Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Comparative studies of microbial populations in the rumen,

duodenum, ileum and faeces of lactating dairy cows
J.C. Frey1, A.N. Pell1, R. Berthiaume2, H. Lapierre2, S. Lee3, J.K. Ha3, J.E. Mendell4 and E.R. Angert4
1 Department of Animal Science, Cornell University, Ithaca, NY, USA
2 Agriculture and Agri-Food Canada, Sherbrooke, Quebec, Canada
3 Department of Agricultural Biotechnology, Seoul National University, Seoul, Korea
4 Department of Microbiology, Cornell University, Ithaca, NY, USA

Keywords
analysis of similarities (anosim), dairy cows,
methanogen, nonmetric multi-dimensional
scaling (MDS), small subunit rRNA, T-RFLP.
Correspondence
Esther R. Angert, Department of
Microbiology, Cornell University, Wing Hall,
Ithaca, NY 14853, USA.
E-mail: era23@cornell.edu
2019 ⁄ 1288: received 18 July 2019, revised 6
October 2019 and accepted 13 October 2019
doi:10.1111/j.1365-2672.2009.04602.x
Abstract
Aims: Understanding factors that influence the composition of microbial popu-
lations of the digestive system of dairy cattle will be key in regulating these
populations to improve animal performance. Although rumen microbes are
well studied, little is known of the dynamics and role of microbial populations
in the small intestine of cows. Comparisons of fingerprints of microbial popu-
lations were used to investigate the effects of gastrointestinal (GI) segment and
animal on community structure.
Methods and Results: Samples from four lactating dairy cows with ruminal,
duodenal and ileal cannulae were collected. Terminal-restriction fragment
length polymorphism (T-RFLP) comparisons of small subunit rRNA genes
revealed differences in microbial populations between GI segments (P < 0Æ05).
No significant differences in either methanogen populations or microbial
community profiles between animals were observed. Quantitative PCR was
used to assay relative changes in methanogen numbers compared to procaryote
rRNA gene numbers, and direct microscopic counts were used to enumerate
total procaryote numbers of the duodenal and ileal samples.
Conclusions: T-RFLP comparisons illustrate significant changes in microbial
diversity as digesta passes from one segment to another. Direct counts indicate
that microbial numbers are reduced by eight orders of magnitude from the
rumen, through the abomasum, and into the duodenum (from c. 1012 to
c. 3Æ6 · 104 cells per ml). Quantitative PCR analyses of rRNA genes indicate
that methanogens are present in the duodenum and ileum.
Significance and Impact of the Study: The contribution of microbial popula-
tions of the small intestine to the nutrition and health of cattle is seldom
addressed but warrants further investigation.

methanogenesis affects animal production, leading to

Introduction
Gastrointestinal (GI) micro-organisms perform vital
functions in dairy cattle. In the rumen, micro-organisms
convert feed into volatile fatty acids and microbial mass,
thus providing nutrients to the animal. The rumen is a
diverse ecosystem that includes bacteria, fungi, protists
and archaea (Hespell et al. 1997; Stewart et al. 1997).
Methanogenic archaea are of particular note, as enteric
considerable (2–12%) energy loss for the animal (Crutzen
et al. 1986; Johnson et al. 1993; Johnson and Johnson
1995). In addition, methane produced both in the rumen
(Crutzen et al. 1986; Moss 1993) and in the hindgut
(Murray et al. 1978; Torrent and Johnson 1994) is a
potent greenhouse gas.
Much of what is known about the function of rumen
micro-organisms is based on pure culture studies

