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RESEARCH ARTICLE
Assessment of microbial diversityalong the feline intestinal tract
using16S rRNA gene analysis
Lauren E. Ritchie, J ¨
org M. Steiner & Jan S. Suchodolski
Gastrointestinal Laboratory, Department of Small Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M
University, College Station, TX, USA
Correspondence: Jan S. Suchodolski,
Gastrointestinal Laboratory, Department of
Small Animal Clinical Sciences, College of
Veterinary Medicine and Biomedical Sciences,
Texas A&M University, 4474 TAMU, College
Station, TX 77843-4474, USA. Tel.: 11 979
458 0933; fax: 11 979 458 4015; e-mail:
jsuchodolski@cvm.tamu.edu
Received 15 July 2008; revised 8 September
2008; accepted 11 September 2008.
First published online November 2008.
DOI:10.1111/j.1574-6941.2008.00609.x
Editor: Julian Marchesi
Keywords
16S rRNA gene; cat; gastrointestinal tract;
microbiota; bacteria; microbial communities.
Abstract
The aim of this study was to describe the microbial communities along the
gastrointestinal tract in healthy cats based on analysis of the 16S rRNA gene.
Gastrointestinal content (i.e. content from the stomach, duodenum, jejunum,
ileum, and colon) was collected from four healthy conventionally raised colony
cats and one healthy specific pathogen-free (SPF) cat. Bacterial 16S rRNA genes
were amplified using universal bacterial primers and analyzed by comparative
sequence analysis. A total of 1008 clones were analyzed and 109 nonredundant 16S
rRNA gene sequences were identified. In the four conventionally raised cats, five
different bacterial phyla were observed, with sequences predominantly classified in
the phylum Firmicutes (68%), followed by Proteobacteria (14%), Bacteroidetes
(10%), Fusobacteria (5%), and Actinobacteria (4%). The majority of clones fell
within the order Clostridiales (54%), followed by Lactobacillales,Bacteroidales,
Campylobacterales, and Fusobacteriales (14%, 11%, 10%, and 6%, respectively).
Clostridiales were predominantly affiliated with Clostridium clusters I (58%) and
XIVa (27%). The intestinal microbiota of the SPF cat displayed a reduced bacterial
diversity, with 98% of all clones classified in the phylum Firmicutes. Further
classification showed that the Firmicutes clones belonged exclusively to the class
Clostridiales and were predominantly affiliated with Clostridium cluster I.
Introduction
It has been recognized that the intestinal microbiota plays an
important role in gastrointestinal health and disease. The
resident bacterial flora has a physiologic effect on gastro-
intestinal motility, the development of the intestinal epithe-
lium, and the immune system (Falk et al., 1998; Hooper
et al., 2001). The microbiota further supplies nutrients such
as vitamins, lactate, and short-chain fatty acids to host
tissues (Macfarlane & Macfarlane, 2003). The residential
bacteria also provide a natural defense mechanism against
invading pathogens (Gibson & Roberfroid, 1995).
In contrast, alterations in the commensal intestinal micro-
biota have been implicated in gastrointestinal disease in
humans and many animal species, including cats (Johnston
et al., 2001; Linskens et al., 2001; Janeczko et al., 2008). It has
been suggested that an ineffective clearance of enteric
pathogens or a loss of tolerance to the residential intestinal
microbiota may be a contributing factor in the pathogenesis
of inflammatory bowel disease (IBD) in humans (Linskens
et al., 2001). In cats, FISH analysis revealed differences in
the composition of the duodenal mucosa-associated micro-
biota between healthy cats and cats with IBD, with the latter
having a significantly higher number of mucosa-associated
organisms belonging to the family Enterobacteriaceae
(Janeczko et al., 2008). Given the impact of the microbiota
on gastrointestinal health, knowledge about the composi-
tion of the gastrointestinal microbiota of healthy cats is
important for future clinical studies exploring differences in
microbial communities between healthy cats and cats with
gastrointestinal disease.
