmunityisreached.Thiswillresultinclinical
coccidiosisandeventuallydeathduetopoornutrientabsorption(Stayer,2017).
SpraycabinetapplicationisthemethodpreferredbyProducerXinordertoprovide
themostuniforminitialvaccinecoverage.Chicksaresubjectedtoaspray
vaccinationonthefirstdayoflifeatthehatchery.Redorgreendyeismixedinwith
thevaccinetotrackapplicationandencouragepreeningamongthechicks.The
chicksinoculatethemselveswiththevaccinebyingestingitviapreeningboth
themselvesandotherbirdsinthesprayboxes(Riley,2017).
DespitetherelianceofProducerXonspraycabinetadministration,thereare
severalothermethodswithwhichtoadministercoccidiosisvaccines.Feedspray
administrationisapopularoptionwherebyaproducermixesthevaccinewithnon-
chlorinatedwaterandthenspraysthemixtureoverthesurfaceofthefeed.This
methodisforchickensfourdaysofage.
Producersalsochoosetovaccinateviaediblegel.Here,gel“pucks”are
scatteredthroughouttransportcratesoronthegrowingsurfaceofthehousewhen
thechicksarrive.Thepucksarebrightlycoloredwhichservestoattractthechicks
andpromotefeeding(Fanatico,2006).Inovoinjectionasamethodofapplication
waspopularintherecentpast.Mechanicalinjectionofcoccidiosisvaccines
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occurredduringthetransferofeggsfromincubatortohatcheratthehatchery.This
techniquehasbecomemuchlesswidelyusedastheeffectivenessofspraycabinets
continuestoimprove.
TurnOut
Inordertomaximizetheeffectsofvaccinesinnewlyinoculatedchicks,
producersconfinethebirdstoasmallareaofthehouseforthefirstfewweeksof
life.Thisareaisknownasthe“broodend”ofthehouseduetothepresenceofa
highernumberofbroodlampscomparedtothe“offend”(Figure6).TheEimeria
strainswithincoccidiosisvaccinestypicallyreleaseoocystsevery5-9days
dependingonthespecies.Bylimitingthegrowingarea,producersareableto
increasethecontactbetweenbirdsandoocyst-ladenfeces.Thegoalofthispractice
istosecondarilyinoculatethebirdsviaeatingofoocystsfromthefloorlitter.
Secondaryinoculationiscriticalinestablishingsuccessfulimmunedevelopmentas
discussedlater.
Birdsaresaidtobe“turnedout”whentheyareallowedaccesstothe“off
end”(previouslyunoccupiedend)ofthehouse.Producerstypicallyturnoutbirds
betweendaystwelveandsixteendependingontherecommendationsofthevaccine
manufacturer.
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Figure6:Floorplanofatypicalchickenhouse.Dimensionsarenottoscaleandwaterdeliverysystems(runparalleltofeedtroughs)arenotpictured.
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ChapterThree:ProblemandObjective
Problem
Notwithstandingthenumerousimprovementsbroughttothepoultry
industrybycoccidiosisvaccines,thesystemisfarfromperfect.Aveterinarian
representingthelocalproducerstatedthattheproblemtheyareexperiencingisa
lackofconsistencyinresultswhenusingthevaccines.ProducerXcontinuesto
observesporadicmortalityduetococcidiosisdespiteutilizationofCoccivac®-D2.
Mortalityratesfluctuatebetweenfarmsandoccasionallybetweenhousesatasingle
farm.
ThemostlikelyexplanationsofunsuccessfulimplementationofCoccivac®-
D2arethateitherA)birdsarenotproperlydevelopingimmunityorB)birdsare
beingoverexposedtothecoccidia.Byturningouttooearlychicksarenotableto
consumeenoughoftheoocystsshedintothelitterbytheirneighbors.Thisfailure
bythebirdstore-infectthemselvesresultsinanunevenpatternofcoccidiacycling
andcanleadtoafailureofearlyresistanceultimatelycausingdeathlaterin
development(Figure7).
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Figure7:Thedevelopmentalprogressionofadaptiveimmunityinvaccinatedchicks.
