Item does not contain fulltext4th Symposium on Gut Microbiology, 21 juni 200

Alen, T.A. van

Ederveen, A.

Fried, J.

Graaf, R.M. de

Hackstein, J.H.P.

Huynen, M.A.

Jouany, J.P.

McEwan, N.R.

Michalowski, T.

Moon-van der Staay, S.Y.

Newbold, J.C.

Pristas, P.

Ricard, G.N.S.

Severing, E.

Staay, G.W.M. van der

Radboud Repository

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Session I: Taxonomic and functional diversity of gut microbes S25
sequence-specific enzymes: (1) a restriction
endonuclease that specifically cleaves (phage)
DNA within the recognition sequence and (2) a
modification methyltransferase that specifically
methylates DNA. It has been shown that a large
number of R-M systems can be found in natural
bacterial populations. The widespread presence
of R-M systems in bacteria would be a conse-
quence of the selective advantage of having a
defense tool against phage infection. The anal-
ysis of R-M activities in seven phenotypically
different rumen strains of Megasphaera elsdenii
revealed the presence of GATC-specific restric-
tion-modification systems (MboI isoschizom-
ers) in all of the strains tested. A complete lack
of other restriction and/or modification enzymes
previously characterized in closely related
Selenomonas ruminantium was confirmed by a
methylation protection assay. Based on the
results of our experiments, it could be assumed
that M. elsdenii, as compared to S. ruminantium,
uses a strategy different from R-M systems for
bacteriophage protection.
Diversity of rumen methanogens from sheep
in Western Australia and Queensland identi-
fied by 16S clone libraries. C. Pimm, A.F.
Toovey, A.J. Williams, B. Winder, S. Rodgers,
K. Smith, A.-D.G. Wright (CSIRO Livestock
Industries, Private Bag 5 Wembley WA 6913,
Australia).
Individual 16S clone libraries were prepared
from the rumen contents of 17 Merino sheep in
Western Australian fed 3 different diets (grazing
pasture vs. oaten hay vs. lucerne hay). Amongst
the 733 clones examined, 65 phylotypes were
found to be similar to cultivated methanogens of
the order Methanobacteriales. The diversity of
rumen methanogens was the greatest in the sheep
grazing pasture. Methanobrevibacter strains SM9,
M6 and NT7 accounted for over 90% of the
733 clones examined, with M6 being more prev-
alent in grazing sheep and SM9 more prevalent
in sheep fed the lucerne-based diet. Five new
species were identified, two of which have very
little sequence similarity to any cultivated meth-
anogens. In contrast, an investigation into the
molecular diversity of rumen methanogens from
pooled rumen fluid contents from five Merino
sheep in Queensland, Australia revealed that
only eight of the 34 phylotypes from 89 clones
were similar to methanogens belonging to the
order Methanobacteriales. The remaining 26 phy-
lotypes represented a new taxonomic order of
methanogens, atypical for the rumen environ-
ment, some 15–20% dissimilar to Methanobac-
teriales.
Implications for disease of colonic bacterial
diversity: A culture independent analysis of
the microbial community from the colon of
individuals with IBD and colon cancer.
P. Scanlan, F. Shanahan, J. Marchesi (Dept. of
Microbiology, University College Cork, Cork,
Eire).
The large intestine is the most heavily colonised
part of the gastrointestinal (GI) tract with num-
bers reaching 1012 bacteria per gram of luminal
contents. Micro-organisms are essential to main-
taining the normal functioning of the gut, and as
such have a profound effect on the health status
of their host from an immunological, dietary and
physiological perspective. Thus, the potential
role bacteria hold in various disease states of the
GI tract is widely appreciated. In this study the
total bacterial diversity and the diversity of spe-
cific bacterial genera of colon cancer (n = 40),
polyp (n = 40) and inflammatory bowel disease
(IBD) patients, on specific probiotic trials, were
analysed. Molecular techniques (ribosomal inter-
genic spacer analysis and denaturing gradient
gel electrophoresis) widely used in the microbial
ecology field were employed to investigate the
bacterial populations at given time points. The
results obtained were compared to those of nor-
mal subjects and are discussed here. The meth-
ods employed illustrated not only considerable
variation between individuals but also intra-
individual variation in certain sub-groupings of
the trials.
Fe-hydrogenases from bovine rumen: a metage-
nomic approach. E. Severing, A. Ederveen,
G.W.M. van der Staay, S.Y. Moon-van der Staay,
R.M. de Graaf, Th.A. van Alen, N. McEwan*,
C.J. Newbold*, J.-P. Jouany#, T. Micha owski*,
P. Pristas*, J. Fried*, G. Ricard*, M.A. Huynen*,
J.H.P. Hackstein (Dept. Evolutionary Microbi-
ology, Fac. Sci., University of Nijmegen, Toer-
nooiveld 1, NL6525ED Nijmegen, The Nether-
lands and the EU projects # ERCULE/*CIMES).