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J.C. Frey et al.
(Hungate 1966; Hespell et al. 1997). While culture-based
methods provide depth of knowledge on individual
microbes, these approaches only describe a fraction of
microbial diversity (Amann et al. 1995; Pace 1997). The
application of cultivation-independent techniques to
describe rumen dynamics (Whitford et al. 1998; Tajima
et al. 1999; Weimer et al. 1999; Tajima et al. 2000;
Kocherginskaya et al. 2001; Tajima et al. 2001; Whitford
et al. 2001; Klieve et al. 2003; Larue et al. 2005; McEwan
et al. 2005; Michelland et al. 2009; Hook et al. 2009;
Welkie et al. 2009) has helped to improve our understand-
ing of the breadth of microbial diversity in the rumen and
has provided descriptions of how subpopulations of rumi-
nal micro-organisms are affected by host animal manage-
ment. With the exception of pathogens shed in the faeces,
microbial populations of the intestinal tract and their rela-
tionship to host diet remain less well characterized.
Terminal-restriction fragment length polymorphism
(T-RFLP) is a gene-based community profiling method
that generates ‘fingerprints’ of populations present in
environmental samples (Liu et al. 1997). Because T-RFLP
is rapid, it can be used to process and compare multiple
samples to follow spatial and temporal changes in micro-
bial diversity. This technique has been used to investigate
community dynamics within one or two defined regions
of the GI tract of pigs (Leser et al. 2000; Högberg et al.
2004), chickens (Gong et al. 2002), mice (Sait et al. 2003;
Kibe et al. 2004) and gorilla (Frey et al. 2006b) and
human faecal samples (Hayashi et al. 2002; Wang et al.
2004; Jernberg et al. 2005).
The goal of the study was to examine changes in
microbial populations in different segments of the GI
tract of dairy cattle. Specifically, T-RFLP analyses of the
small subunit (SSU) rRNA gene were used to characterize
the composition of the microbial community and sepa-
rately the methanogen populations in the rumen, duode-
num, ileum and faeces of lactating dairy cows. Differences
in profiles were statistically analysed for effects of GI seg-
ment, animal and diet.
Materials and methods
Experimental animals
The lactating cows were housed at the Dairy and Swine
Research and Development Center, Sherbrooke, Quebec
and were cared for according to the guidelines of the
Canadian Council on Animal Care (Olfert et al. 1993)
following a protocol approved by the Institutional Animal
Care Committee. Four cows (A–D) were surgically fitted
with ruminal cannulae, closed T-type duodenal cannulae
and open T-type ileal cannulae as described previously
(Berthiaume et al. 2001). Cows A, B, C and D were 62,
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Journal compilation ª 2019 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1982–1993
Bovine GI tract communities
35, 397 and 82 days in milk, respectively, and were pro-
ducing between 36Æ5 (low protein diet) and 38Æ0 (high
protein diet) kg milk per day. The animals weighed
604 ± 82 kg.
Experimental diets
Cows were used in a crossover design with two levels of
protein in the diet over two consecutive 21-day periods.
High and low protein diets were fed to animals based on
the National Research Council requirements (NRC 2001).
The low and high protein treatments provided 75 and
115% of required metabolizable protein, respectively. The
diets were iso-energetic. To achieve a steady state in the
rumen, animals were fed 95% of their previous ad libitum
intake in 12 equal meals per day for 14 days prior to
sampling. The total mixed ration (TMR) on an as-fed
basis consisted of 17Æ4 kg day)1 of grass silage (44Æ2% of
ration), 10Æ2 kg day)1 of corn silage (25Æ9% of ration)
and 11Æ8 kg day)1 of concentrate (29Æ9% of ration). The
concentrate was either high or low in crude protein and
in bypass protein. The nutrient composition of the ingre-
dients and the TMR are listed in Table 1. Samples for the
determination of the microbial communities were taken
on the first period of the crossover. During this time,
cows A and B were fed the low protein diet, while cows
C and D were fed the high protein diet. The dry matter
intake (DMI) of the experimental animals is shown in
Table 2. A two-sample t-test with unequal variances indi-
cated that no differences were observed in the DMI
between animals receiving the two rations (P = 0Æ40).
Cow A received 32Æ4 kg TMR per day, all other cows
received 37Æ2 kg day)1. All animals had access to fresh
water at all times, and each animal received grass hay,
1 kg day)1.
For nutrient analyses, feed samples were lyophilized
and ground to pass a 1 mm screen. For ash determina-
tion, subsamples were ashed at 550C for 12 h in a muffle
furnace. Nitrogen was determined by micro-Kjeldahl
(Association of Official Analytical Chemists 1998). Acid
detergent fibre, neutral detergent fibre, lignin and
nitrogen insoluble in neutral detergent and acid detergent
were determined according to Van Soest et al. (1991).
Nonstructural carbohydrates were also determined (Snif-
fen et al. 1992).
Sample collection
The samples were collected on 17 January 2002 at 9 am.
Approximately, 250 ml of digesta from the rumen,
duodenum, ileum and faeces was collected. Ruminal sam-
ples were collected in plastic beakers, duodenal samples
were collected in 500-ml Nalgene bottles (Nalge Nunc,
1983
Bovine GI tract communities J.C. Frey et al.

Table 1 Chemical composition of feed ingredients on a per cent dry matter basis

Concentrate Total mixed ration

Grass Corn Grass High Low High Low

silage silage hay protein protein protein protein

Dry matter (%) 42Æ6 35Æ8 81Æ9 81Æ9 82Æ1 53Æ6 53Æ4
Neutral detergent fibre 45Æ4 38Æ5 57Æ3 18Æ4 27Æ6 32Æ7 37Æ2
Acid detergent fibre 35Æ9 23Æ8 33Æ6 11Æ0 18Æ5 23Æ4 26Æ5
Lignin 4Æ4 1Æ8 3Æ9 1Æ0 1Æ0 3Æ6 3Æ1
Nonstructural carbohydrate 24Æ7 45Æ5 18Æ4 52Æ5 49Æ8 40Æ2 38Æ7
Starch 2Æ4 26Æ9 4Æ5 45Æ0 36Æ0 19Æ8 25Æ2
Crude protein 18Æ0 9Æ6 14Æ4 20Æ7 11Æ0 17Æ9 13Æ7
Soluble crude protein 7Æ4 6Æ2 4Æ6 3Æ0 2Æ3 6Æ6 6Æ6
Insoluble crude protein 10Æ6 3Æ4 9Æ8 17Æ7 8Æ7 11Æ3 7Æ0
Neutral detergent insoluble crude protein 3Æ7 1Æ3 5Æ2 1Æ3 1Æ7 1Æ8 1Æ8
Acid detergent insoluble crude protein 1Æ2 0Æ8 0Æ7 1Æ1 0Æ8 1Æ3 1Æ0
Ash 9Æ5 3Æ4 7Æ4 5Æ7 6Æ1 6Æ8 6Æ9
Fat 2Æ6 3Æ1 2Æ7 2Æ8 5Æ6 2Æ7 3Æ7
Net energy of lactation (MCal kg)1) 1Æ4 1Æ6 1Æ4
Net energy of maintenance (MCal kg)1) 1Æ4 1Æ6 1Æ4
Net energy of gain (MCal kg)1) 0Æ7 0Æ9 0Æ7