Past studies characterizing the feline intestinal microbiota
have used traditional microbiological culture techniques
(Smith, 1965; Osbaldiston & Stowe, 1971; Itoh et al., 1984;
Johnston et al., 1993, 1999; Papasouliotis et al., 1998). These
studies reported that, in contrast to other mammalian
species, the feline intestinal tract harbors predominantly
facultative and obligate anaerobic bacteria (Johnston et al.,
FEMS Microbiol Ecol 66 (2008) 590–598
c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
1993, 2001; Papasouliotis et al., 1998). Also, these studies
have described bacterial populations present in samples
obtained either from the proximal parts of the small intestine
(i.e. duodenum) or from the colon or feces (Osbaldiston &
Stowe, 1971; Johnston et al., 1993, 1999, 2001; Papasouliotis
et al., 1998). However, to our knowledge, no study has
assessed the microbiota in the ileum of cats. Additionally, no
study has evaluated the bacterial diversity along the entire
gastrointestinal tract of cats.
It has now been recognized that traditional culture-based
techniques, as used in previous studies, fail to accurately
characterize bacterial communities and underestimate bio-
diversity in complex biological ecosystems, such as the
intestine (Itoh et al., 1984; Amann et al., 1995; Langendijk
et al., 1995; Greetham et al., 2002). Recent studies aiming to
characterize the intestinal microbiota in many mammalian
species have used molecular methods based on the amplifica-
tion of the 16S rRNA gene (Greetham et al., 2002; Hold et al.,
2002; Wang et al., 2003; Delgado et al., 2006). It is likely that a
molecular-based approach may identify greater bacterial
diversity in the intestinal tract of cats than reported previously.
Therefore, in the present study, we aimed to characterize the
residential bacteria found in all segments of the intestine of
healthy cats using 16S rRNA gene sequences analysis.
Materials and methods
Sample collection
The protocol for sample collection was approved by the
University Laboratory Animal Care Committee at Texas A&M
University.
Five healthy colony cats (ages 13–18 months), euthanized
for an unrelated project, were used for molecular character-
ization of the intestinal microbial communities. One of the
five healthy cats was a specific pathogen-free (SPF) cat. The
SPF cat was born and raised in a barrier-maintained facility,
theoretically free from all pathogens, until the age of 7 weeks.
After 7 weeks, the cat was transferred to a nonbarrier
laboratory facility. The other four cats were raised conven-
tionally. All five cats were fed Hill’s Science Diet Original
(Hill’s, Topeka, KA) and received no treatment that would be
expected to alter the intestinal microbiota.
Immediately after death, luminal intestinal content was
collected by needle aspiration. All samples were collected
separately, and no samples were pooled. Samples were
transferred into sterile tubes, snap-frozen in liquid nitrogen,
and stored at 80 1C until analysis. It was attempted to
collect samples from several segments along the gastroin-
testinal tract. However, in some cats not all segments could
be sampled due to an insufficient amount of luminal
content. For cats 1 and 2, samples from the jejunum, ileum,
and colon were collected. For cat 3, only a sample from the
colon was available. For cat 4, samples from the duodenum,
ileum, and colon were obtained. From the SPF cat, samples
were collected from the stomach, duodenum, jejunum,
ileum, colon, and rectum.
Extraction of DNA
Each sample was extracted and analyzed separately. DNA
extraction was carried out as described previously, using a
bead-beating method, followed by phenol : chloroform :
isoamylalcohol extraction (Suchodolski et al., 2008). Pur-
ified DNA was stored at 80 1C until further use. A negative
control containing H
2
O instead of a sample was purified in
parallel to each extraction batch to screen for contamination
of extraction reagents.
16S rRNA gene amplification by PCR
Extracted DNA was used as a template for PCR amplification
of a c. 450-bp amplicon of the 16S rRNA gene with universal
bacterial primers F-341 (50-CCTACGGGAGGCAGCAG-30)
and R-786 (50-GACTACCAGGGTATCTAATC-30). Each reac-
tion mixture (25 mL) consisted of a reaction buffer (GeneAmp
10 PCR Gold buffer, Applied Biosystems, Foster City, CA)
[final concentrations: 15 mM Tris-HCl, 50mM KCl, 3mM
MgCl
2
(pH 8.0)], 1.25 U of Taq DNA polymerase [Amplitaq
Gold Low DNA (LD), Applied Biosystems], 50 mMeachofthe
deoxynucleoside triphosphates, 0.24 mMeachprimer,andc.