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Bydelayingturnouttheproducerrunstheriskofexposingthechicksto
excessiveoocystsheddingintheconfinedbroodingarea.Anextremelyhighlevelof
vaccinestrainoocystconsumptioncanultimatelyoverpowerthebirds’developing
immunesystemsandcausedeath.Producersturnoutbirdsinaccordancewiththe
recommendationsofthevaccinemanufacturer;however,asmentionedbefore,
theserecommendationsarebasedonfieldtrialsthatcouldvaryinanumberofways
fromthegrowingenvironmentrelevanttotheproducer(Stayer,2017).Fieldtrials
candeviatefromcommercialoperationsinflockdensity,growingsurfaces,feed
programs,andatmosphericconditionsbasedonlocation(MerckAnimalHealth,
2004).ProducerXfearsthatstudiesperformedinternationallymaydifferso
drasticallyinenvironmentalconditionssoastonotbeaneffectiveguidein
constructingtheirowncoccidiamanagementprogram.Itisforthisreasonthat
ProducerXisinneedofsite-specificcoccidiacyclinginformationtobestdetermine
aturnoutscheduleoptimalfortheirenvironment.
Anadditionalproblemfacedbytheindustryisthatproducersaretypically
unawareoftheseverityofinfectionswithinhousesuntiltheyseehighmortality.
Onlythendotheyadministeranticoccidialsoutsidethetypicalgrowingprogram;
however,bythispointitisoftentoolate(Riley,2017).ProducerXhopesthatby
analyzinglocalcyclingdataandcomparingittovariousmeasuresofproductivity
suchasweightandmortality,theycoulddevelopamethodforearlydetectionof
“problemhouses.”ProducerXalsohopestousethisinformationtoanswer
questionsoftheirownregardingpatternsofcoccidianburdensbetweensexesand
differentlocationswithinthehouses.
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Finally,withinthecommercialchickenindustrythereisnoclearconsensus
onthebestwaytosampleforcoccidia.Themethodmostwidelyusedispoolingof
fecesinordertoobtainahouseaverageasopposedtocollectingindividualfeces
(LongandRowell,1975).Pastresearchhasindicatedthatnodifferenceexistsin
OPGcountsbetweenthetwomethods(Velkersetal.,2010).However,ProducerX
desiresastudyperformedontheirfarmsusingbothmethodsinordertodetermine
ifpoolingsamplescouldoverestimatecoccidiaburdencomparedtothemeanof
individualsamples.
PreviousResearch
ProducerXemployedresearchersMay-Julyof2016toinvestigatevariation
intimingofoocystcyclingwithintheirchickenhouses.Researcherssampledfrom
12farmsonceaweekforchickages8-44days.Althoughtheobjectivewasto
documentpeaksinoocystexcretion,bysamplingonlyonceaweek,theresearchers
wereunabletodefinethepatternofoocystexcretionprecisely.
Objectives
Thisprojectwasstructuredtosatisfytheneedsofacommercialproducer
locatedinsouthMississippi.Mygoalforthisresearchwastoperformafocused
studyonpullets(immaturefemalebreeders)andcockerels(immaturemale
breeders)inordertofillaninformationalgapinthepoultryindustry.Ishortened
thesamplingperiodfromprevioustrialsaswellasdecreasedthenumberof
observedfarmsinordertoallowforagreaternumberofsamplingdays.The
primaryobjectiveofthisprojectwastoobtainanaccuratepictureofEimeriaoocyst
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cyclinginflocksvaccinatedwithCoccivac®-D2aswellasanswerthefollowing
researchquestions:
1. Doeslagtimeof2ndpeakaffect3rdpeakoocystcounts?
2. Do2ndpeakoocystcountspredictbodyweightat4weeks?
3. Do2ndpeakoocystcountspredictmortalityat30days?
4. Do2ndpeakoocystcountsvaluesdifferbysex?
5. Dooocystcountsdifferbetweenthebroodendandoffendatday21?
6. Doespoolingofsamplesover-estimatetheOPGcomparedtothemeanof
individualsamples?
7. Doesinternationalcyclingdatadifferfromlocaldata?
ByansweringthefirstquestionIintendedtodeterminetheimpactofturning
outeithertooearlyortoolate.Anegative“lagtime”fora2ndpeakvalueindicated
thatpeakshedhadoccurredpriortorelease.Previousresearchsuggeststhatinthis
circumstancehigherdensitywouldresultinhigherratesofinfection(Stanleyetal.,
2004).
Bycomparing2ndpeakoocystcountstobirdweightandmortalityatroughly
4weeksofageIattemptedtoidentifyacorrelationthatProducerXcoulduseinthe
futuretopredictmanagementsuccessbeforebirddeath.Instudyingthedifference
betweenmaleandfemaleoocystcountsIhopedtoprovideProducerXwithdata
thattheydesired.
Thecomparisonofbroodendandoffendoocystcountsonday21wasof
particularinteresttotheproducer.PreviousworkbyNewberryandHall(1990)
reportedhigherdensityatthebroodingsitepersistedafterbeingturnedout.