Table 2 Dry matter intake (DMI) of experimental animals
Cow Ration*
A Low
B Low
C High
D High
*Experimental animals were fed ‘low’ or ‘high’ protein diets, which
provided 75 or 115%, respectively, of the NRC dietary requirements
for metabolizable protein.
No differences were observed in the DMI between animals receiving
the two rations (P = 0Æ40).
Rochester, NY, USA) connected to the closed T-cannula,
and ileal samples were collected in Whirl-Pac bags (NAS-
CO, ON, Canada). Faecal samples were obtained from
the animals by rectal grab sampling. Samples were mixed
thoroughly, and subsamples (10 ml) were placed in sterile
15-ml conical tubes for community analysis, heat-treated
at 65C for 15 min to reduce nuclease activity, frozen in
liquid nitrogen and shipped on dry ice to Cornell Univer-
sity, where they were stored at )80C until DNA extrac-
tion.
DNA extraction
Frozen samples were thawed at room temperature and
mixed by inversion. The samples were assessed by micros-
copy to determine cell integrity and whether or not pro-
tozoa were present. For each DNA extraction, 400 ll of a
ruminal, duodenal or ileal sample was transferred into
DMI
1Æ5-ml microcentrifuge tubes using a wide bore pipet.
(kg day)1) Faecal samples (c. 50 mg) were placed in tubes using a
sterile toothpick. Samples were washed three times with
13Æ3 500 ll TE (10 mmol l)1 Tris, 1 mmol l)1 EDTA, pH 6Æ0).
18Æ4
Each sample was extracted in triplicate, as previously
17Æ2
15Æ9
described (Frey et al. 2006a) with bead-beating except the
CTAB ⁄ NaCl step was omitted. Samples were purified with
the GeneClean purification kit according to the manufac-
turer’s instructions (Qbiogene, Carlsbad, CA, USA).
Because little DNA was recovered from duodenal samples,
and these samples failed to amplify, multiple extractions
were performed. All duodenal DNA samples, from a
single cow, were pooled and precipitated. This DNA was
not purified with GeneClean.
T-RFLP analyses
Amplification reactions consisted of HotStarTaq Master
mix (Qiagen, Valencia, CA, USA) containing
1Æ5 mmol l)1 MgCl2, 0Æ2 mmol l)1 of each of the four de-
oxynucleotides, 0Æ2 lmol l)1 of each primer and 500 U of
HotStarTaq DNA polymerase in a final volume of 100 ll.
Primers were synthesized by Integrated DNA Technolo-
gies, Coralville, IA, USA. Universal primers, 5¢ 6-FAM
(carboxyfluorescein) 515F (GTGCCAGCMGCCGCGG-
TAA) (Lane 1991) and unlabeled 1391R (GACGGGCG-
GTGTGTRCA) (Stahl et al. 1988), were used for DNA
amplification in a PTC-200 Thermal Cycler (MJ Research,
Inc., Waltham, MA, USA) using the following
programme: 15 min at 95C followed by 25 cycles of