100 ng of DNA template. To screen for potential contamina-
tion of PCR reagents, a negative PCR control using H
2
O
instead of a DNA template was used. The samples were
amplified in a thermocycler (Mastercycler Gradient, Eppen-
dorf AG, Hamburg, Germany), using the following PCR
protocol: initial denaturing at 95 1C for 3 min, 30 cycles of
denaturation at 95 1C for 30 s, annealing at 54 1Cfor30s,
extension at 72 1C for 1 min, and a final extension at 72 1Cfor
10 min. Two samples (one duodenal and one ileal sample
each) collected from cat 4 did not have enough amplified
DNA after 30 PCR cycles for a successful ligation. Therefore,
these samples were reamplified using the same PCR protocol
with 35 cycles. For all samples, between four and eight
independent PCR reactions were performed. The indepen-
dent PCR reactions for the corresponding compartments of
cat 1 and 2 were analyzed together. PCR products belonging
to the same sample were pooled and concentrated, using the
DNA Clean & Concentrator-5
TM
(Zymo Research, Orange,
CA), following the manufacturer’s instructions. The purity of
the PCR amplicons was assessed on 1% agarose electrophor-
esis gels stained with Gel Red
TM
(Biotium Inc., Hayward, CA).
Cloning of bacterial 16S rRNA gene amplicons
Amplified PCR products were ligated into pCR
s
4-TOPO
s
linearized cloning vectors and the products were
FEMS Microbiol Ecol 66 (2008) 590–598 c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
591Intestinal microbial communities in healthy cats
transformed into chemically competent DH5a
TM
-T1
R
Escherichia coli by heat shock following the manufacturer’s
instructions (TOPO TA, Invitrogen, Carlsbad, CA). Trans-
formed products were grown overnight on Luria–Bertani
(LB) medium with ampicillin (75 mgmL
1
)at371C. The
pCR
s
4-TOPO
s
vector allows direct selection of recombi-
nant cells via disruption of the lethal E. coli gene ccdB. Up to
96 colonies for each sample were randomly selected and
clones were grown for 24 h in 1.4 mL LB broth treated with
ampicillin (75 mgmL
1
) in 96-well blocks (Perfectprep
s
BAC 96, Eppendorf, North America Inc., Westbury, NY).
Plasmid extraction and sequencing of 16S rRNA
genes
Plasmid DNA was purified using the Perfectprep
s
BAC 96
plasmid purification kit (Eppendorf). Plasmid DNA was then
eluted using 50 mL of DNA grade water. The purified inserts
were sequenced using the ABI PRISM BigDye Terminator
Cycle Sequencing Kit (Applied Biosystems), and the products
were analyzed with an automated sequence analyzer (ABI
3100 Capillary Sequencer, Applied Biosystems).
Sequence analysis
Each sequence was edited to exclude the PCR primer-
binding sites, and then tested for possible chimeric struc-
tures using the CHECK_CHIMERA and PINTAIL software available
through the Ribosomal Database Project (RDP). Identified
chimeras were excluded from further analysis.
Cloned sequences were compared with existing 16S rRNA
gene sequences using GenBank and RDP (release 9.59, c.
489 840 16S rRNA gene sequences), and the closest neighbor
for each sequence was obtained. Sequences were aligned using
the multiple sequence alignment program CLUSTAL_Wand the
alignment was inspected and manually adjusted using the
alignment editor in the BIOEDIT software. A PHYLIP distance
matrix was generated and used as the input file for the DOTUR
software to determine operational taxonomical units (OTUs)
(Schloss & Handelsman, 2005). Groups of sequences with
o2% sequence divergence (98% similarity) to each other were
defined as an OTU. Phylogenetic trees were generated based on
the neighbor-joining algorithm using the TREECON software
package (version 1.3b) and the Jukes–Cantor model for
inferring evolutionary distances (Van de et al., 1996). Branch
stability was assessed by bootstrap analysis (100 replicates)
using the algorithms available in the TREECON package. Aquifex
pyrophilus wasusedasanoutgroup.Adendrogramwas
constructed based on the UNIFRAC distance metric, to illustrate
the phylogenetic affiliation of the microbiota from the different
compartments obtained from each cat.