24
Additionally,ProducerXhasnotedthatbirdsdemonstratingseverecoccidiosistend
tobecomemoresedentaryasthediseaseprogresses(Riley,2017).Thiswould
indicatethatbroodendOPGloadcouldbehigherthanthatoftheoff-endresulting
inanunevenreuptakeof3rdgenerationoocystsonday21.
TestingtheresultsofpreviousworkdonebyVelkersetal.(2010)regarding
thecomparisonofpoolingvs.individualsamplinginterestedmeasitcouldserveto
improveresearchwithintheindustry.Finally,bycomparinglocalandinternational
oocystcyclingdataIintendedtodetermineifvariationexistedbetweenregions.
PriorresearchbyAndersonetal.(1976)andWaldenstedtetal.(2001)onthe
effectsoftemperatureandlittermoistureoncoccidiaseemedtolendcredenceto
ProducerX’sdesireforalocalizedstudy.Throughansweringeachofthese
questionsIintendedtoaddtotheexistingknowledgesurroundingcoccidaand
improvecommercialproductivitybothforProducerXandtheindustryasawhole.
25
ChapterFour:Methods
MaterialsandMethods
SampleswerecollectedbetweenthedatesofJuly5thandAugust8th,2017,
frompulletsandcockerelsfromfourdifferentfarms(denotedA,B,C,andD)located
betweenPrentiss(31°35’N;89°52’E)andLaurel(31°41’N;89°7’E)Mississippi.The
temperatureinthehousesacrossallfarmswasregulated(Table3).
Dayoldchicks(Ross708)wereadministeredtheanticoccidialvaccine
Coccivac®-D2byMerck&Co(Kenilworth,NewJersey)viasprayapplication(25ml/
100chicks).Allfarmsutilizedwoodshavingsasagrowingsurface.Modesofwater
distributionvariedslightlybetweenfarms.FarmsA,B,andCuseddrinkingnipples
whichbirdspeckedtoreleasewater.FarmChadatraybelowthenipplesto
preventwetfloorlitter,whilefarmBusedbelldrinkerswherebythechicksdrink
fromashallowcirculartrough.Allfarmsusedachainfeeder(troughsystem)asthe
methodoffeeddispersal.Birdswereofferedfeedadlibitumuntilturnout,atwhich
pointallwereswitchedtoskip-a-dayfeeding.Allfarmsmaintainedanidentical
lightprogramthatcalledfor24hoursoflightfordays0-3,12hoursfordays4-14,
and8hoursfordays15-week20.
Birddensityvariedonlyslightlybetweenfarms.Fullhousemeasurementsat
farmsA,C,andDwere40x400ft.;whereas,farmBhouseswere48x500ft.This
resultedinfemalebrooddensitiesof0.75ft2perbirdandfullhousedensitiesof1.5
ft2inthefirstthreefarmsanddensitiesof0.68ft2and1.36ft2respectivelyinfarmB.
26
Malebrooddensitieswere1.9ft2perbirdandfullhousedensitieswere3.8ft2.Turn
outfrombroodtofullhouseatA,B,andCoccurredonday13whereasfarmD
turnedoutatday14.Birdsweregivenaselectivethiamineantagonist,Amprolium
(Huvepharma,Sofia,Bulgaria)(10ozpergallonofstocksolutionwithstocksolution
meteredinat1:128throughmedicatorforafinalconcentrationof0.0047%),atday
18tolessentheeffectsoftheexistinginfections(Pohl,2012).Throughoutthe
courseofmystudyservicetechsateachfarmrecordedmortalityandaveragebird
liveweight(viaanautomatedscaleatlitterlevel)andprovidedmewiththatdata
weekly.Weightwasrecordedinlbs.asisthestandardforProducerX.
27
AgeinDays Temp AgeinDays Temp AgeinDays Temp AgeinDays Temp
1* 90 8 86 15 82 22 79
2* 89 9 85 16 82 23 79
3* 88 10 85 17 81 24 78
4 88 11 84 18 81 25 78
5 87 12 84 19 80 26 78
6 87 13 83 20 80 27 77
7 86 14 83 21 79 28 77
Table3:Temperatureprogramusedatallofthestudiedfarms.*Days1,2,and3arelittertemperature.Afterthree(3)days,temperatureismeasuredatbirdheight.
