IIIReprod. Nutr. Dev. 46 (2006) III–XXII© INRA, EDP Sciences, 2006

Contents

SESSION I: Microbial diversity in health and disease

Functional analysis of the GI-tract microbiota. C.C.G.M. BOOIJINK, E.G. ZOETENDAL, H. SMIDT, M. KLEEREBEZEM,W.M. DE VOS ......................................................................................................................... S3

Prokaryotic community of the mucosal fraction of human ileum: a metagenomic approach. M.C. LECLERC, P. ROBE, P. MARTEAU, R. NALIN, M. GELFAND, J. DORÉ ........................... S3

Identification DNA-microarray for the monitoring of intestinal bacteria during gastroenteritis. K. KNÖSCHE, T. HAIN, T. CHAKRABORTY, E. DOMANN, T.T. BACHMANN ........................... S3

Distribution of the main functional groups of microorganisms in the gut of IBS subjects. C. CHASSARD, P. MARQUET, C. DEL’HOMME, C. DUBRAY, K.P. SCOTT, H.J. FLINT,

M. DAPOIGNY, G. BOMMELAER, A. BERNALIER-DONADILLE ................................................ S4

Culture-independent analyses of the temporal variation of the dominant faecal microbiota, Clostridium spp., Bacteroides spp., in Crohn’s disease and colon cancer. P.D. SCANLAN, F. SHANAHAN, J.R. MARCHESI ..................................................................... S4

Serine protease autotransporters (SPATE) and autoaggregative adhesins from Escherichia coli are potential virulence factors in inflammatory bowel disease. D.O. KRAUSE, C.N. BERNSTEIN, S. SEPEHRI, R. KOTLOWSKI ............................................... S5

Exploration of the human intestinal microbiota during antibiotic administration using microbiomics approaches. J.K. JANSSON, C. JERNBERG, S. LÖFMARK, C. EDLUND ........................................................ S5

Murine gut microbiome: nature and nurture. M.K. FRISWELL, P. GILBERT, D.G. ALLISON, I.J.STRATFORD, B.A. TELFER,A.J. MCBAIN ........................................................................................................................... S6

Sequence variation among α-toxin of Clostridium perfringens strains isolated from diseased and healthy chickens. L. ABILDGAARD, R.M. ENGBERG, K. PEDERSEN, A. SCHRAMM, O. HØJBERG ...................... S6

The bacterial community of the horse stomach. R.A.M. AL JASSIM, S. DENMAN, J.D. HERNANDEZ, C.M. MCGOWAN, F.M. ANDREWS, C.S. MCSWEENEY ................................................................................................................... S7

Detection and activity of a bacteriocin produced by Lactobacillus strains. F.N. ALLOUCHE, A. HELLAL, A. LARABA .............................................................................. S7

Quantification of protozoal concentrations in the rumen and duodenum of growing lambs using real-time PCR: effect of age. A. BELANCHE, G. DE LA FUENTE, L. CALLEJA, D.R. YAÑEZ-RUIZ, C.J. NEWBOLD,J. BALCELLS, M. FONDEVILA ................................................................................................. S7

Article publié par EDP Sciences et disponible sur le site http://rnd.edpsciences.org ou http://dx.doi.org/10.1051/rnd/200646001

IV Contents

Identification of antibiotic resistant genes in Streptococcus thermophilus using microarray. G. BERRUTI, A.H.A.M. VAN HOEK, H.J.M. AARTS, L. TOSI, L. MORELLI, M. CALLEGARI ........................................................................................................................ S8

Optimizing growth conditions for ruminal ciliates in the in vitro system Rusitec. C.P.J. BONNET, L. MEILE, M. KREUZER, C.R. SOLIVA .......................................................... S8

Isolation of bacteriophage active against E. coli O157:H7 and Salmonella spp. from cattle and swine. T.R. CALLAWAY, T.S. EDRINGTON, A.D. BRABBAN, E.M. KUTTER, R.C. ANDERSON, R.B. HARVEY, D.J. NISBET ..................................................................................................... S9

An animal model that reproduces microbial disruption observed in the gut of IBS subjects: a new animal model for IBS?C. CHASSARD, C. DEL’HOMME, P. MARQUET, M. DAPOIGNY, G. BOMMELAER,A. BERNALIER-DONADILLE ..................................................................................................... S9

A comparison of Clostridium difficile carriage in inflammatory bowel disease and irritable bowel syndrome out-patients. E.M. CLAYTON, M.C. REA, F. SHANAHAN, E.M. QUIGLEY, C. HILL, R.P. ROSS .................. S10

Activity spectrum of reuterin, a broad spectrum antimicrobial produced by Lactobacillus reuteri from glycerol, on intestinal bacteria. V. CLEUSIX, G. LE BLAY, S. VOLLENWEIDER, C. LACROIX ................................................... S10

Lactobacillus reuteri decreases E. coli concentration in the presence of glycerol in an in vitro model of colonic fermentation.V. CLEUSIX, G. LE BLAY, S. VOLLENWEIDER, C. LACROIX ................................................... S11

Characterization of Lactobacillus microbiota from the duodenum of organically farmed pigs. P.S. COCCONCELLI, A. GENTILI, M.L. CALLEGARI ................................................................ S11

Monitoring major ruminal bacteria in sheep during adaptation to Neotyphodium coenophialum-infected tall fescue straw with real-time PCR. M.J.M. DE LORME, S.L. LODGE-IVEY, A.M. CRAIG .............................................................. S11

Phylogenetic analysis of TNT degrading enrichments isolated from ovine rumen fluid. M.J.M. DE LORME, A.M. CRAIG ............................................................................................ S12

Stx2 synthesis in pathogenic Escherichia coli: transcriptional analysis, secretion and activity. T. DE SABLET, Y. BERTIN, A. GOBERT, J.P. GIRARDEAU, C. MARTIN .................................. S12

Enumeration in cattle of verocytotoxigenic E. coli containing ehxA and eaeA virulence genes.S.E. DENMAN, R.A. GILBERT, J. PADMANABHA, C.S. MCSWEENEY ..................................... S13

The impact of lifestyle on the human child intestinal microbiota. J. DICKSVED, H. FLÖISTRUP, A. SCHEYNIUS, J.K. JANSSON ................................................... S13

Development and application of RNA and DNA based DGGE to study the bacterial colonisation of fresh perennial ryegrass in the rumen. J.E. EDWARDS, S.A. HUWS, E.J. KIM, A.H. KINGSTON-SMITH, N.D. SCOLLAN .................... S14

Contents V

A comparison of colon and faeces microbial diversities in horses using biomolecular techniques. C. FAUBLADIER, V. JULLIAND, L. VEIGA, F. CHAUCHEYRAS-DURAND ................................. S14

Molecular phylogeny of the Neocallimasticales (Mycota) based on ribosomal RNA genes: exploring the root of the fungal kingdom. K. FLIEGEROVÁ, K.VOIGT ...................................................................................................... S14

Effect of two energy and two protein sources in sugar cane based diets on the population of rumen ciliate protozoa in water buffalo (Bubalus bubalis) and zebu cattle (Bos taurus indicus). R. FRANZOLIN, F.P. ROSALES, W.V.B. SOARES ..................................................................... S15

Bacterial diversity and antibiotic resistance in colon contents from hooded seals (Cystophora cristata) in the Greenland Sea. T. GLAD, K.M. NIELSEN, L. NORDGÅRD, M.A. SUNDSET ..................................................... S15

Quantification and visualization of the uncultured bacterial groups U2 and U3 from the rumen. H. GOTO, H. YABUKI, T. SHINKAI, Y. KOBAYASHI ............................................................... S16

Molecular characterisation of the colonic mucosal associated flora.G.L. GREEN, J.D. SANDERSON, J. BROSTOFF, B.N. HUDSPITH, D.S. RAMPTON, K.D. BRUCE ............................................................................................................................ S16

Antimicrobial resistance of Escherichia coli and Enterococcus spp. from an integrated, semi-closed, swine and human population. R.B. HARVEY, H.M. SCOTT, W.Q. ALALI, L.D. HIGHFIELD, T.R. CALLAWAY, T.L. POOLE, R.C. ANDERSON, D.J. NISBET ............................................................................ S17

Enteral feeding, compared to parenteral feeding, enhances bacterial gut colonization in neonatal pigs. R.B. HARVEY, K. ANDREWS, R.E. DROLESKEY, K.V. KANSAGRA, B. STOLL, D.G. BURRIN, T.R. CALLAWAY, R.C. ANDERSON, D.J. NISBET ............................................ S17

Plasmid occurrence in local population of Selenomonas ruminantium is not affected by the presence of restriction and modification systems. J. IVAN, P. JAVORSKÝ, P. PRISTAŠ .......................................................................................... S17

Construction and function based screening of metagenomic libraries of the human gut microbiota. B.V. JONES, J.R. MARCHESI ................................................................................................... S18

Use of Ranikhet-inactivated vaccine combined with V4 HR and BCRDV for the prevention of viscerotropic velogenic Newcastle disease in backyard poultry. M.A. KAFI, M.M. AMIN, M.B. RAHMAN ............................................................................... S18

Swedish allergic infants have less diverse intestinal microbiota compared to healthy infants, at their age of one week. C. KARLSSON, M. WANG, C. OLSSON, G. MOLIN, A. WOLD, B. HESSELMAR, R. SAALMAN, I.-L. STRANNEGÅRD, N. ÅBERG, I. ADLERBERTH S. AHRNÉ ........................... S19

VI Contents

Effects of mastication on temporal bacterial colonisation and degradation of fresh perennial ryegrass in the rumen. E.J. KIM, J.E. EDWARDS, R. SANDERSON, A.H. KINGSTON-SMITH, N.D. SCOLLAN, M.K. THEODOROU ................................................................................................................... S19

Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater impacted by swine production facilities.

S. KOIKE, I.G. KRAPAC, H.D. OLIVER, J.C. CHEE-SANFORD, R.I. AMINOV, R.I. MACKIE ............................................................................................................................ S20

The effect of celiac diet on microbes in the colon.J. KOPE NÝ, J. MRÁZEK, K. FLIEGEROVÁ ............................................................................... S20

The incidence of Listeria monocytogenes in meat and meat products, Zagreb area, Croatia. I. KOVA EK, N. KNEŽEVI -JONJI , D. PUNTARI , J. BOŠNIR, B. MATICA, D. KOVA EK, N. URŠULIN-TRSTENJAK .......................................................................................................... S21

Analysis of the temporal stability and diversity of the bacteria in the human gastrointestinal tracts of healthy individuals using DGGE and RAPD PCR.S.F. LAWTON, J.R. MARCHESI ................................................................................................ S21

The antimicrobial peptide Pediocin PA-1 has no effect on common intestinal bacteria compared to nisin and antibiotics. G. LE BLAY, A. ZIHLER, C. LACROIX, I. FLISS ....................................................................... S21

Pediocin PA-1 is active against Listeria innocua in an in vitro model for the terminal ileum. G. LE BLAY, I. FLISS, C. LACROIX ......................................................................................... S22

Assessing the methanogen diversity of the human intestinal flora by targeting the mcrA gene.A. MIHAJLOVSKI, M. ALRIC, J.F. BRUGERE ........................................................................... S22

Evaluation of three techniques for the extraction of DNA from rumen fluid.S. MUETZEL, D. PAILLARD, R.J. WALLACE ............................................................................ S23

Effects of anti-Helicobacter pylori treatment and probiotic supplementation on intestinal microbiota. E. MYLLYLUOMA, T. AHLROOS, L. VEIJOLA, H. RAUTELIN, S. TYNKKYNEN,R. KORPELA ............................................................................................................................. S23

Multiple bacteriocin structural genes are present in animal gut cocci. K. NIGUTOVA, P. JAVORSKÝ, P. PRISTAŠ ................................................................................ S24

Molecular identification of methanogens from sheep from Venezuela.N. OBISPO, X. MA, A.-D.G. WRIGHT ..................................................................................... S24

High prevalence of oral tetracycline resistance genes in Bolivian Amerindians. I. PANTOJA, M. MOJICA, G. VARGAS-PINTO, K.P. SCOTT, A. PATTERSON, H.J. FLINT, M. BLASER, M.G. DOMINGUEZ-BELLO ................................................................................... S24

Detection of novel mosaic tetracycline resistance genes. A.J. PATTERSON, M.T. RINCON, H.J. FLINT, K.P. SCOTT ....................................................... S25

Č

Č Ć Ć Ć Č

Contents VII

Use of rifampicin-resistant Clostridium perfringens strains for infection studies on necrotic enteritis in broiler chickens.K. PEDERSEN, L. BJERRUM ..................................................................................................... S25

Mercury-induced shift in rumen protozoan Entodinium caudatum associated bacterial populations. M. PIKNOVA, J. MRAZEK, S. KISIDAYOVA, Z. VARADYOVA, T. TOTHOVA, P. JAVORSKÝ, P. PRISTAŠ ........................................................................................................ S25

Development of a molecular technique to study the equilibrium of poultry gut microbiota. C. PISSAVIN, I. GABRIEL, C. BUREL, S. MALLET, R. MAURICE, M. LESSIRE, P. FRAVALO ............................................................................................................................ S26

Characterization of the 16S-23S rRNA gene intergenic spacer region to develop probes for tracking probiotics in the reindeer rumen.K.E. PRÆSTENG, R.I. MACKIE, I.K.O. CANN, S.D. MATHIESEN, M.A. SUNDSET .................. S26

Comparative molecular analysis of an ovine ruminal enrichment culture using two different DNA separation techniques. R.M. RATTRAY, A.M. CRAIG ................................................................................................. S27

Helicobacter pylori status in an urban population is inversely associated with asthma. J. REIBMAN, M. MARMOR, M.-E. FERNANDEZ-BEROS, L. ROGERS, G.I. PEREZ-PEREZ, M.J. BLASER ........................................................................................................................... S27

A broad host range integration vector for bioluminescent in vivo imaging of bacteria. C.U. RIEDEL, S.C. CORR, I.R. MONK, C.G.M. GAHAN, C. HILL ........................................... S27

Identification of novel tetracycline resistance genes in the metagenome of the human colon-associated bacterial ecosystem. M.T. RINCON, K. KAZIMIERCZAK, P. YOUNG, D. HENDERSON, K.P. SCOTT, H.J. FLINT ............................................................................................................................... S27

Prevalence of Helicobacter pullorum in broiler chickens reared in northern Italy. M. ROSSI, V. SANGUINETTI, R. GAVIOLI, P. LOZITO, G. MANFREDA, R.G. ZANONI ............ S28

Study of spirillar enteric flora of dogs and cats in Italy. M. ROSSI, V. SANGUINETTI, D. GIACOMUCCI, P. ACUTIS, R.G. ZANONI .............................. S28

Influence of diet and sampling site on the diversity of the bacterial community attached to the rumen epithelium in lambs. S. SADET, C. MARTIN, B. MEUNIER, D.P. MORGAVI ............................................................. S29

Microbial community changes in the intestine of the pre-adolescent turkey. A.J. SCUPHAM ......................................................................................................................... S29

Type A Clostridium perfringens subtypes are differentiated between necrotic enteritis and healthy broiler intestinal isolates and are distributed among and between hosts. G.R. SIRAGUSA, M.G. WISE ................................................................................................... S30

Engineering improved tolerance to stresses encountered in the gastrointestinal tract. R.D. SLEATOR, C. HILL .......................................................................................................... S30

VIII Contents

Antibiotic resistance, resistance genes and transfer in animal gut cocci. V. STOVCIK, P. JAVORSKÝ, P. PRISTAŠ ................................................................................... S31

The prevalence of mobile gene cassettes in indigenous gut microbiota and dairy starter bacteria. J. ŠTŠEPETOVA, E. SEPP, K. TRUUSALU, K. LÕIVUKENE, P. HÜTT, E. SONGISEPP, M. MIKELSAAR ........................................................................................................................ S31

Resistance of Methanothermobacter thermautotrophicus to therapeutics and antimicrobial substances. S. ŠURÍN, L. UBO OVÁ, A. MAJERNÍK, P. MCDERMOTT, J.P.J. CHONG, P. ŠMIGÁ .......... S31

Control of Campylobacter jejuni colonization in chickens treated with probiotics or bacteriocins. E.A. SVETOCH, B.V. ERUSLANOV, V.D. POKHILENKO, Y.N. KOVALEV, L.I. VOLODINA, V.V. PERELYGIN, E.V. MITSEVICH, I.P. MITSEVICH, V.N. BORZENKOV, V.P. LEVCHUK, O.E. SVETOCH, T.Y. KUDRIAVTSEVA, N.J. STERN ....................................... S32

Comparing gut microflora in pigs by using an integrated probing system. N. THANANTONG, A. ANDERSON, S. EDWARDS, O.A.E. SPARAGANO ................................... S32

In vitro evolution of the equine gastric microflora. M. VARLOUD, V. JULLIAND .................................................................................................... S33

Prevalence of two Lactobacillus and Bifidobacterium probiotic strains in the neonatal ileum. R. WALL, G. FITZGERALD, S.G. HUSSEY, C.A. RYAN, M. O’NEILL, R.P. ROSS, C. STANTON ............................................................................................................................ S33

Enrichment in a complex medium of a novel bacterial group from the rumen of wild sika deer as confirmed by real-time PCR assays and FISH detection. H. YAMANO, K. MIYAWAKI, Y. SAWABE, Y. KOBAYASHI ..................................................... S34

Sequencing and annotation of pCY360, a megaplasmid from Clostridium proteoclasticum B316T. C. YEOMAN, W.J. KELLY, J. RAKONJAC, G.T. ATTWOOD ...................................................... S34

Genetic diversity of the microbial community in ileostomy patients. E.G. ZOETENDAL, C.C.G.M. BOOIJINK, S. EL AIDY, H. SMIDT, M. KLEEREBEZEM, W.M. DE VOS ......................................................................................................................... S35

Long-chain fatty acids and fatty aldehydes in rumen bacterial genera Butyrivibrio and Pseudobutyrivibrio. M. ZOREC, R. MARINSEK-LOGAR ........................................................................................... S35

SESSION II: Microbial metabolism of dietary components

Microarray gene expression profiling of Ruminococcus flavefaciens FD-1 grown on cellobiose or cellulose. M.E. BERG, D.A. ANTONOPOULOS, M. RINCON, M. BAND, A. BARI, T. AKRAIKO, A. HERNANDEZ, R. KIM, L. LIU, J. THIMMAPURAM, I. BOROVOK, S. JINDOU, R. LAMED, H.J. FLINT, E.A. BAYER, B.A. WHITE .................................................................. S39

Č Ň Ň

Contents IX

Analysis of Methanobrevibacter ruminantium genome sequence.W.J. KELLY, G.T. ATTWOOD .................................................................................................. S39

Post-genomics approaches to study the functionality of bifidobacteria in infant faeces. E.S. KLAASSENS, R. BOESTEN, F.H.J. SCHUREN, E.E. VAUGHAN, W.M. DE VOS ................ S39

Impact of reducing dietary carbohydrate intake upon the faecal microbial community and metabolites in human volunteers.S.H. DUNCAN, A.M. JOHNSTONE, G.E. LOBLEY, G. HOLTROP, A. BELENGUER, D. BREMNER, M. HRDINOVA, H.J. FLINT ............................................................................... S40

Contribution of ruminal protozoa to duodenal flow of N, CLA and TVA in steers fed silage differing in water-soluble carbohydrates. D.R. YÁÑEZ-RUIZ, N.D. SCOLLAN, R.J. MERRY, C.J. NEWBOLD .......................................... S40

Metabolism of linoleic acid by bacterial species isolated from the human gut: biosynthesis of conjugated linoleic and hydroxy fatty acids. E. DEVILLARD, F.M. MCINTOSH, S.H. DUNCAN, H.J. FLINT, R.J. WALLACE ....................... S41

Effect of polyethylene glycol on in vitro gas production and digestibility of tannin-rich feedstuffs from North Africa arid zone. R. ARHAB, M. RIRA, D. MACHEBOEUF, H. BOUSSEBOUA ..................................................... S41

In vitro ruminal fermentation of some desertic browse species and date palm by-products. R. ARHAB, D. MACHEBOEUF, R. BERGEAULT, H. BOUSSEBOUA ........................................... S42

The diversity and prevalence of conjugated linoleic acid-producing Bifidobacterium species in the human gastrointestinal tract. E. BARRETT , R.P. ROSS , G.F. FITZGERALD , F. SHANAHAN , C. STANTON .......................... S42

Alternative mechanisms of metabolic cross-feeding between human gut bacteria. A. BELENGUER, S.H. DUNCAN, G. CALDER, G. HOLTROP, P. LOUIS, G.E. LOBLEY, H.J. FLINT ............................................................................................................................... S43

The ability of the rumen ciliate Diploplastron affine to digest and use 1,3-β-D-glucans. G. BE ECKI, R. MILTKO, T. MICHA OWSKI ......................................................................... S43

Interaction of buckwheat seed extracts and intestinal microflora regarding changesof bacterial profile of intestinal microflora and antioxidant status of extracts.E. BIEDRZYCKA, L. MARKIEWICZ, M. BIELECKA, R. AMAROWICZ ....................................... S44

Accumulation of rumen biohydrogenation intermediates through grazing of botanically diverse pastures is associated with changes in the rumen protozoal population. C. BOECKAERT , N. BOON, M. LOURENÇO, W. VERSTRAETE, V. FIEVEZ .............................. S44

Rapid screening of bacterial genes coding histidine and tyrosine decarboxylase using PCR. R. BURDYCHOVA, A. SPANOVA, B. RITTICH, T. KOMPRDA ................................................... S44

Identification and characterisation of xylan-degrading bacteria from the human colon. C. CHASSARD, M. ADAMS-LECLERC, A. BERNALIER-DONADILLE ........................................ S45

Molecular quantification of the human gut acetogen, R. hydrogenotrophicus, in faecal samples using real-time quantitative PCR. C. DEL’HOMME, E. DELMAS, Y. LEBLOND, A. BERNALIER DONADILLE ............................... S45

Ł Ż Ł

X Contents

Role of ciliate protozoa in the metabolism of unsaturated fatty acids in the rumen. E. DEVILLARD, F.M. MCINTOSH, C.J. NEWBOLD, K. YOUNG, M. BARBIER, R.J. WALLACE ......................................................................................................................... S46

Differential export of two Ruminococcus albus cellulases by the Tat and Sec translocation pathways of Escherichia coli. J. ESBELIN, C. MARTIN, E. FORANO, P. MOSONI .................................................................... S46

Cross-feeding between Bifidobacterium longum BB536 and acetate-converting, butyrate-producing colon bacteria during growth on oligofructose. G. FALONY, A. VLACHOU, K. VERBRUGGHE, L. DE VUYST .................................................. S47

Bile tolerance mechanisms of Listeria monocytogenes: implications for survival in the host GI tract. C.G. GAHAN, R.D. SLEATOR, M. BEGLEY, C. HILL ............................................................... S47

Comparison of microbial pellets for estimation of microbial protein synthesis in continuous-culture fermenters. A.I.M. GARCÍA, M.J. RANILLA, E.M. ALCAIDE, M.D. CARRO ............................................... S48

Antioxidant activity and total polyphenol content studies in selected products using an in vitro digestion tract model with human faecal flora. M. GUMIENNA, K. GODERSKA, Z. CZARNECKI ....................................................................... S48

Effect of defaunating the rumen on microbial protein synthesis in native Iranian sheep. K.K. JAFARI, M. REZAEIAN, M. ZAHEDIFAR, A. MIRHADI ..................................................... S49

Strain-specific architecture in cellulosome assembly in Ruminococcus flavefaciens. S. JINDOU, I. BOROVOK, M. RINCON, H.J. FLINT, M. BERG, B.A. WHITE, E.A. BAYER, R. LAMED. ......................................................................................................... S49

Monitoring of rumen bacteria attached to ruminally incubated rice straw quantified by real-time PCR. S. KOIKE, H. YABUKI, Y. KOBAYASHI .................................................................................... S49

Molecular identification of carbohydrate-fermenting bacteria in the human colonby RNA-stable isotope probing.P. KOVATCHEVA-DATCHARY, M. EGERT, A.A. DE GRAAF, N.E.P. DEUTZ, A. MAATHUIS, H. SMIDT, W.M. DE VOS, K. VENEMA .................................................................................... S50

Evaluation of the effect of tannin-rich plants on rumen microbial fermentation. C. LONGO, J. HUMMEL, J. LIEBICH, R.S. DIAS, E.F. NOZELLA, S.L.S. CABRAL FILHO, P. BURAUEL, A.L. ABDALLA, K.-H. SÜDEKUM ...................................................................... S50

Butyrate metabolism in human colonic bacteria: characterisation of a novel CoA-transferase.P. LOUIS, C. CHARRIER, H.J. FLINT ........................................................................................ S51

Retention time of substrate affects fermentation characteristics in Rusitec fermenters. M.E. MARTÍNEZ, S. RAMOS, M.L. TEJIDO, M.J. RANILLA, L.A. GIRALDO, M.D. CARRO ............................................................................................................................ S51

NMR study of plant fibre degradation by Ruminococcus albus and Fibrobacter succinogenes. M. MATULOVA, R. NOUAILLE , P. CAPEK, M. PÉAN, A.-M. DELORT, E. FORANO ................ S51

Contents XI

Variations in intermediates and products of linoleic acid biohydrogenation by two different human mixed colonic flora. F.M. MCINTOSH, R.J. WALLACE, E. DEVILLARD ................................................................... S52

The existence and composition of distinctive bacterial communities colonising each of three insoluble dietary substrates in a model of the human colon. E.C. MCWILLIAM LEITCH, A.W. WALKER, S.H. DUNCAN, H.J. FLINT ................................. S52

Isolation of xylanase genes from two new xylan-degrading species from the human colon, Bacteroides sp. and Roseburia intestinalis.C. MIRANDE, C. CHASSARD, A. BERNALIER-DONADILLE, E. FORANO, C. BÉRA-MAILLET .................................................................................................................. S53

Effect of red beet juice and crisps from red beet on the growth of intestinal microorganisms and antioxidant stability. M. NEUMANN, W. GRAJEK ..................................................................................................... S53

Binding and biotransformation of the Fusarium mycotoxin zearalenone to α-zearalenol by lactic acid bacteria. V. NIDERKORN, H. BOUDRA, D.P. MORGAVI ......................................................................... S54

Ability of lactic acid bacteria to bind or/and metabolize N-nitrosodimethylamine (NDMA) connected with growth stages of the bacteria. A. NOWAK, Z. LIBUDZISZ ....................................................................................................... S54

Influence of heterocyclic aromatic amines (HCA) on growth and survival of Lactobacillus strains. A. NOWAK, Z. LIBUDZISZ ....................................................................................................... S54

Activation of pro-estrogens from hops by intestinal bacteria. S. POSSEMIERS, S. BOLCA , A. HEYERICK, W. VERSTRAETE ................................................. S55

Proteomics-based investigation of major metabolic pathways of a human gut anaerobe, Fusobacterium varium. J. POTRYKUS , S.L. BEARNE, R.L. WHITE .............................................................................. S55

Effects of dilution rate on methane production and fibre degradability in Rusitec fermenters fed a high-concentrate diet. S. RAMOS, M.E. MARTÍNEZ, M.J. RANILLA, M.L. TEJIDO, L.A. GIRALDO, M.D. CARRO ........................................................................................................................... S56