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J.C. Frey et al.
denaturation (30 s at 94C), annealing (30 s at 55C) and
extension (1 min at 72C). A final elongation step
(10 min at 72C) was included. Genomic DNA (1 ll)
extracted from each sample was used in individual ampli-
fication reactions. Methanogen-specific primers (Wright
and Pimm 2003), 5¢ 6-FAM Met86F (GCTCAGTAACA
CGTGG) and Met1340R (CGGTGTGTGCAAGGAG),
were used as described previously but with an annealing
temperature of 50C. A 10 ll aliquot of each 100 ll
reaction was fractionated on a 0Æ8% TAE agarose gel
(Sambrook et al. 1989). The remaining 90 ll was purified
with the Qiaquick purification kit (Qiagen). PCR prod-
ucts were eluted from the purification column with 23 ll
10 mmol l)1 Tris, pH 8Æ0. For each sample, the products
from three independent amplifications were pooled prior
to enzymatic digestion.
Restriction enzymes were selected based on in silico
digestions with SSU rRNA sequences available in
GenBank from ruminal micro-organisms (Hespell et al.
1997; Stewart et al. 1997). PCR products obtained
with universal SSU rRNA gene primers were digested with
either HaeIII or MseI, and PCR products obtained with
methanogen-specific primers were digested with AciI,
AluI, HhaI or MseI. All restriction endonucleases were
purchased from New England Biolabs, Beverly, MA, USA,
and reactions were performed as specified by the manu-
facturer. Digests were incubated overnight.
Each digested sample (0Æ5 ll) was mixed with 9Æ5 ll of
deionized formamide, containing 0Æ05 ll an internal size
standard (GeneScan 500 LIZ) and analysed on an ABI
3730 DNA Analyzer (Applied Biosystems Inc., Foster City,
CA). T-RF lengths were determined using the 2nd Order
Least Squares size-calling method with heavy smoothing
available from ABI GeneMapper software package ver.
3.0 (Applied Biosystems) (Frey et al. 2006a).
Statistical analysis
T-RFLP data (peak height, area and presence ⁄ absence)
were analysed as described previously (Rees et al. 2004)
using Primer ver. 5.2.9 software (PRIMER-E Ltd, Plym-
outh, UK). The relative abundance of each T-RF was
calculated by dividing the height or area of each T-RF
by the total height or total area of all community T-RFs
(measured in relative fluorescent units). Data were stan-
dardized by omitting peaks contributing <1% of the
total fluorescent units. The Bray–Curtis similarity matrix
was calculated for height, area and presence ⁄ absence
data. For each parameter, nonmetric multi-dimensional
scaling (MDS) was used to ordinate the similarity data
and create a two-dimensional configuration of the sam-
ples. One hundred random restarts were used for each
MDS plot. Points clustering together in a MDS plot rep-
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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1982–1993
Bovine GI tract communities
resent samples that are similar in species composition
(Clarke and Gorley 2001; Clarke and Warwick 2001).
Analysis of similarities (anosim) was used to test the
null hypotheses that there were no differences in
community structure between segments, cows or diets.
Global R sample statistics were computed for each com-
parison. Global R values near zero indicate that the null
hypothesis is true, whereas values near 1 indicate that
replicates within sites are more similar to each other
than replicates from different sites (Clarke and Gorley
2001; Clarke and Warwick 2001). If global R statistics
indicated that groups differed, pairwise differences were
investigated further. Species contributions to similarity
(SIMPER) were used to assess similarities between seg-
ments, cows and diets and to compute the average con-
tribution of individual T-RFs to the average dissimilarity
between samples.
Quantitative PCR
16S rRNA genes were amplified from Bacillus subtilis
PY79 (with primers 8F and 1492R) and Methanospirillum
hungatei JF-1 (with primers Met86F and Met1340R) as
described previously. The PCR products were cloned into
pCR2.1-TOPO plasmid vector (Invitrogen, Carlsbad, CA)
and used to transform Escherichia coli TOP10 competent
cells following manufacturers instructions. Plasmid DNA
was recovered using the QIAprep Spin Miniprep kit
(Qiagen) and quantified spectrophotometrically. Tenfold
serial dilutions ranging from 2 · 107 to 200 copies per ll
were used as quantification standards.
Quantitative PCR (qPCR) assays were used to determine
the relative abundance of methanogen 16S rRNA genes to
total bacterial plus archaeal 16S rRNA genes in the rumen,
duodenum, ileum and faeces of cow D. All qPCR samples
were prepared on ice and contained 1X Power SYBR Green
PCR Master mix (Applied Biosystems) and 900 nmol l)1 of
the appropriate forward and reverse primers and analysed
with the ABI 7300 Sequence Detection System. Quantifica-
tion standards were run in duplicate, GI tract samples and
negative controls were run in triplicate. Primers PRK341F
and PRK806R (Yu et al. 2005) were used to enumerate
total procaryote 16S rRNA genes. Thermal cycling condi-
tions were as follows: 10 min at 95C, followed by 40 cycles