28
Sampling
Twomethodsofsamplecollectionwereusedthroughoutthecourseofthis
projectinordertomeettheresearchneedsofbothacommercialpoultryproducer
andmythesis.Thecompanyrequiredcoccidiancountsfrombothmalesandfemales
onmultiplefarms.Alsoofinteresttothecompanywaswhetherthenumberof
oocystsdifferedbetweenthe“broodend”andthe“offend.”Theproducerrequired
thatindividualfecesfromeachpoultryhousebecombinedsothatasinglehouse-
widemetricofinfectionwasobtained.Whilepooledsamplesallowedservice
techniciansandmanagerstotracktheprogressionofEimeriaacrossawide
geographicarea,pooledsamplesheldlittlestatisticalpowerforhypothesistesting
formythesisresearch.Forthatreasonasecondsamplingmethodusingoocyst
countsoffecesfromhaphazardlychosenindividualchickensatasinglefarmwas
employed.SamplesatfarmsA,B,C,andDwerecollectedaccordingtotheschedule
showninTable4.
Throughoutthestudyeachfarmwasvisitedataconsistenttimeofdayto
eliminatevariationduetothecircadianpatternofoocystshedding(Boughton,
1933;BrawnerandHill,1999;Villanúaetal.,2006),asfollows:B-7:00,D-8:00,A-
10:00,C-11:00.
AtfarmsA,B,andC,10freshfecalsampleswerecollectedoffofthelitterby
handfromboththebroodendandoffend(followingturnout)ofeachhouse.Cecal
feceswerenotcollected(Villanúaetal.,2006).Theuseofaheadlampdramatically
easedtheprocessoffindingandidentifyingfreshsampleswithinthehouses.Ten
samplesfromeachendwereplacedintoasinglelabeledWhirl-Pak®,(Stamford,
29
Connecticut)fortransportationtothelab.Samplesfromeachfarmwerestoredina
cooleruntiltheywereprocessedinthelab.
AtFarmDpooledsampleswerenotdividedbetweenbroodendandoffend
asmyfocuswastocomparepoolingvs.individualsampling.Therefore,ateachof
thefourhousesatFarmD,tenindividualfemaledroppingswerecollectedand
packagedseparatelyforcomparisontopooledcounts.
30
Farm 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
A X X X X X X X X X X X
B X X X X X X X X X
C X X X X X X X X X X X
D X X X X X X X X X X X X X X X X
Table4:Daysoffecalsampling(X)atfourfarmsacrosschickages7-28days.
31
OocystCounts
Fecalsampleswereexamineddailywithinhoursofcollection.Whirl-Paks
containingapooltypesampleweremassagedinordertohomogenizethecontents
ofeachpacket.5.00goffeceswasremovedandaddedtoabeakercontaining45.0
mLofasupersaturatedNaClinwatersolution.Thismixturewasthensealedwitha
capandshakeninordertobreakapartfecesandreleaseoocytes.Thebeakerwas
setasidefor10minutestoallowflotationofoocysts.Duringthistimeadditional
beakerswereprepared.
Aftersittingfor10minutesaPasteurpipettewasusedtotransferliquidfrom
thesurfaceofthesolutiontoonechamberofaMcMastercountingslide.Samples
werethenallowedtositinthechamberforanother10minutestoallowoocytesto
floattothegridsurface.Oocystswerecountedindividuallyunderamicroscopeat
100xmagnification.Ahandheldtallyclickerwasusedinordertocountlarge
numbersofoocystsacrossthegriddedMcMasterslide.Theviewingareaonthese
slideswasdividedintosixcolumnsallowingforeaseofcounting.Whenindividual
columncountsexceeded2000,threecolumnswerecountedandthetotalwas
multipliedbytwotoobtainanaccurateestimationofthetotal.Thismethodwas
chosentoestimatehighnumbersasopposedtoanotherdilutionduetotheresults
ofworkdonebyDunnandKeymer(1986),Pereckieneetal.(2007),andCringoliet
al.(2004),showingthatthemostaccuratecountsareobtainedatdilutionsof1:10-
1:15.
Oocystspergramoffeces(OPG)wasdeterminedusingthefollowing
equationOPG=n(d/v)wherenequalsthenumberofoocystscounted,disthe
32
dilutionfactor(10inthecaseofthisstudy)andvisthevolumeoftheareacounted
(0.15ml).Theindividualfecalsampleswerecountedinthesameway;however,a
slightlydifferentpreparationwasneededtoachievea1:10dilutionwiththesmaller
fecalsizes.Forthesesamples,0.5gramsoffeceswasmixedwith4.5mLofsalt
watersolutioninabeaker.
InternationalData
InadditiontowhatIcollectedIwasprovidedwithunpublisheddatafrom
EgyptandChinabyasourcewhoseidentityIamnotatlibertytoshare.My
objectiveincollectingthesedatawastotestforvarianceinoocystcyclingbetween
foreignfarmsandProducerX.However,duetothevarioussamplingschedulesand
methodsofcollectionutilizedinthegatheringoftheinternationaldata,itwasnot
comparablestatistically.InsteadIconductedadescriptivecomparisonofmeanOPG
nearestto14days,thetimeofidealpeakshed(day13inChinaandday14inEgypt
andProducerX).