Metabolism of the pyrrolizidine alkaloid senecionine by an enrichment of ovine ruminal microbes as detected by high-performance liquid chromatography and mass spectrometry. R.M. RATTRAY, J.M. DURINGER, A.M. CRAIG ...................................................................... S56

Novel structural and catalytic elements in the Ruminococcus flavefaciens cellulosome revealed by genome analysis.M. RINCON, I. BOROVOK, M.E. BERG, D.A. ANTONOPOULOS, R. KIM, L. LIU, J. THIMMAPURAM, S. JINDOU, R. LAMED, H.J. FLINT, E.A. BAYER, B.A. WHITE ................ S57

Polysaccharidases isolated from the rumen metagenome. M.T. RINCON, D. HENDERSON, J.C. MARTIN, K.P. SCOTT, H.J. FLINT ................................. S57

XII Contents

Transcriptome analysis of the cluster XIVa human colonic anaerobe Roseburia inulinivorans. K.P. SCOTT, J. MARTIN, J. POTRYKUS, G. CAMPBELL, M. RINCON, G. RUCKLIDGE, C.-D. MAYER, H.J. FLINT ....................................................................................................... S58

Localization of cellulolytic rumen bacteria on the plant fibrous materials determinedby a newly developed fluorescent in situ hybridization protocol. T. SHINKAI, Y. KOBAYASHI .................................................................................................... S58

Detection and quantification of chitinases in the human fecal chitinolytic bacterium Clostridium paraputrificum J4. J. ŠIM NEK, G. TISHCHENKO, H. BARTO OVÁ ...................................................................... S58

Fermentation activity of intestinal Lactobacillus strains and end products in the presence of fructooligosaccharides. K. LI EWSKA ......................................................................................................................... S59

The influence of non-enzymatically glycosylated potato proteins on the survival and metabolic activity of chosen bacteria.D. WI TECKA, K. MARCINIAK-DARMOCHWA , H. KOSTYRA, A. WI TECKI ...................... S59

Metabolism of the food associated carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) by human intestinal microbiota. L. VANHAECKE, T. VAN DE WIELE, W. VERSTRAETE ............................................................ S60

Determination of breakdown pathway of tryptophan and indole acetic acid to skatole by Clostridium scatologenes and swine manure slurry. T.R. WHITEHEAD, N. PRICE, M.A. COTTA ............................................................................. S60

SESSION III: Microbial interactions with the host

Comparison of direct-fed microbial and antibiotic supplementation on innate and adaptive immune characteristics of weanling pigs. M.E. DAVIS, D.C. BROWN, M.S. DIRAIN, H.D. DAWSON, C. MAXWELL, T. REHBERGER ......................................................................................................................... S63

Intestinal microbial composition affects the ratio of digestive ileal brush border enzyme activity to gene expression in the gnotobiotic pig.B.P. WILLING, A.G. VAN KESSEL ........................................................................................... S63

Interactions between the mucin-degrader Akkermansia muciniphila and host cells in the GI tract. M. DERRIEN, E.G. ZOETENDAL, E. NORIN, W.M. DE VOS .................................................... S63

Engineering of the human commensal Bacteroides ovatus for the controlled in situ delivery of immunomodulatory proteins. Z.Z.R. HAMADY, M.D. FARRAR, T. WHITEHEAD, K.T. HOLLAND, J.P. LODGE, S.R. CARDING .......................................................................................................................... S64

U° Ň

Ś Ż

Ś Ą Ł Ś Ą

Contents XIII

Probiotic bacteria impede Listeria monocytogenes infection and modulate the mucosal immune response. S.C. CORR , C. HILL , C.G.M. GAHAN ................................................................................... S64

Enterohaemorrhagic Escherichia coli inhibit nitric oxide synthesis by activated human intestinal epithelial cells. M. VAREILLE, T. DE SABLET, A. DURAND, C. MARTIN, A.P. GOBERT ................................. S65

Resident intestinal bacteria direct expression of a bactericidal lectin. H.L. CASH, C.V. WHITHAM, C.L. BEHRENDT, L.V. HOOPER ................................................ S65

Characterisation of the effects of lactobacilli on the integrity of intestinal epithelial cells. R.C. ANDERSON, A.L. COOKSON, R.D. HURST , L.A. CASSIDY, W.C. MCNABB, N.C. ROY ................................................................................................................................ S66

A tool for studying adherence of intestinal pathogens on intestinal mucus – opportunities for screening products for pathogen inhibition in various hosts. J. APAJALAHTI, O. SIIKANEN, L. PURKAMO, M. LAURAEUS .................................................. S66

Effect of inoculation with intestinal bacteria on bodyweight and intestinal inflammationin the interleukin-10 knockout mouse. M.P.G. BARNETT, S.T. ZHU, A. COOKSON, R. BROADHURST, B. KNOCH, W.C. MCNABB, N.C. ROY ...................................................................................................... S67

Effect of selected strains of lactic acid bacteria on non-specific cellular and humoral defence mechanisms of healthy and Salmonella-infected chickens. M. BIELECKA, A.K. SIWICKI, E. BIEDRZYCKA, R. WÓJCIK, W. SMORAGIEWICZ, A. OR OWSKI, A. MAJKOWSKA .............................................................................................. S67

Probiotic bacteria in early defence against viral infection. T. BOTI , M. IVEC, S. KOREN, M. JAKOBSEN, H. WEINGARTL, A. CENCI ........................... S68

Adaptation of adhesion test on model epithelial cells for anaerobic bacteria. T. EPELJNIK, M. NARAT, B. LAH, B. DOLENEC, R. MARINŠEK-LOGAR ............................... S68

Microbial extinctions and modern diseases. M.G. DOMÍNGUEZ-BELLO ...................................................................................................... S68

Probiotic bacteria alter intestinal absorption and metabolism of aflatoxin B1 in vitro. S. GRATZ , H. MYKKÄNEN, H. EL-NEZAMI ............................................................................ S69

Inhibition of human atopic dermatitis like skin lesion by the administration of Lactobacillus johnsonii NCC533 (La1) during weaning period in a model mouse, NC/Nga – the predicted inhibitory mechanism of lesion by La1 from gene expression analyses. R. INOUE, M. OTSUKA, A. NISHIO, Y. NAKAMURA, Y. FUKUSHIMA, K. USHIDA ................. S69

Rose hip and lactobacilli used in an ischemia-reperfusion model in mice to suppress the intestinal injury. M. JAKESEVIC, Å. HÅKANSSON, D. ADAVI, S. AHRNÉ, B. JEPPSSON, G. MOLIN .................. S70

Serological and genetic markers in Crohn’s disease patients and their healthy relatives. M. JOOSSENS, S. JOOSSENS, N. VAN SCHUERBEEK, K. CLAES, P. RUTGEERTS,S. VERMEIRE ........................................................................................................................... S70

Ł

Č Č

Č

XIV Contents

Study of pathogenesis of Helicobacter pylori in Iran.P. KHAKI, S.M. BIDHENDI, H. ELIASI, M. ALIMORADI ........................................................... S70

Comparison of the influence of propolis and virginiamycin on broiler performance and immune response to ND vaccine. S.M.M. KIAEI, M. MODIRSANEI, H. BOZORGMEHERIFARD, B. MANSOORI, B. GHOLAMIAN. A. GHALYANCHI, M. RABANI ...................................................................... S71

Time-course of the progression of intestinal inflammation in interleukin-10 knockout mice inoculated with intestinal bacteria. B. KNOCH , M.P.G. BARNETT, S.T. ZHU, R. BROADHURST, A. COOKSON, W.C. MCNABB, S.O. KNOWLES, C.E. UGARTE, N.C. ROY .................................................... S71

Isolation and partial purification of chicken intestinal mucus for use in the study of attachment of Lactobacilli to the poultry GI tract. K. MCGREEGHAN-CROSBY, J.K. THOMPSON, C. NICHOLSON, M.A. COLLINS ...................... S72

Cloning of the murC gene in order to study its putative role in attachment of Lactobacilli to poultry GI tract epithelium. K. MCGREEGHAN-CROSBY, J.K. THOMPSON, M.A. COLLINS ................................................ S72

The aerobic/anaerobic cell culture model for studies on bacterial adhesion. A. OLEJNIK, M. LEWANDOWSKA, W. GRAJEK ........................................................................ S73

Intestinal epithelial cell responses to Lactobacillus plantarum. S. PAVAN , J.J.M. VAN DE SANDT, K. VENEMA , M. KLEEREBEZEM ..................................... S73

Daily oral supplementation with L. paracasei CNCM I-2116 leads to a down- modulation of milk protein hypersensitivity in mice.S. PECQUET, S. CHIBANI-CHENNOUFI, F. ROCHAT, A. MERCENIER ....................................... S73

An immunoblot for the determination of antibodies against Lactobacilli. A.-L. PRANGLI, M. UTT, R. UIBO ........................................................................................... S74

Mannan induced changes in cytokine expression and growth of enteropathogenic E. coli-challenged broilers. P. SINGBOOTTRA, F.W. EDENS, A. KOCHER ........................................................................... S74

Interleukin-18 levels in the small bowel after oral infection with Salmonella. A. SPLICHALOVA, I. TREBICHAVSKY, I. RYCHLIK, Y. MUNETA, Y. MORI, I. SPLICHAL ............................................................................................................................. S75

The preventive effect of Enterococcus faecalis cell preparation (EC-12) against the experimental infection of porcine edema disease. T. TSUKAHARA , N. NAKANISHI, C. KAMEUE, K. NAKAYAMA, N. MATSUBARA, M. HAMASAKI, K. USHIDA ...................................................................................................... S75

Modulating mucin dynamics using functional carbohydrates. Z. UNI, A. SMIRNOV ................................................................................................................ S76

Oral dose of Lactobacillus plantarum and Megasphaera elsdenii increased intestinal IgA secretion, colonic butyrate and growth of colonic mucosa in piglets.K. USHIDA, R. INOUE, N. SHIMOJO, S. TAKAHASHI, C. KAMEUE, T. TSUKAHARA ................ S76

Contents XV

Adhesion of Bifidobacterium strains to intestinal mucus and its influence on competitive exclusion of Escherichia coli from the intestine. E. WASILEWSKA, M. BIELECKA ............................................................................................. S77

Disappearance of purified plasma IgG from the rumen. Y.J. WILLIAMS, S. POPOVSKI, S. REA, A.D.G. WRIGHT ........................................................ S77

SESSION IV: Nutritional manipulation

Effects of mannan-oligosaccharide-based yeast cell wall preparations on the prevalence of antibiotic resistant bacteria in axenic culture of intestinal bacteria: are there strategies for decreasing the prevalence of antibiotic resistant bacteria in the intestinal tract? S.M. SCHEUREN-PORTOCARRERO, M.M. NEWMAN, K.A. DAWSON, C.A. MORAN .............. S81

SAFEWASTES’ by-products as potential preventives of intestinal adhesion of bacteria.P.M. BECKER, S. GALLETTI, J. VAN DER MEULEN ................................................................ S81

Effect of diet and protozoa on the community composition of feed-adherent bacteria in the rumen. D.P. MORGAVI, D. GRAVIOU, M.J. RANILLA, C. MARTIN ..................................................... S81

Inulin and Lactobacillus amylovorus supplemented to human gut microbiota lower the microbial bioactivation of dietary aromatic contaminants to estrogenic metabolites. T. VAN DE WIELE, L. VANHAECKE, H. JACOBS, W. VERSTRAETE ........................................ S82

Quantification of faecal Bifidobacterium and Lactobacillus species in infants receiving a prebiotic infant formula. M. HAARMAN, B. STAHL, G. BOEHM, J. KNOL ...................................................................... S82

PCR-DGGE and real-time PCR analyses of total bacteria, Bifidobacteria and Lactobacilli populations in human subjects consuming calcium gluconate. K. ANDERSON, J. CHEN, Z. YU, J. JENKINS, P. COURTNEY, M. MORRISON ........................... S83

Effect of antibiotics in diets and level of feeding on caecal microbial biodiversity in lactating does as estimated by DGGE. L. ABECIA, N.R. MCEWAN, J. BALCELLS, G.E. LOBLEY, M. FONDEVILA ............................. S83

Microbial biodiversity in the caecum of litters from lactating does given antibiotics in the diet to manipulate their digestive population. L. ABECIA, N.R. MCEWAN, J. BALCELLS, E. SOLANAS, G.E. LOBLEY, M. FONDEVILA ........................................................................................................................ S84

Rumen microbial population characteristics and detection of transgenic DNA in rumen bacteria from sheep fed Bt176 maize for three years. G. ACUTI, M. TRABALZA-MARINUCCI, E. FORANO, P. MOSONI, L. MUGHETTI, S. COSTARELLI, C. RONDINI ................................................................................................... S84

Detection of recombinant maize DNA in rumen contents of dairy cows and in mixed ruminal cultures. G. ACUTI, P. MOSONI, M. TRABALZA-MARINUCCI, C. ANTONINI, E. FORANO ..................... S85

XVI Contents

A new promising promoter for the production of prebiotic polysaccharides from sucrose or maltose using recombinant bacteria and yeasts. T. ALAMÄE, K. TAMMUS, J. LASHMANOVA, T. VISNAPUU .................................................... S85

Effect of oral nitroethane administration on ruminal nitroethane reduction and methane production in cattle. R.C. ANDERSON, G.E. CARSTENS, E.G. BROWN, J.L. MCREYNOLDS, L.J. SLAY, T.R. CALLAWAY, D.J. NISBET ................................................................................................ S86

The microbiological contamination of grains used in the production of probiotics for broiler chickens. J. BIERNASIAK, M. PIOTROWSKA, K. SLIZEWSKA, Z. LIBUDZISZ ........................................... S86

Survival of probiotics in a dynamic artificial gastrointestinal system.S. BLANQUET, S. DENIS, V. ROUSSEAU, F. PAUL, S. HOLOWACZ, G. GARRAIT, B. HÉBRARD, E. BEYSSAC, M. ALRIC ..................................................................................... S87

Bioavailability of phytoestrogens from soy and hops. S. BOLCA , S. POSSEMIERS, A. HEYERICK, D. DE KEUKELEIRE, H. DEPYPERE, M. BRACKE, I. HUYBRECHTS, S. DE HENAUW, W. VERSTRAETE ........................................... S87

Effects of a gluco-oligosaccharide on performance and gut microflora in weaned piglets. M.L. CALLEGARI, S. SOLDI, F. ROSSI, M. MORLACCHINI, P. GATTI, L. MORELLI ............................................................................................................................. S87

A combination of inulin and type 3 resistant starch alters bacterial fermentation characteristics in the rat cecum and colon. D.J. CAMPBELL, P.R. HEAVY, F. BROUNS, A. ADAM, S. SILVI, C. COUDRAY, R.M. HUGHES, I.R. ROWLAND ................................................................................................ S88

Fermented liquid feed and fermented cereal grains – microbial composition and effect on the gastrointestinal ecology of piglets. N. CANIBE, O. HØJBERG, B.B. JENSEN ................................................................................... S88

In vivo and in vitro characterisation of antibiotic safety and competitive exclusion properties of Lactobacillus salivarius and Enterococcus faecium. A.J. CARTER , R.M. LA RAGIONE, V. PERRETON, M. ADAMS, M.J. WOODWARD ................ S89

Fungal colonisation of alkali treated wheat straw in the rumen of sheep. A.S. CHAUDHRY ...................................................................................................................... S89

The effect of two different types of arabinoxylanoligosaccharides on the level of human faecal bifidobacteria determined using real-time PCR.L. CLOETENS, V. DE PRETER, K. SWENNEN, T. VAN DE WIELE, W. VERSTRAETE, W.F. BROEKAERT, C.M. COURTIN, J.A. DELCOUR, P. RUTGEERTS, K. VERBEKE ................. S90

Molecular and functional characterisation of new Lactobacillus isolates from Bulgarian dairy products. S.T. DANOVA, R.N. ALEKSANDROVA, I.N. ILIEV ................................................................... S90

Effect of dietary intervention with different pre- and probiotics on intestinal bacterial enzyme activities. V. DE PRETER, L. CLOETENS, E. HOUBEN, P. RUTGEERTS, K. VERBEKE .............................. S91

Contents XVII

Effect of short- and long-term dietary intake of oligofructose-enriched inulin on the colonic fate of NH3 in healthy volunteers. V. DE PRETER, L. CLOETENS, P. RUTGEERTS, K. VERBEKE .................................................. S91

Effect of short- and long-term administration of Lactobacillus casei Shirota, oligofructose-enriched inulin and their synbiotic combination on the colonic fate of ammonia. V. DE PRETER, L. CLOETENS, P. RUTGEERTS, K. VERBEKE .................................................. S92

Effect of crude linseeds, extruded linseeds or linseed oil on digestion and fatty acid metabolism in dry cows. M. DOREAU, E. AUROUSSEAU, S. GACHON, F. GLASSER, J. NORMAND, G. CHESNEAU, C. MARTIN ...................................................................................................... S92

Effect of essential oils on degradation of starch-rich substrates in a rumen simulating fermenter (Rusitec). S.M. DUVAL, R.J. WALLACE, C.J. NEWBOLD ........................................................................ S93

Fermented feed for laying hens. R.M. ENGBERG, N.F. JOHANSEN, B.B. JENSEN ...................................................................... S93

Effect of a Saccharomyces cerevisiae supplementation on microbial profiles and fermentation patterns in faeces of horses under transportation. C. FAUBLADIER, F. CHAUCHEYRAS-DURAND , L. VEIGA , V. JULLIAND ............................... S93

Capric acid concomitantly affects rumen methanogenesis and biohydrogenation of linoleic and linolenic acid.V. FIEVEZ, C. BOECKAERT, G. BRUGGEMAN, K. DESCHEPPER .............................................. S94

Monitoring the bacterial population changes in the rumen of the camel associated with change in diet by real-time PCR. M.B. GHALI, P.T. SCOTT, G.A. ALHADRAMI, R.A.M. AL JASSIM ......................................... S94

Post-weaning maturation of rabbit caecal microbial communities: impact of live yeast intake. T. GIDENNE, N. BENNEGADI-LAURENT, V. MONTEILS, G. FONTY ........................................ S95

Digestive fate of Saccharomyces cerevisiae CBS 493 94, fed at 3 different concentrations to horses. J. GOBERT, G. BERTIN, V. JULLIAND ..................................................................................... S95

Shifts of rumen microbial population detected by real-time PCR when methanogens are inhibited.Y.Q. GUO, J.X. LIU, W.Y. ZHU .............................................................................................. S96

Effects of urea and controlled release non-protein nitrogen on ruminal fermentation and microbial protein synthesis in rumen-simulating cultures. G.A. HARRISON, J.M. TRICARICO, K.A. DAWSON ................................................................. S96

Effect of allicin on fermentation and microbial populations in the rumen simulating fermentor Rusitec.K.J. HART, S.E. GIRDWOOD, S. TAYLOR, D.R. YÁÑEZ-RUIZ, C.J. NEWBOLD ...................... S97

Recovery and phylogenetic analysis of the fumarate reductase gene from the bovine rumen. K. HATTORI, H. MATSUI ......................................................................................................... S97

XVIII Contents

Bellis perennis tested as new antiprotozoal feed additive in vivo achieved a persistent partial defaunation in sheep. E.M. HOFFMANN, N. SELJE, R.J. WALLACE, K. BECKER ....................................................... S97

Coarse structured feed stimulates members of the genera Lactobacillus and Mitsuokella as well as propionate and butyrate producers in the pig stomach. O. HØJBERG, L.L. MIKKELSEN, B.B. JENSEN ......................................................................... S98

Fatty acid composition of rumen protozoa: a link with chloroplast ingestion? S.A. HUWS, E.J. KIM, M.R.F. LEE, A.K. KINGSTON-SMITH, N.D. SCOLLAN ........................ S98

Effect of forage type and level of fish oil inclusion on bacterial diversity in the rumen. S.A. HUWS, M.R.F. LEE, S. MUETZEL, R.J. WALLACE, N.D. SCOLLAN ................................ S99

Monitoring of bioactive peptides isolated from artisanal Balkan yogurts. I. IVANOVA, I. TZVETKOVA, N. TZEKOVA, M. DALGALARRONDO, T. HAERTLE .................... S99

Characterization and possible probiotic influence of Bacillus smithii strain LTN074 in a murine model. E. JÕGI, I. SUITSO, E. TALPSEP, P. NAABER, A. NURK ........................................................... S99

Changes in rumen pH and population density of Streptococcus bovis in sheep during dietary transition. F.M. JONES, N.D. COSTA, K.R. GUEST, P.E. VERCOE, R.H. JACOB, J.M. SNOWDEN, A.-D.G. WRIGHT ..................................................................................................................... S100

Determination of the effective dose of Saccharomyces cerevisiae CBS 493.94 used as microbial additive for horses. J.-P. JOUANY, B. MEDINA , V. JULLIAND, G. BERTIN ............................................................. S100

Dietary essential oil supplementation enhanced intestinal immunocompetence in young broiler chicks. H. KETTUNEN, A. OUWEHAND, H. SCHULZE, N. RAUTONEN ................................................. S101

Probiotics in treatment of patients with Crohn’s disease and ulcerative colitis. P. KOHOUT, D. TUÈEK, V. ZBOØIL, Z. BENEŠ ........................................................................ S101

The influence of dairy season on the safety of sheep milk.M. LADYKOVÁ ........................................................................................................................ S101

Diet modulates resilience of the gut ecosystem in human microbiota associated rats. F. LEVENEZ, V. ROCHET, M. SUTREN, S. DROINEAU, A. FOUSSIÉ, P. GUILLAUME, S. RABOT, M. LECLERC, J. DORÉ ............................................................................................ S102

Dietary carbohydrate source determines molecular fingerprints of the rat fecal microbiota. T.R. LICHT, M. HANSEN, M. POULSEN, L.O. DRAGSTED ....................................................... S102

Effect of gas pressure and buffer composition on in vitro ruminal fermentation. D. MACHEBOEUF, B. LASSALAS, R. BERGEAULT, D.P. MORGAVI ......................................... S102

Dose-response effect of diallyl disulfide on ruminal fermentation and methane production in vitro. D. MACHEBOEUF, B. LASSALAS, M.J. RANILLA, M.D. CARRO, D.P. MORGAVI .................... S103

Contents XIX

Combination of feed additives to meet production and environmental targets in ruminants - in vitro optimization. D. MACHEBOEUF, M.J. RANILLA, M.D. CARRO, M.L. TEJIDO, B. LASSALAS, D.P. MORGAVI ........................................................................................................................ S103

The influence of chestnut tannins on growth and enzymatic activities of selected rumen bacteria.R. MARINSEK LOGAR, T. EPELJNIK ...................................................................................... S104

Effect of probiotics, prebiotics and synbiotics as functional food supplements on gut microflora of rats. L. MARKIEWICZ, A. MAJKOWSKA, M. BIELECKA .................................................................. S104

Effects of non-antibiotic additives on the microbial equilibrium of broiler chicken intestine. B. MASSIAS, M. ARTURO-SCHAAN, A.-M. ELIE, K. BEBIN, V. HOCDE, M. DENAYROLLES, M.C. URDACI ........................................................................................... S105

Role of protozoa and antiprotozoal agents on CLA biohydrogenation in mixed ruminal microorganisms. N. MCKAIN, R.J. WALLACE ................................................................................................... S105

Survivability of L. delbrueckii ssp. bulgaricus and S. thermophilus in the human gastrointestinal tract. M.L. MICHAYLOVA, ZH.P. DIMITROV, L. VLACHKOVA, P. PETROVA ................................... S106

Effects of probiotics on body weight gain, body height, diarrhea occurrence and health condition of newborn calves. M.R. MOKHBER-DEZFOULI, P. TAJIK ..................................................................................... S106

Behaviour of live yeast Biosaf® Sc 47 during digestive transit in dairy cows. V. MONTEILS, L. CAUQUIL, E. AUCLAIR, C. BAYOURTHE ..................................................... S106

Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates. P. MOSONI, F. CHAUCHEYRAS-DURAND, C. BÉRA-MAILLET, E. FORANO ............................ S107

Molecular analysis of the intestinal microbiota of weaning piglets fed with diets supplemented with xylo-oligosaccharides. P. MOURA, F. SIMÕES, S. MARQUES, L. ALVES, F. GÍRIO, J.P.B. FREIRE, M.P. ESTEVES ......................................................................................................................... S107

Evaluation of a natural dietary strategy for controlling the severity of pathogenic protozoal infections in the gastrointestinal tract. L. NOLLET, R. MURPHY ......................................................................................................... S108

DNA stability and transformability of feed derived DNA in the gastrointestinal tract of rats. L. NORDGÅRD , T. TRAAVIK, K.M. NIELSEN .......................................................................... S108

The effect of L. plantarum and L. fermentum on the microflora in the digestive system of calves.V. OBERAUSKAS, A. SEDEREVICIUS, R. ZELVYTE, R. SUTKEVICIENE, I. MONKEVICIENE ................................................................................................................... S108

Č

XX Contents

Ruminal acetogens in dairy cows: dietary effects and quantification of E. limosum. K. OLSSON, P. EVANS, K.N. JOBLIN ....................................................................................... S109

The effect of yeast on Listeria innocua survival in the digestive tract of sheep. A.M. OLVERA-RAMÍREZ, N.R. MCEWAN, F.M. MCINTOSH, C.J. NEWBOLD ......................... S109

In vitro activity of essential oils towards intestinal microbes. A.C. OUWEHAND, H. KETTUNEN, H. SCHULZE, N. RAUTONEN ............................................. S110

Functionality of the pig gastrointestinal microbiota in response to alternatives for in-feed antimicrobials. O. PÉREZ, W. PELLIKAAN, M. VERSTEGEN, H. SMIDT, W.M. DE VOS .................................. S110

Yeast cell wall preparations prevent the attachment of enteropathogenic Escherichia coli on broiler gut mucus. S. PEURANEN, A. KOCHER, K.A. DAWSON ............................................................................. S111

Effect of human milk on the growth of bifidobacteria: stimulation of Bifidobacterium bifidum, inhibition of Bifidobacterium animalis. V. RADA, J. NEVORAL, E. VLKOVÁ, I. TROJANOVÁ, J. KILLER JI Í ...................................... S111

The effects of organic acids and essential oils on methane production in vitro. M.J. RANILLA, M.D. CARRO, M.L. TEJIDO, D. MACHEBOEUF, B. LASSALAS, D.P. MORGAVI ........................................................................................................................ S111

Antiprotozoal properties of some plants from Iran. M. REZAEIAN, R. NINGRAT, R.J. WALLACE ........................................................................... S112

Growth of Butyrivibrio fibrisolvens JW11 and Fusocillus P-18 in the presence of fish oil, fatty acids and their derivatives. A.J. RICHARDSON, R.J. WALLACE .......................................................................................... S112

Recovery of Lactobacillus casei DN-114 001Ri in the intestinal tract of healthy humans after consumption of fermented milk. V. ROCHET, L. RIGOTTIER-GOIS, F. LEVENEZ, J. CADIOU, A. MOGENET, J.-L. BRESSON, N. GOUPIL-FEUILLERAT, J. DORÉ .................................................................. S113