of denaturation (30 s at 94C), annealing (30 s at 49C)
and extension (1 min at 72C). A final dissociation step
was added to confirm PCR product specificity. Primers,
S-P-MArch-0348-S-a-17 and S-D-Arch-0786-A-a-20
(Sawayama et al. 2006), were used to enumerate methano-
gen 16S rRNA genes. Thermal cycling conditions were as
earlier except annealing (30 s at 58C). Data were compiled
and analysed using the Sequence Detector Software package
(ABI).
1985
Bovine GI tract communities
Cell counts using DAPI
To enumerate microbial cells, samples were stained with
4¢,6-diamindino-2-phenylindole (DAPI). DAPI forms
fluorescent complexes when bound to double-stranded
DNA. Only DAPI stained particles (cells containing
DNA) were counted. To determine microbial cell densi-
ties, 1 g of sample was added to 9 ml of phosphate buf-
fered saline (PBS). Cells were fixed by adding
paraformaldehyde to 1% and incubating at room temper-
ature for 5 min. The cells were washed once with PBS
and resuspended in PBS to a total volume of 10 ml. The
cells were mixed well, and 10 ll of the sample was
removed to a clean microcentrifuge tube containing 90 ll
of water and DAPI (2 lg ml)1). The sample was stained
for 15 min at room temperature, mixed with 10 ml water
and filtered on to a black 0Æ2 lm nucleopore filter (What-
man Inc., Florham Park, NJ, USA). The filter was then
mounted in glycerol on a microscope slide with a cover
glass. Slides were viewed using an Olympus BX51 epifluo-
rescence microscope with filters appropriate for viewing
DAPI fluorescence. Sample dilutions were adjusted as
needed so that fields contained 100–300 cells. For each
sample, ten fields of cells were randomly selected, photo-
graphed and counted.
Results
T-RFLP analysis with universal primer set
Forty-six HaeIII and 30 MseI T-RFs were resolved in
electropherograms. HaeIII generated T-RF sizes (in
bases): 53, 54, 69, 70, 71, 72, 73, 101, 102, 112, 128,
132, 133, 151, 217, 218, 224, 226, 227, 228, 229, 246,
292, 294, 323, 331, 333, 339, 340, 377, 378, 379, 380,
381, 382, 410, 411, 412, 413, 414, 415, 461, 468, 469,
498 and 499. MseI generated T-RF sizes (in bases): 35,
36, 38, 55, 56, 74, 75, 76, 77, 78, 88, 89, 90, 103, 119,
230, 231, 297, 346, 347, 348, 349, 353, 355, 356, 357,
436, 437, 439 and 440. The numbers of dissimilar T-RFs
for the rumen, duodenum, ileum and faeces were 20,
26, 27 and 23 with HaeIII; 17, 20, 11 and 16 with MseI.
The MseI-generated T-RFs 78, 103 and 439 were found
in all segments of all cows whereas no common T-RFs
were observed in HaeIII profiles.
In all cases except one (AluI dataset), analysis of PCR
product relative abundances was determined by peak area
data. Height or presence ⁄ absence data yielded the same
statistical conclusions as area, and therefore, the peak area
dataset was used for data ordination and pairwise com-
parisons. The MDS plots obtained from area data for the
MseI and HaeIII for segment (rumen, duodenum, ileum
and faeces), cow (A–D) and diet (high or low protein)
1986 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1982–1993
J.C. Frey et al.
MseI HaeIII
Segment Segment
Cow Cow
Diet Diet
Figure 1 Multi-dimensional scaling plots of the gastrointestinal (GI)
segments, cows and diets for terminal-restriction fragment length
polymorphism community profiles generated with the universal primer
set and restriction enzymes MseI (left) and HaeIII (right). Clustering of
the data points in the segment plots indicates that microbial
communities within GI segments were more similar to each other
than those between segments. The stress of the plots generated with
MseI and HaeIII were 0Æ14 and 0Æ15, respectively. The following
symbols represent data for each segment: rumen ( ), duodenum
(s), ileum ( ) and faeces ()); cow: A (+), B ( h ), C (·) and D ( ) or
diet: low protein diet (s) and high protein diet (d).
are shown in Fig. 1. GI segments clustered together, indi-
cating that T-RF communities within segments were more
similar than between segments. This observation is in
agreement with previous reports comparing the bacterial
communities associated with the bovine rumen and faeces
(Michelland et al. 2009). Animal and diet MDS plots
showed no distinct pattern.
Supporting the clustering patterns obtained with MDS
plots, differences in T-RF community structure were
observed between segments as determined by anosim
(Table 3). The overall significance level of the sample
statistic was set at P < 0Æ05. Pairwise differences were
observed between the rumen and ileum, the rumen and
faeces, the duodenum and ileum, and the ileum and
faeces (P < 0Æ05) when MseI was used (Table 4). No
differences were detected between the rumen and duode-
num (P = 0Æ114) or the duodenum and faeces
(P = 0Æ057). Pairwise differences between all segments
were observed with the enzyme HaeIII (P < 0Æ05). No
differences in community profiles were observed between
different cows or diets (P > 0Æ05) with either enzyme. In
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J.C. Frey et al. Bovine GI tract communities