StatisticalAnalysis
Spearman’sCorrelationwasusedinordertoexaminetherelationship
between2ndpeaklagtimeand3rdpeakoocystcounts.Additionally,differences
withinthe3rdpeakvaluesweretestedforwiththeMannWhitneyUTest.
Spearman’sCorrelationwasuseddetermineif2ndpeakoocystcountsaffectedbird
mortalityorweightatroughlyfourweeksofage.AMannWhitneyUTestwasused
toinvestigatedifferencesin2ndpeakoocystcountsbetweensexes.AWilcoxon
SignedRankTestcomparedtheOPGcountsofbroodendsandoffendsacross10of
thehouses.Lowindependentsamplesizepreventedastatisticaltestfrombeing
33
performedonthedifferencesbetweenpooledsamplesandindividualaverages.
Lastly,asmentionedpreviously,internationaldatawasnotsuitableforstatistical
analysis.Therefore,adescriptivecomparisonofmeanOPGattimeofpeakshedwas
performed.
34
ChapterFive:ResultsEffectofLagTimeon3rdPeakOPGValues
ByexaminingpeakdatachronologicallyIwasabletodeterminethelagtime
betweenthedayof2ndpeakshedandtheturnouttime(day14)acrossallhouses.
ThirdpeakOPGvaluesofhouseswithlagtimes≤0(peakbeforeturnout)were
comparedtothosewithlagtimes>0(peakafterturnout)(Figure8).Duetogapsin
sampling,3rdpeakscouldnotbeidentifiedinfiveofthehouses.Resultsindicated
thatlagtimedoesnotpredictthemagnitudeof3rdpeaksheds.Thiswasevidenced
byalackofsignificantdifferencein3rdpeakvaluesbetweenthegroups(Mann
WhitneyUTest,U=14,n1=4,n2=9,p=0.60)aswellasalackofcorrelation
betweenindividuallagtimesandtheirrespectiveOPGvalues(Spearman
Correlation,rho=0.14,n=13,p=0.65).
35
Figure8:LagtimedidnotattributesignificantvariationbetweenmeanOPGvaluesofthetwogroups(MannWhitneyUTest,U=14,n1=4,n2=9,p=0.60).Errorbarsshowstandarddeviation.
Figure9:ThirdpeakOPGvaluesshowednocorrelationwithlagtime(SpearmanCorrelation,rho=0.14,n=13,p=0.65).
-100000
-50000
0
50000
100000
150000
200000
250000
300000
350000OPG
LagTime≤0LagTime>0
0
50000
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-3.00 -2.00 -1.00 0.00 1.00 2.00 3.00
OPG
LagTime(Days)
36
Effectof2ndPeakOPGValuesonBodyWeightat4Weeks
Ananalysisoftherelationshipbetween2ndpeakOPGvaluesandweightdata
foreachrespectivehouseindicatedthattherelationshipwassignificant.Bodymass
atonemonthofageisnegativelycorrelatedwith2ndpeakOPGvalues(Spearman
Correlation,rho=-0.568,n=18,p=0.016)(Figure10).
Figure10:Housesinwhichbirdsshedmore2ndpeakoocystsproducedbirdswithsmaller28dayliveweights(SpearmanCorrelation,rho=-0.568,n=18,p=0.016).
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 100000 200000 300000 400000 500000 600000 700000 800000 900000
28DayBodyMass(lbs)
2ndPeakOPG
37
Effectof2ndPeakOPGValuesonMortality
Itwasdeterminedthattheintensityof2ndpeakOPGshedwasnotassociated
withpercentmortalityat30days(SpearmanCorrelation,rho=-0.212,n=18,p=
0.398).Interestingly,thehousewiththehighestpercentmortality(0.069)hadan
OPGvalue(274,467)thatrankedonly11thhighestoutofthe18observed(Figure
11).
Figure11:SecondpeakOPGvaluesdidnotshowacorrelationwithpercentmortalityat30days(SpearmanCorrelation,rho=-0.212,n=18,p=0.398).
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0 100000 200000 300000 400000 500000 600000 700000 800000 900000
ProportionofM
ortalityat30Days
2ndPeakOPG
38
InfluenceofSexon2ndPeakOPGValues PooledOPGvaluesobtainedfromall14houseswereanalyzedtodetermine
themagnitudeofthe2ndpeakineachdataset(Figure12).Thesevalueswere
groupedbysexoftheirrespectivehousesandcompared.Resultsindicatedthatsex
ofaninfectedbirddidnotinfluencetheintensityofthe2ndshedevent.Despitethe
factthatthemeanfemaleOPGvalue(330,656)waslargerthanthemalevalue
(253,700),thedifferencewasnotstatisticallysignificant(MannWhitneyUTest,U=
50,n1=12,n2=6,p=0.213).