Statistical analysis
The coverage of the individual clone libraries (i.e. the
probability that any additional analyzed clone is different
from any previously obtained single clone) was calculated
according to Good (1953) using the formula [1 (n/N)] 100,
where nis the number of molecular species represented by one
clone and Nis the total number of sequences. Bacterial diversity
was calculated using the Shannon–Weaver diversity index,
defined as Sp
i
ln(p
i
), where p
i
is the proportion of individual
bacteria found in a certain species (Atlas & Bartha, 1998).
Nucleotide sequence accession numbers
The 16S rRNA gene sequences obtained were deposited into
the GenBank database with accession numbers EU877804–
EU877912.
Results
A total of 1332 clones were selected randomly. Of these, 1071
clones contained an insert with a sequence of adequate quality.
Sixty-three of these clones were identified as possible chimeras
and excluded from further analysis. A total of 109 OTUs,
representing a total of 1008 clones, were used for subsequent
phylogenetic analysis. Table 1 summarizes the number of
analyzed samples, analyzed clones, identified OTUs, coverage,
and bacterial diversity for each intestinal segment.
Twenty-one OTUs (19%) showed o98% similarity to
existing 16S rRNA gene sequences in the NCBI database.
Five different bacterial phyla were identified, with the
majority of OTUs being classified as Firmicutes, followed
by Proteobacteria, Bacteroidetes,Fusobacteria, and Actino-
bacteria, respectively.
Actinobacteria
A total of 21 clones representing seven OTUs were identified
within the phylum Actinobacteria. Two clones were isolated
from the jejunum (10%), 11 clones were isolated from the
Table 1. Number of analyzed samples, selected clones, identified OTUs,
coverage, and bacterial diversity index (H) constructed from samples
obtained from various segments of the feline intestinal tract
No. of
samples
No. of
clones
No. of
OTUs Coverage
H
w
Stomach 1 91 5 97.8 0.9
Duodenum 2 93 7 96.8 1.4
Jejunum 2 172 25 93.1 2.2
Ileum 3 261 49 92.8 3.0
Colon 4 322 84 87.1 3.0
Rectum 1 75 16 92.0 2.0
According to Good (1953).
w
Shannon–Weaver diversity index.
FEMS Microbiol Ecol 66 (2008) 590–598
c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
592 L.E. Ritchie et al.
ileum (52%), and eight clones were isolated from the colon
(38%). One OTU showed 98% similarity to Actinomyces
hyovaginalis AF489584 and representative clones were ob-
tained from the jejunum (two), ileum (six), and colon (two).
Bacteroidetes
A total of 95 clones were classified within the phylum
Bacteroidetes representing 17 OTUs (Supporting Informa-
tion, Fig. S1). The majority of clones were isolated from the
ileum and colon (43% and 50%, respectively), followed by
the rectum (5%) and the jejunum (o2%). Four different
bacterial families were identified: Bacteroidaceae,Porphyro-
monadaceae,Prevotellaceae, and Rikenellaceae. A total of 54
clones representing 10 OTUs were identified within the
family Bacteroidaceae. A total of 35 clones representing four
OTUs were classified within the family Prevotellaceae. The
Rikenellaceae and Porphyromonadaceae families were repre-
sented by one and two OTUs, respectively.
Firmicutes
The majority of all clones (82%) were classified within the
phylum Firmicutes representing 67 OTUs. Figures S2 and S3
show the OTUs classified within the bacterial class Clostri-
diales, and Fig. S4 illustrates the OTUs identified within the
Bacilli and Mollicutes class.
A total of 754 clones were classified in the bacterial class
Clostridiales representing 56 different OTUs. Clones belonging
to this bacterial class were affiliated with six different Clostri-
dium clusters:clustersI,III,IV,XI,XIVa,andXIVb.The
percentages of the 16S rRNA gene sequences affiliated with
these clusters are illustrated in Fig. 1. A total of 541 clones
representing 13 OTUs were affiliated with Clostridium cluster I.