In vitro growth inhibition of Helicobacter pylori and Campylobacter jejuni by Lactobacillus salivarius. K.A. RYAN, Y. LI, P.W. O’TOOLE .......................................................................................... S113

Composition and temporal stability of oral and intestinal microbiota during probiotic ingestion. M. SAARELA, J. MAUKONEN, M.-L. SUIHKO, J. MÄTTÖ ........................................................ S113

Cultured milk replacer with prophylactic influence on intestinal diseases. A. SARKINAS ........................................................................................................................... S114

Dietary essential oil supplementation can affect broiler performance and digesta microbial community. H. SCHULZE, H. KETTUNEN, A.C. OUWEHAND, N. RAUTONEN ............................................. S114

Ř

Contents XXI

Continuous pH measurements in the rumen of sheep to test the anti-acidotic efficacy of nettles (Urtica dioica). N. SELJE, E.M. HOFFMANN, P. LAWRENCE, K. BECKER ........................................................ S115

Microbial composition and metabolite concentration in fermented liquid diets for pigs. A. SHLIMON, N. CANIBE, O. HØJBERG, B.B. JENSEN ............................................................. S115

Ultrasonic modification of alginate matrices for the develop of probiotic delivery systems. A.I. SIDOROV, O.V. MANAENKOV, E.M. SULMAN, L.E. SMIRNOVA,V.F. VINOGRADOV .................................................................................................................. S116

Influence of Bifidobacterium bifidum on mineral release from bread in the process of enzymatic digestion in vitro. K.A. SKIBNIEWSKA, B. NALEPA, A. BABUCHOWSKI .............................................................. S116

Effect of organic acids and monolaurin on Escherichia coli, Salmonella spp. and Clostridium perfringens. E. SKRIVANOVA, M. MAROUNEK , G. DLOUHA ..................................................................... S116

The influence of probiotics on growth of turkeys. L. SOL IANSKA, D. VLADÁROVÁ ........................................................................................... S117

Effect of feeding processed karanj (Pongamia glabra) meal on the microbial protein synthesis and immune response of growing lambs. N.M. SOREN, V.R.B. SASTRY, T.K. GOSWAMI, S.K. SAHA ................................................... S117

Bacterial community change and Streptococcus suis disappearance in piglet gut after oral administration of probiotic S1. Y. SU, W.Y. ZHU, W. YAO ..................................................................................................... S117

Dynamic single fermenter model for study of survival and growth of probiotics in the human upper gastrointestinal tract. I. SUMERI , L. ARIKE, S. ADAMBERG, K. ADAMBERG , L. LAPP , T.-M. LAHT,T. PAALME .............................................................................................................................. S118

Effects of exogenous fibrolytic enzymes on in vitro ruminal fermentation of grass hay and its cell wall. M.L. TEJIDO, L.A. GIRALDO, M.D. CARRO, J.M. TRICARICO, M.J. RANILLA ....................... S118

Effects of dietary nitrogen source and amino acid composition on intestinal microbiota. M. TOKURA, Y. JOJIMA, M. MORI, T. KOBAYASHI, Y. HONGOH, T. INOUE, M. OHKUMA, R. FUDO ............................................................................................................ S119

Relationships between gut microbial species and energy metabolism in broiler chickens. V.A. TOROK, K. OPHEL-KELLER, R.J. HUGHES ..................................................................... S119

Effects of live yeasts on the fatty acid biohydrogenation by ruminal microflora. A. TROEGELER, J.-P. MARDEN, C. BAYOURTHE, R. MONCOULON, F. ENJALBERT ......................................................................................................................... S120

The trial of fermented carrot juice technology development. M. TRZASKOWSKA, D. KOLOZYN-KRAJEWSKA ...................................................................... S120

Č

XXII Contents

New generation probiotics: potential enhancers of rumen function. N.D. WALKER, A. AMEILBONNE, E. FORANO, F. CHAUCHEYRAS-DURAND, B. DULL ................................................................................................................................... S120

Germination and conjugation of Bacillus thuringiensis in the gut of gnotobiotic rats. A. WILCKS, L. SMIDT, L. ANDRUP, M. BAHL, B.M. HANSEN, N.B. HENDRIKSEN,T.R. LICHT ............................................................................................................................... S121

Encapsulated fumaric acid as a means of decreasing ruminal methane emissions. T.A. WOOD, R.J. WALLACE, A. ROWE, J. PRICE, D.R. YANEZ, S.P. WILLIAMS, C.J. NEWBOLD.......................................................................................................................... S121

Physiological and microbial status of rats fed a diet containing grapefruit flavonoids and inulin. M.WRÓBLEWSKA, E. BIEDRZYCKA ......................................................................................... S122

Real-time PCR and PCR-DGGE analyses of stool microbiomes from infants fed human breast milk, infant formula, or formula with added fructo-oligosaccharides. Q. XIA, Z. YU, T. PREMARAJ, T. WILLIAMS, M. MORRISON .................................................. S122

Microbial numbers and diversity in the rumen of sheep supplemented with fumaric acid. D.R. YÁÑEZ-RUIZ, G. BE ECKI, R.J. WALLACE, C.J. NEWBOLD ......................................... S123

Daidzein affects the composition of gastrointestinal bacterial community of piglets. Z.-T. YU, W.Y. ZHU, W. YAO ................................................................................................ S123

Yakult Symposium

Diversity and function of the human gut microbiota.H.J.M. HARMSEN, L.C.M. WILDEBOER-VELOO, G.C. RAANGS, R.H.J. TONK, S.P. VAN TONGEREN, J.E. DEGENER, G.W. WELLING ............................................................ S127

From phylogenetics to metagenomics – insight in gut microbial functionalityJ. DORÉ .................................................................................................................................... S127

The gut microflora and colorectal cancer risk. I. ROWLAND ............................................................................................................................ S128

Microbially-induced inflammation and colon cancer.E.M. EL-OMAR ........................................................................................................................ S128

Authors alphabetical list ......................................................................................................... S133

Ł Ż

S3Reprod. Nutr. Dev. 46 (2006) S3–S35© INRA, EDP Sciences, 2006

O-1 Functional analysis of the GI-tractmicrobiota. C.C.G.M. Booijinka,b, E.G.Zoetendala,b, H. Smidta,b, M. Kleerebezema,W.M. De Vosa,b (a Wageningen Centre for FoodScience (WCFS), The Netherlands, b Laboratoryof Microbiology, Wageningen University, TheNetherlands).

The human gastrointestinal (GI) tract provideshome to a complex microbial community, col-lectively termed microbiota. Though majoreffort has been put into describing the diversityand stability of the GI tract microbiota, its func-tionality has only been investigated at the isolatelevel. In this study, cDNA Amplified FragmentLength Polymorphism (cDNA-AFLP; Bachemet al., 1996 Plant J. 9: 745–753) was used as anRNA fingerprinting technique to investigate thedominant transcripts expressed by the micro-biota. In short, cDNA-AFLP visualizes tran-script diversity after selective amplification ofspecific restriction fragments obtained fromcDNA fragments synthesized from total RNA.Since no prior sequence knowledge about thegenomic diversity is necessary for application ofthis method, we hypothesized that it should alsobe applicable to complex consortia of mostlyuncultured micro-organisms, such as thosefound in the human GI tract. cDNA-AFLP wasused to compare RNA and MicrobExpressenriched mRNA extracted from fecal samplescollected at different time points. Subsequently,sequence analysis was performed to trace thecorresponding expressed genes. This resulted inthe identification of cDNAs that were character-ized by sequence analysis. This revealed that35% of them could encode prokaryotic proteinsbelonging to several classes that will be dis-cussed. A minor fraction was assigned witheukaryotic origin. Furthermore, comparison ofthe temporal profiles revealed a relatively vari-able microbial transcriptome, while 16S rRNAgene-targeted PCR-DGGE profiles remainedstable during this period. This study is the firstto show that cDNA-AFLP can be applied forfunctional analysis of complex microbial eco-systems.

O-2 Prokaryotic community of the mucosalfraction of human ileum: a metagenomicapproach. M.C. Leclerca, P. Robeb, P.Marteauc, R. Nalinb, M. Gelfandd, J. Doréa

(a INRA, Jouy-en-Josas, France, b Libragen,

Toulouse, France, c HEGP, Paris, France,d ITTP, Moscow, Russia).

A fosmid library of 20000 clones was con-structed from an ileum segment obtained from a50 years old individual. The sample was checkedfor its adequate sampling and representation ofa “healthy” individual. To determine whetherour metagenomic library adequately representsthe dominant bacteria in the mucosal ecosystemand to compare this approach with the PCRbased techniques, the same DNA was used to do16S rDNA PCR-based molecular inventories,for both Eubacteria and Archaea. Ninety-sixclones from the metagenomic library were ran-domly chosen, sub-cloned and full inserts weresequenced at CNS (French National SequencingCenter, Evry, France). Phylogenetic assignmentof 84 fosmid inserts, with an average insert sizeof 40 kb, showed that 50 were part of the BPP(Bacteroides-Prevotella-Porphyromonas) phy-lum and 34 were members of the Firmicutes.Such composition compared well with the 16SrDNA diversity of the molecular inventory per-formed on the same DNA. However, the propor-tion of the phyla slightly differed between thePCR and the metagenomic approach, suggestingbias of the PCR or/and large insert cloning pro-cedure. Annotation of 96 × 40 kb-fosmid insertshas been providing preliminary data on domi-nant genes of the ileum mucosa ecosystem.Among this set of putative 4 000 genes, 700 pro-teins have been so far identified. The presenceof 30 genes coding for fibre-degrading enzymesor its regulation were detected within fosmidinserts belonging to BPP and Firmicutes phyla.This work demonstrates that the metagenomicapproach can provide information on the func-tions of cultured and uncultured prokaryoticorganisms of the ileum mucosa. A few examplesof post-genomic strategies for studying the dis-tribution and expression of these enzymes willbe discussed.

O-3 Identification DNA-microarray for themonitoring of intestinal bacteria during gas-troenteritis. K. Knöschea, T. Hainb, T.Chakrabortyb, E. Domannb, T.T. Bachmanna

(a Institute for Technical Biochemistry, Univer-sity of Stuttgart, Allmandring 31, 70569 Stutt-gart, b Institute for Medical Microbiology, Uni-versity of Giessen, Frankfurter Str. 107, 35392Giessen, Germany).

S4 Session I: Microbial diversity in health and disease

Bacterial gastroenteritis is one of the majorreported infectious diseases in Germany, andinternationally three to five billion cases of acutediarrhea occur yearly. Diarrheal diseases canquickly reach epidemic proportions, especiallyin cases of food-borne pathogens. Due to thisfact enteritic pathogens have to be reported to thepublic health departments. Microarrays enable aparallel and fast identification of the particularpathogen and do not rely on cultivation of theorganisms, which is often problematic in thecase of intestinal bacteria. In the frame of theGerman national project PathoGenoMik, wedeveloped an oligonucleotide microarray toidentify 18 bacterial species and 13 genera com-prising the most prevalent gastroenteritis-caus-ing bacteria. Additionally, the array includesidentification probes for the most important res-ident intestinal bacteria and common probioticstrains used for medical treatment. Therefore,the microarray enables the monitoring of theaetiopathology of inflammatory bowel diseasesand the assessment of the therapeutical successwith probiotic strains, which were shown inpractical applications to be effective therapeu-tics. Following the multiple probe concept, wedesigned specific probes for the 16S and 23SrRNA genes applying the ARB software envi-ronment. For the validation of the array, clinicalisolates were used and the results were con-firmed by sequencing. The future impact of themicroarray will be greatly enhanced by the com-bination with other DNA-microarray modulesdeveloped in PathoGenoMik (ESBL, quinoloneresistance, pathoadaptive mutations, and para-sitic protozoa) and the extension to a quantitativedetection system to monitor population dynam-ics and spread of antibiotic resistance.

O-4 Distribution of the main functional groupsof microorganisms in the gut of IBS subjects.C. Chassarda, P. Marqueta, C. Del’hommea, C.Dubrayb, K.P. Scottc, H.J. Flintc, M. Dapoignyd,G. Bommelaerd, A. Bernalier-Donadillea (a INRA,Unité de Microbiologie, 63122 Saint-Genès-Champanelle, France, b Département de Phar-macologie clinique, CHU, Place Henri Dunant,63000 Clermont-Ferrand, France, c RowettResearch Institute, Bucksburn, Aberdeen, AB219SB, UK, d Service d’Hépato-Gastroentérolo-gie, Hôtel-Dieu, 63000 Clermont-Ferrand,France).

Irritable bowel syndrome (IBS) is a commongastrointestinal disorder characterized byabdominal pain and disturbances in bowel func-tion. The causes of IBS are poorly known. Manyfactors have been proposed as possible reasonsbehind IBS, but recent studies suggest that theintestinal microbiota may play an important rolein this pathology. An alteration in faecal flora ofIBS patients has thus to be considered. In thiscontext, we investigated the distribution of themain functional groups of micro-organisms inthe gut of IBS subjects compared to that inhealthy volunteers. All IBS patients selected forthe study fulfilled the Rome II criteria for IBS.Both IBS and asymptomatic subjects ate an occi-dental diet including 10–15 g of dietary fibres.Faecal samples from both groups of volunteerswere collected and processed within a few hoursfor analyses based on microbial cultivation.Extensive variation was observed in the gas-trointestinal microbiota of IBS subjects com-pared to asymptomatic subjects. IBS patientsshowed important disturbances in microbialcommunities implicated in H2 and lactatemetabolism. In particular, the number of sul-phate-reducing bacteria was significantly higherin IBS subjects compared to healthy ones (P <0.05). Furthermore, the population levels of lac-tic acid bacteria (bifidobacteria, lactobacilli)as well as lactate-utilizing bacteria were signif-icantly lower in IBS than in healthy volunteers.Disruption affecting functionally importantgroups of micro-organisms was therefore evidentin the gut of IBS subjects. These observationswill be completed by studying the distribution ofthe predominant groups of micro-organismsidentified by molecular methods in faecal sam-ples from IBS and healthy subjects.

O-5 Culture-independent analyses of the tem-poral variation of the dominant faecal micro-biota, Clostridium spp., Bacteroides spp., inCrohn’s disease and colon cancer. P.D. Scanlan,F. Shanahan, J.R. Marchesi (Alimentary Pharm-abiotic Centre/Department of Microbiology,University College Cork, Cork, Ireland).

The human gastrointestinal microbiota showshost-specific diversity, temporal stability andsignificantly contributes to maintenance of ahealthy gut. However, in inflammatory boweldisease (IBD) and colon cancer this microbiota

Session I: Microbial diversity in health and disease S5

has been implicated as a contributory factor tothe illness. The aim of the present study was tocompare bacterial dynamics in Crohn’s disease(CD) and colon cancer patients with a controlgroup. This aim was achieved using a cultureindependent method to assess the temporal sta-bility, relative diversity and similarity of thedominant fecal microbiota, Clostridium coc-coides group, Bacteroides spp. and Bifidobacte-rium spp. in all individuals. Fecal samples werecollected from CD and colon cancer individualsover several time points. DNA from the fecalsamples was isolated and denaturing gradientgel electrophoresis (DGGE) profiles were gen-erated for the different microbial groups by spe-cifically targeting different regions of the 16SrRNA gene. The species profiles were comparedon the basis of similarity (Sorenson’s pair-wisesimilarity coefficient) and also various indicesof diversity. Temporal stability and diversitywas variable for both disease states when com-pared to the control group. Significant altera-tions observed in group-specific profiles for twofunctionally important mutualistic groups ofbacteria in the gut indicate that the changes inthese microbiota have implications for the host’sgut health through loss of functionality, espe-cially butyrate production.

O-6 Serine protease autotransporters (SPATE)and autoaggregative adhesins from Escheri-chia coli are potential virulence factors ininflammatory bowel disease. D.O. Krause,C.N. Bernstein, S. Sepehri, R. Kotlowski (Uni-versity of Manitoba, Winnipeg, Manitoba R3Y2N2, Canada).

Inflammatory bowel disease is a chronic diges-tive tract disease in humans made up of Crohn’sdisease and ulcerative colitis. The root of the dis-ease is thought to lie at the intersect of a micro-bial antigen, a susceptible human genome, anda dysfunctional immune system. Culture inde-pendent methods can be used to identify bacteriathat are uniquely associated with inflammatorybowel disease, and serve as a prelude to targetedcultivation. Isolation of bacteria is important,because ultimately the virulence factors associ-ated with disease must be studied. We utilized84 biopsies from 46 controls and IBD patientsdrawn from a unique population-based case-control study. Ribosomal intergenic spacer anal-

ysis (RISA) was conducted to identify uniquespecies in biopsy tissue, and PCR analysis wasused to identify virulence factors. RISA analysisidentified unique bands in IBD biopsies whichwere identified as Escherichia coli. Targetedculture revealed a significantly higher number(4 logs) of Enterobacteriacea in IBD biopsies(P < 0.05). There were also significantly (P <0.05) more serine protease autotransporters(SPATE) and adhesions in the E. coli from IBDbiopsies. Together, the autoaggregative proper-ties of the adhesions and the proteolytic andmucolytic activities of the SPATE’s mightexplain the varied phenotypes of IBD. This is thefirst study in which SPATE’s have been associ-ated with IBD.

O-7 Exploration of the human intestinalmicrobiota during antibiotic administrationusing microbiomics approaches. J.K. Janssona,C. Jernbergb, S. Löfmarkb, C. Edlundb

(a Department of Microbiology, Swedish Uni-versity of Agricultural Sciences, 750 07, Upp-sala, Sweden, b Department of Laboratory Med-icine, Karolinska University Hospital, 141 86,Stockholm, Sweden).

The use of molecular tools to characterize com-plex microbial communities, microbiomics, hasgreat potential to increase our understanding ofthe microbial ecology of the human gut and theinfluence of diet, disease, antibiotics and probi-otics on the gut microbiota. We aimed to studythe impact of clindamycin administration for7 days on the human intestinal microbiota usingmicrobiomics approaches. In a preliminarystudy, the composition of the intestinal microbi-ota of healthy subjects returned more rapidly topre-treatment levels in those that simultaneouslyingested a probiotic as assessed by terminalrestriction fragment length polymorphism(T-RFLP) analysis of DNA extracted from fecalsamples. In a long-term study, disturbances inthe composition of the human fecal microbiotaof 4 healthy subjects, compared to 4 control sub-jects, were observed after 7 day clindamycinexposure for up to 2 years as assessed byT-RFLP using both general and Bacteroidesspecific primers. Although the total bacterialcommunity only exhibited a transient changeduring clindamycin administration, there was asevere disruption of the specific Bacteroides

S6 Session I: Microbial diversity in health and disease

community. Even after 2 years post-treatmentthere was no sign of a return of the Bacteroidescommunity to its original composition prior toclindamycin administration. Some Bacteroidesclones that were typed by rep-PCR were shownto become resistant after treatment. In addition,increases in several antibiotic resistance genes,such as ermF, were found by real-time PCR inDNA extracted from the fecal samples of treatedindividuals. The results of these studies demon-strate that a combination of microbiomicsapproaches can define changes in the composi-tion of the gut microbiota during antibiotic treat-ment. In the future, molecular fingerprintingtools may be used as a means to type the intes-tinal microbiota of individuals in order to designindividually optimized drug treatments and dietsto maintain a healthy gut environment.

O-8 Murine gut microbiome: nature and nur-ture. M.K. Friswell, P. Gilbert, D.G. Allison, I.J.Stratford, B.A. Telfer, A.J. McBain (School ofPharmacy and Pharmaceutical Sciences, Uni-versity of Manchester, M13 9PL, UK).

Little is known of the impact of immune statusor individual genotype on microbiome compo-sition. We have used molecular ecologicalapproaches to characterise the gut microbiomeof various mouse strains, bred and housed in acommon environment, to test the hypothesis thatgenotype and immune status dictate the eubac-terial community faecal flora. Faecal sampleswere collected from different mouse strains,identically fed and housed and inbred within thesame facility. Mouse strains (3–7 individuals)comprised: C3H (control strain); MF1 (T celldeficient); C57 (geriatric); CD1 (immunodefi-cient); GFEC (immunocompetent). C3H andMF1 strain mice were moved after weaning(4 weeks old), and as adults (8 weeks) to a dif-ferent common environment and faecal florawas monitored. All samples were analysed byPCR-DGGE, utilising primers specific for theV2-V3 region of eubacterial 16S rDNA. Hierar-chical dendrograms (cluster analysis) based onDNA sequence analysis were constructed toobjectively compare microbial community fin-gerprints. Dominant DGGE bands were excisedand sequenced for identity. Each mouse strainhad a unique eubacterial faecal finger print, clus-tering individuals within hierarchical dendro-

grams (80–100% homology), whereas differentstrains converged at 60–65%. Sequence analysisshowed dominant microflora to include Bacter-oides, Eubacterium and Campylobacter. Faecalflora was conserved within strains from adult-hood regardless of environment, yet animalsrelocated to different environments at weaningshowed temporary dysbiosis leading to a new,stable microflora at eight weeks. These modifiedfloras were again strain specific. Results clearlyindicate that murine genotype and immune sta-tus, together with the microbial challenge fromtheir environment during maturation, affect theadult microbiome.

P-1 Sequence variation among α-toxin ofClostridium perfringens strains isolated fromdiseased and healthy chickens. L. Abildgaarda,R.M. Engberga, K. Pedersenb, A. Schrammc, O.Højberga (a Danish Institute of Agricultural Sci-ences, Department of Animal Health, Welfareand Nutrition, PO Box 50, 8830 Tjele, Denmark,b Danish Institute for Food and VeterinaryResearch, Denmark, c University of Aarhus,Institute of Biological Sciences, Department ofMicrobiology, Denmark).

Necrotic enteritis is a severe gastrointestinal dis-ease in broiler chickens caused by C. perfringensproducing α-toxin (phospholipase C); a key vir-ulence factor in the pathogenesis of gas gangrenein humans as well. The gene sequence and struc-ture of the α-toxin may influence the pathogen-esis. The α-toxin was recently reported to behighly conserved in C. perfringens isolated fromdiseased chickens. Variations were observed inonly nine out of the 398 amino acid (aa) posi-tions. We sequenced the α-toxin gene from fur-ther 60 strains of C. perfringens, isolated fromdiseased as well as healthy chickens. Overall thesequences showed close similarity (> 98% on theamino acid level) to the formerly reportedchicken isolate sequences. Variations wereobserved in 23 aa positions, leading to 26 differ-ent α-toxin sequence types among the 60 iso-lates. Moreover, the gene sequence from threeisolates had an insertion of 834 nucleotides at the5’ end corresponding to the N-terminal domainof the enzyme encompassing the catalytic site.The three strains were all isolated from healthybirds, but were different strains (different pulsed-field gel electrophoresis profiles). Studies are

Session I: Microbial diversity in health and disease S7

being carried out to reveal whether these isolatesproduce α-toxins with phospholipase activity. Inconclusion, a higher degree of variation in the aasequences of α-toxin than reported earlier fromchicken studies was found. The variation couldnot be connected to an outbreak of disease andit remains to be clarified, if phospholipase activ-ity of the toxin is influenced by the observed var-iations.

P-2 The bacterial community of the horsestomach. R.A.M. Al Jassima, S.E. Denmanb,J.D. Hernandeza, C.M. McGowana, F.M.Andrewsc, C.S. McSweeneyb (a School of Ani-mal Studies, Faculty of Natural Resources, Agri-culture, and Veterinary Science, The Universityof Queensland, Gatton Campus QLD 4343, Aus-tralia, b CSIRO Livestock Industries, St. Lucia,4067 Australia, c College of Veterinary Medi-cine, The University of Tennessee, USA).

Culture dependent and independent techniqueswere used to study the bacterial diversity of thehorse stomach. Samples of stomach contents andlining were obtained from twenty horses postmortem and all samples were processed by:(1) culturing onto a pre-reduced modified agarMRS medium for enumeration and isolation oflactic acid producing bacteria and (2) extractionof genomic DNA and analysis by denaturinggradient gel electrophoresis (DGGE). Plugswere taken from DGGE bands for cloning andfurther DNA analysis and sequencing. The num-bers of colony forming units (CFU) in stomachcontents cultured on MRS-agar medium aver-aged 2.3 × 107. With the exception of Lactoba-cillus salivarius, which was detected in bothtechniques, other isolates belonging to the genusLactobacillus grown on MRS medium were dif-ferent from those belonging to the same genusin the clone library. Cultured bacteria clusteredwith bacterial species belonging to the generaLactobacillus, Streptococcus and Clostridium,while DGGE clones belonged to the genera Pro-pionibacterium, Clostridium, Lactobacillus, Pre-votella, Pasteurella, and Pseudomonas. Otherfewer clones were closely related to Escheri-chia, Actinobacillus, Moraxella, Rhodococcus,Veillonella, Legionella and Eubacterium. Resultsof this study demonstrated that lactic acid pro-ducing and spore forming bacteria are capable ofsurviving the acidic conditions of the stomach

and are associated with the contents and the lin-ing of both the healthy and ulcerated stomachs.This is the first report on the bacterial diversityof the stomach of the horse. Work is in progressto identify bacterial changes associated withchanges of diet and management of thorough-bred horses and the possible involvement of bac-teria and their products in the pathogenesis ofstomach ulceration in horses.

P-3 Detection and activity of a bacteriocinproduced by Lactobacillus strains. F.N.Allouche, A. Hellal, A. Laraba (LaboratoireBioénergie et Environnement, Centre de dével-oppement des Énergies Renouvelables, BP 62,route de l’Observatoire Village Céleste, Bouza-reah, Algeria).

Ten strains of Lactobacillus isolated from rawcow milk and commercial dairy starter wereshown to be antagonistic towards several food-borne pathogens. These strains produced andexcreted an antibacterial substance in MRSbroth which inhibited the growth of Bacillussubtilis, Staphylococcus, Escherichia coli andPseudomonas aeruginosa. The study of growthkinetics and excretion permitted to choose onlyone strain of Lactobacillus acidophilus as amodel for production and purification of bacte-riocin. The bacteriocin of Lactobacillus acido-philus Lba1 was isolated and purified by acidextraction and reversed-high-performance liq-uid chromatography. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated a homogeneous protein withan estimated molecular weight of 30 000 Da. Apreliminary characterisation showed that theinhibitory substance was heat-labile; activitywas detected at pH 5. Bacteriocinogenic activitywas destroyed by autoclaving at 121 °C for20 min. Bacteriocin was stable at 45 °C and overthe pH range 5 to 6. It was sensitive to proteolyticenzymes, indicating that the active moiety of theinhibitor was proteinaceous in nature. Theseproperties suggested that the inhibitory sub-stance could be considered as a bacteriocin-likesubstance.

P-4 Quantification of protozoal concentra-tions in the rumen and duodenum of growinglambs using real-time PCR: effect of age. A.

S8 Session I: Microbial diversity in health and disease

Belanchea, G. De la Fuentea, L. Callejaa, D.R.Yañez-Ruizb, C.J. Newboldb, J. Balcellsa, M.Fondevilaa (a Departamento de ProducciónAnimal y Ciencia de los Alimentos, Universidadde Zaragoza, Spain, b Institute of Rural Sciences,University of Wales, Aberystwyth, UK).

