Methods in Gut Microbial Ecology for Ruminants

220 Pages·2005·5.199 MB·English

by  Adrian R. Egan (auth.), Harinder P.S. Makkar, Christopher S. McSweeney (eds.)

METHODSINGUTMICROBIALECOLOGYFORRUMINANTS
Methods in Gut Microbial Ecology
for Ruminants
Editedby
HARINDERP.S.MAKKAR
AnimalProductionandHealthSection
JJJointFAO/IAEADivisionofNuclearTechniquesinFoodandAgriculture
InternationalAtomicEnergyAgency
VVVienna,Austria
CHRISTOPHERS.McSWEENEY
CSIROLivestockIndustries
QueenslandBiosciencePrecinct
StLucia,Queensland,Australia
AC.I.P.CataloguerecordforthisbookisavailablefromtheLibraryofCongress.
ISBN-10 1-4020-3790-2(HB)
ISBN-13 978-1-4020-3790-0(HB)
ISBN-10 1-4020-3791-0(e-book)
ISBN-13 978-1-4020-3791-7(e-book)
PublishedbySpringer,
P.O.Box17,3300AADordrecht,TheNetherlands
www.springeronline.com
Printedonacid-freepaper
(cid:1)C 2005InternationalAtomicEnergyAgency
Nopartofthisbookmaybereproduced,storedinaretrievalsystem,ortransmitted
inanyformorbyanymeans,electronic,mechanical,photocopying,microfilming,recording,
orotherwise,withoutwrittenpermissionfromthePublisher,withtheexception
ofanymaterialsuppliedspecificallyforthepurposeofbeingentered
andexecutedonacomputersystem,forexclusiveusebythepurchaserofthework.
PrintedintheNetherlands.
Contents
Foreword vii
Introduction ix
EditorialNote xi
Part1. Designingin-vivoMicrobialEcologyStudies
1.1. Experimentaldesignsforrumenmicrobiology 03
AdrianR.Egan
Part2. ClassicalMethodsforIsolation,Enumeration,Cultivation
andFunctionalAssaysofRumenMicrobes
2.1. Rumenbacteria 23
ChristopherS.McSweeney,StuartE.DenmanandRoderickI.Mackie
2.2. Bacteriophages 39
AtholV.Klieve
2.3. Methanogenicarchaea 47
KKKeithN.Joblin
2.4. Anaerobicfungi 55
MichaelK.Theodorou,JayneBrookmanandAnthonyP.J.Trinci
2.5. Ciliateprotozoa 67
BurkA.Dehority
Part3. PCR-BasedMethodsforAnalysisofPopulations
andGeneExpression
3.1. Nucleicacidextraction,oligonucleotideprobesandPCRmethods 81
ZhongtangYuandRobertJ.Forster
3.2. Quantitative(real-time)PCR 105
StuartE.DenmanandChristopherS.McSweeney
vi Contents
Part4. MolecularFingerprintingTechniquesforGenotypicAnalysisof
PureCulturesandMicrobialCommunities
4.1. Denaturinggradientgelelectrophoresis 119
SvetlanaA.Kocherginskaya,IsaacK.O.CannandRoderickI.Mackie
4.2. Bacteriophagepopulations 129
AtholV.KlieveandRosalindA.Gilbert
4.3. Anaerobicfungalpopulations 139
JJJayneL.BrookmanandMatthewJ.Nicholson
4.4. RAPD,RFLP,T-RFLP,AFLP,RISA 151
StuartE.Denman,MakotoMitsumoriandChristopherS.McSweeney
Part5. DNACloneLibrariesofMicrobialCommunities
5.1. 16S/18SribosomalDNAclonelibraryanalysisofrumenmicrobial
diversity 163
André-DenisG.Wright,KiyoshiTajimaandRustamI.Aminov
Part6. UseofSmallSubunitRibosomalRNADirectedOligonucleotide
ProbesforMicrobialPopulationStudies
6.1. Northernblotanalysistoinvestigatetheabundanceofmicro-organisms 177
DenisO.Krause
6.2. Wholecellprobingwithfluorescentlylabelledprobesforinsitu
analysisofmicrobialpopulations 191
LindaL.Blackall
6.3. Combinedfluorescenceinsituhybridizationandmicroautoradiography
(FISH-MAR) 201
ManeeshaP.Ginige
Part7. GenomicAnalysisofMicrobialEcosystems
7.1. Metagenomicanalysisofthemicrobiomesinruminants
andotherherbivores 209
MarkMorrison,SarahE.Adams,KarenE.Nelsonand
GraemeT.Attwood
ChapterwiseKeywords 221
KeywordIndex 223
Foreword
Asaresultofvarioushumanactivities,suchasincreaseinhumanpopulation,decrease
inarablelandduetosoildegradation,urbanization,industrializationandassociated
increaseinthedemandforlivestockproducts,dramaticchangesareoccurringinthe
globalruminantlivestocksector.Thesechangesincludeshiftinthesizeofregional
livestock populations and in the types of management and feeding systems under
wwwhichruminantlivestockareheld,andincreaseddemandofawiderrangeofquality
attributes from animal agriculture, not just of the products themselves but also of
the methods used in their production. The livestock sector will need to respond to
newchallengesofincreasinglivestockproductivitywhileprotectingenvironmentand
humanhealthandconservingbiodiversityandnaturalresources.