ł 
S26 Session I: Taxonomic and functional diversity of gut microbes
The evolution of eukaryotic Fe-hydrogenases is
still poorly understood. It has remained unclear
until now as to whether these hydrogenases rep-
resent an old eukaryotic heritage or whether they
were acquired by bacterial-to-eukaryote gene
transfer. Here we describe the recovery of a set
of DNA sequences encoding the H-cluster of Fe-
hydrogenases from rumen ciliates. The rumen
ciliates were isolated from the rumen fluid of a
cow by electromigration. The DNA of the total
rumen ciliate population was purified and used
to amplify the H-clusters of Fe-hydrogenases by
PCR with degenerated primers. For the identifi-
cation of the corresponding ciliates, PCR was
performed on DNA from type-strain ciliates.
Phylogenetic studies revealed the presence of a
monophyletic group of eukaryotic Fe-hydroge-
nases in the bovine rumen. Supported by the EU
Contract QLRI-CT-2000-01455 “ERCULE”
and Contract QLK3-2002-02151 “CIMES”.
Study of two simple techniques for cryopreser-
vation of rumen ciliate protozoa. E. Nsabimanaa,
D. Macheboeufa, C.J. Newboldb, J.-P. Jouanya
(a INRA, CR de Clermont/Theix, 63122 St-
Genès-Champanelle, France; b University of
Wales, Aberystwyth, UK).
Rumen ciliates are difficult to cryopreserve. The
two-step freezing technique, which has been
applied successfully (Nsabimana et al. 2003,
Appl. Environ. Microbiol., 69, 3826–3832), is
complex and needs a special equipment to be
performed. Two simple techniques using
Bicell and M.Frosty freezing cells and allow-
ing the respective cooling rates of 0.5 °C/min
and 1 °C/min, were tested for their ability to
cryopreserve the following ciliate species:
Dasytricha ruminantium, Entodinium cauda-
tum, Epidinium ecaudatum caudatum, Eud-
iplodinium maggii, Isotricha intestinalis, Isot-
richa prostoma, Metadinium medium. The
ciliates were isolated from sheep monofaunated
with each species. Centrifuged fresh rumen fluid
was used as the freezing and thawing media.
Equilibration with DMSO (0.56 M) was set at
25 °C or 30 °C for 5 or 10 min before freezing
in the two tested freezing cells. Tubes containing
the ciliates were plunged into liquid nitrogen
when the freezing cells reached the temperature
–80 °C. The survival rate (SR) of ciliates was
determined from their motility. After two weeks
of storage in liquid nitrogen, the highest SR were
obtained with D. ruminantium (100%), I. pros-
toma (100%), I. intestinalis (100%), E. ecauda-
tum caudatum (87%) for the two freezing cells.
Bicell allowed a higher SR for M. medium (87
vs. 80%) and for E. maggii (94 vs. 76%), while
M.Frosty gave better results for E. caudatum
(64 vs. 40%). In conclusion, the two simple
freezing techniques Bicell and M.Frosty can be
applied to cryopreserve the rumen ciliates with
an acceptable rate of survival. This project was
supported by EU infrastructure grant ERCULE
(QLRI-CT-2000-01455) www.ercule.com.
The effect of temperature on the in vitro via-
bility of the mixed rumen protozoal popula-
tion. G. de la Fuentea, M. Pérez-Quintanab, J.A.
Cebriánc, M. Fondevilaa (a Departamento de
Producción Animal y Ciencia de los Alimentos y,
Universidad de Zaragoza, Spain; b Departamento
de Química y Biología, Universidad de Matanzas,
Matanzas, Cuba; c Departamento de Bioquímica
y Biología Molecular y Celular; Universidad de
Zaragoza, Miguel Servet 177, 50013 Zaragoza,
Spain). 
Survival of rumen protozoa is reduced at low
temperatures, making their storage in refrigera-
tion difficult and hampering their study. A
mixed rumen protozoal population (type A)
from sheep was anaerobically diluted 1:100 in
culture medium and kept at 38, 15 and 5 °C for
2, 4 or 6 h to determine the effect of temperature
on cell viability, according to an estimation of
membrane damage by a double-stain fluores-
cence method (5 tubes per treatment). Temper-
ature was reduced from 38 °C to 15 °C at
3 °C·min–1 and from 15 to 5 °C at 0.6 °C·min–1.
Apparently, all genera responded to both stains,
and Ophryoscolex showed an effect of temper-
ature (80.3, 55.4 and 54.5% viability at 38, 15
and 5 °C; P < 0.01). However, only Entodinium
cells were in a number high enough to be strictly
compared. The viability of the Entodinium spe-
cies in the rumen inoculum was 93.8 ± 2.69, and
remained between 90.5 and 79.8% when culti-
vated at 38 °C for 2 to 6 h. The proportion of non
damaged cells after 2 h at 15 or 5 °C did not
diminish (87.3 and 86.2%; P > 0.05). However,
viability was reduced (P < 0.05) to 61.3 and
59.9 % after 6 h at 15 °C and 5 °C (s.e.m. = 3.78).
The viability of Entodinium was reduced at
refrigeration temperatures; around 60% of the


English

Fe-Hydrogenases from  bovine rumen: a metagenomic approach

Concerns and Responses to Food Safety, Health and Environmental IssuesNational audienc

van Der Staay, G.W.M.