Although protozoa contribute 50% of the micro-bial mass in the rumen, the contribution of pro-tozoa to microbial flow to the duodenumremains unknown. The presence of protozoa inrumen contents and their contribution to micro-bial N in duodenal chymus was studied in3 groups of 5 Rasa Aragonesa lambs that wereslaughtered at 30 (only milk-fed lambs),45 (weaning lambs, being fed on milk, concen-trates and straw) and 90 (end of fattening period,given concentrates and straw) days of age. Bac-terial and protozoal standards were extractedfrom rumen contents by differential centrifuga-tion and sedimentation-filtration, and theirpurity was 97.92 ± 1.23 and 61.48 ± 9.89%,respectively. Genes encoding bacterial 16SrDNA and protozoal 18S rDNA subunits werequantified by real-time PCR. Total rumen bac-terial DNA was 16.05 ± 6.02, 22.52 ± 3.70 and19.11 ± 2.65 ng DNA/g FM for 30, 45 and 90 dlambs and similar among groups for the chymussamples (5.32 ± 6.88 ng DNA/g FM). ProtozoalDNA appeared in the rumen of 2 milk-fed lambs(3.03 ± 0.81 ng DNA/g FM), was almost nil atweaning and could only be detected in the rumenof 2 out of 5 of the fattened lambs (11.82 ±1.73 ng DNA/g FM), showing that optimal envi-ronmental conditions for protozoa are slowlyacquired after weaning with a high individualvariability. Estimations by real-time PCR werevalidated by microscopy counts. Protozoal DNAin the chymus was in concordance to rumen val-ues. Estimated contribution of protozoa DNA toduodenum was only significant in 90 days fau-nated lambs (3.53 ± 0.93 ng DNA/g FM).

P-5 Identification of antibiotic resistant genesin Streptococcus thermophilus using microar-ray. G. Berrutia, A.H.A.M. van Hoekb, H.J.M.Aartsb, L. Tosia, L. Morellia, M. Callegaria

(a UCSC, Istituto di Microbiologia, Piacenza viaEmilia Parmense 84, 29110 Piacenza, Italy,b RIKILT, Institute of Food Safety WageningenUniversity, Wageningen, The Netherlands).

The increase of antibiotic resistant (AR) popu-lations in the food chain community is now wellrecognized as a source of antimicrobial resist-ance determinants for the gut flora and is gener-ally caused by horizontal gene transfer. In orderto increase the understanding of the molecularbiology and ecology of AR determinants andtheir hosts, our interest should not only be lim-ited to pathogenic bacteria but also take accountof bacteria coming from food-related environ-ments, like S. thermophilus, for which very fewdata are available. These bacteria might not rep-resent a clinical risk in themselves, but they canbe vehicles of AR genes. Here we report theassessment of a 50 and 60-mer oligonucleotideDNA-based microarray for the identification ofAR genes in a significant number of S. ther-mophilus strains. The AR genes represented bythe oligonucleotides on the microarray belongto: aminoglycoside, extended spectrum β-lacta-mase (ESBL), chloramphenicol, macrolidelincosamides and streptogramin (MLS) group,sulfonamide, tetracycline, trimethoprim andvancomycin. All strains were obtained from theUCSC culture collection, except for 11 strainsthat were isolated from raw milk during thestudy, and all originated from dairy products. Tovalidate the results obtained by microarray ana-lysis all isolates were subjected to PCR and MICtesting on Iso-sensitest medium (with lactose1% w/v) based on a microdilution method. tetSand ermB genes were found and sequenced indifferent S. thermophilus isolates. Although forstreptomycin very high MIC values wereobserved no streptomycin related genes werefound by microarray analysis.

P-6 Optimizing growth conditions for rumi-nal ciliates in the in vitro system Rusitec.C.P.J. Bonneta, L. Meileb, M. Kreuzera, C.R.Solivaa (a Institute of Animal Science, AnimalNutrition, Switzerland, b Institute of Food Sci-ence and Nutrition, Laboratory of Food Biotech-nology, ETH Zurich, 8092 Zurich, Switzerland).

The Rumen Simulation Technique (Rusitec)was shown to maintain ruminal ciliates during> 15 days of in vitro incubation. In order to guar-antee high and stable ciliate numbers, there isstill a need to improve Rusitec conditions andoperation protocol. Therefore, two Rusitec trialswere conducted investigating the effect of dif-ferent modifications on ciliate growth. In the

Session I: Microbial diversity in health and disease S9

first trial, carried out with bovine ruminal fluidand lasting for 15 days, three modifications weretested; (i) polyurethane sponge was bedded as amatrix into the fermenters to protect the ciliatesfrom being washed out of the system, (ii) thepore size of the feed bags was increased from100 to 200 µm, and (iii) the buffer flow rate washalved while keeping the buffering capacityconstant. In the second trial, lasting for 10 days,the effect of two incubation media on the growthof isolated or cryo preserved/revitalized Ento-dinium spp. was tested in the presence of liveruminal bacteria. One medium consisted ofdefaunated bovine ruminal fluid, the other oneof the ciliate cultivation medium of Dehority,commonly used in batch cultures. No conclusiveeffects on ciliate growth of sponge addition, var-ied feed bag pore size or reduced buffer flow ratewere observed. The larger bag pore size, how-ever, resulted in an increased apparent feed deg-radation and higher fermentation gas formation.Additionally, an increased bacterial populationsize was found with the larger bag pore size.Entodinium spp. were successfully cultivated inboth media, but growth conditions were morefavorable in the Dehority medium than withdefaunated ruminal fluid.

P-7 Isolation of bacteriophage active againstE. coli O157:H7 and Salmonella spp. fromcattle and swine. T.R. Callawaya, T.S. Edring-tona, A.D. Brabbanb, E.M. Kutterb, R.C. Ander-sona, R.B. Harveyb, D.J. Nisbeta (a USDA/ARS,Food and Feed Safety Research Unit, CollegeStation, TX, USA, b Evergreen State College,Olympia, WA, USA).

Bacteriophage are fairly common members ofthe gastrointestinal microbial ecology. How-ever, the incidence of bacteriophage in mam-mals has not been examined. Bacteriophagehave been proposed as potential strategies toreduce foodborne pathogenic bacteria, espe-cially E. coli O157:H7 and Salmonella spp. Ifphage are to be used in this fashion, we mustunderstand what role they play in the microbialecology of the gut. From a regulatory aspect, theincidence of phage is necessary informationprior to the approval process. Therefore the cur-rent study was designed to determine the inci-dence of phage active against E. coli O157:H7and Salmonella in the feces of commercial feed-

lot cattle and commercial finishing swine in theUnited States. Fecal samples (n = 60) were col-lected from each of four feedlots in two GreatPlains states and from each of four commercialswine finishing operations in one state. Samples(n = 6) were collected from 10 randomly selectedpens throughout the operation. The total numberof isolates collected from each species was n =240. Salmonella and E. coli O157:H7 werefound in 3.8% and 11.7% of the cattle fecal sam-ples, respectively. Bacteriophage active againstE. coli O157:H7 were isolated from 15% of theindividual cattle fecal samples in 55% of the cat-tle pens. Swine results were similar for Salmo-nella spp. and phage active against Salmonellaspp. Results indicate that bacteriophage arefairly widespread across commercial productionfacilities in both cattle and swine, indicating thatthey could potentially be used as a food safetyintervention strategy.

P-8 An animal model that reproduces micro-bial disruption observed in the gut of IBS sub-jects: a new animal model for IBS? C.Chassarda, C. Del’hommea, P. Marqueta, M.Dapoignyb, G. Bommelaerb, A. Bernalier-Donadillea (a INRA, Unité de Microbiologie, 63122 Saint-Genès-Champanelle, France, b Ser-vice d’Hépato-Gastroentérologie, Hôtel-Dieu,63000 Clermont-Ferrand, France).

Irritable bowel syndrome (IBS) is the most com-mon chronic gastrointestinal disorder. However,the origins of IBS remain not clearly understood.Many factors have been proposed as possiblereasons behind IBS, recent studies suggestingthat intestinal microbiota may play an importantrole in this pathology. We recently evidencedimportant disruption of different functionalgroups of microorganisms in the gut of IBS sub-jects. Further investigations on mechanismsinvolved in the physio-pathology of IBS, as wellas those concerning new IBS therapy, wouldneed suitable in vivo models. Our objective wasto develop an animal model able to reproduce themicrobial disruption observed in the intestinalflora of IBS subjects and to analyse the effect ofthis flora on the host. Germ-free rats were inoc-ulated with faecal microflora from either IBSpatients fulfilling the Rome II criteria or healthysubjects. Distribution of the main functionalgroups of microorganisms establishing in the gut

S10 Session I: Microbial diversity in health and disease

of human-flora associated (HFA) rats and totalgas excretion by these animals were analysedand compared for each, IBS and healthy HFArats. Similar disruptions in functional groups ofmicroorganisms were shown in IBS-HFA florarats and in IBS subjects, disturbances in micro-bial communities involved in H2 and lactatemetabolism being evidenced in the gut of themodel as well as IBS subjects. In addition, anextensive increase in H2-excretion of IBS-HFArats was observed compared to healthy-HFArats. IBS-HFA rats could thus be considered asan animal model reproducing microbial disrup-tions characterizing IBS gut flora. Further stud-ies are underway to evaluate the impact of thisIBS gut flora on the animal host.

P-9 A comparison of Clostridium difficile car-riage in inflammatory bowel disease and irri-table bowel syndrome out-patients. E.M.Claytona,b,c, M.C. Reaa,b, F. Shanahanb,d, E.M.Quigleyb,d, C. Hillb,c, R.P. Rossa,b (a MooreparkFood Research Centre, Teagasc, Fermoy, Co.Cork, Ireland, b Alimentary Pharmabiotic Cen-tre, National University of Ireland, Cork, Ire-land, c Department of Microbiology, NationalUniversity of Ireland, Cork, Ireland, d Depart-ment of Medicine, Cork University Hospital,Cork, Ireland).

Irritable bowel syndrome (IBS) is a commongastrointestinal (GI) disorder characterized byabdominal pain and disturbances in bowel func-tion. The causes of IBS are poorly known andsymptoms vary from constipation to diarrhoea.The condition is diagnosed in the absence ofdetectable clinical pathology. Crohn’s diseaseand ulcerative colitis are idiopathic inflamma-tory disorders known collectively as inflamma-tory bowel disease (IBD). Both disorders maypresent conditions ranging from excessivebowel movements to vomiting and weight loss.Toxigenic Clostridium difficile is the leadingidentified cause of diarrhoea and colitis associ-ated with antibiotic therapy. It has been welldocumented that the organism spreads nosoco-mially and causes hospital outbreaks of C. diff-icile-associated diarrhoea (CDAD). Certain indi-viduals can become asymptomatic carriers oftoxigenic C. difficile, whilst others developsevere colitis and multiple relapses dependingon the speed of the immune response. The aimof this study was to determine, whether patients

suffering from IBD and IBS have a higher car-riage rate of C. difficile than asymptomatichealthy volunteers. C. difficile was selected fromstool samples on Cycloserine Cefoxitin EggYolk (CCEY) agar and phenotypic isolates wereconfirmed by 16S rDNA sequencing. Thegenetic relatedness of C. difficile at a strain levelwas characterized by genomic fingerprinting(PFGE). Toxicity testing of the resultant isolatesdemonstrated that up to 53% of C. difficile fromIBS and IBD patients were toxigenic comparedto only 10% of those from healthy people.

P-10 Activity spectrum of reuterin, a broadspectrum antimicrobial produced by Lacto-bacillus reuteri from glycerol, on intestinalbacteria. V. Cleusix, G. Le Blay, S. Vollenwei-der, C. Lacroix (ETH-Zentrum, 8092 Zurich,Switzerland).

Reuterin produced from glycerol by Lactobacil-lus reuteri, a normal inhabitant of the humanintestine, is a broad spectrum antimicrobialagent. Reuterin is active against intestinal path-ogens, yeasts, fungi, protozoa and viruses, but itseffect upon the commensal intestinal bacteria isunknown. The aim of this study was to test theactivity of reuterin against a representative panelof intestinal bacteria. Reuterin was producedwith cells of L. reuteri in pure glycerol solution(85% yield). After cell elimination by centrifu-gation, the supernatant was filter-sterilized,lyophilized and reuterin was purified with ace-tone/ethyl acetate on a silica gel column. Purityof reuterin was checked with the tryptophane-HCl assay, using acrolein as standard. Minimalinhibition (MIC) and minimal bactericidal con-centrations (MBC) of reuterin were determinedfor different strains belonging to Bifidobacte-rium (6), Bacteroides (3), Lactobacillus (6),Eubacterium (2) Gram-positive cocci (2),Clostridium coccoides (4), Escherichia coli andListeria (2), using a twofold serial dilutionmicroplate assay under anaerobic conditions. Asupplemented BHI broth, reduced with titaniumcitrate and allowing a high growth for all testedbacteria, was used for the activity test. The mostresistant bacteria to reuterin were the 2 strains ofL. reuteri (MICs 30–50 mM) followed by theother lactobacilli and Clostridium clostridio-forme (MICs 15–40 mM). E. coli, Ruminococ-cus productus, Bifidobacterium breve and Liste-ria innocua exhibited intermediary sensitivities

Session I: Microbial diversity in health and disease S11

(MICs: 7.5–15 mM), followed by the other Bifi-dobacterium spp., Streptococcus salivarius and2 of the 3 Bacteroides tested (MICs 1.9–3.8 mM). The most sensitive strains to reuterinwere Bacteroides vulgatus and Clostridium dif-ficile (MICs 1.9 mM). Our results suggest thatreuterin could be used to selectively inhibit anumber of intestinal pathogens such as Clostrid-ium difficile, while preserving health-promotingcommensal bacteria of the human gastrointesti-nal tract.

P-11 Lactobacillus reuteri decreases E. coliconcentration in the presence of glycerol in anin vitro model of colonic fermentation. V.Cleusix, G. Le Blay, S. Vollenweider, C. Lac-roix (ETH-Zentrum, 8092 Zurich, Switzerland).

Lactobacillus reuteri is a well-known probioticculture that produces, in the presence of glyc-erol, a potent broad spectrum antimicrobial sub-stance named reuterin, active against manyintestinal pathogens in vitro. Moreover, L. reu-teri has been shown to prevent intestinal infec-tions in vivo. However, the mechanism of actionand the potential implication of reuterin are notknown. In this study, we tested the effect ofL. reuteri on the gut microbiota and its capacityto secrete reuterin in situ using a novel in vitrocolon model mimicking conditions of theascending colon. A fecal sample collected froma healthy person was immobilized in polysac-charide gel beads under anaerobic conditions.Beads were inoculated in two reactors, run inparallel for 60 days and continuously fed with anutritive medium simulating the adult ilealchime. The effects of not adding or adding daily108 cfu·mL–1 of L. reuteri without and with dif-ferent glycerol concentrations (0, 10 and100 mM) were tested on the major intestinalbacteria populations in effluent samples withfluorescence in situ hybridization. Short chainfatty acids, 1,3-propanediol and reuterin concen-trations were quantified by HPLC. The adjunc-tion of 100 mM glycerol induced an increase in1,3-propanediol concentrations in the presence(15.6 ± 4.2 vs 1.9 ± 0.5 mM) or absence (37.0 ±4.3 vs 2.1 ± 0.7 mM) of L. reuteri, whereas noreuterin was ever detected. Moreover, glycerolinduced a decrease in E. coli populations in thepresence of L. reuteri, whereas the adjunction ofL. reuteri alone had no effect. These data suggest

that the inhibition of E. coli by L. reuteri in thepresence of glycerol could derive from the in situproduction of reuterin despite its absence ofdetection in the fermented medium. Indeed, reu-terin is a very reactive compound that is usuallynot detectable in complex media.

P-12 Characterization of Lactobacillusmicrobiota from the duodenum of organi-cally farmed pigs. P.S. Cocconcellia,b, A.Gentilia, M.L. Callegaria,b (a Centro RicercheBiotecnologiche, Università Cattolica del SacroCuore, Via Milano 24, 26100, Cremona, Italy,b Istituto di Microbiologia, Università Cattolicadel Sacro Cuore, Via Emilia Parmense 84, 29100Piacenza, Italy).

The Lactobacillus population of intestinalscraped samples collected from three organi-cally farmed pigs was studied using molecularbiology techniques, including cultivation inde-pendent DGGE analysis, and an isolation basedapproach, where strains were taxonomicallyidentified by species-specific PCR, ARDRAand 16S rDNA sequencing. The dominant spe-cies detected by DGGE analysis were L. reuteri,L. crispatus, L. vaginalis and L. fermentum,while L. johnsonii, L. reuteri and L. mucosaewere identified using cultivation-based approaches.RAPD fingerprint demonstrated that each sub-ject harboured a unique collection of lactoba-cilli. Strains were analysed by physiologicalassays in order to evaluate their probiotic capac-ity. The resistance of strains to synthetic gastricjuice and the ability to adhere to the intestinalepithelium was also evaluated. A hydrophobic-ity test and the presence of genetic determinantscoding for important adhesion factors wereassessed to determine potential colonisationability. A susceptibility test performed on iso-lated lactobacilli to antimicrobials of clinicaland veterinary importance revealed that 31% ofthe analysed strains were sensitive to all 11 anti-biotics tested.

P-13 Monitoring major ruminal bacteria insheep during adaptation to Neotyphodium coen-ophialum-infected tall fescue straw with real-time PCR. M.J.M. De Lorme, S.L. Lodge-Ivey,A.M. Craig (College of Veterinarian Medicine,Oregon State University, Corvallis, OR 97330,USA).

S12 Session I: Microbial diversity in health and disease

Tall fescue toxicosis is a disease caused by ergotalkaloids produced by grass/straw-infesting fun-gal epiphytes. Sheep have a tolerance above thatof other grazing animals to fescue toxicosis. Thisincreased tolerance supports the belief that thereare unique microorganisms or groups of micro-organisms in the rumen of sheep capable ofdetoxifying alkaloids, primarily ergovaline, pro-duced by the fungal endophyte, Neotyphodiumcoenophialum. Six major sheep ruminal bacteriawere monitored using real time PCR and sixgroup specific primer sets as well as a universalbacterial primer set during the feeding and adap-tation to Neotyphodium coenophialum-infectedtall fescue straw. Serum prolactin levels weremonitored as indicators of toxicosis. Serum pro-lactin was decreased by approximately 72% insheep exposed to 500 ppb of ergovaline as com-pared to the control group, indicating clinicalfescue toxicosis. Prevotella bryantii B14 and theStreptococcus group (S. bovis, S. caprinus,S. equines) were detected in low levels throughthe entire feeding period and were not differentbetween treatment groups. Selenomonas rumi-nantium-Mitsuokella multiacida JCM6582 wasthe most abundant organism found in the sam-ples and Eubacterium ruminantium (ATCC17233) was undetectable in most samples overall sampling times. Ruminococcus flavefaciens(ATCC 19208T) sequence was detected in mod-erate levels through the entire feeding period andlevels were approximately the same betweentreatment groups. Ruminococcus albus wasdetected in low levels through the entire feedingperiod and was not affected by treatment. Theseresults shows no change in the bacterial popula-tions monitored, indicating that either thedetoxification does not cause a population shiftor the organism(s) responsible for the detoxifi-cation has yet to be identified.

P-14 Phylogenetic analysis of TNT degradingenrichments isolated from ovine rumen fluid.M.J.M. De Lorme, A.M. Craig (College of Vet-erinarian Medicine, Oregon State University,Corvallis, OR 97330, USA).

The clean up and degradation of munitionswastes is a growing concern as environmentalrestrictions increase. One of the major munitionswastes in the United States is 2,4,6-trinitrotolu-ene (TNT). Anaerobic microbes found in the

rumen are well equipped with reductiveenzymes that are often responsible for the rapidreduction of TNT in bioremediation systems.Previous work has shown that whole bovinerumen fluid is capable of rapidly degrading TNTin vitro. Two TNT-degrading enrichments werecultivated from ovine rumen fluid in order toassess the TNT degradation potential of ovineruminal bacteria. Each enrichment was capableof degrading 100 ppm TNT in less than 24 h. Full16S rDNA was amplified from the enrichmentsusing universal eubacterial primers, then clonedand sequenced in order to identify individualmembers. In one enrichment designated 3–1,91% of clones had high sequence similarity(99%) to Enterococcus faecium and E. durans.The remaining clones had high similarity toLactobacillus species with the strongest associ-ation to L. mucosae (99%). The other enrich-ment, designated 4–1, produced only onesequence type with a 99% similarity to Strepto-coccus bovis. These results indicated that morethan one species of bacteria from the rumen iscapable of degrading TNT. We feel it may bepossible to use sheep as bioreactors for the biore-mediation of munitions wastes. However, invivo studies and assessments of toxicity in thehost animal are still to be addressed.

P-15 Stx2 synthesis in pathogenic Escherichiacoli: transcriptional analysis, secretion andactivity. T. De Sablet, Y. Bertin, A.P. Saint-Gobert, J.P. Girardeau, C. Martin (INRA Cler-mont-Ferrand-Theix, Unité de Microbiologie,63122 Saint-Genès-Champanelle, France).

Shiga-toxin producing Escherichia coli (STEC)are associated with food-borne infections caus-ing human diseases, ranging from uncompli-cated diarrhoea to hemorrhagic colitis (HC) andhaemolytic-uremic syndrome (HUS). Cattle andother ruminants are the main reservoir of STECstrains. Shiga-toxins (Stx) are the most criticalvirulence factors responsible for the principalmanifestations of HUS and HC, and it has beensuggested that the varied capacity of STECstrains to cause serious disease in humans isassociated with the type and/or amount of Stxproduced. We determined the stx2 alleles andcytotoxicity of a total of 42 Stx2-producingSTEC obtained from patients or from healthycattle. Nine strains belong to the seropathotype

Session I: Microbial diversity in health and disease S13

A, containing O157:H7 LEE-positive strains,which are the most frequently associated withsevere outbreaks in the world. The remainingstrains belong to seropathotype C that includemainly non-O157:H7, LEE-negative strains fre-quently associated with sporadic cases of HUS.Then, a subset of 24 strains was analyzed for stx2transcription and Stx2 secretion, with and with-out mitomycin C induction. The stx2 allele wasmainly associated with the most pathogenichuman strains belonging to the O157:H7 sero-type. In contrast, stx2-vha and stx2-vhb weremainly associated with strains belonging to theless virulent seropathotype C. In addition, ourresults suggest that the higher virulence ofstrains producing the Stx2 variant could berelated to the higher expression of the gene andto the higher secretion of its product, in basal aswell as in induced conditions.

P-16 Enumeration in cattle of verocytotoxi-genic E. coli containing ehxA and eaeA viru-lence genes. S.E. Denman, R.A. Gilbert, J.Padmanabha, C.S. McSweeney (CSIRO Live-stock Industries, Queensland Bioscience Pre-cinct, Queensland 4067, Australia).

In order to enumerate putative enterohaemor-rhagic E. coli populations present in bovine sam-ples, a method based on DNA hybridisation andhydrophobic grid membrane filter (HGMF)technology was developed. In contrast to previ-ously published HGMF methods, which use sin-gle DNA probes to detect virulence factors suchas the shiga toxin genes, this method utilises sev-eral DNA probes and colour detection, allowingfor the identification of multiple virulence geneswithin bacterial isolates on replicate HGMFmembranes. Molecular probes were developedfor EHEC virulence factors encoded by thegenes for enterohemolysin (ehxA), intimin(eaeA), as well as the shiga toxin genes, stx1 andstx2. In this way, it was possible to identifyE. coli strains incorporating any combination ofthese virulence genes, thus enabling the specificenumeration of potential EHEC strains, contain-ing a full genetic complement for enterohaemo-lysin, intimin and at least one shiga toxin gene.HGMF analysis of these populations also indi-cated that diet did have an effect on the preva-lence of these E. coli subpopulations, howeverthe incidence and overall concentrations of fae-

cal populations were low (< 3 log10 MPN per gfaeces). Within the detection limits of HGMFanalysis, results also indicated that the hide andcarcase of animals found to shed putative EHECin their faeces, were not subsequently contami-nated with these organisms.

P-17 The impact of lifestyle on the humanchild intestinal microbiota. J. Dicksveda, H.Flöistrupb, A. Scheyniusc, J.K. Janssona

(a Department of Microbiology, Swedish Uni-versity of Agricultural Sciences, Uppsala, Swe-den, b Institute of Environmental Medicine,Karolinska Institute, Stockholm, Sweden,c Department of Medicine, Clinical Immuno-logy and Allergy Unit, Karolinska Institute andUniversity Hospital, Stockholm, Sweden).

The use of molecular tools has recently enableddetailed exploration of the composition of thehuman intestinal microbiota. To date, however,few studies have taken the known large inter-individual variation into consideration and moststudies are based on few analyzed individuals. Inaddition, factors that contribute to the microbialcomposition in the human intestine are stillunknown. In this study we used the molecularprofiling approach, terminal-restriction frag-ment length polymorphism (T-RFLP), in com-bination with multivariate statistics, to assess themicrobial community structure in fecal samplesof 90 children from three European countries. Inparticular, we studied how certain lifestylesimpact on the microbial community composi-tion. We found significant differences in thediversity of the fecal microbiota in children fromfamilies with an anthroposophic lifestyle com-pared to children living on farms. In addition, weobserved that several of the characteristics asso-ciated to the anthroposophic lifestyle, such as arestricted use of antibiotics and a diet mainlybased on biodynamically or organically pro-duced food items, also correlated with a highermicrobial diversity. Lactic acid bacteria andrelated species were also specifically monitoredby T-RFLP. The lactic acid bacterial communi-ties in the gut could be divided into two groupsdepending on the presence or absence of specificdominant populations. However, the functionalsignificance of these dominant populations iscurrently not known. In conclusion, the ecologyof the intestinal tract requires more exploration

S14 Session I: Microbial diversity in health and disease

to understand the mechanisms that drive themicrobial composition and to couple composi-tion to function.

P-18 Development and application of RNAand DNA based DGGE to study the bacterialcolonisation of fresh perennial ryegrass in therumen. J.E. Edwards, S.A. Huws, E.J. Kim,A.H. Kingston-Smith, N.D. Scollan (Institute ofGrassland and Environmental Research, PlasGogerddan, Aberystwyth, SY23 3EB, UK).

Currently, the temporal sequence by whichrumen bacteria colonise fresh plant material ispoorly understood. Analysis of populationchanges by molecular techniques has been lim-ited by dominant amplification of chloroplast-derived 16S rDNA. Development of a newprimer pair for profiling bacterial populations byDenaturing Gradient Gel Electrophoresis (DGGE)has resolved this, enabling investigation of bac-terial colonisation of fresh perennial ryegrass inthe rumen, using both RNA and DNA as molec-ular markers. Sequence analysis of the dominantbands in DGGE profiles of fresh plant materialconfirmed that the new primers detected 16SrDNA from epiphytic bacteria, not chloroplasts.The methodology was applied to perennial rye-grass previously incubated in sacco in therumens of two grazing, cannulated cows, andmachine washed. Duplicate polyester bags gaveconsistent DGGE profiles. The plant epiphyticsamples formed a distinct group from the rumenincubated samples, although RNA and DNAbased profiles clustered differently within this.Rumen incubated samples clustered togetherregardless of which molecular marker was used,and no differences between the colonising bac-terial populations were observed at the 5, 10, 15or 30 min time points. These results demonstratethat the process of bacterial colonisation of freshforage in the rumen can be studied using eitherDNA or RNA. While population changes relat-ing to colonisation of perennial ryegrass byrumen bacteria occurred in the first 5 min, pop-ulation diversity was then consistent until 30 minof ruminal incubation. It is possible that changesin population sizes were also occurring over thisperiod of apparent stability.