Four OTUs were affiliated with the Clostridium perfringens
subgroup, and one OTU was affiliated with Sarcina ventriculi
AF110272 (Clostridium subcluster Ia). One OTU was affiliated
with Clostridium cluster III. Six OTUs were affiliated with
Clostridium cluster IV, and two OTUs were affiliated with
Clostridium cluster XI. A total of 54 clones were affiliated
with Clostridium cluster XIVa representing 20 OTUs. Three
OTUs were affiliated with Clostridium cluster XIVb.
A total of 70 clones representing 10 different OTUs were
classified within the class Bacilli.Thirty-fourcloneswere
isolated from the jejunum (48%), nine from the ileum (13%),
and 27 from the colon (39%). Further classification showed
that 65 clones (92%) representing eight OTUs were classified
in the bacterial order Lactobacillales. One OTU consisting of
one clone from the colon was classified in the class Mollicutes.
Fusobacteria
A total of 26 clones representing four OTUs were classified
within the phylum Fusobacteria. Four clones were isolated
from the jejunum (15%), four from the ileum (15%), and
18 from the colon (69%). One OTU in this phylum had
99% similarity to Fusobacterium equinum AJ295750, and its
representative clones were predominantly obtained from the
colon (10), followed by the jejunum (three) and the ileum
(two), respectively. One OTU had 99% similarity to Fuso-
bacterium russii X55409 and had one representative clone
from the jejunum, ileum, and colon each.
Proteobacteria
A total of 78 clones representing 14 OTUs were classified
within the phylum Proteobacteria (Fig. S5). The majority of
clones were isolated from the duodenum (41%), followed by
the ileum, colon, jejunum, and rectum (32%, 19%, 5%, and
3%, respectively). Four bacterial classes were identified:
Betaproteobacteria,Deltaproteobacteria,Epsilonproteobacteria,
and Gammaproteobacteria. The two predominant classes were
Gammaproteobacteria and Epsilonproteobacteria. Within the
class Gammaproteobacteria, six OTUs were identified consist-
ing of 13 clones from the colon, five from the ileum, and two
from the jejunum. The Epsilonproteobacteria class was com-
prised of four OTUs representing 32 clones isolated from
the duodenum and 14 clones from the ileum. OTUs from the
duodenum were only classified in the class Epsilonproteobac-
teria. Two OTUs were classified as Betaproteobacteria.One
Fig. 1. Classification of 16S rRNA gene sequences belonging to the
different Clostridium clusters in the four conventionally raised cats (A)
and the healthy SPF cat (B).
FEMS Microbiol Ecol 66 (2008) 590–598 c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
593Intestinal microbial communities in healthy cats
OTU from the ileum was classified as a Deltaproteobacteria,
with 99% similarity to Desulfovibrio piger AF192152.
Comparison of phylogenetic groups between
the SPF cat and the conventionally raised cats
Three bacterial phyla were identified in the SPF cat: Firmi-
cutes,Proteobacteria, and Bacteroidetes, with 98% of clones
classified within the phylum Firmicutes. Two OTUs be-
longed to the phylum Bacteroidetes, class Bacteroidales.
Three OTUs fell in the phylum Proteobacteria. Two of these
were classified as Gammaproteobacteria and one was classi-
fied as Betaproteobacteria. Further classification showed that
the Firmicutes clones belonged exclusively to the class
Clostridiales.Clostridium cluster I was the most predomi-
nant cluster (96%), followed by cluster XIVa (2%) and
cluster XIVb (1%) (Fig. 1). Less than 1% of sequences were
affiliated with Clostridium clusters III and IV. Sequences
affiliated with Clostridium cluster I were isolated from all
compartments while sequences affiliated with clusters III,
XIVa, and XIVb were only isolated from the rectum.
Sequences affiliated with Clostridium cluster IV were iso-
lated from the colon.
In contrast, five different bacterial phyla were identified in
the four conventionally raised cats: Firmicutes (67%), Proteo-
bacteria (14%), Bacteroidetes (10%), Fusobacteria (5%), and
Actinobacteria (4%). Further classification within the phylum
Firmicutes showed clones in three bacterial classes: Clostridiales,
Bacilli,andMollicutes. Clones isolated from the conventionally
raised cats were predominantly affiliated with Clostridium
clusters I (58%) and XIVa (27%) (Fig. 1). Sequences belonging
to Clostridium cluster I were isolated from three compartments
(i.e. jejunum, ileum, and colon). The number of clones
affiliated with cluster I increased along the intestinal tract,
with the highest number of clones isolated from the colon.