Figure12:Onaverage,femalesdemonstratedhigherlevelsof2ndpeakOPG.However,thesedifferenceswerenotsignificant(MannWhitneyUTest,U=50,n1=12,n2=6,p=0.21).Errorbarsshowstandarddeviation.
0
100000
200000
300000
400000
500000
600000
Female Male
OPG
39
OPGCountsinBroodEndvs.Off-End
PooledOPGsampleswereobtainedfromboththebroodendandtheoffend
ateachhouseandcomparedonday21(Figure13).Althoughthemeanoff-endOPG
count(19,766)washigherthanthatofthebroodend(13,946),thedifferencefailed
toachievestatisticalsignificance(WilcoxonSignedRankTest,n=10,p=0.41).
Figure13:OffendmeanOPGvaluesweregreaterthanthatofthebroodendonday21.However,nostatisticalsignificancewasobserved(WilcoxonSignedRankTest,n=10,p=0.41).Errorbarsshowstandarddeviation.
-5000
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
BroodEnd OffEnd
OPG
40
Poolingvs.IndividualSampling
AsseeninFigure14,themeanOPGvalueofindividualsampleswashigherin
allexceptthethirdhouse.Lowindependentsamplesizepreventedanystatistical
testsfrombeingperformed.Itisworthpointingoutthatonday15(peakshedin
manyhouses)thedifferencebetweensamplingmethodswasmorepronounced
(Figure15)(Figure16),however,thiscouldbeattributedtochance.Arandom
resamplingofindividualdatafromhouse1onday15(Figure16)demonstrateda
decreaseinstandarddeviationassamplesizeincreased.
Figure14:MeanOPGwashigheramongindividualsamplesthanthatofpooledsamples.Errorbarsshowstandarddeviation.
-150000
-100000
-50000
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150000
200000
250000
300000
350000
Ind.Avg Pool Ind.Avg. Pool Ind.Avg. Pool Ind.Avg. Pool
House1 House2 House3 House4
OPG
41
Figure15:DifferencebetweenindividualaveragesandpooledOPGaverageswaspronouncedaroundtimeofpeakshedatday15.Errorbarsshowstandarddeviation.
Figure16:Randomre-samplingofindividualoocystcountsfromHouse1onday15suggeststhataminimumofsixsamplesareneededinordertoobtainausefulestimationofactualcoccidianload.Thepooledsamplevalueonthisdayisrepresentedbythehorizontallineat352,400.Errorbarsshowstandarddeviation.Thebarat10showsnoerrorbarsasresamplingofallindividualvaluesalwaysresultsinthesamemean.
-100000
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700000
Ind.Avg.
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Pool IndAvg.
Pool
House1 House2 House3 House4
OPG
352400
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700000
800000
1 2 3 4 5 6 7 8 9 10
OPG
NumberSubsampled
42
AssessmentofInternationalData
TheproducerinChinasufferedmuchhigher2ndpeakmeanOPG(301,600)
thaneithertheproducerinEgypt(142,443)orProducerX(111,777).
Figure17:MeanOPGvaluescomparedacrossregionsatroughly2weeksofage(day13inChina,day14inEgyptandUSA(ProducerX)).Errorbarsshowstandarddeviation.
0
100,000
200,000
300,000
400,000
500,000
600,000
Egypt China USA
OPG
43
ChapterSix:Discussion
EffectofLagTimeon3rdPeakOPGValues
Mydatasuggestthatlagtimeof2ndpeakoocystshedhasnocorrelationwith
themagnitudeof3rdpeakshed.Thisisinterestingduetothecommonbeliefinthe
industrythatwhenbirdsexperiencea2ndpeakbeforeturnout(negativelagtime)
theyareoverlyexposedtothevaccinationstrainEimeria.Thislargeparasiticload
exceedswhatthechicksdevelopingimmunesystemcancopewithandleadsto
severecoccidiosis.Thisbeliefisbasedontheassociationofhigherdensitywith
higherinfectionrates(Stanleyetal.,2004).Inthecontextofthisstudy,this
relationshipwouldhavemeantthathouseswithlagtimes≤0wouldhaveshown
significantlyhighershedvaluesduringthethirdpeak.Toavoidsuchsituations
producershaveinvestedsubstantialtimeandfundsintoresearchaimedattracking
oocystcountsinordertomodifyprogramssothatturnoutoccursroughlyoneday
beforepeakshed.However,theabsenceofacorrelationbetweenlagtimeand3rd
peakshedshowninmystudyindicatesthattheindustryisperhapsmistakenly
attributingobservedvariationwithinoocystshedcountstotheimpropertimingof
turnout.Myresultssuggestthatoptimizingturnouttime(achievinglagtimes>0)
maynotbetheoptimalmethodofachievinguniformflockresponsetococcidiosis
vaccines.