P-19 A comparison of colon and faeces micro-bial diversities in horses using biomolecular

techniques. C. Faubladiera, V. Jullianda, L.Veigaa,b, F. Chaucheyras-Durandb,c (a ENE-SAD, Dijon, b Lallemand Animal Nutrition,Blagnac, c Unité de Microbiologie, INRA,Saint-Genès-Champanelle, France).

Some limitations in the knowledge of the micro-bial diversity in the equine digestive ecosystemare linked to the difficulty in studying non cul-tivable micro organisms. New techniques basedon molecular analysis could be used to describethe variety of microbial populations and to studytheir evolution depending on the different com-partment. Our objective was to evaluate the use-fulness of TTGE (Temporal Temperature GelElectrophoresis) and RISA (Ribosomal Inter-genic Spacer Analysis) to compare colonic andfaecal microbial diversities in horses. Fourmature geldings, fitted with a right ventral coloncannula, received a morning meal providing aminimum of 200 g/100 kg BW of starch. Afterfourteen days of adaptation to the diet, faecal andcolonic contents were collected five hours afterthe morning meal in order to perform biomo-lecular analysis (RISA and TTGE). DNA wasextracted, amplified by PCR and separated onadapted gels. Similarity analysis of the electro-phoresis gels was performed using the programDiversity database BIO RAD. Preliminary datahighlighted strong individual differences inmicrobial profiles. Similarity indexes from com-parison of banding patterns have been deter-mined for two animals. The microbial profileswere almost identical between faecal andcolonic content for one horse (95–100% similar-ities with RISA; 77–95% with TTGE) but weredifferent for the other one (23% with RISA; 41%with TTGE). More analyses are in progress. Theresults obtained in this study will demonstrate iffaeces are representative of the colon microbialdiversity. Furthermore, using RISA and TTGE,it will be possible to compare the impact of dif-ferent factors on the microbial diversity in bothcolonic and faecal ecosystems.

P-20 Molecular phylogeny of the Neocalli-masticales (Mycota) based on ribosomal RNAgenes: exploring the root of the fungal king-dom. K. Fliegerováa, K. Voigtb (a Institute ofAnimal Physiology and Genetics Academy ofSciences of Czech Republic, Prague, Czech

Session I: Microbial diversity in health and disease S15

Republic, b Institute of Microbiology, Friedrich-Schiller University, Jena, Germany).

Anaerobic fungi inhabit the gastrointestinal tractof herbivores, especially ruminants, and make asignificant contribution to rumen metabolism,particularly to the digestion of plant structuralmaterial. Anaerobic fungi exhibit two basicforms, with nuclear migration throughout thehyphal mass for polycentric species (Anaeromy-ces, Orpinomyces) and with concentration ofnuclear material in a zoosporangium for mono-centric species (Neocallimastix, Piromyces,Caecomyces). All genera of rumen fungi havebeen assigned to the family Neocallimasticaceaeof the class Chytridiomycetes. However, gutfungi differ significantly from other chytrids inmany aspects, and the relationship betweenrumen anerobic fungi and aerobic chytrids is notanswered satisfactorily. Also chytridiomycetefungi are well known to be direct descendantsfrom the common ancestor of all true fungi andare therefore at the root of recent fungi. We aregoing to present a comparative phylogeneticanalysis of rumen anaerobic Chytridiomycetes(Neocallimasticales) with special emphasis onpolycentric genera in comparison to aerobicchytridiomycetous and zygomycetous fungibased on all available 18S and 28S rDNAsequence data. We have investigated partial SSUrDNA fragments using primer pair NS1 and NS2and partial LSU rDNA fragments using theprimer pair NL1 and NL4. Sequence analysis ofthese spacers has been applied to thirteen strainsof gut anaerobic fungi (Orpinomyces, Anaero-myces, Neocallimastix) and eleven strains of aer-obic chytrids and zygomycetes (Mucor,Hyphochytrium, Allomyces, Blastocladiella,Catenaria, Phlyctochytrium, Nowakowskiella,Monoblepharis). Since chytridomycetes arebelieved to be the most basal lineage of the king-dom Mycota, our investigations aim at the phy-logenetic reconstruction of the root of the Fungi.At the same time we own a reliable tool formolecular taxonomical identification of obligatebiotrophic anaerobic fungi, for which cultiva-tion in artificial culture is rather tedious.

P-21 Effect of two energy and two proteinsources in sugar cane based diets on the pop-ulation of rumen ciliate protozoa in waterbuffalo (Bubalus bubalis) and zebu cattle (Bos

taurus indicus). R. Franzolin, F.P. Rosales,W.V.B. Soares (Universidade de Sao Paulo,FZEA, Pirassununga-SP, Brasil).

The true role of the protozoa population in therumen still is not clear, since wide differencesoccur among ruminant animal species, feedingsystems and environmental conditions aroundthe world. These organisms have survived in therumen for thousands of years and knowledge oftheir function may provide a key to improvinganimal production and preservation of the envi-ronment. The effect of diet upon both rumen pro-tozoa concentrations and generic distribution isobvious. In general, Entodinium species repre-sent more than 80% of the population for mostdomestic ruminants even under grazing condi-tions. However, our previous studies haveshown that a different population is generallyfound in water buffalo. Four rumen fistulatedbuffaloes and four zebu cattle, in two 4 × 4 LatinSquare design experiments, were fed withchopped sugar cane (60% DM) and a 2 × 2 fac-torial schedule of energy sources (corn grain vs.citrus pulp) and protein (urea vs. soybean meal).Zebu cattle had higher rumen protozoa concen-trations than buffalo. Generic composition wassignificantly different between buffalo and cat-tle; average percentage compositions were Ento-dinium (61.0% vs. 84.9%), subfamily Diplodi-niinae (28.6% vs. 1.4%), Epidinium (5.3% vs.0.0%) and Dasytricha (2.6% vs. 11.5%), respec-tively. Percentages of Entodinium and Diplodi-niinae species were higher in cattle fed groundcorn as the energy source, whereas the same spe-cies were higher in buffalo when urea was theprotein source. Sugar cane based diets main-tained much higher percentages of Dasytricha inthe rumen of cattle. Comparing the concentra-tion and composition of rumen ciliate popula-tions in these two animal species confirms theprevious observations that the fauna of waterbuffalo differs considerably from that of otherruminants under similar feeding and environ-mental conditions.

P-22 Bacterial diversity and antibiotic resist-ance in colon contents from hooded seals(Cystophora cristata) in the Greenland Sea. T.Glada, K.M. Nielsena, L. Nordgårdb, M.A.Sundsetc (a Department of Pharmacology andInstitute of Pharmacy, University of Tromsø,

S16 Session I: Microbial diversity in health and disease

Tromsø, Norway, b Norwegian Institute of GeneEcology, Tromsø, Norway, c Department ofArctic Biology and Institute of Medical Biology,University of Tromsø, Tromsø, Norway).

Hooded seals are abundant in the arctic/subarc-tic regions of the North Atlantic Ocean. Theyprey on a variety of pelagic fish, squid and tosome extent crustaceans. Once a year they gatherat particular breeding sites, one of them being thepack ice of the Greenland Sea, to give birth andmate. In this study, colon content was collectedfrom six female hooded seals harvested duringa three weeks expedition to the Greenland Seain March–April 2004. Samples were processedwithin 15 min of sampling. Both aerobic andanaerobic bacteria in the colon content of the sixanimals were enumerated. Numbers of anaero-bic bacteria ranged between 8.1 (± 1.1) × 108 and5.2 (± 1.2) × 109 cfu·mL–1, and numbers of aer-obic bacteria between 1.2 (± 0.3) × 108 and3.1 (± 1.1) × 109 cfu·mL–1. Further, we searchedfor aerobic and anaerobic bacteria with tetracy-cline and ampicillin resistance determinants. Noanaerobic tetr and ampr isolates were detectedafter 72 h growth (detection limit > 20). Aerobictetr isolates were found in all samples, the num-bers ranging from 4.8 (± 3.7) × 102 to 1.8(± 1.5) × 103 cfu·mL–1. Aerobic ampr isolateswere found in three of the samples, the numbersranging between 1.4 (± 1.7) × 101 and 1.6 (±0.3) × 104 cfu·mL–1. A selection of phenotypicalampr isolates (n = 208), as well as communityDNA isolated directly from the colon contents,were investigated for the presence of ampicillinresistance gene blaTEM by PCR. No blaTEMgenes were detected. We are currently making16S rDNA clone libraries based on colon con-tents from three seals. A number of clones willbe analysed by DNA sequencing in order todescribe the bacterial diversity in the colon con-tents of hooded seals.

P-23 Quantification and visualization of theuncultured bacterial groups U2 and U3 fromthe rumen. H. Goto, H. Yabuki, T. Shinkai, Y.Kobayashi (Graduate School of Agriculture,Hokkaido University, Japan).

Recent studies on phylogeny of fiber-attachingrumen bacteria have revealed the presence ofseveral groups of uncultured bacteria. We devel-

oped real-time PCR assays and a fluorescencein situ hybridization (FISH) protocol for quan-tification and visualization of two of such uncul-tured groups (U2 and U3, Koike et al., FEMSMicrobiol. Lett. 229: 23–30, 2003) in the rumen.The PCR primers were designed to amplify par-tial 16S rDNA of U2 and U3, and specificity ofthe PCR was confirmed by sequencing theamplicons. The real-time PCR assays for thesegroups were developed and tested for their accu-racy and sensitivity. The quantifiable range ofU2 and U3 of these newly developed assays was102 to 109 copies of 16S rDNA at pure culturelevel. The assay values were highly reproduciblewith minimum intra- and inter- assay variations(< 14%). A DNA probe was designed for U2 anda FISH protocol was developed for detecting U2attaching to fibrous materials. The copy numberof 16S rDNA of U2 was higher in the solid phasethan in the liquid phase of rumen digesta bothfrom sheep and cattle (P < 0.05). Proportions ofU2 and F. succinogenes of total bacteria werehigh enough to be representatives in the rumen(0.6–1.9 and 4.0–6.1%, respectively). Rumi-nally incubated milled orchardgrass hay stemwas used for the quantitation and visualizationof U2. U2 showed the maximum at 12 h (× 109/g)and then decreased gradually. The same trendwas observed by FISH detection. F. succino-genes showed the maximum population size at24 h, generally exceeding the population size ofU2. These results suggest that U2 could be oneof the major members of the fibrolytic consor-tium in the rumen.

P-24 Molecular characterisation of thecolonic mucosal associated flora. G.L. Greena,J.D. Sandersona, J. Brostoffa, B.N. Hudspitha,D.S. Ramptonb, K.D. Brucea (a Life Sciences,King’s College London, London, UK, b QueenMary School of Medicine and Dentistry, Insti-tute of Cellular and Molecular Science, London,UK).

Despite the significant roles the bacterial com-munity of the large intestine plays in health anddisease, the mechanisms involved remain poorlyunderstood. We have used Denaturing GradientGel Electrophoresis (DGGE) of phylogeneti-cally-informative 16S rRNA genes amplifiedfrom DNA extracted directly from colonic biop-sies to profile mucosa associated bacterial flora.

Session I: Microbial diversity in health and disease S17

We focused on eighteen patients who showed nosigns of gastrointestinal disease at colonoscopy.Analysis showed that the bacterial communitiespresent at adjacent sites (2–5 cm apart) withinindividuals were highly similar both in terms ofthe presence and absence of bands (Mean Sim-ilarity Coefficient (MSC) 99%, Standard Devi-ation (SD) 1.9%), and in the relative band inten-sities. Two hundred and seven matched bandswere determined within the profiles from adja-cent biopsies, the mean difference in the relativeintensities was 1.5% (SD 1.9%). The number ofbands detected in the profiles from each individ-ual ranged from 9 to 23 and MSC for the com-munities studied was 40% (SD 14%), indicatingthat the majority of species differed betweenindividuals. Subsequently, it was found that thebacteria associated with the mucosa sampledfrom different regions of the large intestine (rec-tum, sigmoid, transverse and descending colonand caecum) was highly similar within an indi-vidual (MSC 98%, SD 1.4%). This workstrongly suggests that the dominant bacterialspecies present throughout the large intestinevary little between different colonic locations.Studies such as these are important in order toprovide additional insights into the bacterialpopulations that are involved in the complexinterplay between these micro-organisms andthe gastrointestinal tract.

P-25 Antimicrobial resistance of Escherichiacoli and Enterococcus spp. from an inte-grated, semi-closed, swine and human popu-lation. R.B. Harveya, H.M. Scottb, W.Q. Alalib,L.D. Highfieldb, T.R. Callawaya, T.L. Poolea,R.C. Andersona, D.J. Nisbeta (a Food and FeedSafety Research Unit, Agricultural ResearchService, USDA, College Station, TX 77845,USA, b Department of Veterinary IntegrativeBiosciences, Texas A&M University, CollegeStation, TX 77843, USA).

Abstract withdrawn.

P-26 Enteral feeding, compared to parenteralfeeding, enhances bacterial gut colonizationin neonatal pigs. R.B. Harveya, K. Andrewsa,R.E. Droleskeya, K.V. Kansagrab, B. Stollb,D.G. Burrinb, T.R. Callawaya, R.C. Andersona,D.J. Nisbeta (a FFSRU, ARS, USDA, College

Station, TX 77845, USA, b Children’s NutritionCenter, ARS, Houston, TX 77845, USA).

Total parenteral nutrition (TPN) has been asso-ciated with mucosal atrophy, impaired gut bar-rier function, and translocation of luminal bac-teria with resultant sepsis in preterm humaninfants. Currently, we examined the effects ofenteral (ENT) or TPN treatments on transloca-tion events in neonatal pigs and on colonizationand composition of microbiota in the neonatalgut. Newborn, colostrum-deprived pigs (< 24 hold) were fitted with intravenous catheters andwere fed either ENT (n = 13) or TPN (n = 13)for 7 d. After 7 d of treatment, pigs were eutha-nized and samples were collected for bacterialculture from the blood, intestinal tract andorgans. ENT pigs had an increased number ofbacterial genera isolated, higher concentrationsof bacteria (cfu·mL–1), and increased coloniza-tion of all segments of the intestinal tract com-pared to the TPN pigs. Translocation of bacteriafrom the intestinal tract to tissues or blood wassimilar (8 of 13) for both groups. The ENT grouphad 1/13 positive for Clostrium difficile toxin Awhereas the TPN group had 5/13. We concludedthat ENT favored increased bacterial concentra-tions comprised of more speciation in the gas-trointestinal tract compared to TPN, and thatTPN-treated piglets were at higher risk ofcolonization by toxin-expressing strains ofC. difficile.

P-27 Plasmid occurrence in local populationof Selenomonas ruminantium is not affectedby the presence of restriction and modifica-tion systems. J. Ivan, P. Javorský, P. Pristaš(Institute of Animal Physiology of Slovak Acad-emy of Sciences, Šoltésovej 4-6, Košice 04001,Slovakia).

Restriction and modification systems (RMS) inbacteria primarily act to protect the organismfrom foreign DNA invading the cell. However,only few RMS were tested for the protection invivo, and there is limited data on the effect ofRMS on the horizontal gene transfer frequency.In our previous work, a complex population ofRMS systems was detected in local populationof the rumen anaerobe Selenomonas ruminan-tium, when at least 12 different RMS phenotypeswere observed. Simultaneously, in more than

S18 Session I: Microbial diversity in health and disease

60% of studied strains, multiple plasmid DNAswere detected in size ranging from 1.4 kb tomore than 40 kb. The aim of this work was toanalyze the correlation between RMS occur-rence and the presence of small plasmidspSRD191 (1.4 kb), pSRD192 (2.3 kb) andpSRD194 (4.1 kb) in S. ruminantium. The threeplasmids were cloned into Escherichia coli plas-mids and analyzed for the presence of restrictionsites. No significant correlation was found betweenthe occurrence of plasmids and the presence ofRMS in S. ruminatium, indicating that the dis-tribution (spreading) of plasmids is not affectedby the presence of RMS.

P-28 Construction and function based screen-ing of metagenomic libraries of the humangut microbiota. B.V. Jones, J.R. Marchesi (Ali-mentary Pharmabiotic Center, University Col-lege Cork, Cork, Ireland).

A highly diverse and complex microbial ecosys-tem resides within human gastro-intestinal (GI)tract. This community has been found to consistof > 1000 species of bacteria. It is estimated that70–80% of these are uncultured. The unculturedfraction of the gut microbiota is likely to containnovel metabolic capabilities with the potential toinfluence human health. To access the metaboliccapabilities of the human gut microbiota, large-insert metagenomic libraries were constructed,and high through-put function driven screeningimplemented. 12 functional screens have beenoptimised to detect clones producing protease,lipase and esterase, DNase, cellulase, bile salthydrolase, bacteriocins, quorum-sensing signalmolecules, low pH tolerance, oxalate degrada-tion, siderophores, antibacterial and antifungalactivity. To date a total of 517 000 clones havebeen examined using 8 of these screens, andclones positive for production of protease, lipo-lytic enzymes, cellulase, DNase, oxalate degra-dation, and tolerance of low pH have been iden-tified. In total 882 positive clones were isolated.End sequencing data from protease positive andoxalate degrading clones indicated that theseclones originated from a wide range of bacterialgroups present in the gut. Further characterisa-tion of clones exhibiting protease productionindicated that the optimum pH varied betweenpH 6 and pH 7, while no activity was observedat pH 8. Ability to degrade host derived proteins

was assessed but no activity against fibrin, elas-tin, or collagen was detected. These results sug-gest that the human gut metagenome is a richsource of novel enzymes and bioactive agents,which can be accessed though functional screen-ing of metagenomic libraries.

P-29 Use of Ranikhet-inactivated vaccinecombined with V4 HR and BCRDV for theprevention of viscerotropic velogenic New-castle disease in backyard poultry. M.A. Kafi,M.M. Amin, M.B. Rahman (Department ofMicrobiology and Hygiene, Faculty of Veteri-nary Medicine, Bangladesh Agricultural Uni-versity, Mymensingh-2202, Bangladesh).

The performance of V4HR (asymptomaticstrain) of Newcastle disease Virus after primaryand secondary vaccination as well as its efficacyadministered with mesogenic (M) live andRanikhet-inactivated vaccine were investigated.Similarly comparison of F strain vaccine knownpopularly as BCRDV in Bangladesh was alsoaccomplished. To perform this, 180 day-oldchicks were divided into six groups, A, B, C, D,E and F, that were vaccinated with differentschedule except group F, which served as unvac-cinated control. The birds were reared separatelywith proper hygienic management and feed ad-libitum. ND vaccinations were carried out at 7(1 week) 28 (4 weeks), 63 (9 weeks) and 126(18 weeks) days of age via ED with V4HR andBCRDV while in case of mesogenic (M) live andRanikhet-inactivated vaccine i/m route was fol-lowed. The HI titre manifested a protectivepotentiality against ND when interpreted toprobable value of serum neutralization (SN). Itwas also observed that the birds of group Aadministered repeatedly 4 times with V4HRexhibited a HI titre of 40 to 80 with a GMT of49.25 in the last occasion. It was observed thatgroup B, where the birds had three successiveinoculations of V4HR followed by mesogenic(M) live, the HI titres in most cases were at 40to 80 with a GMT of 42.87. In case of group C,the HI titres were found to be 160 to 320 with aGMT of 242.51 of sera samples collected aftervaccination with Ranikhet inactivated vaccinepreceded by three successive administration ofV4HR. The birds of group D, which had threesuccessive inoculations of BCRDV followed bymesogenic (M) live vaccine, the HI titre of sera

Session I: Microbial diversity in health and disease S19

samples collected on last occasion varied from80 to 160, of which the GMT was 98.49. In caseof group E, the vaccine used was Ranikhet-inac-tivated vaccine where sera samples exhibited HItitre of 80 to 160 with a GMT of 113.14. Theunvaccinated control group manifested no suchmeasurable both for the HI titre and GMT on142 days age of birds. From this study it may beconcluded that the vaccination schedule consist-ing of either V4HR or BCRDV followed by mes-ogenic live vaccine or Ranikhet inactivated vac-cine provided protective potentiality of HI titres.The V4HR when administered alone for maxi-mum of three successive administrations pro-duced HI presence of protective potentiality.The mesogenic (M) live vaccine when adminis-tered after three successive inoculation ofBCRDV had a comparatively higher level of HIantibody than similar schedule with V4HR. Vac-cination schedule consisting of three successiveinoculation of V4HR followed by RanikhetInactivated vaccine elucidated highest than sim-ilar schedule with BCRDV.

P-30 Swedish allergic infants have less diverseintestinal microbiota compared to healthyinfants, at their age of one week. C. Karlssona,M. Wanga, C. Olssonb, G. Molina, A Woldc, B.Hesselmard, R. Saalmand, I-L. Strannegårdd, N.Åbergd, I. Adlerberthc, S. Ahrnéa (a Laboratoryof Food Hygiene, Department of Food Technol-ogy, Engineering and Nutrition, PO Box 124,Lund 22100, Sweden, b Department of Surgery,Universitetssjukhuset MAS, 20502, Malmö,Sweden, c Department of Clinical Bacteriology,Gothenburg University, 41346 Göteborg, Swe-den, d Department of Paediatrics, GothenburgUniversity, 41685 Göteborg, Sweden).

The prevalence of allergy is rising, especially indeveloped countries. The intestinal microflora isconsidered important for a correct maturation ofthe immune system in children. Thus it is of greatinterest to investigate how the composition ofthe flora differs between healthy individuals andpatients with immunological diseases. The aimof the present study was to analyse differencesin the composition of the dominating intestinalmicrobiota in Swedish allergic (diagnosed at18 months) and healthy infants, at their age ofone week. DNA was extracted from faecal sam-

ples and the 16S rRNA genes were amplifiedwith universal primers. The samples were ana-lysed with Terminal Restriction FragmentLength Polymorphism (T-RFLP). In this studywe showed that the microbiota of healthy infantsgenerated a statistically significant highernumber of peaks compared to allergic infants,when the AluI enzyme was used (P = 0.003).Also the Shannon and Weiner diversity indexwas significantly higher in healthy infants (P =0.005). Furthermore, T-RFLP was proved to bea suitable method for analysing the diversity ofthe intestinal microbiota in infants. Results fromthis study showed that a high bacterial diversityseems to be one of the important parameters fornot developing allergy.

P-31 Effects of mastication on temporal bac-terial colonisation and degradation of freshperennial ryegrass in the rumen. E.J. Kim, J.E.Edwards, R. Sanderson, A.H. Kingston-Smith,N.D. Scollan, M.K. Theodorou (Institute ofGrassland and Environmental Research, PlasGogerddan, Aberystwyth, SY23 3EB, UK).

Understanding of interactions between fresh for-age and microbes during microbial colonisationand degradation of masticated feed boli in therumen is limited. Our objective was to examinetemporal changes in bacterial colonisation anddegradation of fresh perennial ryegrass (PRG) inthe rumen and the influence mastication has onthese processes. Samples of freshly-cut PRG andmasticated PRG bolus material were incubatedin polyester bags in the rumen of two grazingcows for 0, 0.25, 0.5, 1, 2, 4, 8, 12 or 24 h, thenmachine-washed and freeze-dried. With bolusmaterial, 20.1% (s.e. 3.05) of the dry matter(DM) was immediately soluble, whilst, therewas a lag of 1.3 (s.e. 0.11) h before any net DMloss was observed with intact forage. Totalgenomic DNA was extracted from the freshmaterials and residues, and analysed by 16SrDNA based Denaturing Gradient Gel Electro-phoresis (DGGE) to profile the colonised bacte-rial populations. Cluster analysis of DGGE datashowed that plant epiphytic samples clustereddistinctly from residues following rumen incu-bation. Bacterial colonisation was evident within15 min. Rumen incubated samples groupedtogether in both animals, however, intact PRG

S20 Session I: Microbial diversity in health and disease

from early incubations ( 2 h) clustered consist-ently within this. The delay in onset of degrada-tion of intact PRG, compared to PRG bolus,shows the contribution of mastication to theprocess of ruminal degradation of forage. Dif-ferences in DGGE banding patterns betweenintact and bolus material indicated that mastica-tion was associated with a difference in the col-onising bacterial population. Further analysis ofthe colonising protozoa and anaerobic fungi is inprogress.

P-32 Monitoring and source tracking of tetra-cycline resistance genes in lagoons andgroundwater impacted by swine productionfacilities. S. Koikea, I.G. Krapacb, H.D. Olivera,J.C. Chee-Sanfordc, R.I. Aminovd, R.I. Mackiea

(a Department of Animal Sciences, Universityof Illinois, Urbana-Champaign, USA, b IllinoisState Geological Survey, Champaign, IL 61821,USA, c USDA Agricultural Research Service,USA, d Rowett Research Institute, AberdeenAB21 9SB, UK).

Antibiotics are routinely used in the livestockindustry and a concern is that farm-generatedresistant bacteria can easily migrate into soil,ground- and surface- waters via seepage fromwaste handling lagoons and from land applica-tion of manure. To monitor the dissemination ofresistance genes into the environment, we deter-mined the occurrence of tetracycline resistancegenes (Tcr) in groundwater underlying twoswine confinement operations, by establishing awell network around the lagoons at each of twofacilities. Groundwater and lagoon sampleswere collected at six sampling times from 2000through 2003. Total DNA was extracted andPCR was used to detect seven Tcr [tet(M), tet(O),tet(Q), tet(W), tet(C), tet(H) and tet(Z)]. Theconcentration of Tcr was quantified by real-timeqPCR. To confirm the tet gene source in ground-water, comparative analysis of tet(W) genesequences was performed on groundwater andlagoon samples. All seven Tcr persisted ingroundwater during the three-year monitoringperiod at both sites. At Site A, the level of detec-tion frequency and concentration for Tcr wascorrelated with other inorganic contaminantsassociated with animal waste, confirming thatseepage from the lagoon influenced the distribu-

tion of Tcr in groundwater. Comparative analy-sis of tet(W) sequences revealed that theimpacted groundwater contained almost identi-cal gene sequences (99.8% identity) with thatfound in the lagoon. This result supports the dis-semination of Tcr from the lagoon into ground-water. However, novel sequence clusters ofresistance genes were also found. This supportsthe hypothesis that the source of resistance genesis multifactorial and is not limited to swinemanure.

P-33 The effect of celiac diet on microbes inthe colon. J. Kope ný, J. Mrázek, K. Fliegerová(Laboratory of Anaerobic Microbiology, Insti-tute of Animal Physiology and Genetics, CzechAcademy of Sciences, Víde ská 1083, 142 20,Czech Republic).