Table 3 Global R statistics generated with ANOSIM analyses for segment, cow and diet*

Segment Cow Diet

Presence ⁄ Presence ⁄ Presence ⁄

Area Height absence Area Height absence Area Height absence

HaeIII 0Æ693à 0Æ686à 0Æ522à )0Æ130 )0Æ141 )0Æ069 )0Æ093 )0Æ070 )0Æ003
MseI 0Æ616à 0Æ575à 0Æ624à )0Æ040 )0Æ040 0Æ015 )0Æ095 )0Æ108 )0Æ131
AciI§ 0Æ135 0Æ135 0Æ047 )0Æ076 )0Æ063 )0Æ013 )0Æ098 )0Æ109 )0Æ101
AluI§ 0Æ264 0Æ272 0Æ235 0Æ528à 0Æ401à 0Æ187 0Æ150 0Æ102 )0Æ052
HhaI§ 0Æ282 0Æ264 )0Æ014 )0Æ123 )0Æ130 )0Æ012 )0Æ057 )0Æ002 )0Æ019
MseI§ 0Æ51à 0Æ514à 0Æ356à )0Æ194 )0Æ193 )0Æ133 )0Æ066 )0Æ066 )0Æ034

T-RFLP, terminal-restriction fragment length polymorphism.

*Global R statistics were computed to assess differences between segment (rumen, duodenum, ileum and faeces), cow (A–D) and diet (high and
low protein).
Enzymes used in T-RFLP with the universal primer set.
àStatistical differences of the sample statistic were observed (P < 0Æ05).
§Enzymes used in T-RFLP with the methanogen primer set.

Table 4 ANOSIM between segment pairwise comparisons of T-RFLP*

Microbial MseI profiles Microbial HaeIII profiles Methanogen MseI profiles

R statistic P-value R statistic P-value R statistic P-value

Rumen–duodenum 0Æ417 0Æ114 0Æ427 0Æ029 NA NA

Rumen–ileum 0Æ865 0Æ029 0Æ938 0Æ029 0Æ958 0Æ029
Rumen–faeces 0Æ500 0Æ029 0Æ740 0Æ029 )0Æ12 1Æ0
Duodenum–ileum 0Æ906 0Æ029 0Æ948 0Æ029 NA NA
Duodenum–faeces 0Æ563 0Æ057 0Æ552 0Æ029 NA NA
Ileum–faeces 0Æ615 0Æ029 0Æ781 0Æ029 0Æ958 0Æ029

NA, not applicable, as duodenal methanogen profiles were not obtained; T-RFLP, terminal-restriction fragment length polymorphism.
*R statistics and corresponding P-values were computed to assess pairwise differences between each segment (rumen, duodenum, ileum and
faeces).

