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Applied and Environmental Microbiology, August 1999, p. 3738-3741, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of Ruminococcus
flavefaciens as the Predominant Cellulolytic Bacterial Species of
the Equine Cecum
Veronique
Julliand,1,*
Albane
de
Vaux,1
Liliane
Millet,2 and
Gerard
Fonty2
Laboratoire associé de Recherches Zootechniques
INRA-ENESAD, 21036 Dijon Cedex,1 and
Laboratoire de Microbiologie, INRA, Centre de Recherches de
Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle,2 France
Received 8 February 1999/Accepted 10 May 1999
 |
ABSTRACT |
Detection and quantification of cellulolytic bacteria with
oligonucleotide probes showed that Ruminococcus
flavefaciens was the predominant species in the pony and donkey
cecum. Fibrobacter succinogenes and Ruminococcus
albus were present at low levels. Four isolates, morphologically
resembling R. flavefaciens, differed from ruminal strains
by their carbohydrate utilization and their end products of cellobiose fermentation.
| |
TEXT |
Microbial degradation and
fermentation of plant polymers into nutrients is a major function of
the equine intestinal ecosystems. Moreover, incomplete fiber
utilization can lead to illness and even death of the animal
(2). However, information on the fibrolytic community in
these nonruminant herbivores is very scarce. Fibrobacter succinogenes, Ruminococcus flavefaciens, and
Ruminococcus albus, the three main ruminal
cellulolytic bacterial species, have been identified based on
morphological criteria (4, 7). F. succinogenes has been demonstrated with a specific oligonucleotide
probe in a pony (19). Bacteroides sp.,
Bacillus cellulosae dissolvens (7),
Clostridium sp., Eubacterium sp., and
Butyrivibrio fibrisolvens have been observed. Protozoa do
not seem to play an important role in cellulosis (23), but
fungi appear to be strong cellulose degraders (15).
The objectives of our study were to determine the size of the
cellulolytic bacterial community in the donkey and pony ceca by culture
methods, to detect and quantify with oligonucleotide probes the three
major cellulolytic bacterial species usually found in the rumen, to
characterize the dominant cellulolytic bacterial strains, and to
compare them with ruminal strains.
Three donkeys and three ponies, cecally fistulated, were fed a 70%
lucerne-orchard hay and 30% concentrate (43% barley, 40% beet pulp,
10% soybean meal, 5% molasses, 3% minerals) (22) given in
two equivalent daily meals. The total cecal contents (200 ml),
collected before the morning meal into CO2-saturated flasks, were serially diluted in an anaerobic mineral solution (5) under O2-free CO2
(12). Total viable counts of bacteria were determined in
roll tubes on a complete agar medium (18), and the numbers
of cellulolytic bacteria were estimated as the most probable number in
broth (11) containing a strip of filter paper (Whatman no.
1) as the sole energy source. Culture methods were based on those
described by Hungate (12) and Fonty et al. (8),
except that the pH was adjusted to 7.3 before autoclaving and a mixture
of ruminal fluid and cecum liquor (1:1) replaced ruminal fluid.
Cellulolytic bacteria were also detected and quantified with
oligonucleotide 16S rRNA probes. Total RNA was extracted from 50 mg of
lyophilized cecal sample after disruption of bacterial cells with
zirconium beads (6). The general procedure for RNA isolation
and quantitation was based on those previously described (1, 24,
25). The probes S-D-Bact-0338-a-A-18 (which targets eubacteria),
S-S-F.succ-0650-a-A-20 (F. succinogenes),
S-S-R.alb-0196-a-A-18 (R. albus), and S-S-R.fla-1269-a-A-20
(R. flavefaciens) (21) were labeled at their 5'
ends with T4 polynucleotide kinase (Eurogentec) and
[
-32P]ATP (ICN). The hybridized 16S RNAs were
visualized by exposure of the membranes to Hyperfilm MP (Amersham) for
24 h and quantified by liquid scintillation counting (Tricarb 2000 CA; Packard).
Cellulolytic bacterial strains were isolated from the highest-dilution
tubes in the most-probable-number assay, serially diluted, and
inoculated (12) into a solid medium (11)
containing cellobiose (4 g liter
1) as the sole energy
source. Colonies grown after 3 days were then transferred to cellulose
broth (11). Purity was checked under a phase-contrast
microscope. Four representative strains (AB and AD for donkeys and PA
and PB for ponies) from the isolates collection were phenotypically
characterized and compared with strains FD1 and 007 of R. flavefaciens. Their ability to utilize carbohydrates (see Table 2)
was determined by using a semisynthetic broth (20)
containing 4 g of mono-, di-, or trisaccharides per liter or
1 g of polysaccharides per liter. Cultures were considered positive when the growth was maintained after three subcultures. The
end products of cellobiose fermentation were analyzed by high-pressure liquid chromatography HPLC (14) after 72 h of
incubation. Oligonucleotide probes targeting the 16S rRNA of R. flavefaciens, R. albus, and F. succinogenes
(1, 24, 25) were used for the presumptive identification of
the four isolates. RNA extraction of the bacterial cultures and
hybridization with the probes were performed as described above for the
cecal samples. RNA of each species was extracted from 25 mg of
bacterial pellets obtained after centrifugation of a 24-h culture on
0.2% cellobiose.