Celiac disease is a digestive disorder, caused byan immune reaction in the small intestine, result-ing in damage to the surface of the small intestineand an inability to absorb several grain proteins.The allergens are grain glutens (prolamins) –glutein and gliadin from wheat, barley, rye andoat. Some speculate that celiac disease has beenaround since humankind switched from a forag-ing diet of meat and nuts to a cultivated dietincluding grains such as wheat or barley. Celiacdisease can be cured with a gluten-free diet. Thedirect effect of celiac diet on the intestinalmicrobes was not described. In healthy individ-uals, we studied the effect of celiac diet andceliac diet combined with chitosan. Based onDGGE analysis of digesta, it was found thatceliac diet significantly influences the structureof the gut bacterial population. Microbial metab-olism was changed as well as short chain fattyacids were increased and pH decreased. Appli-cation of celiac diet with chitosan (3 g per day)changed the structure of the bacterial populationand their metabolism even more. Beside that,chitosan increased total counts of fecal chitino-lytic bacteria and decreased the body weight ofthe treated persons. We described the effect ofceliac diet in indicated childhood celiac diseasepatients. The changes in SCFA, pH and micro-bial population correlated with the trendsobtained with healthy individuals. We havesequenced DNA fragments of bacteria present inthe lumen as well as the in the adhered popula-tion to intestinal biopsy samples. Under in vitroconditions, we have observed that isolated

č

ň

Session I: Microbial diversity in health and disease S21

prolamins inhibited the growth of some anaero-bic bacteria. Degradability of prolamins wasalso tested.

P-34 The incidence of Listeria monocytogenesin meat and meat products, Zagreb area,Croatia. I. Kova eka, N. Kneževi -Jonji a, D.Puntari a, J. Bošnira, B. Maticaa, D. Kova ekb,N. Uršulin-Trstenjakc (a Zagreb Institute of Pub-lic Health, Mirogojska 16, Zagreb, Croatia,b Biotechnical Faculty, University of Biha ,Bosnia and Herzegovina, c High MedicineSchool, Mlinarska 38, Zagreb, Croatia).

Listeria monocytogenes is a pathogen involvedin several outbreaks and sporadic cases of food-borne illness associated with the consumption ofcontaminated foods. It can be isolated from dairyproducts, meat and meat products and otherfoods. Its wide-spread existence in differentfoods is related to its ability to grow and survivein unfavourable conditions. This study exam-ined the incidence of L. monocytogenes in meatand meat products in the Zagreb area, Croatia.In meat products, L. monocytogenes is oftenaffected by several types of stress caused by theprocessing treatment, like heating, freezing anddrying. Recovering stressed L. monocytogenesfrom food is of great importance, because sub-lethally injured bacteria can recover and regaintheir pathogenicity. A total of 202 samples wereexamined, from stores, butcher shops and res-taurants. L. monocytogenes was isolated fromapproximately 23% of raw meat and thermallytreated products, meaning the danger of diseaseis slim, but a good thermal processing is by allmeans necessary. L. monocytogenes was alsofound in meat by-products for end-use, i.e. inthose products which will get no further thermaltreatment. One needs to exercise extreme cau-tion with these products, since they are beingkept on refrigerator temperature, which allowsL. monocytogenes to multiply. Therefore, suchhigh-risk food items need to be regularlyinspected for the existence of this rare pathogen.

P-35 Analysis of the temporal stability anddiversity of the bacteria in the human gas-trointestinal tracts of healthy individualsusing DGGE and RAPD PCR. S.F. Lawton,J.R. Marchesi (Alimentary Pharmabiotic Cen-

tre, Department of Microbiology, UniversityCollege Cork, Ireland).

The microbial diversity of the human gastroin-testinal (GI) tract is generally thought to be con-stant over time, but the bacteria that are meta-bolically active may vary considerably withtime. The aim of this project is to analyse thesemicrobes using culture independent approachessuch as Denaturing Gradient Gel Electrophore-sis (DGGE) and Random Amplified Polymor-phic DNA (RAPD), using RNA as an indicatorof metabolic activity and DNA as an indicator ofdiversity. Samples were collected from fourindividuals over a six month period. Nucleicacids were isolated and cDNA synthesised fromthe RNA, and both gDNA and cDNA were usedas a template for PCR. DGGE was used to com-pare gDNA and cDNA using both universal andgroup specific primers for the 16S rDNA gene,in order to analyse the temporal stability anddiversity of microbes in the GI tract of healthyindividuals. Initial DGGE results displayed dif-ferences between an individual’s gDNA andcDNA, thus indicating that microbial diversityand microbial metabolic activity may be dissim-ilar. Genomic DNA was used for RAPD analy-sis. In the RAPD approach there were notabledifferences in diversity between individuals, butalso within these individuals, over time.

P-36 The antimicrobial peptide Pediocin PA-1has no effect on common intestinal bacteriacompared to nisin and antibiotics. G. Le Blay,A. Zihler, C. Lacroix, I. Fliss (ETH-Zentrum,8092 Zurich, Switzerland).

Bacteriocins are anti-microbial peptides natu-rally produced by many micro-organisms,including lactic acid bacteria (LAB). Theyinclude a wide variety of peptides and proteinsin terms of size, microbial targets, and mecha-nisms of action. Although for some broad-spec-trum bacteriocins such as nisin and pediocin, theantimicrobial activity against human and animalpathogens has been largely studied, the inhibi-tory effects toward intestinal bacteria have neverbeen reported. The aim of this study was to testthe sensitivity of the most representative intes-tinal bacteria to broad-spectrum LAB bacterioc-ins and to compare data with antimicrobialeffects of common antibiotics. Twenty-one

č ć ćć č

ć

S22 Session I: Microbial diversity in health and disease

common intestinal bacteria were grown in sup-plemented brain heart infusion (BHI) mediumand their sensitivities to nisin Z (102 µg·mL–1),nisin A (102 µg·mL–1), and pediocin PA-1(205 µg·mL–1) were evaluated using the agardiffusion test. The antibiotic sensitivity of intes-tinal strains was tested using disc assays. NisinA and nisin Z exhibited very similar antimicro-bial spectra, inhibiting at different levels allGram-positive intestinal bacteria tested, exceptStreptococcus salivarius. In contrast, pediocinPA-1 exhibited high activity against Listeria butshowed no detectable effect on intestinal bacte-ria. Most intestinal bacteria were sensitive topenicillin (10 µg), ampicillin (10 µg), chloram-phenicol (30 µg) and tetracycline (30 µg) (19 of21), whereas high resistance was observed againstoxacillin (1 µg) and streptomycin (10 µg). Incontrast to antibiotics and nisins that exhibiteda large spectrum of activity, pediocin PA-1 hada much narrower spectrum, suggesting its poten-tial utility in the treatment of intestinal infections.

P-37 Pediocin PA-1 is active against Listeriainnocua in an in vitro model for the terminalileum. G. Le Blay, I. Fliss, C. Lacroix (ETH-Zentrum, 8092 Zurich, Switzerland).

Listeria monocytogenes is responsible for severefood-born infections, which can be life-threat-ening especially for infants and elderly popula-tions. The emergence of antibiotic-resistantpathogens has stimulated the search for newstrategies, such as the use of bacteriocins, to pre-vent or cure food-born infectious diseases in theintestine. In this study, we evaluated the efficacyof partially purified pediocin PA-1 from Pedio-coccus acidilactici to inhibit Listeria innocua(used as a model for the pathogen, L. monocy-togenes) under physiological conditions of theterminal ileum during a continuous in vitro fer-mentation. A fecal sample collected from ahealthy person was immobilized in gel beads andcultivated for 30 days in a continuous stirredtank reactor, fed with a nutritive medium simu-lating the ileal chime (pH 7.5). After reaching apseudo-steady state (day 19), the reactor wasinoculated with L. innocua at a final concentra-tion of 107 cfu·mL–1. The concentration ofL. innocua in the reactor was followed during8 h by plating on selective medium. Commensalpopulations were monitored with fluorescencein situ hybridization and short chain fatty acid

concentrations by HPLC. Three bacteriocinconcentrations (2, 3 and 5 MIC) were tested onthe disappearance kinetics of L. innocua in thecontinuous reactor. Our data showed a dose-dependent effect of pediocin PA-1. The adjunc-tion of pediocin at 5 MIC induced a disappear-ance of listeria below 2.5 Log cfu·mL–1 in5 h, whereas the adjunction of 3 and 2 MICinduced listeria concentrations of respectively3.2 and 3.7 Log cfu·mL–1 after 8 h, compared to4.5 Log cfu·mL–1 with the control listeria with-out adjunction of pediocin. In addition, pedi-ocin-PA1 had minimal effects on the balance ofcommensal bacteria. Our study suggests thatpediocin PA-1 could be a potential candidate toprevent or treat listeria infection, as an alterna-tive or complement to antibiotherapy.

P-38 Assessing the methanogen diversity ofthe human intestinal flora by targeting themcrA gene. A. Mihajlovski, M. Alric, J.F.Brugere (ERT-CIDAM, Université d’Auver-gne, Clermont-Ferrand, France).

The human colon includes a wide variety ofmicro-organisms which perform an intense met-abolic activity. Hydrolytic and fermentativebacteria produce important quantities of H2, re-utilized by 3 groups of hydrogenotrophs: theSulphate-Reducing Prokaryotes (SRP), the ace-togenic bacteria and the methanogenic micro-organisms, exclusively members of the archaeadomain. Up until now, colonic methanogens arerepresented by Methanobrevibacter smithii(CH4 production from H2 and CO2), and alsosometimes by Methanosphaera stadtmanae (CH4production from H2 and methanol), both belong-ing to the class Methanobacteriaceae. They areknown to be quantitatively variable betweenpeople, in an age- and nutrition-dependent man-ner, some people being non-carriers. To date,their presence is mainly determined by a breath-test (CH4 is detected in breath when there are atleast 107 methanogens per gram of faeces) and/or by culture-dependent techniques (whichprobably do not recover all the micro-organismspresent in samples). Molecular tools targetingthe SSU rRNA (or gene) can avoid these draw-backs. An alternative way for the characterisa-tion of metabolically-defined species is to targeta specific molecular marker involved in themetabolic pathway of interest. In various eco-systems, this approach has led to a successful

Session I: Microbial diversity in health and disease S23

detection of methanogens using the mcrA gene.It encodes the α sub-unit of the methanogen-spe-cific methyl-CoM reductase, a α2β2γ2 enzymecatalysing the last step of methanogenesis.Stools have been collected from 6 healthy vol-unteers and primers designed from an alignmentof known mcrA sequences. PCR amplificationsfrom stools were positive for 5 of the 6 samples.Cloning is now being performed, followed byRFLP studies. The sequencing of each RFLPpattern will permit to better identify the diversityof the colonic methanogenic flora.

P-39 Evaluation of three techniques for theextraction of DNA from rumen fluid. S.Muetzel, D. Paillard, R.J. Wallace (RowettResearch Institute, Bucksburn, Aberdeen AB219SB, UK).

A critical step in microbial community analysisis the choice of an accurate way to extract DNAfrom environmental samples. Indeed, there is anincreasing number of DNA extraction kits avail-able for various types of environmental samples.In this study, two commercial DNA extractionkits (Qiagen Stool kit and Qbiogene FastDNASoil kit) and a phenol/chloroform-based extrac-tion modified from a previously published study(Whitford et al., 1998, Anaerobe 4: 153–163)were compared and modified to optimise yieldand purity of the DNA extracted. QuantitativePCR was subsequently used to evaluate theextraction efficiency for DNA from three recal-citrant organisms commonly present in therumen: Butyrivibrio sp., Ruminococcus albusand Streptococcus bovis. The Qiagen Stool kit isthe only method that relies only on a chemicallysis. Although the yield of DNA appeared to behigh, the lysis of small Gram-positive bacteriawas poor. The Soil kit yielded very good qualityDNA but comparatively low amounts of DNA.A three-fold increase of the mechanical lysistime improved the DNA recovery by 250%. Thephenol/chloroform technique initially resultedin poor quality DNA that had to be further puri-fied on a column. Quantitative PCR specific forthree Gram-positive bacteria of the rumen dem-onstrate that the Stool kit was not appropriate toextract DNA from ruminal cocci. The phenol/chloroform technique was more appropriate forButyrivibrio spp., but not for the two other spe-cies. These experiments demonstrate that amechanical lysis is needed in order to extract

DNA from cocci, but do not suggest a singlemethod for all organisms.

P-40 Effects of anti-Helicobacter pylori treat-ment and probiotic supplementation on intes-tinal microbiota. E. Myllyluomaa,b, T.Ahlroosc, L. Veijolad, H. Rauteline,f, S. Tynkky-nenb, R. Korpelaa,b,c (a Institute of Biomedicine,Pharmacology, University of Helsinki, Helsinki,Finland, b Foundation for Nutrition Research,Helsinki, Finland, c Valio Ltd, Research andDevelopment, Helsinki, Finland, d Departmentof Bacteriology and Immunology, HaartmanInstitute, University of Helsinki, Helsinki, Fin-land and Herttoniemi Hospital, Helsinki, Fin-land, e Department of Bacteriology and Immu-nology, Haartman Institute, University ofHelsinki, Helsinki, Finland, f HUSLAB, Hel-sinki University Central Hospital Laboratory,Helsinki, Finland).

Antimicrobials may disrupt the balance of themicrobiota by causing quantitative and qualita-tive changes such as decrease in colonizationresistance against pathogens. The aims of thisstudy were to evaluate the effect of recom-mended antimicrobial treatment of H. pyloriinfection, consisting of clarithromycin, amoxi-cillin and lansoprazole, on intestinal microbiotaand the ability of probiotic combination admin-istration to prevent treatment-induced altera-tions in the intestinal microbiota. Faecal sampleswere obtained from 39 H. pylori infectedpatients randomized into two treatment groupsreceiving treatment with placebo or treatmentwith probiotics. Nineteen H. pylori negative vol-unteers were also included into the study as acontrol group. Samples were collected before,during and after the treatment, and the microbi-ota was analyzed by fluorescent in situ hybridi-sation, quantitative PCR and culturing. Thequantities of predominant bacterial groups werealtered significantly after the antimicrobial treat-ment in both groups and disturbances werefound even after 9 weeks of the treatment (P <0.001). Probiotics slightly counteracted theeffects of anti-H. pylori treatment, monitored assignificantly less alterations in the total numbersof aerobes (P < 0.001) and lactobacilli/entero-cocci (P < 0.05). At the baseline, the composi-tion of the microbiota between H. pylori positivevs. negative individuals was different as far as

S24 Session I: Microbial diversity in health and disease

clostridia and the total number of aerobes wereconsidered (P < 0.05). This study suggests thatthe recommended treatment for H. pylori infec-tion induces significant disturbances in the intes-tinal microbiota. The probiotic supplementationcould have some potential in inhibiting themicrobiota alterations.

P-41 Multiple bacteriocin structural genesare present in animal gut cocci. K. Nigutova,P. Javorský, P. Pristaš (Institute of Animal Phys-iology, Slovak Academy of Science, Košice,Slovakia).

Bacteriocins are antimicrobial peptides and pro-teins that inhibit or eliminate the growth of thetarget, usually phenotypically close organisms.In recent years there has been considerable inter-est in bacteriocins produced by lactic acid bac-teria (LAB), especially by Streptococcus andEnterococcus strains. Enterococcal and strepto-coccal isolates obtained from the gastrointesti-nal tract of different animals were analysed forbacteriocin-like activity (BLA) production andresistance by overlay test. The production ofBLA was tested by a simple method on severalindicator microorganisms. About 40% of thetested strains showed BLA production in thistest. The occurrence of bacteriocin-like activi-ties among the rumen bacteria suggests that bac-teriocin production and resistance of ruminalbacteria to bacteriocins may play an importantrole in general ecology of the rumen ecosystem.The occurrence of selected bacteriocin structuralgenes encoding enterocin A, enterocin B, ente-rocin P, enterolysin A, and cytolysin was ana-lysed by PCR analysis and compared with dataon bacteriocin production and sensitivity. Everytested strain showed the presence of at least a sin-gle bacteriocin structural gene, and up to fourstructural genes were found in a single isolate.Cytolysin and enterocin B homologues were themost frequently detected, while enterocin A wasfound to be the least common. The bacteriocingenes in Gram-positive bacteria are located onplasmids, but the location on chromosomalDNA has been reported for several other bacte-riocins. The high frequency of bacteriocin struc-tural genes in the tested strains indicated the pos-sibility of horizontal gene transfer of bacteriocinstructural genes. The results of this study shouldbroaden the knowledge of bacteriocin-like activ-

ity production and resistance among Gram-pos-itive bacteria.

P-42 Molecular identification of methano-gens from sheep from Venezuela. N. Obispoa,X. Mab, A.-D.G. Wrightb (a Instituto Nacional deInvestigaciones Agrícolas (INIA) Edificio 3área universitaria de la UCV, Maracay Apartadode Correo 4653, Aragua, Venezuela, b CSIROLivestock Industries, Private Bag 5, Wembley,WA 6025 Australia).

The diversity of rumen methanogens in sheepfrom Venezuela was investigated at the molec-ular level by using individual 16S rRNA genelibraries. These libraries were prepared from therumen contents obtained from 10 sheep. A totalof 76 clones were examined by using HaeIII andBcnI digestion to create riboprints. The riboprintpatterns have revealed 16 phylotypes, five ofwhich represent existing strains and 11 werenew. Among the new strains, three belong to thegenus Methanobrevibacter and were detected bythe BcnI digestion. Denaturing gradient gel elec-trophoresis (DGGE) analysis was also applied tothe same samples. The results of DGGE profileindicated that variations of methanogen at themolecular level exist between individual sheep.Of the 14 DGGE bands detected, four sheep hadnine prominent bands, one sheep had only fourprominent bands, and the remaining five sheephad between four to nine bands. A detailed studyof the comparison of the methods of individual16S rRNA gene libraries and the use of DGGEanalysis in methanogen molecular diversity isunder way.

P-43 High prevalence of oral tetracyclineresistance genes in Bolivian Amerindians. I.Pantojaa, M. Mojicab, G. Vargas-Pintob, K.P.Scottc, A. Pattersonc, H.J. Flintc, M. Blaserd,M.G. Dominguez-Belloa (a University of PuertoRico, San Juan, Puerto Rico, b Hospital Bolivi-ano Japones, Sucre, Bolivia, c Rowett ResearchInstitute, Aberdeen, UK, d New York Univer-sity, New York, NY, USA).

Indigenous bacteria are an important reservoir ofantibiotic resistance genes. The potential forconjugal transfer to pathogens limits the utility

Session I: Microbial diversity in health and disease S25

of antibiotics in treating infections. The level ofcirculation of Tetracycline resistance (tetR)genes in third world countries with relativelylow antibiotic usage has not been assessed. Inthis study, we aimed to assess the presence ofribosomal protection gene tet(M) in patients andhealthy rural subjects in Bolivia. We obtainedoral swabs from 58 Bolivian Amerindians; 40were symptomatic patients consulting the Gas-troenterology service and 18 were asymptomaticfrom rural communities. All volunteers pro-vided informed consent of participation. DNAwas extracted and subjected to amplification of16S rRNA genes and tet(M) genes. BacterialDNA was present in 51 of 58 oral samples, illus-trated by amplification of 16S rDNA. Tet(M)amplified in 81% and 70% of symptomatic andasymptomatic subjects, respectively (P = 0.413).Oral tetR genes were present in a high proportionof subjects from a rural community in Bolivia,suggesting there might be a selective factor otherthan antibiotics favouring the persistence ofresistance determinants in oral bacteria.

P-44 Detection of novel mosaic tetracyclineresistance genes. A.J. Patterson, M.T. Rincon,H.J. Flint, K.P. Scott (Rowett Research Institute,Bucksburn, Aberdeen, UK).

Tetracycline resistance (Tcr) has become themost common type of bacterial antibiotic resist-ance, mainly as a consequence of the intenseselection pressure exerted by the excessive useof tetracycline, not only in human and veterinarymedicine but also in animal feeding practices.Approximately 40 TcR genes conferring resist-ance by four different mechanisms, ribosomalprotection (RP), efflux, enzymatic inactivationand modification of the rRNA target, have beendescribed. Recently, various forms of mosaicgenes, presumably arising by recombinationbetween wild-type ribosome protection typegenes, have been discovered. In this study, weamplified TcR genes in a wide range of humansaliva, human faecal, and animal faecal samples,using full-length tet(O) primers. Sequence anal-ysis of these products illustrated that mosaicgenes represented 78% and 46% of sequences,in pig and human faecal samples, respectively.The genes identified included novel combina-tions of tet(O), tet(W) and tet(32). It appearsfrom this study that constant antibiotic exposuredrives the evolution of these genes, selects for

the bacteria carrying them, and results in the pre-dominance of mosaic genes in certain environ-ments. We are investigating the evolutionaryadvantages of mosaic TcR genes over their wild-type counterparts, but there is evidence that theyconfer increased levels of antibiotic resistance totheir bacterial hosts.

P-45 Use of rifampicin-resistant Clostridiumperfringens strains for infection studies onnecrotic enteritis in broiler chickens. K.Pedersen, L. Bjerrum (Danish Institute for Foodand Veterinary Research, Department of Poul-try, Fish and Fur Animals, Hangøvej 2, 8200Aarhus N, Denmark).

Abstract withdrawn.

P-46 Mercury-induced shift in rumen proto-zoan Entodinium caudatum associated bacte-rial populations. M. Piknovaa, J. Mrázekb, S.Kisidayovaa, Z. Varadyovaa, T. Tothovaa, P.Javorskýa, P. Pristaša (a Institute of AnimalPhysiology of Slovak Academy of Sciences,Košice, Slovakia, b Institute of Animal Physiol-ogy and Genetics, Academy of Sciences of theCzech Republic, Praha, Czech Republic).

The rumen protozoal population is characterizedby multiple complex protozoa-bacteria interac-tions. Heavy metals, including mercury, can beinhibitory to the growth of both protozoa andbacteria present in the rumen. The aim of thepresent study was to elucidate the role of bacte-ria-protozoa interactions in response to stressevoked by an increased mercury level. Duringlong term in vitro cultivation of Entodinium cau-datum with an increased dose of mercury, themercury resistance of the E. caudatum cultureincreased from an initial level of less than 0.5 µgper mL to more than 10 µg per mL. No changesof the chemical status of mercury were detectedin the protozoal culture, indicating the absenceof detoxification – mercury reductase – activi-ties. DGGE analysis of associated bacterial pop-ulations revealed a clear shift of the eubacterialpopulation structure towards hydrogen sulphideproducing bacteria. Very limited changes wereobserved in the archaebacterial population struc-ture. The data obtained indicated that free living

S26 Session I: Microbial diversity in health and disease

bacteria protect protozoal cells and their archae-bacterial endosymbionts by eliminating mer-cury into its insoluble form.

P-47 Development of a molecular techniqueto study the equilibrium of poultry gut micro-biota. C. Pissavina, I. Gabrielb, C. Burela, S.Malletb, R. Mauricea, M. Lessireb, P. Fravaloa

(a French Agency of Food Safety, BP 53, 22440Ploufragan, France, b National Institute of Agro-nomic Research, 37380 Nouzilly, France).

The recent changes in the European rules lead tomodifications in the feeding and rearing condi-tions of poultry. Little is known about the ecol-ogy of the poultry gut. The standard bacteriolog-ical methods enable only the study of a minorityof the gut microbiota. Since a decade, severalmolecular methods, all based on the amplifica-tion of the 16S rDNA gene, were applied to thestudy of different ecological niches. The aim ofthis study was to estimate the relevance of theCE-SSCP to be used as an epidemiological tool.Among several tested DNA regions, we retainedthe V3 region as the PCR reaction target. Ourresults showed that this method gives reproduc-ible data. The fingerprints of caeca, ileum andstool samples exhibited from about 25 to40 bands. The pattern of the ileum is closer tothat of the cloacal stool than that of the caeca.The variability between individuals was weak.However, fingerprint of pooled samples may bea better indicator of the animal health inside ahusbandry. In order to estimate the variabilityinside and between housings, we comparedmolecular gut patterns of animals coming fromthe same hatchery and reared under the sameconditions (zootechnical parameters control,feeding), in parallel in two separate husbandries.The numeration of specific flora (total aerobicbacteria, coliforms, lactic bacteria) was per-formed by using conventional methods. Only aslight difference was observed for the coliformcounts comparing the caeca and the cloacalstools. The fingerprint of the total flora revealedvery slight variabilities inside the two housings.However, more differences were detected whencomparing one housing to the other. In conse-quence, this method gives gut flora fingerprintsthat may be useful to analyse the effect of varioustreatments on the equilibrium of gut flora.

P-48 Characterization of the 16S-23S rRNAgene intergenic spacer region to developprobes for tracking probiotics in the reindeerrumen. K.E. Præstenga,b, R.I. Mackieb, I.K.O.Cannb, S.D. Mathiesenc, M.A. Sundseta(a Department of Arctic Biology, University ofTromsø, Norway, b Department of Animal Sci-ences, University of Illinois at Urbana-Cham-paign, Urbana, IL 61801, USA, c Department ofArctic Veterinary Medicine, The NorwegianSchool of Veterinary Science, Norway and Nor-dic Saami Institute, Guovdageaidnu, Norway).

Reindeer in northern Norway are free-rangingand experience large seasonal variations in feedquality and abundance. Warming of the Arcticregion with periods of subsequent thawing andfreezing of snow in winter occasionally exposereindeer to starvation, resulting in a reducednumber of rumen bacteria and death. Supple-mentary feeding has become more common inthe Saami reindeer husbandry to alleviate theeffects of starvation and weight loss. Use ofruminal probiotics during these periods mayhelp prevent digestive disorders and increaseanimal welfare. Phenotypic screening and sub-sequent 16S rDNA sequencing of 51 rumen bac-terial isolates from reindeer revealed that 7 ofthese were novel (< 97% sequence identity topreviously described strains). Three fibrolyticbacterial isolates (strain 8/94-32 and 8/9293-9from Norwegian reindeer, and strain E14 fromSvalbard reindeer) were selected for a probioticto increase plant cell wall digestibility. Thesewere both CMC-active and cellulolytic. No inhi-bition was found between the strains whengrown in co-culture. The 16S rDNA sequence ofstrain 8/94-32 confirmed a 98% identity toRuminococcus flavefaciens, and 8/9293-9 andE14 98–99% identity to Butyrivibrio fibrisol-vens. Specific oligonucleotide probes to detectand quantify the probiotic strains are beingdeveloped based on 16S-23S rDNA intergenicspacer region (ITS) sequences (489–626 bp).Analysis of the ITS region of B. fibrisolvensstrains (n = 10) isolated from reindeer, strainE14, R. flavefaciens FD-1 and R. albus Ra8,showed heterogeneity between the strains. Het-erogeneity was also found among ITS sequencesfrom the same isolates, indicating that B. fibri-solvens and R. flavefaciens have multiple ITSregions. Probe specificity will be evaluatedbased on sequence comparison with the data-base, as well as hybridization with a range of

Session I: Microbial diversity in health and disease S27

rumen bacterial isolates. Real-time qPCR prim-ers will be developed and validated for use infeeding trials to track the probiotic strains andevaluate their ability to establish in the rumen.

P-49 Comparative molecular analysis of anovine ruminal enrichment culture using twodifferent DNA separation techniques. R.M.Rattray, A.M. Craig (Oregon State University,Corvallis, Oregon, USA).

Abstract withdrawn.