44
Effectof2ndPeakOPGValuesonBodyWeightat4Weeks
ThenegativecorrelationbetweenOPGvaluesandbodyweightsobserved
withinmystudyparalleledtheresultsofpreviousresearch(Preston-Mafhamand
Sykes,1970).However,mymethodoftestingwassomewhatnovel.Ratherthan
trackbodyweightsandOPG’stogetherovertime,myresearchdemonstratedthatby
assessingthecoccidiaburdenatpeakcycleonecanpredictthefuturetrendsinlive
weight(SpearmanCorrelation,rho=-0.568,n=18,p=0.016).Byidentifyingthis
negativecorrelationIhave,inessence,providedtheproducerwiththeabilityto
determinethelong-termproductivityofahousewithinthefirsttwoandahalf
weeksoflife.Theimplicationsofthisfindingwarrantfuturestudyandpotentiallya
modificationofcurrentgrowingprograms.Forinstance,iffurtherresearch
indicatedthatthistrendinweightlosscontinuestoday60or90thenimplementing
thenecessaryanticoccidialmeasuresimmediatelyupondetectionof“problem
houses”onday14couldresultinmajorimprovementstoproductivity.
Effectof2ndPeakOPGValuesonMortality
Althoughacorrelationbetween2ndpeakOPGandpercentmortalitywasnot
observed,myresultindicatingthatanegativecorrelationexistsbetweenbody
weightand2ndpeakOPGvalidatestheneedforfurtherresearch.Decreasesinlive
weightwithinahouseserveasanindicationthattheresidentEimeriahave
proliferatedtothepointofsignificantlyaffectingnutrientabsorptionwithinthe
birds.Ifthetrendinweightlossidentifiedinthisstudywasfoundtocontinuethen
housewideanorexiaandmortalitywouldsoonfollow.Therefore,Ibelievethata
correlationbetween2ndpeakOPGvalueandmortalitymayverywellexist.
45
However,Icontendthatbycomparingpeakstomortalitydataat30daysrather
thanlaterinthelifespan,IfailedtoallowtheEimeriaadequatetimeinwhichto
causesignificantmortality.Bothstudieswouldhavebeensignificantlyimprovedby
comparing2ndpeakOPGtomortalityandbodyweightnotonlyat30days,butalso
atdays60and90inordertoobservethelongtermramificationsoflarge2ndpeak
oocystcounts.
InfluenceofSexon2ndPeakOPGValues Previousstudiesontheinfluenceofsexasitrelatestoparasiticburdenhave
indicatedthatmalevertebratestypicallyexpressdecreasedimmunefunctiondueto
theproductionoftestosterone,andthus,increasedparasiticload(Sainoetal.,1995;
Poulin,1996;Zuketal.,1996).Mydataindicatedthatnosignificantdifferencein
oocystcountsexistedbetweensexesatProducerX.Inthisstudyitappearsasifthe
males’naturaltendencytosuffergreaterparasiticinfectionwasmadeless
significantbytheeffectsofhigherdensityonthefemales.Itwasstatedin“Materials
andMethods”thatmalebroodandoffenddensities(1.9ft2,3.8ft2)fellbelowthoseof
theaveragefemalehouse(0.72ft2,1.43ft2).AspointedoutbyStanleyetal.(2004),
whenbirddensityisdecreasedsotooisthelittermoisture.Thisservestoreduce
theamountofmicroorganismsinthelitter(Waldenstedtetal.,2001)andlikely
explainwhynosignificantdifferencewasdetected.
OPGCountsinBroodEndvs.Off-End
ThelackofsignificantdifferenceinOPGcountsfromthebroodendandoff-
endonday21indicatedthatbirdsweredispersinguniformlyfollowingturnout.As
mentionedpreviously,workbyNewberryandHall(1990)suggestedthatbirds
46
tendedtostayclosertothebroodendduringthistime.However,myresults
demonstratedthatthiswasnotthecaseforProducerX.Theirhousesseemto
resemblethosestudiedbyPrestonandMurphy(1989),whichshowedbirdstendto
flowthroughagrowingareawithoutpreferenceforonelocation.Theresultsofthis
studywereimportanttoProducerXastheydemonstratedthatunevendistribution
ofoocystswithinthehouseonday21wasnotcausingtheinconsistenciesin
mortalitytheyobserved.Thesefindings,aswellasthelackofcorrelationbetween
lagtimesand3rdpeakshed,furtherthevalidityofmypropositionthatturnouttime
anditsresultingeffectsplayalesssignificantroleinproductivitythanpreviously
thought.