Themicro-organismsinthedigestivetractsofruminantlivestockhaveaprofound
influenceontheconversionoffeedintoendproducts,whichcanimpactonthean-
imal and the environment. As the livestock sector grows particularly in developing
countries, there will be an increasing need to understand these processes for bet-
ter management and use of both feed and other natural resources that underpin the
developmentofsustainablefeedingsystems.
Untilrecently,knowledgeofruminantgutmicrobiologywasprimarilyobtainedus-
ingclassicalculture-basedtechniques,suchasisolation,enumerationandnutritional
characterization, which probably only account for 10–20% of the rumen microbial
population.Newgene-basedtechnologiescannowbeemployedtoexaminemicrobial
diversitythroughtheuseofsmallsub-unitribosomalDNAanalysis(e.g.16SrDNA)
andtounderstandthefunctionofcomplexmicrobialecosystemsintherumenthrough
metagenomicanalysis.Thesetechnologieshavethepotentialtorevolutionizetheun-
derstandingofrumenfunctionandwillovercomethelimitationsofclassical-based
techniques, including isolation and taxonomic identification of strains important to
efficientrumenfunctionandbetterunderstandingoftherolesofmicro-organismsin
relationtoachievinghighproductivityanddecreasingenvironmentalpollutants.
ThisbookhasbeenproducedbytheJointFAO/IAEADivisionofNuclearTech-
niqueinFoodandAgriculture,IAEAVienna,AustriaincollaborationwiththeCSIRO
Livestock Industries, Brisbane, Australia. It gives a comprehensive up-to-date ac-
countofthemethodologiesandtheprotocolsforconventionalandmodernmolecular
vii
H.P.S.MakkarandC.S.McSweeney(eds.),MethodsinGutMicrobialEcologyforRuminants,vii.
©2005IAEA.PrintedintheNetherlands.
viii Foreword
techniques that are currently in use for studying the gut microbial ecology of ru-
minants. Each chapter has been contributed by experts in the field. The techniques
and procedures described are also relevant and adaptable to other gastrointestinal
ecosystems and the microbiology of anaerobic environments in general. The fu-
tureofruminantgutmicrobiologyresearchisdependentupontheadoptionofthese
molecular-based research technologies, and the challenge at present is the use of
thesetechnologiestoimproveruminantproductionanddecreaseenvironmentpollu-
tantsthroughabetterunderstandingofmicrobialfunctionandecology.Itishopedthat
thisbookwillequipthereadersbetterinordertomeetthisunprecedentedchallenge.
JamesD.Dargie ShaunG.Coffey
Director,JointFAO/IAEADivision Chief,CSIROLivestockIndustries
ofNuclearTechniquesinFoodandAgriculture Brisbane,Australia
VVVienna,Austria
Introduction
Currentapproachestotheevaluationofdigestibilityandthenutritivevalueoffeed
resourcesusingconventionalinvitrofeedevaluationandanimalstudieshaveresulted
in a large body of information about nutrient composition, digestion kinetics and
digestibility. However, these techniques are unable to describe the microbial mech-
anisms involved in digestion by ruminants and other herbivores, and are unlikely
to result in the development of new feeding strategies. Conventional culture-based
methods of enumerating and identifying rumen bacteria are being rapidly replaced
bythedevelopmentofnucleicacid-basedtechniquesthatcanbeusedtocharacterise
complex microbial communities. Ruminant nutritionists and microbiologists have
recognized the importance of molecular microbial ecology, but many have found it
difficulttoemploythemostappropriatetechniquesbecausetheyarenotfamiliarwith
the methods. In addition, this field is developing very rapidly and even researchers
withexperienceinmolecularmicrobialecologyfinditdifficulttokeepabreastwith
theincreasingnumberoftechniquesandalternatives.