P-50 Helicobacter pylori status in an urbanpopulation is inversely associated with asthma.J. Reibman, M. Marmor, M.-E. Fernandez-Beros, L. Rogers, G.I. Perez-Perez, M.J. Blaser(Departments of Medicine and EnvironmentalMedicine, NYU School of Medicine, and VAMedical Center, New York, NY 10016, USA).

Reduced exposures to gastrointestinal microbi-ota may provide one explanation for the increasein asthma prevalence, consistent with the“hygiene hypothesis.” Since the prevalence ofthe gastric colonizer Helicobacter pylori isdiminishing in human populations, and the pres-ence of these bacteria in a host is inverselyrelated to gastroesophageal reflux disease(GERD), we hypothesized that gastric coloniza-tion with H. pylori is inversely related to thepresence of asthma. Adult asthma cases (n =347) and controls (n = 214) were identified froman ongoing asthma registry in New York Cityand serum IgG antibodies to whole cell antigen(H. pylori+) or the immunodominant CagA anti-gen (CagA+) were measured. Seropositivity tothe H. pylori or CagA antigens was present in47.0% of the study population. After adjustmentfor potential confounding, asthma was associ-ated with atopy, as expected, and inversely asso-ciated with CagA seropositivity (OR = 0.65,95% CI = 0.42–1.0). Age of onset of asthma wassignificantly older (median = 21 years) amongindividuals with cagA+ strains compared toH. pylori individuals (median = 10 years). Thesedata are consistent with the hypothesis thatH. pylori colonization, especially with cagA+

strains, is protective against asthma presentationin this population. One possible mechanism mayinvolve cagA+ H. pylori induction of atrophic

gastritis, which protects against GERD bydecreasing gastric acidity, and thus might pro-tect against one of its potential consequences.

P-51 A broad host range integration vectorfor bioluminescent in vivo imaging of bacte-ria. C.U. Riedel, S.C. Corr, I.R. Monk, C.G.M.Gahan, C. Hill (Alimentary Pharmabiotic Cen-tre, University College Cork, College Road,Cork, Ireland).

Bioluminescent imaging of bacterial infectionsand contaminations has recently been applied toa range of different organisms. However, themolecular tools to label different bacteria havebeen highly specific. Here, we report the con-struction of a broad host range integration vectorfor bioluminescent labeling of both Gram posi-tive and Gram negative bacteria. A constitutiveand highly active promotor was synthetisedbased on the L. lactis consensus sequence. Tofurther enhance promotor activity the 5’ untrans-lated region of the hly promotor of L. monocy-togenes was introduced. This hybrid syntheticpromotor was cloned upstream of the promotor-less synthetic luxABCDE operon of P. lumines-cence. After functionality was shown in vitro inE. coli and L. monocytogenes the luxABCDEoperon was cloned together with the syntheticpromotor into a broad host range integration vec-tor based on the temperature sensitive repliconof pGh9::ISS1. The resulting vector was thenused to generate integration mutants of a rangeof bacterial genera as diverse as Escherichia,Salmonella, Listeria, and Lactobacillus. Suita-ble integrants were analysed in vitro for anydefects in growth and virulence and the site ofintegration was identified. Suitable biolumines-cent strains were then used to study infection orcolonisation of murine models as well as con-tamination of food products. Bioluminescencewas detected in intact animals, disected organs,and food products and was shown to correlateclosely with colony forming units in infectedorgans. In conclusion, a new tool for biolumi-nescent labeling was developed suitable for thenon-invasive monitoring of a wide range of bac-teria.

P-52 Identification of novel tetracyclineresistance genes in the metagenome of the

S28 Session I: Microbial diversity in health and disease

human colon-associated bacterial ecosystem.M.T. Rincon, K. Kazimierczak, P. Young, D.Henderson, K.P. Scott, H.J. Flint (RowettResearch Institute, Greenburn Road, Bucks-burn, Aberdeen AB21 9SB, UK).

Tetracycline resistance (TcR) genes are wide-spread in nature due to the extensive use of tet-racyclines as therapeutic antimicrobial agents inveterinary and human medicine and as feed addi-tives in farm animals. TcR genes are often asso-ciated with chromosomal mobile elements orplasmids that greatly enhance their probabilityof transfer between different bacterial hosts. Inthis work we report the use of a metagenomicapproach for the analysis of TcR genes in ahuman colon-associated bacterial ecosystem. Ametagenome library containing the collectivegenome of bacteria isolated from a faecal sampleof a volunteer who had been on long-term tetra-cycline therapy was constructed using a fosmidvector. Approximately 4000 clones with anaverage DNA insert size of 35 kb, equivalent toapproximately 35 bacterial genomes, werescreened for the occurrence of TcR genes. A totalof 88 clones exhibiting resistance to tetracyclineat 10 µg·mL–1 were isolated for further analysis.PCR-based analysis carried out using gene-spe-cific primer sets revealed the tet(O/32/O) mosaicgene to be the most abundant known TcR gene,followed by tet(W), tet(O) and tet(Q). Manyclones appeared to contain multiple TcR genes,with tet(O/32/O) and tet(W) the most commoncombination. Sequencing of selected clones alsorevealed the existence of a further new TcR geneclosely linked to tet(O/32/O). Computer analy-sis indicates that the newly identified genebelongs to the efflux pump type of TcR genes,similar to tet(A). Extensive analysis of one insertrevealed that the tandem tet(O/32/O) and newefflux pump TcR genes are located in a mobileelement similar to a previously characterizedtransposon in Enterococcus faecalis that carriesa cluster of genes implicated in resistance to van-comycin, although vancomycin resistance geneswere not detected in this case. In conclusion, themetagenome approach provided a powerfulmethod for gaining information on both knownand previously unknown TcR genes.

P-53 Prevalence of Helicobacter pullorum inbroiler chickens reared in northern Italy. M.

Rossia, V. Sanguinettia, R. Gaviolia, P. Lozitoa,G. Manfredab, R.G. Zanonia (a Department ofVeterinary Public Health and Animal Pathol-ogy, University of Bologna, Italy, b Departmentof Food Science, University of Bologna, Italy).

During 2005, in order to establish the prevalenceof Helicobacter pullorum in broiler chickens, atotal of 155 caecum contents collected from ani-mals intensively reared in 31 poultry farms(5 animals per farm) were sampled at the slaugh-terhouse. A modified Steele-McDermott mem-brane filter method was used to isolate H. pullo-rum. Each fresh sample was diluted 1:4 in amixture containing 25 mL Brain Heart Infusion,75 mL horse serum and 7.5 g glucose. Three hun-dred microlitres of this suspension were inocu-lated on a membrane filter (0.65 µm) previouslyapplied on the surface of a Brucella Agar 5%sheep blood plate. After one hour of incubationat 37 °C in increased H2-microaerophilic atmos-phere, the filter was removed and the plate wasincubated up to 7 days. Small, white-greyish col-onies of gram-negative, slightly curved rod bac-teria were preliminary identified as H. pullorumby a PCR assay based on 16S rRNA, and thensubjected to a RFLP-PCR assay to distinguishH. pullorum from H. canadensis. According tothe PCR and PCR-RFLP results, 127 out of155 animals examined were positive for H. pul-lorum (82%) and 100% of farms resultedinfected. All positive samples showed a highnumber of colonies (> 50 cfu/plate) phenotypi-cally consistent with H. pullorum in the mediaof first isolation, which suggests that this micro-organism, when present, colonizes the broilerchicken caecum at an elevated load.

P-54 Study of spirillar enteric flora of dogsand cats in Italy. M. Rossia, V. Sanguinettia,D. Giacomuccia, P. Acutisb, R.G. Zanonia(a Department of Veterinary Public Health andAnimal Pathology, University of Bologna,Ozzano Emilia, Bologna, Italy, b State Veteri-nary Institute of Piedmont, Liguria and AostaValley, Torino, Italy).

During 2002, in order to define the prevalenceof Campylobacter spp, enteric Helicobacter spp.and Anaerobiospirillum spp., a survey of190 dogs and 84 cats, either healthy or affected

Session I: Microbial diversity in health and disease S29

by gastroenteric disease, was carried out. Eightystrains of Campylobacter, 92 strains of Helico-bacter and 30 strains of Anaerobiospirillumwere isolated using different selective mediasuch as Skirrow, Blaser-Wang and CCDA. Theidentification of the genus was carried out byspecific PCR. Campylobacter was isolated from53 dogs and from 27 cats, Helicobacter from65 dogs and 18 cats and Anaerobiospirillumfrom 27 dogs and 3 cats. A polyphasic approachwas used to identify the species of both Campy-lobacter and Helicobacter. Seventeen C. jejuni,33 C. upsaliensis, 2 C. helveticus and one C. larifrom dogs and 7 C. jejuni, 6 C. upsaliensis and14 C. helveticus from cats were identified usingphenotypic tests and species-specific PCR.Whole cell protein profile analysis, phenotypictests, 16S rDNA PCR-RFLP and a phylogeneticstudy of partial groEL sequences were used toidentify 22 H. canis and 14 H. cinaedi in dogsand 12 H. canis and 2 H. cinaedi in cats. “Heli-cobacter rappini” with a typical flexispira mor-phology in TEM was found in 37 dogs and 5 cats.The protein profiles of these isolates were sim-ilar to that of the strain “Flexispira rappini”ATCC 49308, forming together a distinct clusterat the 82% similarity level. There was no statis-tically significant correlation found between theisolation of Campylobacter or Helicobacter andthe presence of gastroenteric symptoms in bothdogs and cats.

P-55 Influence of diet and sampling site on thediversity of the bacterial community attachedto the rumen epithelium in lambs. S. Sadet, C.Martin, B. Meunier, D.P. Morgavi (INRA,Theix, 63122 Saint-Genès-Champanelle, France).

The bacteria attached to the rumen epithelium(bacterial epimural community, BEC) are aubiquitous component of the rumen microbialecosystem, still their biological role is not wellknown. In order to better understand the func-tions of BEC, the effect of diet and sampling siteon its diversity was studied using PCR-DGGEwith universal eubacterial 16S rDNA primers.Eight lambs were allocated into two groups fedforage or a concentrate-rich diet. After slaugh-ter, 5 different sites of the rumen epithelium(dorsal (D1, D2), ventral (V), caudal (C) and lat-eral (LA) area) were sampled, as well as rumencontent of the liquid (LP) and solid (SP) phases.

Clustering analysis of DGGE profiles showedthat neither diet type nor sampling site had anyinfluence on BEC structure. For diet type, a con-founding animal effect cannot be discarded dueto the experimental design. However, for bothLP and SP samples separate clusters wereobtained for each diet. This result suggests thatthe bacterial population from the rumen contentswas more affected by diet than BEC. Further-more, diversity indexes based on DGGE opera-tional taxonomic units showed no differencesbetween diets or among sampling sites, exceptfor V, which tended to be more diverse than D1(P = 0.1) for the Shannon index (H). BEC diver-sity was, irrespective of sampling site, equallydistributed within the rumen of individuallambs. However, BEC structure differed amonganimals and seems to be influenced by the host.Future work will look at assessing the diversityof BEC functional groups and link them up withtheir physiological activity under different feed-ing conditions.

P-56 Microbial community changes in theintestine of the pre-adolescent turkey. A.J.Scupham (National Animal Disease Center,USDA, Agricultural Research Service, Ames,IA 50010, USA).

Colonization of the intestine by opportunisticpathogens can be inhibited by the native flora,but factors such as antibiotic use and age can dis-rupt the equilibrium, providing a susceptibleenvironment. Thus, identifying periods of innatesusceptibility in the poultry intestine is impor-tant for both animal health and food safety con-cerns. To monitor development of intestinalcommunities, cecal droppings from individualmale broad-breasted turkeys (n = 5 and 6 birdsper trial) were sampled weekly (from day ofhatch through 18 weeks of age). AutomatedRibosomal Intergenic Spacer Analysis (ARISA)community profiles identified a continualincrease in both bacterial and fungal speciesrichness with a slight decrease during weeks 16–18. Sorensen’s analysis of the fingerprint dataand MEGA 3.0 dendrograms describing com-munity similarity identified a period of commu-nity transition at week 12 in the two trials. Bac-terial libraries representing weeks 9, 11, 12 and14 were sequenced. While both ARISA and

S30 Session I: Microbial diversity in health and disease

LIBSHUFF analyses indicated a significant dif-ference in the species represented in the two tri-als, a predominance of Clostridia-like species atweek 9 was replaced by a predominance ofBacteroides-like species in weeks 11, 12 and 14.B. uniformis prevalence in week 11 libraries(85% and 65%) of both trials provides compel-ling evidence that it may act as a vanguard, pre-paring the gut environment for colonization byother Bacteroides species. The dynamics ofintestinal communities in maturing poultry sug-gest a continuous susceptibility to colonizationby pathogens, with a period of exceptionalpotential around week twelve. Bacteroides uni-formis has been identified as a potentially impor-tant microbe associated with maturation of theintestinal community and protection from colo-nization by opportunistic pathogens.

P-57 Type A Clostridium perfringens subtypesare differentiated between necrotic enteritisand healthy broiler intestinal isolates and aredistributed among and between hosts. G.R.Siragusaa, M.G. Wiseb (a Agricultural ResearchService, United States Department of Agricul-ture, Russell Research Center, 950 CollegeStation Road, Athens, Georgia, 30605, USA,b Current address: Bacterial Barcodes, Inc.,Athens, Georgia, 30605, USA).

We have consistently observed a differentialrep-PCR pattern distribution between gut iso-lates of type A Clostridium perfringens from anecrotic enteritis disease-state origin and thoseof healthy flock mates of commercially reareddrug-free (antibiotic growth promotant free)broiler chickens. From a drug free broiler isolatelibrary, we previously found and reported fivemajor rep subtypes of Cl. perfringens amongstbroiler gut isolates as well as environmental iso-lates obtained from darkling beetle adult and lar-vae and spent litter. Those findings indicated asubtype of Cl. perfringens which was dominatedby disease-state derived Cl. perfringens strains.Here, a library of Cl. perfringens gut isolatesobtained from healthy and necrotic broilerchickens from 3 different drug-free farms (fedno ionophoric anticoccidials or antibioticgrowth promotants yet receiving an anti-coccid-ial vaccine) were subtyped using rep-PCR andanalyzed with DiversiLab v 3.1 analytical soft-ware. We present data which suggest that within

a single broiler chicken intestinal tract can befound not only disease state linked Cl. perfrin-gens isolates but healthy-state isolates. Diseasestate isolates from these three farms were highlyrelated (> 90% similarity) based on rep-PCR pat-terning. Overall, our data suggest that strainsexist which in some manner might contribute tothe opportunistic mechanism of necrotic enteri-tis in drug-free broilers. The individual or com-bined pathogenic potential of these isolatesremains to be tested in an appropriate broilerchicken necrotic enteritis disease model.

P-58 Engineering improved tolerance tostresses encountered in the gastrointestinaltract. R.D. Sleator, C. Hill (Department ofMicrobiology and Alimentary PharmabioticCentre University College Cork, Ireland).

The objective of this study was to engineer Lis-teria monocytogenes strains with a significantlyimproved ability to tolerate stresses encounteredin the external environment and during gastroin-testinal transit, thus, improving Listeria’s effi-cacy as a potential vaccine and drug deliveryplatform. Using a directed evolution approach,based on a random mutagenesis strategy involv-ing the E. coli XL1-Red mutator strain, we gen-erated a mutant variant of the listerial betL gene(designated betL*), encoding a secondarybetaine uptake system. The mutant betL* pro-motes a dramatic increase in resistance to anumber of biologically relevant stresses whenexpressed in a variety of different surrogatehosts. Using a luciferase (Lux) reporter systemin combination with the IVIS Imager System(Xenogen Corporation, Alameda, CA), wetracked betL* expression, in real time, both invitro under various environmental stresses andin vivo in animal models of infection. In eachcase strains expressing betL* demonstrated amarked improvement over those expressingwild type betL, both in terms of gene expressionand bacterial growth. Sequence analysis of themutated gene revealed a single nucleotide dele-tion in the spacer region between the –10 and–35 promoter elements upstream of the betLcoding region. This deletion presumably intro-duces a conformational ‘twist’ in the putativepromoter, thereby increasing its transcriptionaloutput. Furthermore, the betL* mutation appearsto counter the heretofore unreported ‘twisted’

Session I: Microbial diversity in health and disease S31

cell morphology observed using scanning elec-tron microscopy of L. monocytogenes grown atelevated osmolarities. It is possible to selectivelyimprove genes required for bacterial stress sur-vival both inside and outside of the host. Suchmutated genes systems may ultimately be usedfor the construction of more physiologicallyrobust bacterial based vaccine and drug deliveryplatforms.

P-59 Antibiotic resistance, resistance genesand transfer in animal gut cocci. V. Stovcik,P. Javorský, P. Pristaš (Institute of AnimalPhysiology of Slovak Academy of Sciences,Šoltésovej 4–6, Košice 04001, Slovakia).

Enterococci have emerged as important patho-gens responsible for various infections, particu-larly of respiratory infections not only inhumans. Recent surveys have found widespreadresistance to various antimicrobial agents, espe-cially macrolides, tetracyclines, and in recentyears also to beta-lactam antibiotics. The aim ofthe present study was to analyze antibiotic resist-ance, resistance genes and transfer in animal gutcocci. Three hundred rumen or faecal isolateswere tested for tetracycline, erythromycin andampicillin resistance. High resistance frequencywas observed for tetracycline and erythromycin(40% of isolates). Eight (4%) isolates showedresistance to ampicillin due to lactamase produc-tion. Rumen isolates showed different resistanceprofiles compared to faecal isolates. TetM andTetL determinants were found to be predomi-nant in tetracycline resistant isolates, whereas allerythromycin resistant isolates possessed ermBdeterminant. No known bla gene was detected inampicillin resistant isolates. All resistance deter-minants were found to be transmissible with rel-atively high frequency. BOX-PCR fingerprint-ing indicated that both clonal spread of resistantstrain(s) and horizontal gene transfer are respon-sible for resistance transfer in animal gut cocci.The gastrointestinal tract of animals and theenterococci inhabiting it could serve as a reser-voir of mobile resistance determinants availablefor resistance spreading.

P-60 The prevalence of mobile gene cassettesin indigenous gut microbiota and dairystarter bacteria. J. Štšepetova, E. Sepp, K.

Truusalu, K. Lõivukene, P. Hütt, E. Songisepp,M. Mikelsaar (University of Tartu, Departmentof Microbiology, Ravila 19, 50411, Tartu,Finland).

The integrons are mobile gene cassettes that con-tain the determinants of site-specific recombina-tion systems to capture genes. Up to now, thesegenes are associated with transferable antimi-crobial resistance. However, there are few dataon the prevalence of integrons in indigenous gutmicrobiota and dairy starter strains. The aim ofthe present study was to estimate the prevalenceof the Int1 gene in indigenous E. coli, Lactoba-cillus and Bifidobacterium isolates and in starterlactic acid strains. A total of 30 indigenous E.coli isolates were collected from stool of 10 anti-biotic naïve infants (age < 1 y, born in 1998) andanother 30 isolates from stool of 9 elderly people(age > 65 y, born before 1940). Additionally,11 Lactobacillus and Bifidobacterium sp. ofhuman origin and 7 food starter lactic acidstrains were included. Microbial DNA wasextracted using the QIAamp DNA Mini Kit. Int1was detected by PCR with primers Int1-F andInt1-R according to Leverstein-van Hal. Theprevalence of the Int1 gene was significantlyhigher in the samples of infants as compared toelderly people (16/30 vs. 5/30, P < 0.01). PCRproducts of Int1 were detected also in 3/7 starterlactic acid bacteria and in 3/11 lactobacilli andbifidobacteria. The integrons of tested bacteriavaried in the size of the PCR amplicons. Theprevalence of IntI genes in indigenous E. coliisolates was higher in late birth cohorts. At thesame time, IntI genes can be found also indifferent food starter lactic acid bacteria andamong indigenous Lactobacillus/ Bifidobacte-rium sp. isolates. Their role in carriage andtransfer of antibiotic resistance or some othergenes needs elucidation.

P-61 Resistance of Methanothermobacterthermautotrophicus to therapeutics and anti-microbial substances. S. Šurína, L. ubo ováa,A. Majerníka, P. McDermottb, J.P.J. Chongb,P. Šmigá a (a Institute of Animal Biochemistryand Genetics, Slovak Academy of Sciences,90028 Ivanka pri Dunaji, Slovakia, b Depart-ment of Biology, the University of York, York,YO10 5WY, UK).

Č ň

ň

S32 Session I: Microbial diversity in health and disease

Methanoarchaea are found as microbial com-mensals in the guts of insects to humans. How-ever, a role for these commensals in the humandigestive tract and their potential for the gener-ation and dispersal of drug resistance is onlypoorly understood. Methanothermobacter ther-mautotrophicus strain ∆H is relatively easy toculture in the laboratory, has been extensivelystudied and is a very close relative of Methano-sphaera stadmanae and Methanobrevibactersmithii, two known human methanogenic com-mensals. We have used M. thermautotrophicusto screen for genetic mutants resistant to differ-ent therapeutics such as the antibiotic neomycin,the uncoupler TCS or the potassium-retainingdiuretic amiloride. Application of amilorideresulted in the isolation of colonies of spontane-ous mutants at a frequency of 10–6, a rate similarto that observed for neomycin resistance. Strainsresistant to both these drugs have been stablycultivated in selective and non-selective growthmedium for several months. The growth rate ofresistant strains was lower than that of the wildtype. However, methanogenesis was elevated inboth resistant strains. Moreover, the resistantphenotypes display similar changes in their pro-tein spectra when compared to those of the wildtype. Interestingly, the phenotypes of bothresistant strains indicate the presence of defectsin their bioenergetic processes. In particular,ATP synthesis and ion transport were affectedby mutations. Here we propose a direct mecha-nism of amiloride resistance based on biochem-ical data. This study reveals how different, com-monly used therapeutics can cause specificresistant phenotypes in methanogens, which cangenerate increased amounts of methane com-pared to wild type.

P-62 Control of Campylobacter jejuni coloni-zation in chickens treated with probiotics orbacteriocins. E.A. Svetocha, B.V. Eruslanova,V.D. Pokhilenkoa, Y.N. Kovaleva, L.I. Volodinaa,V.V. Perelygina, E.V. Mitsevicha, I.P. Mitsevicha,V.N. Borzenkova, V.P. Levchuka, O.E. Svetocha,T.Y. Kudriavtsevaa, N.J. Sternb (a State ResearchCenter for Applied Microbiology, Obolensk,Russia, b USDA, ARS, RRC, PMSRU, Athens,GA, USA).

Chicken intestinal material was screened fordiverse bacterial genera, and isolates wereassessed for antagonism (zones of inhibition)

against Campylobacter jejuni, a bacterial com-mensal in poultry. Isolates from these generawere used to treat chickens in an attempt to con-trol colonization by Campylobacter. Individualand groups of antagonistic isolates (probiotics)were gavaged into naïve chicks to protect againstCampylobacter challenge. Only when Campy-lobacter challenges approached a chick CD50%, were prophylactic probiotic treatmentseffective. Unless the treatment was 100% effec-tive, as birds aged, Campylobacter numbersapproached untreated control birds' levels. Com-mercial flocks may be exposed to Campylo-bacter at various levels anytime during produc-tion. To understand the mechanism enablingcompetitive exclusion, two of our most promis-ing antagonists, Lactobacillus salivarius NRRLB-30514 and Paenibacillus polymyxa NRRLB-30509, were further studied. We character-ized distinct bacteriocins (antimicrobial pep-tides) produced by each of the isolates. Whenbacteriocins were purified and fed to colonizedchickens, the subject birds became free ofCampylobacter, or manifested at least a one-mil-lion fold reduction in levels when compared tothe untreated control animals. Whereas treat-ments with probiotics were at best only modestlyeffective in controlling Campylobacter coloni-zation, orally administered bacteriocins pro-vided substantial reductions of Campylobacter.We believe that in vivo bacteriocin activity pro-vides the central explanation for competitiveexclusion within the intestinal tract.

P-63 Comparing gut microflora in pigs byusing an integrated probing system. N.Thanantong, A. Anderson, S. Edwards, O.A.E.Sparagano (School of Agriculture, Food andRural Development, University of Newcastleupon Tyne, NE1 7RU, UK).

The gastrointestinal tract of mammals harboursa complex microbial community in which manyspecies still have not been characterized. Themicroflora in a gut is in constant evolution butneeds to stay in equilibrium with the other spe-cies, including autochthonous and non-autoch-thonous species. Changes in diet and farrowingevents were monitored in sows while weaningtimes and feeds were followed in piglets. Amacro-array-based method was developed tosimultaneously screen Lactobacillus, Strepto-coccus and Bifidobacterium species in different

Session I: Microbial diversity in health and disease S33

parts of the pig gut (ileum, caecum and colon),rectal samples were also collected. The method-ology is based on a 16S-based PCR coupled witha reverse line blot hybridisation technique usingseveral species-specific biotinylated probes,which have been shown to be highly specific.Chemiluminescence was used as the detectionmethod. No differences in the microflora popu-lation targets were observed between boars andgilts or between replicated experiments butchanges in the diet had some impact on the pres-ence of Lactobacillus and Streptococcus spe-cies, while samples collected during farrowingtimes showed a higher number of species in thegut of all processed sows. The presence or notof fructo-oligosaccharides (FOS), used as die-tary additives to improve enteric health in pigsalso showed a shift between Lactobacillus aci-dophilus and Streptococcus hyointestinalis pop-ulations.

P-64 In vitro evolution of the equine gastricmicroflora. M. Varlouda, V. Julliandb (a EVIA-LIS, 56250 Saint-Nolff, France, b ENESAD,21000 Dijon, France).

Microbial concentrations in the gastric contentsof conscious horses have been shown to increasefrom 5.7 to 7.2 log10 cfu·mL–1 during the firstpostprandial hour (Varloud et al., 2004). Thisexperiment was set to bring explicative data ofthis phenomenon through an in vitro evaluationof the postprandial evolution of the equine gas-tric microflora. Four mature geldings (429 kgBW) were adapted to a hay and pellets based dietduring 3 weeks. Gastric contents collectedbefore (C1) and 1 h after the meal (C2) werediluted before stomacher treatment and filtra-tion. This inoculum (23 mL) was added toground pelleted feed (0.7 g, 20% starch) before-hand hydrated with dilution medium (47 mL) inhermetic flasks. Blanks without inoculum orwithout substrate were tested. After incubationat 38 °C in a shaking water bath, fermentationswere stopped after 60, 120 and 210, and 60 and210 min, for C1 and C2, respectively. The pHwas measured immediately. The concentrationsof total anaerobes, lacticolytic bacteria, Strepto-cocci and Lactobacilli were investigated in theraw gastric contents and after in vitro incubation.Lactate and volatile fatty acid concentrationswere evaluated at the end of the incubation.Between 60 and 210 min of incubation, the total

anaerobic population increased from 5.1 to6.1 log10 cfu·mL–1 with C1 and from 5.8 to7.1 log10 cfu·mL–1 with C2. In the same time,the lacticolytic population increased from 3.9 to5.9 log10 cfu·mL–1. The concentrations ofLactobacilli and Streptococci increased moreslowly during the incubation. This in vitro devel-opment of the population was not equivalent tothat previously observed in vivo. Multiplicationof bacteria within the chyme may not explainentirely the in vivo increase of bacterial concen-tration.