Sequences affiliated with Clostridium cluster XIVa were
predominantly isolated from the colon (Fig. 2).
Spatial differences within the feline intestinal
tract
Because of the above-described differences in the microbiota
between the SPF cat and the four conventionally raised cats,
the SPF cat was excluded for this part of the analysis. Also,
as mentioned above, not all intestinal segments could be
sampled from all four conventionally raised cats. Therefore,
a separate analysis was performed describing only the
intestinal segments for which samples were obtained from
at least two individual cats (i.e. jejunum, ileum, and colon).
Fourteen different bacterial orders were identified in these
three intestinal segments. The proportions of the predomi-
nant bacterial orders within the selected compartments are
shown in Fig. 3. The majority of clones were classified within
the order Clostridiales. The second most predominant order
was Lactobacillales in the jejunum and Bacteroidales in the
ileum and colon. Clones classified within the order Bacteroi-
dales were predominantly isolated from the distal intestine
(i.e. ileum and colon), with only one clone isolated from the
proximal portion of the intestine (i.e. jejunum).
Comparison of the clone libraries using UNIFRAC
analysis
The dendrogram constructed based on the UNIFRAC distance
metric revealed that the samples tended to cluster by the
Fig. 2. Distribution of the two predominant Clostridium clusters I and
XIVa in the four conventionally raised healthy cats.
Fig. 3. Percentages of 16S rRNA gene sequences belonging to the
major phylogenetic lineages in the compartments in which samples were
obtained from at least two conventionally raised cats.
FEMS Microbiol Ecol 66 (2008) 590–598
c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
594 L.E. Ritchie et al.
individual cats rather than the collection site (Fig. 4). A
separate analysis was performed to analyze the phylogenetic
similarities of the OTUs that were characterized within the
phylum Firmicutes. The results of this analysis were similar
to the overall UNIFRAC analysis, where the clone libraries
clustered by individual cat, rather than by the intestinal site
from where the sample was obtained.
Discussion
The intestinal bacterial microbiota of the feline gastrointest-
inal tract was characterized using comparative 16S rRNA
gene analysis. This study illustrated that phylogenetic simi-
larities were found between the different compartments
obtained from each cat. The microbiota in different intest-
inal compartments tended to be more similar within the
individual than between corresponding compartments of
different cats. These findings are similar to the results of one
study that analyzed the phylogenetic composition of human
colon and fecal samples and reported that samples clustered
by the individual they came from, and not by the biopsy site
(Eckburg et al., 2005).
Firmicutes were the most abundant phylum in the feline
intestinal tract (825 clones) and were also the most diverse
(67 OTUs). This finding is consistent with previous studies
that used microbiological culture techniques to analyze the
microbiota in the proximal portion of the small intestine
(i.e. duodenum and jejunum) and the colon in healthy cats.
Clostridium spp. were the most common bacterial species
identified in duodenal aspirates, occurring in over 90% of
cats (Papasouliotis et al., 1998; Johnston et al., 2001). The
predominant bacterial species in the jejunum were Enter-
ococcus spp., Streptococcus spp., and Lactobacillus spp., which
all belong to the phylum Firmicutes (Osbaldiston & Stowe,
1971). The most common bacterial species isolated in
colonic and fecal samples were Enterococcus spp. and Lacto-
bacillus spp. (Osbaldiston & Stowe, 1971; Itoh et al., 1984).
Within the phylum Firmicutes,Clostridiales was the most
abundant bacterial class (754 clones), representing 56 OTUs.
These OTUs were affiliated with six different Clostridium
clusters, of which clusters I and XIVa were predominant.
Clones affiliated with Clostridium cluster I were identified in
all intestinal segments, with the highest number of clones
identified in the colon. This finding differs from studies in
humans that reported that very few clones obtained from
the colon were affiliated with Clostridium cluster I (Hold
et al., 2002; Wang et al., 2003; Delgado et al., 2006).