The concentrations of total viable bacteria (4.2 × 108 ± 1.7 × 108 and 5.7 × 108 ± 2.4 × 108 CFU
ml1 in the ceca of ponies and donkeys, respectively) and
of cellulolytics (1.6 × 107 ± 0.4 × 107 and 1.3 × 107 ± 0.6 × 107 bacteria ml1) showed no significant
differences between the two animal species. Hybridization of rRNA from
the cecal contents with specific probes revealed the presence of the
three major ruminal cellulolytic bacterial species (Table
1). In all the animals, R. flavefaciens was the dominant species. F. succinogenes
was also detected, whereas the population size of R. albus
was so small that the amount of detectable 16S rRNA was at the
detection limit (ca 1 ng/50 mg of freeze-dried cecal contents). When
cultured on cellobiose, the equine strains exhibited long chains of
gram-variable cocci resembling R. flavefaciens. The
carbohydrates used by these isolates are given in Table
2. The four strains fermented cellobiose
within the first 24 h of culture and produced mainly acetate,
formate, and ethanol but no malate or fumarate, which differentiated
them from the ruminal strains FD1 and 007 of R. flavefaciens
(Table 3). RNAs extracted from the four
strains hybridized with the oligonucleotide probe S-S-R.fla-1269-a-A-20
targeting R. flavefaciens but not with probes
S-S-F.succ-0650-a-A-20 and S-S-R.alb-0196-a-A-18.
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TABLE 1.
Quantification and percentage of R. albus,
R. flavefaciens, and F. succinogenes 16S rRNA in
cecal contents of ponies and donkeys targeted with specific
oligonucleotide probes
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TABLE 3.
Major end products of cellobiose fermentation for the
ruminal strains R. flavefaciens 007 and FD1 and the four
cecal isolates
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|
Small differences in total viable and cellulolytic counts found between
donkeys and ponies suggest that the animal species and other animal
factors have a limited impact on the size of the microflora in equines.
For the pony cecum, our counts were close to those usually reported in
the literature (10, 16, 17, 23) but much higher than those
of Goodson et al. (10) (104 bacteria
g1). The total and cellulolytic bacterial concentrations
in the ceca of equines are about 100-fold lower than those in the
rumens (9, 13, 16, 17, 26). Cellulolytic bacteria
represented a small percentage of the total anaerobic bacteria in
donkeys (2.3%) and ponies (3.8%), reared under our conditions; these
percentages are lower than that found by Kern et al. (9%)
(16) but similar to those estimated with probes. The three
major cellulolytic species commonly found in the rumen were also
present in the equine cecum. This finding is novel since these species
had never been previously detected together in the equine cecum. The
predominance of R. flavefaciens demonstrated with probes was
consistent with the presumptive morphological identification of our
strains. The proportion of F. succinogenes we observed was
markedly lower than the 12% of total bacterial 16S rRNA found by Lin
and Stahl (19) in the cecum of a pony. The diet given to the
animals might explain this discrepancy. The equine strains of
Ruminococcus exhibited metabolic and fermentative
differences from the ruminal strains of R. flavefaciens. The
four strains hybridized with probe S-S-R fla-1269-a-A-20, which
suggested that the isolates are genetically closely related to R. flavefaciens. These characteristics allowed us to assign cecal
isolates AB, AD, and PA to the genus Ruminococcus.
Characteristics of the 16S rDNA determined by restriction fragment
length polymorphism reported elsewhere (7a) showed genetic
differences between cecal and ruminal strains. These differences could
be due to an evolutionary adaptation of the ruminococci to their
ecosystem, which has previously been reported for strains of F. succinogenes with the creation of two new lineages (Fibro-A and
Fibro-B) (19). Further studies are required to determine
whether the relative proportions of the three detected species,
particularly the preponderance of R. flavefaciens, are
generally observed in the equine or whether the equine bacterial
cellulolytic community is more diverse. Understanding whether its
structure depends on the diet or on other factors linked to the animal
characteristics could allow optimization of lignocellulosic compound
utilization in equines and therefore contribute to healthier equines.
| |
ACKNOWLEDGMENTS |
We thank R. Hutkins for kindly reviewing this paper.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire
associé de Recherches Zootechniques INRA-ENESAD, BP 1607, 21036 Dijon Cedex, France. Phone: 33.(0)3.80.77.25.59. Fax:
33.(0)3.80.77.25.84. E-mail: v.julliand{at}enesad.fr.
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Applied and Environmental Microbiology, August 1999, p. 3738-3741, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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