Moon-van Der Staay, S.Y.

de Graaf, R.M.

van Alen, T.A.

Mcewan, N.

Newbold, C.J.

Jouany, J Pierre

Ricard, G.

HAL Descartes

Fe-hydrogenases from bovine rumen : a metagenomic approach

NARCIS 

EDP Sciences OAI-PMH repository (1.2.0)

Concerns and Responses to Food Safety, Health and Environmental Issues

Radboud Repository (Radboud Univ.)

Gilbert, R. A.

Sheperd, J.

Klieve, A. V.

Nolan, J. V.

Newbold, C. J.

Wallace, R. J.

University of Queensland eSpace

Session I: Taxonomic and functional diversity of gut microbes S19
after the administration and comparison of 4 dif-
ferent diets. Thirty-two growing pigs were ran-
domly distributed into four treatments. The ani-
mals were fed ad libitum for 6 weeks with a
commercial diet (15% barley, 28% soya 44,
54% corn and vegetable oil (basal diet, A). Fine-
sized corn of basal diet (2.5 mm) was changed
to 4 mm corn (B treatment), and sugar beet pulp
(8%) or bran (10%) were added to the basal diet
in treatment C and D respectively. After the
experimental period, the animals were slaugh-
tered and the GI tract was sampled. Digesta sam-
ples were diluted and fixed with formaldehyde
prior to analysis. Fluorescent in situ hybridisation
(FISH) was used to quantify bacterial popula-
tions by using five probes: EUB338, EREC482,
RFLA729 plus RBRO730 and FPRA645 to
quantify total bacteria and the predominant
groups of low G+C Gram positive bacteria
belonging to clusters IV and XIVa in samples of
rectum digesta. Total bacteria were 1.98E11 ±
6.63E10 bacteria·g–1 fresh matter and cluster
XIVa and ruminococci were the predominant
bacterial groups in all treatments. Faecalibacte-
rium spp. represented 1.3 ± 0.3% of the total
bacterial population. The administration of these
diets did not significantly change the bacterial
populations of the pigs and each diet maintained
high proportions of the cluster IV and XIVa
groups. 
Effect of dietary changes on the bacteri-
ophage population in the rumen of sheep.
R.A. Gilberta, J. Shepherdb, A.V. Klievec, J.V.
Noland, C.J. Newboldb, R.J. Wallaceb (a CSIRO
Livestock Industries, Queensland Bioscience
Precinct, St Lucia, Qld 4067 Australia; b Rowett
Research Institute, Bucksburn, Aberdeen AB21
9SB, UK; c Animal Research Institute, Queens-
land Department of Primary Industries, Moorooka,
Queensland 4105, Australia; d University of
New England, Armidale NSW 2351, Australia).
The effect of alternating the diet from a hay-
based to a silage-based diet on the rumen phage
population of four cannulated sheep was exam-
ined in a 2 × 2 factorial design experiment. Two
animals were fed each diet during two 20 d feed-
ing periods, each consisting of an initial 14 d
feed adaptation period and 6 d monitoring
period, in which rumen fluid samples were col-
lected on days 15 and 20, immediately before
feeding and again at 2 h and 6 h after feeding.
Phage DNA extracted from rumen fluid samples
was used to determine rumen phage concentra-
tion, and phage population diversity was deter-
mined by pulsed field gel electrophoresis sepa-
ration of phage DNA. In addition, rumen bacteria
and protozoa were enumerated and pH, and con-
centrations of volatile fatty acids, ammonia and
L-lactate, were determined. No significant dif-
ferences occurred in either the size or diversity
of the rumen phage population between sheep
fed silage or hay-based diets. These findings
contrast with previous studies that have shown
the size of the rumen phage population to vary
dramatically between different dietary regimes.
Other parameters of fermentation and microbial
composition suggested that the rumen microbial
population may not have altered significantly
between the two diets and therefore did not cause
major changes in the size and relative composi-
tion of the rumen phage population.
Functional and ecological characterization
of newly isolated Fibrobacter succinogenes
strains in relation to their phylogeny. T.
Shinkai, N. Matsumoto, Y. Kobayashi (Gradu-
ate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan).
Fibrobacter succinogenes strains were newly
isolated from the rumen of sheep receiving
orchardgrass hay, orchardgrass pasturage, alfalfa
hay or rice straw. Then, rumen solid, liquid and
ruminally incubated forage stems were the bac-
terial sources. Thirty-three strains in total were
obtained mainly from ruminally incubated for-
age stems. Most of these (29 of 33) were affili-
ated with group 1, based on their partial 16S
rDNA sequences. Four strains were classified
into group 2, and then two of these formed a
novel sub-group, the branching of which was
supported by an 82% bootstrap value. Only these
two strains produced yellow pigments when cul-
tured with Avicel. Group 1 strains showed faster
growth to a higher extent than group 2 strains
when cultured with Avicel. Although there were
variations in number and molecular size of
CMCases that were detected on SDS-polyacry-
lamide gel by activity staining, group 2 strains
possessed 112kD CMCase as a common enzyme.
Meanwhile, strains belonging to groups 1 and 2,
when grown on Avicel, showed no particular