Table 5 SIMPER average pairwise similarities between segments* between segments are displayed in Table 5. The highest
average dissimilarities between segments were contributed
Microbial Microbial
by T-RF sizes of 218, 339, 380, 412 and 468 in analyses
MseI HaeIII Methanogen
profiles profiles MseI profiles with the enzyme HaeIII, which accounted for at least
30% of the average dissimilarities between segments. Four
Rumen–duodenum 45Æ8 43Æ2 NA T-RF sizes with the enzyme MseI (55, 56, 103 and 439)
Rumen–ileum 41Æ2 22Æ9 76Æ9
accounted for at least 40% of the average dissimilarities
Duodenum–ileum 26Æ8 17Æ7 NA
between segments.
Rumen–faeces 37Æ8 27Æ4 97Æ5
Duodenum–faeces 32Æ1 27Æ9 NA
Ileum–faeces 36Æ9 19Æ5 76Æ5
T-RFLP analysis with methanogen-specific primer set
NA, not applicable, as duodenal methanogen profiles were not
All duodenal samples produced community profiles when
obtained.
universal amplification primers were used: MseI n = 9,
*Average pairwise similarities (percentages) were computed between
each segment (rumen, duodenum, ileum and faeces). 10, 9 and 11 T-RFs and HaeIII n = 16, 13, 11 and 14 T-
RFs for cows A, B, C and D, respectively. However,
summary, both restriction enzymes yielded the same con- amplification with the methanogen-specific primers
clusions; microbial diversity differed among segments, but Met86F and Met 1340R yielded no PCR product in the
not between animals or between diets. duodenal samples of all four cows. Therefore, the duode-
SIMPER analysis showed that similarities between num was excluded from statistical analysis of the metha-
segments were low (<50%). Average pairwise similarities nogen T-RFLP dataset.

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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1982–1993 1987
Bovine GI tract communities J.C. Frey et al.

When the area, height and presence ⁄ absence data were MseI
analysed for each enzyme, global R statistics revealed no
Segment
differences between diets (Table 3). However, when the
segment and animal data were analysed, discrepancies
between the four enzymes investigated were observed.
The T-RFLP profiles generated with the enzymes AciI,
AluI and HhaI indicated that methanogen communities
did not differ among segments, whereas MseI profiles
showed that segments varied (Table 3). Moreover, the
AluI profiles showed that cows varied when area and
height data were used in anosim analyses, but when the
data were transformed to presence ⁄ absence only, no
differences were observed (P = 0Æ187, Table 3). Therefore, Cow
we concluded no cow differences in methanogen profiles
for the enzyme AluI.
In pairwise comparisons, the MseI methanogen profiles
between the rumen and ileum and the ileum and faeces
were dissimilar (P < 0Æ05), although the methanogen
populations in the rumen and faeces were alike (Table 4).
The MDS plots of the T-RFLP data generated with meth-
anogen-specific primers and MseI are shown in Fig. 2.
Correspondingly, SIMPER analysis indicated that average
pairwise similarities between the rumen and ileum and Diet
the ileum and faeces were lower than between the rumen
and faeces (Table 5). The highest average dissimilarities
were contributed by MseI T-RFs 98, 100, 310 and 469,
which accounted for at least 50% of the average dissimi-
larities between segments.
Quantitative PCR was used to assess the relative abun-
dance of methanogen 16S rRNA genes in the rumen,
duodenum, ileum and faecal samples from one of the test
animals (Table 6). In contrast to the T-RFLP analysis, the
qPCR primer set was able to detect methanogen
Figure 2 Multi-dimensional scaling plots of the gastrointestinal
sequences in the duodenum. We speculate that this is segments, cows and diets for terminal-restriction fragment length
because of the better sensitivity of the qPCR assay and polymorphism community profiles generated with the methanogen
smaller amplicon length. The relative abundance of meth- primer set and restriction enzyme MseI. Segment data points cluster-
anogen to total bacterial plus archaeal 16S rRNA genes ing close together illustrate that the methanogen communities were
was found to be 5Æ0, 1Æ0, 4Æ5 and 1Æ3% in the rumen, more similar within groups than between groups. The stress of each
duodenum, ileum and faeces, respectively. Direct counts plot was zero. The following symbols represent data for each seg-
ment: rumen ( ), ileum ( ) and faeces ()); cow: A (+), B (h), C (·)
of DAPI stained cells from all cows averaged 3Æ6 · 104
and D ( ) or diet: low protein diet (s) and high diet (d).
and 1Æ5 · 107 procaryote cells per ml in the duodenum
and ileum, respectively. DNA is rapidly degraded in the
bovine ileum (Smith and McAllan 1971); therefore, the nity structure along the length of the GI tract of cows.
qPCR results should represent cell-bound DNA. We were able to use the appreciable amount of SSU
rRNA sequence data available from ruminal micro-
organisms to select restriction enzymes for T-RFLP
Discussion analyses. Using a universal amplification primer set, we
T-RFLP studies have been used to describe community predicted that HaeIII and MseI would yield a high num-
diversity associated with the GI tracts of a variety of ani- ber of dissimilar T-RFs, 20 and 22, respectively. Experi-
mals (Leser et al. 2000; Gong et al. 2002; Sait et al. 2003; mentally, these digests yielded 46 and 30 unique T-RFs.
Högberg et al. 2004; Kibe et al. 2004; Frey et al. 2006b). Once again, this points to the value of culture-indepen-
To the best of our knowledge, this is the first report using dent approaches to the study of microbial diversity.
T-RFLP to characterize the changes in microbial commu- Unfortunately, the universal primers employed in this