Poolingvs.IndividualSampling
Mydata,thoughnottestedstatistically,seemstodeviatefromtheresults
determinedbyVelkersetal.(2010).FromstudyingFigures14-16itappearsasif
theaverageofindividualsamplestendstobegreaterthanpooledaverages.A
potentialexplanationofthisoccurrencelieswithintheexperimentalmethods
involvedinpooling.BymixingtenfeceswithinasingleWhirl-Pak®,theimpactofa
samplewithamassivecoccidialoadbecomesslightlydiluted.Thiseffectdoesnot
occurwhenreadingindividualsamplesandaveragingthem,thus,resultinginhigher
meanOPGnumbers.Additionally,bysubsamplingandtakingthestandarddeviation
ofsuccessiveaverages,Ishowedthatobtainingasmallnumberofindividual
samples(1-5)willresultinhighlyvariableaveragesthatmaynotaccuratelyreflect
theimpactofcoccidiaontheaveragebird.IfasimilarstudywastobeperformedI
wouldsuggestthatresearcherstake10individualsamplesfromallhousesacross
47
multiplefarmsandcomparethemtopools.Thecommercialpoultryindustry
generallyreliesonthepoolingmethodtoachieveahousewidemetricofcoccidia
data.Therefore,resultsindicatingthatthismethodislessaccuratecouldhavea
largeimpact.Itshouldbenotedthatthetimeassociatedwithcountingindividual
sampleswouldmorethanlikelyplayabiggerroleindecidingbetweenmethods
withintheindustryifthedifferencewasfoundtobeminiscule.
AssessmentofInternationalData
LargevariationinmeanOPGcountswereobservedamongEgypt,China,and
ProducerXintheUnitedStates.However,themannerinwhichforeigndatawere
obtainedpreventedtheuseofstatisticalanalysis.Thestudyofthesedataallowed
metoseefirsthandtheinconsistenciesthatpervadetheindustryinregardsto
monitoringEimeria.Daysofcollectionvariedgreatlyandinalmostallcasesleft
multiplegapsbetweenshedscompletelyuntested.Additionally,equationsusedto
deriveOPGvalues,typesofcountingslidesused,timeofcollection,andother
importantinformationwasoftenleftout.Withcoccidiosisbeingoneofthemost
expensiveproblemsforthecommercialpoultryindustryitisabsolutelynecessary
forastandardizedprotocoltobeadopted.Suggestionsofstandardsfortest
challenging,measuringvaccineefficacy,andotherEimeriarelatedissueshavebeen
madebyWilliamsandCatchpole(2000)aswellasotherprominentvoiceswithin
theindustry.Practiceofsuchaprotocolwouldimprovethequalityofdataforboth
theindividualfarmerandtheindustryasawhole.ThevariationIobservedcould
potentiallyindicatesubstantialdifferencesincoccidianumbersglobally.This
48
findingfurthersupportsthenecessityofconsistentlyobtained,statisticallypowerful
data.
Conclusion
Myresearchhasshownthatmeasuring2ndpeakOPGshedholdspromiseasa
meansofdetectingfuturedamagetoproductivityduetococcidiosis.Additionally,
mydataindicatesthattheeffectofturnouttimeonthevariationofoocystcounts
andmortalityobservedwithinthefirstfewweeksoflifeisnotassignificantaswas
previouslythought.Asaresult,ProducerXisnowattemptingtoimprovetheirearly
anticoccidialmeasurestoimproveproductionratherthanfocusingonturnouttime.
Asmentionedpreviously,theindustryasawholemustmodifyandimprove
inconsistentmethodsofdatacollection.Byadoptingastandardprotocolforthe
observationofEimeriawithincommerciallyraisedflocks,producersworldwidewill
beabletoquicklyandaccuratelycomparetheirdata.
Furthermore,advancementsintechnologymustbeimplementedtobetter
identify,enumerate,andeliminatespeciesofEimeriawithinhouses.Improvements
inmitochondrialsequencing,researchintoautomatedcounting,anddevelopmentof
newanticoccidialdrugsallholdpotentialasmeanstodecreasetheeffectof
coccidiosisinthecommercialpoultryindustry.
49
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