Clostridium cluster XIVa was the most diverse cluster (20
OTUs) in the feline intestinal tract, with clones isolated
predominantly in the distal intestine (i.e. colon). Similar to
our results, the majority of isolated clones from the colon of
humans and horses were also affiliated with Clostridium
cluster XIVa (Daly et al., 2001; Wang et al., 2003). Clones
affiliated with Clostridium cluster IV could only be obtained
from the colon and were not isolated in any other segment
of the feline intestinal tract.
Lactobacillales were another major constituent of the
phylum Firmicutes and were predominantly isolated from
the jejunum and colon. This is similar to a previous study
that reported Lactobacillus spp. to be a predominant bacter-
ial group in the jejunum and colon of cats when analyzed by
culture-based methods (Osbaldiston & Stowe, 1971).
Clones belonging to the orders Bacteroidales and Fuso-
bacteriales were isolated mainly from the ileum and colon
and were only sporadically found in the jejunum but not in
the duodenum. Previous studies are consistent with our
findings where Bacteroides spp. have been reported to be a
common bacterial group in the feline large intestine (Osbal-
diston & Stowe, 1971; Itoh et al., 1984). However, in
contrast, Bacteroidetes and Fusobacteria have been routinely
cultured from duodenal aspirates using culture-based meth-
ods (Papasouliotis et al., 1998; Johnston et al., 2001). In our
study, duodenal samples were analyzed only from two cats,
and this may explain why these bacterial groups were not
represented in our clone libraries. Interestingly, Fusobacteria
Cat 4-D
Cat 4-I
Cat 4-C
Cat 1&2-J
Cat 1&2-C
Cat 1&2-I
Cat 3-C
SPF-R
SPF-C
SPF- I
SPF-D
SPF-J
SPF-S
>50%
>70%
>99%
>99%
<50%
>50%
>50%
>90%
>90%
>90%
>90%
Fig. 4. Dendrogram illustrating the phylogenetic affiliation of the micro-
biota from the different sample locations (S, stomach; D, duodenum; J,
jejunum; I, ileum; C, colon; and R, rectum). The samples tended to cluster
by the individual cats rather than the collection site. The dendrogram
was constructed using the UNIFRAC distance matrix, a phylogenetic
method for comparing microbial communities. Bootstrap values shown
at the branches are based on 100 replicates.
FEMS Microbiol Ecol 66 (2008) 590–598 c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
595Intestinal microbial communities in healthy cats
has not been reported to be a bacterial group commonly
isolated from human and equine intestinal samples using
16S rRNA gene analysis (Suau et al., 1999; Daly et al., 2001;
Hold et al., 2002; Wang et al., 2003; Eckburg et al., 2005;
Delgado et al., 2006).
Clones classified in the phylum Proteobacteria were more
commonly isolated in the small intestine compared with the
large intestine, and 32% of all clones obtained from the
duodenum were classified as Epsilonproteobacteria.Ina
previous study using culture-based techniques, Escherichia
spp. were isolated from the colon of all six cats evaluated
(Osbaldiston & Stowe, 1971). In contrast, only one clone
classified as an E. coli-like organism was isolated in our
study, and only from the ileum. This absence of Proteobac-
teria from the colon is consistent with human studies where
it has been reported that these bacteria are only found in low
numbers in the human colon and feces using 16S rRNA gene
clone libraries (Suau et al., 1999; Hold et al., 2002; Wang
et al., 2003; Eckburg et al., 2005; Delgado et al., 2006).
Clones classified in the phylum Actinobacteria were pre-
dominantly isolated from the ileum and colon. No Bifido-
bacterium spp. were detected in our clone libraries. In
contrast, previous studies using culture-based techniques
have reported Bifidobacterium spp. to be present in feline
feces. In one study, Bifidobacterium spp. were cultured from
feces in 60% of healthy cats (Itoh et al., 1984). Also, another
study utilizing FISH analysis of fecal extracts reported the
detection of Bifidobacterium spp. in 90% of healthy cats and
64% of cats with IBD (Inness et al., 2007). One potential
explanation for these differences between studies could be
the differences in the methodologies used. For example, it
has been reported that Bifidobacterium spp. are uncom-
monly detected in 16S rRNA gene libraries, probably due to
bias in the commonly used universal primers or PCR
protocols (Wilson & Blitchington, 1996; Hold et al., 2002;
Wan g et al., 2003; Suchodolski et al., 2008). Therefore, a 16S
rRNA gene approach may underestimate the presence of
Bifidobacterium spp. In contrast, traditional bacterial culture
techniques may have led to an overestimation of the diversity
of Bifidobacterium spp. due to insufficient selectivity of culture
media (Greetham et al., 2002). Future studies using genus-
specific primers will be useful for the accurate characterization
of Bifidobacterium spp. in the feline intestinal tract.