ª 2009 The Authors

1988 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1982–1993
J.C. Frey et al.
Table 6 Relative abundance of 16S rRNA genes for each segment*
Segment Rumen
Methanogen 21 500 ± 70Æ7
Total procaryotic 432 000 ± 23 000
Methanogen to procaryotic (%) 5Æ 0
*qPCR numbers for methanogen and total 16S rRNA genes for samples obtained from cow D. For each segment, the same quantity of DNA was
assayed. Different DNA quantities were assayed for each segment therefore only the relative rRNA gene copy numbers within a segment are
provided. Values shown are the mean ± standard deviation for triplicate assays, except the rumen methanogen values represent duplicates.
study, and limits imposed by the 500 LIZ standard, are
not ideal for recovering T-RFs for known 18S rRNA
genes (Moreira and López-Garcia 2002). Our analysis of
potential eucaryal T-RFs recovered compared to the dis-
tribution of microbial eucaryotes in the samples con-
firmed this observation. Therefore, additional studies
focused on eucaryal diversity would be needed to deter-
mine dynamics of these populations.
We used comparative analyses to document major
changes in microbial populations as digesta traversed the
GI tract and to test effects of diet and cow on microbial
diversity. We followed the approach of Rees et al. (2004)
and used (i) nonmetric MDS to visualize patterns in
T-RFLP data, (ii) anosim to assess statistical significance,
(iii) SIMPER to determine the contribution of individual
T-RFs to differences between samples, and (iv) dispersion
indices to investigate heterogeneity. We found the first
three analyses to be very effective (Figs 1 and 2 and
Tables 3, 4 and 5). Dispersion indices, however, varied
within a dataset depending on whether area, height or
presence ⁄ absence was used. Therefore, no conclusions
were based on dispersion indices.
Culture-based analyses (Mackie and Gilchrist 1979;
Leedle et al. 1982) and cultivation-independent surveys
(Tajima et al. 2000, 2001; Kocherginskaya et al. 2001;
Klieve et al. 2003; Wright et al. 2004; Larue et al. 2005)
have indicated that ruminal micro-organisms are affected
by diet. In this study, energy levels were similar in the
two diets, but there were postruminal differences in the
amount of available nitrogen. Although our knowledge of
the nutrient requirements and supply of the hindgut
microbes is limited, it is likely that their requirements are
similar to those of the ruminal bacteria. The nitrogen to
meet the requirements of micro-organisms of the hindgut
is supplied from microbial and dietary protein as well as
from saliva and was assumed to be adequate. Apparently,
this was the case because no differences in microbial
populations attributable to diet were observed (Figs 1 and
2 and Table 3).
Individual animals harbour distinctive and stable gut
microbiota (Zoetendal et al. 1998; Leser et al. 2000; Simp-
son et al. 2000, 2002; Vanhoutte et al. 2004). Previous
ª 2019 The Authors
Journal compilation ª 2019 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2019) 1982–1993
Bovine GI tract communities
Duodenum Ileum Faeces
5600 ± 1120 35 200 ± 2960 18 200 ± 1930
552 000 ± 21 300 780 000 ± 175 000 1 430 000 ± 60 300
1Æ0 4Æ5 1Æ3
studies showed that animal variation accounted for
greater differences in cellulolytic microbial populations
than did diet (Weimer et al. 1999; Koike and Kobayashi
2001). Remarkably, all datasets in this study indicated
T-RFLP community profiles did not vary with our experi-
mental animals. We believe this is due in part to the feed-
ing regime employed in which a steady state was reached,
thus minimizing animal variation over time. This may
dampen the feeding cycle shifts in microbial populations
such as those observed by others in studies of the rumen
(Welkie et al. 2009). For the methanogen populations
conclusions were not as clear. The AluI dataset indicated
less variation in methanogen populations within cows
than between cows when area and height data were used
in anosim (Table 3). However, when T-RF pres-
ence ⁄ absence was considered, the data did not support
this conclusion. Because relative abundance of T-RFs does
not reflect relative microbial abundance because of biases
in PCR amplification and DNA extraction (Frey et al.
2006a), we chose to base our conclusions on consistency
between all datasets.
In ruminants, the pH of digesta varies considerably in
different segments of the GI tract. The pH of intestinal
samples from the cows of this study were duodenum (pH
2–3) and ileum (pH 7–8). Between the rumen (pH 5Æ8–
6Æ8) and the duodenum, digesta passes through the acidic
abomasum that typically has a pH of 2–3. Lysis and enzy-
matic digestion of micro-organisms passing out of the
rumen provide dietary protein to the host. Microbial den-
sities plummet between the rumen (with c. 1012 cells per
ml) and the duodenum (c. 104 cells per ml). Therefore,
we expected to observe differences in T-RFLP fingerprints
between the rumen and the duodenum. Surprisingly,
pairwise differences between the rumen and duodenum
were observed with HaeIII only (Table 4), when signifi-
cance levels of 0Æ05 were used. Clarke and Warwick
(2001) stressed that the interpretation of anosim analyses
should be based on R-values, not significance levels that
can be affected by the number of replicates. The meaning
of R-values have been classified as follows: R > 0Æ75
groups are well separated, R > 0Æ50 groups are overlap-
ping but clearly different, and R < 0Æ25 groups are barely
1989
Bovine GI tract communities
separable (Clarke and Gorley 2001). Although the signifi-
cance levels varied in pairwise comparisons between the
rumen and duodenum microbial population fingerprints
(P = 0Æ114 for MseI and P = 0Æ029 for HaeIII), the
R-values were very similar, R = 0Æ417 and 0Æ427 for MseI
and HaeIII, respectively (Table 4).
It is noteworthy that the microbial population finger-
prints of the rumen and duodenum were more similar
than any other pairwise comparisons, as indicated in the
MDS plots and SIMPER analyses. This may have been
caused by carryover from the rumen into the duodenum.
However, if only spatial closeness was responsible for this
effect, we would also expect low R-values in pairwise
comparisons between the duodenum and ileum, and
ileum and faeces, which were not observed (Table 3).
Clearly, other factors, such as inadequate time for enzy-
matic digestion of micro-organisms passing into the duo-
denum, must be at play.
Although it was difficult to select restriction enzymes
that would discriminate among the ruminal methanogens,
the T-RFLP profiles using methanogen-specific primers
were able to detect and resolve 4–9 T-RFs. In comparative
analyses, no differences in the methanogen profiles
because of either diet or animal were found. There were
discrepancies observed between the datasets (Table 3),
which makes it impossible to resolve whether methanogen
populations varied by segment. As our system could not
detect subtle changes in phylogenetically distinct popula-
tions that account for <1% of the T-RFLP profiles,
further investigation is required to address this inconsis-
tency.
We used qPCR to determine the number of 16S rRNA
genes of methanogens relative to total 16S rRNA genes in
the GI tract samples of one of the cows in this study.
Methanogen sequences comprise 5% of the sequences in
the rumen sample. These numbers are in good agreement
with other culture-independent qPCR results (Hook et al.
2009). Previous studies by Smith and Hungate (1958)
indicate that culturable methanogens exist in the rumen
at densities as great as 108 cells per ml. Correcting for
multiple rRNA operons in some bacterial genomes, if our
qPCR numbers for methanogens represent c. 1% of the
estimated 1012 resident rumen microbes, it suggests that
upward of 1010 methanogens may be present. These num-
bers allude to the diversity of bovine methanogens that
remain uncultured. We could not detect methanogen 16S
rRNA genes in duodenum samples using our T-RFLP pri-
mer set; however, we were able to detect methanogen
genes in the qPCR analysis. This may reflect the lower
amount and state of DNA extracted from microbes in the
duodenum. Furthermore, the qPCR analyses of these
samples indicated that methanogen 16S rRNA genes were
a smaller proportion of total 16S rRNA gene sequences
1990 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1982–1993
J.C. Frey et al.
when compared to the rumen or ileum samples. Total
microbial numbers decrease by c. eight orders of magni-
tude as contents pass from the rumen, through the acidic
abomasum, and into the duodenum. It is noteworthy that
methanogen DNA was detected in the duodenum as
methanogens are generally considered to be sensitive to
drastic changes in pH. The relative proportion of metha-
nogen sequences increase in the ileum suggesting that
methanogens proliferate in the intestinal tract of cows.
Previous studies have also detected methanogen 16S
rRNA in the cecum and colon of ruminants (Lin et al.
1997). Intestinal methanogens are often overlooked in
studies of ruminant microbiota but our data indicate that
methanogens are present in the small intestine. Although
these populations are small relative to those in the rumen,
the impact of methanogens on the microbial ecology of
the intestinal tract should be considered.
Using comparative studies of T-RFLP fingerprints, we
found that in dairy cows the microbial community struc-
ture changed as digesta passed from the rumen, into the
small intestine and out of the animal. Animal differences
were not observed in this study, possibly because of the
12 times per day feeding regime, although this tantalizing
result needs to be confirmed in studies with larger num-
bers of animals. Methanogen 16S rRNA genes were
detected in all four animals, in the rumen and intestinal
samples. These results suggest that postabomasal metha-
nogen populations are present in cows although their
impact on animal performance remains to be determined.
Acknowledgements
We gratefully acknowledge the technical assistance of
Sylvie Provencher, Sherbrooke, Quebec and Jennifer
Nelson, Cornell University. We thank Stephen Zinder,
Suzanna Bräuer and Hinsby Cadillo-Quiroz for providing
methanogen genomic DNA.
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