This manual is written by an expert group of scientists interested in ruminant
digestionandgutmicrobiology.Themostrecentandup-to-datemethodsinmolecular
microbialecologywithspecialemphasisonruminantsarecollatedandinterpretedin
thisbook.Themethodswillprovidethereadersaneasyaccesstomoleculartechniques
thataremostrelevantandusefultotheirareaofinterest.Theauthorshaveattempted
towriteinarecipe-likeformatdesignedfordirectpracticaluseinthelaboratoryand
also to provide insight into the most appropriate techniques, their applications and
the type of information that could be expected. These aspects have been supported
byinclusionoftherelevantliterature.
The contents of the manual are presented in a sequence that recognizes the key
elementsinstudyinggutmicrobialecology.Thefirstchapterprovidesaperspective
onhowtodesignanimaltrialsinwhichmicrobialecologyisstudied.Oftenthepower
of the new molecular techniques is diminished by an inappropriate design in terms
of animal number, sampling frequency, location and replication. The second chap-
ter describes the classical culture-based methods for studying rumen microbes, as
thesemethodsareoftenapre-requisitetoemployingmoleculartechniques.Chapters
3–6provideinformationonthebasicunderpinningtechniquesandtheprotocolsin
ix
H.P.S.MakkarandC.S.McSweeney(eds.),MethodsinGutMicrobialEcologyforRuminants,ix–x.
©2005IAEA.PrintedintheNetherlands.
x Introduction
molecular ecology, such as DNA extraction from environmental samples, the poly-
merasechainreaction(PCR),oligonucleotideprobeandprimerdesignandDNAfin-
gerprintingamongstothers.Theapplicationofthesetechniquestomicrobialdetection
andidentificationarediscussed.Specializedtechniquessuchasdenaturinggradient
gelelectrophoresis(DGGE)and16S/18SribosomalDNAlibrariesforstudyingcom-
plex communities that contain unculturable organisms are also described. Many of
thesetechniquesareusedtoidentifyandenumeratethepopulationoforganismsthat
arepresentinasample.However,thefieldisrapidlymovingtoafunctionalanalysis
ofthemicrobesinanecosystem,andsomeofthemethodsbeingemployedtomeasure
genesexpressionaredescribedinChapter3.InChapter6,knowledgeaboutlocation
and spatial relationships of micro-organisms in their natural environment that are
oftenessentialforunderstandingthefunctionoftheseorganismsarediscussed.The
final chapter deals with metagenomic technologies, which provide the potential to
captureandstudytheentiremicrobiome(thepredominantgenomes)fromacomplex
microbial community, such as the rumen. The rapid high-throughput technologies
developed in mapping the human genome are now being deployed to study micro-
bialecosystems.Anexplosionofknowledgeinthefieldofmicrobialecologyisnow
expected.
The editors wish to acknowledge the contributions made by all the authors who
participated in the publication of this manual. They have spent considerable time
gathering information from many sources into a focussed document that enables
thereadertounderstandhowtechniqueshaveevolvedandthecontextinwhichthe
methodsshouldbeappliedtoaddressspecificissuesrelatingtogutmicrobialecology.
We believe that this manual will ‘demystify’ the methods in molecular microbial
ecologyforreaders,whoarenoviceinthefieldbutareexcitedbytheprospectsofthe
technology.Itwouldalsobeinvaluablefortheexperiencedworkersstrivingforgiving
newdimensiontotheirresearch–expandingtheworkinotherfieldsandinitiating
cross-cutting activities. This manual is seen as the first step towards understanding
andmanipulatinggutmicro-organismsasitisexpectedthatthetechniquesandthe
methodologiesassociatedwiththestudyofmolecularmicrobialecologywillcontinue
togrowandevolve.Akeychallengeforthefuturewillbethesimplificationofthese
techniques, so that these become tools of routine use in nutritional, environmental
andecologicallaboratories.
HarinderP.S.Makkar ChristopherS.McSweeney
AnimalProductionandHealthSection CSIROLivestockIndustries
JJJointFAO/IAEADivision QueenslandBiosciencePrecinct
InternationalAtomicEnergyAgency StLucia,Queensland,Australia
VVVienna,Austria
Editorial Note
The use of particular designations of countries or territories does not imply any
judgementbythepublisher,andtheIAEA,astothelegalstatusofsuchcountriesor
territories,oftheirauthoritiesandinstitutionsorofthedelimitationoftheirbound-
aries.
Thementionofnamesofspecificcompaniesorproducts(whetherornotindicated
asregistered)doesnotimplyanyintentiontoinfringeproprietaryrights,norshould
itbeconstruedasanendorsementorrecommendationonthepartoftheIAEA.
Allpatents,registeredtrademarksandownershipforproducts,reagentsandpro-
cessesmentionedinthispublicationshouldberespected.
xi
H.P.S.MakkarandC.S.McSweeney(eds.),MethodsinGutMicrobialEcologyforRuminants,xi.
©2005IAEA.PrintedintheNetherlands.