In this study, we analyzed samples obtained from the
stomach, duodenum, jejunum, ileum, colon, and rectum of
an SPF cat. Characterization of the bacterial microbiota in
these samples revealed differences when compared with the
intestinal microbiota of four conventionally raised healthy
colony cats. Clones obtained from the SPF cat were pre-
dominantly classified within the phylum Firmicutes (98%),
and this proportion was markedly higher compared with the
other cats (67%). Further classification showed that clones
obtained from the SPF cat belonged exclusively to the class
Clostridiales. In contrast, clones belonging to the classes
Clostridiales,Bacilli, and Mollicutes were identified in the
healthy non-SPF cats. Additionally, 93% of clones obtained
from the SPF cat were affiliated with Clostridium cluster I,
and all clones obtained from the stomach and duodenum
were affiliated with this cluster. In contrast, only 58% of
clones obtained from the four healthy non-SPF cats were
affiliated with Clostridium cluster I. In addition, 27%
of clones obtained from the healthy non-SPF cats were
affiliated with Clostridium cluster XIVa, compared with 3%
of clones obtained from the SPF cat. Unfortunately, only one
SPF cat was analyzed in this study, which makes it difficult
to conclude whether the intestinal microbiota of SPF cats is
generally different from conventionally raised cats.
To our knowledge, only one culture-based study has
compared the intestinal microbiota between conventionally
raised and SPF cats (Itoh et al., 1984). Clostridium spp. were
significantly higher in cesarian section-derived, barrier-main-
tained cats compared with conventionally raised cats. The
SPF cats displayed a microbial community diversity that was
generally similar to the conventionally raised cats, with seven
bacterial classes identified (Itoh et al., 1984). This is in
contrast to the present study, where only four bacterial classes
were identified in the SPF cat, with 98% of all clones classified
as Clostridiales. Differences in the methods used (i.e. bacterial
culture vs. 16S rRNA gene analysis), diets, or environmental
differences in the housing facility may explain the different
results of both studies. Given the fact that SPF cats are
commonly used in immunological studies, and the microbial
communities are believed to play an important role in the
development of the host immune system, the potential
differences in the intestinal microbial communities between
SPF cats and conventionally raised cats warrant further
characterization (Brown et al., 1991; Falk et al., 1998).
In conclusion, the molecular approach described in this
study facilitated the next step in understanding the complex
phylogenetic diversity of the microbial communities in the
feline intestinal tract, and allowed the identification of several
previously uncharacterized bacterial phylotypes. Further stu-
dies are warranted to characterize the intestinal microbiota in
diseased cats using 16S rRNA gene clone libraries.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
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Published by Blackwell Publishing Ltd. All rights reserved
597Intestinal microbial communities in healthy cats
Fig. S1. Phylogenetic tree showing the affiliation of
OTUs isolated from the feline gastrointestinal tract for
Bacteroidetes.
Fig. S2. Phylogenetic tree showing the affiliation of OTUs
isolated from the feline gastrointestinal tract with the class of
Clostridiales.
Fig. S3. Phylogenetic tree showing the affiliation of OTUs
isolated from the feline gastrointestinal tract with Clostri-
dium cluster XIVa.
Fig. S4. Phylogenetic tree showing the affiliation of OTUs
isolated from the feline gastrointestinal tract with the classes
Bacilli and Mollicutes.
Fig. S5. Phylogenetic tree showing the affiliation of OTUs
isolated from the feline gastrointestinal tract with Proteobacteria.
Please note: Wiley-Blackwell is not responsible for the
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should be directed to the corresponding author for the article.
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598 L.E. Ritchie et al.