P-65 Prevalence of two Lactobacillus and Bifi-dobacterium probiotic strains in the neonatalileum. R. Walla,b,c, G.F. Fitzgeraldb,c, S.G.Husseyd,e, C.A. Ryand, M. O’Neille, R.P.Rossa,b, C. Stantona,b (a Teagasc, MooreparkFood Research Centre, Fermoy, Co. Cork, Ire-land, b Alimentary Pharmabiotic Centre, Cork,Ireland, c Department of Microbiology andd Department of Paediatrics and Child Health,Erinville Hospital, University College, Cork,Ireland, e Mayo General Hospital, Castlebar,Mayo, Ireland).

The purpose of this study was to investigate thecolonization of the ileum with lactobacilli andbifidobacteria using samples obtained from twoinfants. Initially, ileostomy samples were exam-ined from a preterm infant at 50 and 74 days oldby plating its caecal contents on Lactobacillusand Bifidobacterium selective agar. Twenty ofthe resulting Lactobacillus isolates were genom-ically fingerprinted by pulsed field gel electro-phoresis and found to be a single strain. 16SrDNA sequencing identified this strain as Lacto-bacillus paracasei, which was recovered at bothtime points. Indeed, this strain dominated thesmall intestine throughout the study period(24 days). In contrast, culturable bifidobacteriawere absent. In the other ileostomy case, twocecal samples were taken from a term infant at24 and 31 weeks old and a fecal sample at44 weeks following ileostomy reversal. In thiscase, lactobacilli were not detected in any of thesamples, however, bifidobacteria were obtainedwhen the ileostomy had been reversed. 16SrDNA sequencing revealed two different strains;Bifidobacterium animalis subsp. lactis andB. scardovi. Interestingly, the B. animalis subsp.lactis strain exhibited the same PFGE pattern as

S34 Session I: Microbial diversity in health and disease

the well-defined probiotic strain B. animalissubsp. lactis BB12, while the L. paracasei strainisolated in the first study was identical toL. paracasei NFBC 338 probiotic strain. Thisstudy demonstrates the prevalence of two differ-ent probiotic strains in the upper intestinal tractat an early stage of human life.

P-66 Enrichment in a complex medium of anovel bacterial group from the rumen of wildsika deer as confirmed by real-time PCRassays and FISH detection. H. Yamano, K.Miyawaki, Y. Sawabe, Y. Kobayashi (GraduateSchool of Agriculture, Hokkaido University,Sapporo 060-8589, Japan).

Phylogenetic analysis of rumen microbiota inwild sika deer, and quantitation of some bacte-rial groups were previously attempted. Throughphylogenetic analysis, unknown bacterial groupA was found in the CFB (Cytophaga Flavo-bacter Bacteroides) phylum. According to real-time PCR quantitation, the population size ofgroup A showed a 17-fold increase from summerto winter. This result suggested that in the rumenof wild sika deer, the unknown bacterial groupA may have important roles in digestion offibrous diet (mainly bark) in winter. The presentstudy was aimed at enriching this novel uncul-tured bacterial group in a complex medium forfuture isolation and physiological analysis. Asthe first step, we screened a carbon source suit-able for enrichment of this group. Also, deerrumen fluid was assessed as a supplement to acomplex medium. The enrichment was evalu-ated by real-time PCR assays and FISH detec-tion. Of 13 carbon sources tested, powdered barkof Japanese oak was most effective in enrich-ment of the group A. Supplementation of deerrumen fluid also successfully stimulated thegrowth of this group. After 5 days incubationin a complex medium, population size of theunknown group A drastically increased (× 104–5).Relative proportion of the group A in total bac-teria also increased from 0.9% to 4.1% duringthe incubation. The enriched group A was con-firmed by sequencing of the products of group-specific PCR. In addition, FISH detection con-firmed the growth of this group. These resultssuggest that the yet uncultured group A is culti-vable in the complex liquid medium containing

habitat-stimulating components from the origi-nal deer rumen digesta.

P-67 Sequencing and annotation of pCY360,a megaplasmid from Clostridium proteoclas-ticum B316T. C. Yeomana,b, W.J. Kellya, J.Rakonjacb, G.T. Attwooda (a Metabolism andMicrobial Genomics, Food and Health Group,AgResearch Ltd., Grasslands Research Centre,PB 11008, Palmerston North, New Zealand,b Institute of Molecular Biosciences, MasseyUniversity, Palmerston North, New Zealand).

The genome of Clostridium proteoclasticumB316T consists of 4 replicons; a ~ 3.2 Mb chro-mosome and three megaplasmids of 360, 290and 190 kb. The largest megaplasmid, desig-nated pCY360, has been completely sequencedand annotated. GLIMMER analysis identified400 open reading frames (ORFs), but only 15%of these show significant sequence similarity topreviously described genes of known function.A putative origin of replication (oriR) was iden-tified containing a replication initiation protein,RepB, a plasmid partitioning protein, ParA, andtwo large inverted repeats. ORFs related to con-jugative transfer functions and type IV pilus for-mation, including members of both the matingpair formation (mpf) complex and the relaxo-some, as well as the coupling protein TraG, havebeen identified and most are clustered togetherin a 35 kb region. pCY360 also contains ninegenes described in Koonin’s minimal gene set,fulfilling Ochmans definition of a miniaturechromosome, however, all nine genes are alsofound on the C. proteoclasticum chromosome.Ten transposase genes are present (7 belong tothe IS4, IS200 or IS605 transposase families)and some share homology with transposases onboth the chromosome and the co-resident 290 kbmegaplasmid, suggesting that transposon-medi-ated gene shuttling has occurred. The function ofpCY360 is unknown, but it appears to contributeto proteins expressed at the cell membrane/sur-face, as 19% of its ORFs are predicted to containone or more transmembrane regions. Analysis ofcodon usage indicates that pCY360 is similar tothe host chromosome, indicating that it hasresided within C. proteoclasticum for a signifi-cant period of time.

Session I: Microbial diversity in health and disease S35

P-68 Genetic diversity of the microbial com-munity in ileostomy patients. E.G. Zoetendala,b,C.C.G.M. Booijinka,b, S. El Aidya,b, H.Smidta,b, M. Kleerebezemb, W.M. De Vosa,b

(a Laboratory of Microbiology, WageningenUniversity, The Netherlands, b WageningenCentre for Food Sciences, Wageningen, TheNetherlands).

The microbial biomass in our gastrointestinal(GI) tract consists of a myriad of microbes thatare pivotal to health and disease. In recent years,16S ribosomal RNA (rRNA)-based approachesprovided a phylogenetic framework of morethan 1 200 microbial species inhabiting our GItract and gave insight into the temporal, spatialand inter-individual microbial diversity (Zoe-tendal et al., 2006). Nevertheless, most studieshave focused on the colon, while insight intostructure, dynamics and functionality of thesmall intestinal microbiota remains limited. Toaccess the small intestinal microbiota withoutusing interfering procedures, effluent samplesfrom eight ileostomy patients were collected.Since these individuals have their colonremoved, the intestinal content leaves their bodyat the end of the ileum via a stoma, providingnon-invasive access to small intestinal content.PCR-DGGE of 16S rRNA genes indicated thatthe bacterial community structure in these efflu-ent samples is less complex and less stable intime compared to colonic samples. Moreover,differences between morning and afternooncommunities were often observed. From foureffluent samples of one individual a metagen-omic library consisting of 25000 fosmids wasconstructed. 16S rRNA approaches indicatedthat this library was representing the actualmicrobial diversity reasonably well. In addition,database hits with bacterial genera that are oftendescribed as inhabitants of the small intestinewere frequently encountered when analyzingend-sequences of randomly selected fosmids.The details of this analysis will be discussed atthe conference.

P-69 Long-chain fatty acids and fatty alde-hydes in rumen bacterial genera Butyrivibrioand Pseudobutyrivibrio. M. Zorec, R.Marinšek-Logar (University of Ljubljana, Bio-technical Faculty, Zootechnical Department,Domzale, Slovenia).

The analysis of long-chain fatty acids and fattyaldehydes is a frequently used chemotaxonomicmethod in bacteria and it was applied to evaluateheterogeneity in the group of rumen bacteriaphylogenetically related to the type strainsButyrivibrio fibrisolvens, B. hungatei, Clostrid-ium proteoclasticum, Pseudobutyrivibrioruminis and P. xylanivorans. Long-chain fattyacids and fatty aldehydes of 62 strains weretransesterified with HCl in methanol and theirmethyl esters analysed by gas chromatographyusing non-polar column Equity-1 (Supelco).According to the long-chain fatty acid and fattyaldehyde profiles strains formed 4 similaritygroups, but to the type strain C. proteoclasticumno similar strain was found. The major fattyacids and fatty aldehydes were C16:0, DMAC18:1 c11, DMA C16:0 and C18:1 c11 in B.fibrisolvens group, C16:0, DMA C14:0, DMAC16:0, C14:0 and DMA i-C15:0 in P. ruminisgroup, a-C17:0, C16:0, i-C16:0, DMA i-C16:0,DMA a-C15:0, DMA a-C17:0 and DMA C16:0in B. hungatei group, and C16:0, DMA C18:1c11, DMA C16:1 c9 and DMA C16:0 inP. xylanivorans group. The P. ruminis groupwas characterized by a high proportion ofstraight saturated fatty acids and fatty aldehydesand the B. hungatei group by a high proportionof branched fatty acids and fatty aldehydes.Strains within the groups showed similarity alsoin the type and quantity of fermentation prod-ucts. This study defines the characteristic pro-files of long-chain fatty acids and fatty alde-hydes for Butyrivibrio and Pseudobutyrivibriospecies, enabling rapid, accurate and simpleidentification of new isolates and a more properdelineation of bacterial species.

S39Reprod. Nutr. Dev. 46 (2006) S39–S60© INRA, EDP Sciences, 2006

O-9 Microarray gene expression profilingof Ruminococcus flavefaciens FD-1 grownon cellobiose or cellulose. M.E. Berga, D.A.Antonopoulosa, M.T. Rinconb, M. Banda, A.Baria, T. Akraikoa, A. Hernandeza, R. Kima,L. Liua, J. Thimmapurama, I. Borovokc, S.Jindouc, R. Lamedc, H.J. Flintb, E.A. Bayerd,B.A. Whitea (a University of Illinois, Urbana, IL61801, USA, b Rowett Research Institute,Bucksburn, Aberdeen AB21 9SB, UK, c TelAviv University, Ramat Aviv 69978 Israel,d The Weizmann Institute of Science, Rehovot76100, Israel).

The 4× draft genome of the Ruminococcus fla-vefaciens FD-1 sequencing project has 1143assembled contigs averaging 3686 bp (rangingfrom 205 to 31132 bp). In silico analysis of thecellulosomal organization and componentsrevealed a cluster of ORFs for the putative scaf-foldin proteins, ScaA, ScaB and ScaC that aresimilar to those of strain 17, except for thenumber of cohesins in ScaA and ScaB. In addi-tion, an unprecedented number (~ 190) of puta-tive dockerin motif-containing ORFs (fourmajor phylogenetic clusters) are associated withprotein families, including xylanases, cellu-lases, proteinases, serpins, surface-anchoringproteins and unknown activities. An interestingcontig (4.67 kb) appears to encode a modularprotein termed “superzyme”, which has twoGH_11 domains, a GH_10 domain, a CBM22module, a dockerin, and a polysaccharidedeacetylase separated by glutamine/asparagine-rich linkers. Microarray expression profilingallows us to analyze which of the strain FD-1cellulosome-associated genes are expressed inresponse to growth on either cellobiose or cel-lulose. EndB-type dockerins seem to be themost common in the up-regulated contigs. Themulti-domain gene families of the dockerin,containing ORFs or contigs with the highest up-regulation (3.5 to 23-fold) were superzyme(10 to 23-fold), CE_3/CBM_22/CesA, RicinB-like lectin CBM/CBM_6/lipase GDSL, alpha-glucosidase, GH_30/CBM_22/CE_3/Ricin-likeCBM/PL_11, and ScaC/ScaA/ScaB (3.5-fold).Multi-domain cellulosomal components with adockerin sequence are highly correlated withup-regulation in response to cellulose.

O-10 Analysis of Methanobrevibacter rumi-nantium genome sequence. W.J. Kelly, G.T.

Attwood (Metabolism and Microbial Genom-ics, Food and Health Group, AgResearch Ltd,Grasslands Research Centre, Palmerston North,and Pastoral Greenhouse Gas Research Consor-tium, New Zealand).

The genome of Methanobrevibacter ruminan-tium, a prominent methanogen in New Zealandruminants, is being sequenced as part of a Pas-toral Greenhouse Gas Research Consortium-funded project to mitigate greenhouse gases.The genome is approximately 3.0 Mb with aG+C content of 33.62%. At least 60 genes areinvolved in methanogenesis and hydrogentransfer. Comparison of the genome sequencewith Methanobacterium thermoautotrophicumand Methanosphaera stadtmanae indicatesmethanogenesis gene organisation is conservedwithin the Methanobacteriales. UnlikeM. stadtmanae, M. ruminantium has genes forcitrate synthase and isocitrate dehydrogenase,indicating that the oxaloacetate to oxoglutaratepathway can operate via citrate, aconitate andisocitrate, as well as via malate, fumarate andsuccinate. The M. ruminantium genome appearsto encode many large surface proteins that con-tain repeat sequences also found in M. stadtma-nae proteins. The characteristics of these pro-teins indicate that they may mediate associationwith other rumen microbes. More than 50 genesare involved in the synthesis and export ofexopolysaccharides, which is in agreement withprevious observations that this organism pro-duces a capsule. Approximately 50% of theORFs identified by GLIMMER analysis haveno known function. Determining the function ofthese new genes will improve our understandingof the biology of this organism and help defineits role in methane formation in the rumen.

O-11 Post-genomics approaches to study thefunctionality of bifidobacteria in infant fae-ces. E.S. Klaassensa, R. Boestenb, F.H.J.Schurenb, E.E. Vaughana, W.M. De Vosa

(a Laboratory of Microbiology, WageningenUniversity, The Netherlands; b TNO, Zeist, TheNetherlands).

Following birth, the human gastrointestinaltract is rapidly colonised by microorganismsthat play a role in the nutrition, physiology andhealth of the host. Bifidobacteria are especiallypredominant in the infant colon. In this study,

S40 Session II: Microbial metabolism of dietary components

we have applied post-genomic technologies,including metaproteomics and bifidobacterialcommunity transcript profiling, to obtain under-standing of the activity of commensal bifidobac-teria in infant faecal microbiota. The metapro-teomes of infant faecal microbiota weredetermined by extraction and subjection of theproteins to two dimensional (2D) gel electro-phoresis. These 2D gels revealed changes of thenumber and intensity of protein spots in time, butthe patterns remained similar for each individ-ual. In-gel digestion of protein-spots andsequencing of the peptides revealed a proteinwhich shares high similarity to bifidobacterialtransaldolases, testifying for the activity of bifi-dobacteria. In addition, transcript profiling ofthe infant faecal microbiota was performed.Total RNA was hybridised to a DNA microarraycomprising clones covering the genomes of sev-eral bifidobacterial species. Significantly hybrid-ising clones were sequenced and compared withthose in the public databases. While somesequences were found to be ribosomal RNA, themajority showed similarity to protein-encodinggenes predicted to be involved in vitaminproduction, stress response, sugar transportand metabolism, and housekeeping functions.Remarkably, similarity was observed to anoperon involved in the utilisation of milk oli-gosaccharides and mucin sugars, indicating thefunctionality of bifidobacteria. To the best of ourknowledge these are the first metaproteomesfrom human faecal bacteria, as well as commu-nity transcriptomes of commensal bifidobacte-ria from the human intestinal faecal ecosystem.

O-12 Impact of reducing dietary carbohy-drate intake upon the faecal microbial com-munity and metabolites in human volunteers.S.H. Duncana, A.M. Johnstonea, G.E. Lobleya,G. Holtropb, A. Belenguera, D. Bremnera, M.Hrdinovac, H.J. Flinta (a Rowett Research Insti-tute and b BioSS, Bucksburn, Aberdeen AB219SB, UK, c Institute of Animal Physiology,Prague, Czech Republic).

Ketogenic (“Atkins-type”) diets are popularstrategies for weight loss and involve very lowintakes of carbohydrates (< 20 g·d–1). Reasona-ble intakes of fermentable dietary carbohy-drates, however, are considered beneficial to guthealth through microbial production of butyrate,a major energy source for colonocytes that may

also protect against cancer and other bowel dis-eases. The objective of this study was to inves-tigate the impact of two high protein diets, dif-fering in carbohydrate supply (20 g·d–1 and170 g·d–1), on colonic bacterial communitiesand their fermentation products. Male, obesevolunteers (17) consumed a maintenance diet for3 days followed by 4 weeks on each of the exper-imental diets. Faecal samples were collected atthe end of each dietary regime. Total faecalSCFA concentrations decreased significantly(P < 0.001) with reduced carbohydrate intake.This decrease was particularly marked forbutyrate, which declined as a proportion of totalfaecal SCFA (P < 0.001). The composition of thefaecal microbiota, studied using a panel of ten16S rRNA-targeted FISH probes, also changedwith carbohydrate intake. Notably, the propor-tion of bacteria belonging to the Roseburia/Eubacterium rectale group of butyrate-produc-ing bacteria decreased approximately fourfoldbetween the maintenance diet and the diet withthe lowest carbohydrate intake (P < 0.001). Itcan be concluded that this group is particularlydependent on fermentable carbohydrates tooccupy a nutritional niche in the colon, and isalso probably the main contributor to butyrateformation. Interestingly, clostridial cluster XIVabacteria other than Roseburia/E. rectaleincreased as the carbohydrate supply decreased(P < 0.025). Although a sufficient supply ofbutyrate is generally considered to be necessaryto maintain gut health, it has not been establishedwhether changes of the duration and magnitudeshown here are likely to have significant conse-quences for gut health.

O-13 Contribution of ruminal protozoa toduodenal flow of N, CLA and TVA in steersfed silage differing in water-soluble carbohy-drates. D.R. Yáñez-Ruiza, N.D. Scollanb, R.J.Merryb, C.J. Newbolda (a Institute of Rural Sci-ences, University of Wales Aberystwyth, Llan-badarn Campus, SY233AL, Aberystwyth, UK,b Institute of Grassland and EnvironmentalResearch, Plas Gogerddan, SY233ED, Aberyst-wyth, UK).

There is currently increasing interest in the fattyacid composition of ruminant products such asmeat and milk, particularly the content of con-jugated linoleic acid (cis-9, trans-11 CLA).CLA is derived from CLA produced in the

Session II: Microbial metabolism of dietary components S41

rumen and synthesis in meat or milk from theruminally derived precursor trans-vaccenic acid(C18:1-trans-11; TVA). To date the productionof CLA and TVA within the rumen has been con-sidered solely from the perspective of bacterialmetabolism and it has been suggested that pro-tozoa are of minor importance. This experimentwas designed to estimate the quantitative contri-bution of rumen protozoa to the total N, CLA andTVA to the duodenum in steers fed two silagediets (CS, control silage and HS, silage high inwater soluble carbohydrates). Protozoal duode-nal flows were estimated by using 18S rDNAdetermined by real-time PCR as a marker forprotozoal biomass. By Denaturing Gradient GelElectrophoresis we established that rumen pro-tozoa populations were similar to those flowingto the duodenum. Estimated duodenal flow ofprotozoa represented 12 and 15% of the total Nflowing to the duodenum. Protozoa accountedfor 49.0 and 63.6% of the cis-9, trans-11 CLA,on the CS and HS diets, respectively, while 39and 40% of the total TVA reaching the duode-num was also of protozoal origin. These resultsdemonstrate the important contribution of pro-tozoa in the flow of CLA and TVA to the duo-denum and suggest that the role of protozoa inruminal biohydrogenation merits further inves-tigation.

O-14 Metabolism of linoleic acid by bacterialspecies isolated from the human gut: biosyn-thesis of conjugated linoleic and hydroxyfatty acids. E. Devillard, F.M. McIntosh, S.H.Duncan, H.J. Flint, R.J. Wallace (RowettResearch Institute, Bucksburn, Aberdeen AB219SB, UK).

Conjugated linoleic acids (CLA) confer a broadrange of health-promoting properties. The maindietary sources of CLA in the human diet areruminant products. CLA, mainly the cis-9,trans-11 isomer, are formed by ruminal bacteriaas intermediates in the biohydrogenation of lino-leic acid (LA). LA is isomerised to CLA, whichis then hydrogenated to trans-vaccenic acid(TVA), then to stearic acid (SA). We recentlyconfirmed that the human faecal flora also usesLA to form CLA, TVA and SA. In this study, weinvestigated the role of 30 major colonic bacte-rial species (low G+C content cluster IV andXIVa and high G+C content cluster) in LAmetabolism. When the bacteria were cultivated

in a medium containing LA, and the total fattyacids were extracted and analysed by gas chro-matography, it emerged that 26 strains metabo-lised LA. Measurement of the linoleate isomer-ase activity of the bacteria indicated that only9 strains converted LA to CLA. In culture, thesame 9 strains produced CLA and/or TVA asfinal metabolites. Among the other 17 LA-metabolising strains, 11 of them produced a 10-hydroxy fatty acid, while the remaining 6 strainsproduced various C18:1 fatty acids. This studysuggests that some human gut bacteria producecis-9, trans-11-CLA and TVA from LA by a bio-hydrogenation mechanism similar to the onedescribed for ruminal bacteria. However, mostof the strains studied here seemed to metaboliseLA to form other products, such as hydroxy fattyacids. It would be interesting to study the metab-olism of these hydroxy fatty acids, which couldbe precursors of the multitude of CLA isomersproduced when mixed faecal flora is incubatedwith LA. This is particularly important since ithas been recently shown that specific CLA iso-mers have potential local effects on colon health.

P-70 Effect of polyethylene glycol on in vitrogas production and digestibility of tannin-rich feedstuffs from North Africa arid zone.R. Arhaba, M. Rirab, D. Macheboeufa,H. Boussebouab (a Unité de Recherches sur lesHerbivores, INRA, 63122 Saint-Genès-Cham-panelle, France, b Laboratoire de Génie Micro-biologique et Applications, Université Men-touri, 25000 Constantine, Algeria).

The aim of the present study was to investigatethe effect of polyethylene glycol on in vitro gasproduction, estimated in vitro organic matterdigestibility (OMD) and metabolisable energy(ME) of four forages and two date palm by-prod-ucts widely utilized by North African farmers.The feedstuffs evaluated include two families ofplants: Gramineae (Aristida plumosa and Dan-thonia froskalaii) and Leguminoseae (Genistasaharae and Astragalus gombiformis), and twodate palm by-products: racemes and leaves. Phy-tochemical analysis showed that all feedstuffscontained antinutritional factors such as ster-oids, saponins and tannins. Date palm leaves hadthe highest concentration of extractable phenolsand of total and condensed tannins; 61.8, 49.1 and36.2 g·kg–1 DM, respectively. A. plumosa wasthe forage with the lowest concentration of phe-nols and total tannins, 5.4 and 3.2 g·kg–1 DM,

S42 Session II: Microbial metabolism of dietary components

respectively, whereas G. saharae had the lowestconcentration of condensed tannins, 0.7 g·kg–1

DM. Addition of polyethylene glycol (PEG)increased gas production (20.87–25.03 mL/200 mg DM), OMD (47.25–50.83 g/100 g DM)and ME (8.96–9.62 Mj·kg–1 DM), without andwith PEG respectively (P < 0.05). The bestimprovement for gas production (16.87,28.76 mL/200 mg DM), OMD (44.69, 54.32%)and ME (8.73, 10.21 MJ·kg–1 DM), with andwithout PEG, respectively, was recorded forAristida plumosa. The lowest increment valuesof gas production and ME were noted for Astra-galus gombiformis, whereas the lower incre-ment of OMD was observed for Danthoniafroskalaii. These results indicate that the posi-tive effect of PEG on the degradation of thesesubstrates was more pronounced in forages withlow tannin content than in those with high tanninconcentration.

P-71 In vitro ruminal fermentation of somedeserts browse species and date palm by-prod-ucts. R. Arhaba, D. Macheboeufa, R. Bergeaulta,H. Boussebouab (a Unité de Recherches sur lesHerbivores, INRA, 63122 Saint-Genès-Cham-panelle, France, b Laboratoire de Génie Micro-biologique et Applications, Université Men-touri, 25000 Constantine, Algeria).

The present study was carried out to assess thein vitro rumen fermentation and production offermentation end-products of four perennialbrowse species: Aristida plumose, Danthoniafroskalaii, Genista saharae and Astragalusgombiformis, and two date by-products:racemes and leaves, comparatively to vetch-oathay. The samples were fermented for 72 h in abatch fermenter with ruminal fluid of sheepusing the pressure transducer technique. At theend of fermentation, pH, volatile fatty acids andammonia production were measured. Therewere wide variations among forages in crudeprotein, neutral detergent fibre (NDF), aciddetergent fibre (ADF) and lignin. Crude proteincontent varied from 25 g·kg–1 DM in racemes to125 g·kg–1 DM for Astragalus gombiformis. TheNDF and lignin ranged from 586.1 to 824.4 g·kg–1

DM and 43.6 to 142.4 g·kg–1 DM, respectively.Polyphenolic content varied between foragesand families. The by-products had the highestvalues for total extractable phenols, total andcondensed tannins. However, the Gramineae

forages had the lowest values (P < 0.001). Theforages produced a lower gas volume than vetch-oat hay (P < 0.001). The highest value observedin forages was for Danthonia froshkalaii(6.08 mmol·g–1 DM) and the lowest for datepalm leaves (2.08 mmol·g–1 DM). The molarproportion of total volatile fatty acids was influ-enced by the forage species (P < 0.001). The ace-tate to propionate ratio was highest for Astra-galus gombiformis (3.69) and lowest forracemes (2.01). For ammonia production, thehighest value was observed for Astragalusgombiformis and the lowest value for vetch-oathay. However, the mean ammonia productionfor date palm by-products was negative. Thisresult could indicate the formation of complexesbetween tannins and protein.

P-72 The diversity and prevalence of conju-gated linoleic acid-producing Bifidobacteriumspecies in the human gastrointestinal tract. E.Barretta,b, R.P. Rossa,b, G.F. Fitzgeraldb,c, F.Shanahanb,d, C. Stantona,b (a Teagasc Biotech-nology Centre, Moorepark Food Research Cen-tre, Fermoy, Co. Cork, Ireland, b AlimentaryPharmabiotic Centre, Cork, Ireland, c Depart-ment of Microbiology and d Department of Med-icine, University College Cork, Ireland).

The aim of this study was to identify intestinallyderived culturable bifidobacteria strains withability to bioconvert free linoleic acid to conju-gated linoleic acid (CLA) and to determine theirprevalence and diversity among human popula-tions. Samples were collected from 28 healthyneonates, 10 healthy adults and 20 elderly adultsinfected with Clostridium difficile, and plated oncys-MRS containing mupirocin to pre-select forbifidobacteria. The isolates were screened forCLA biosynthesis, spectrophotometrically at awavelength of 233 nm, following incubation inthe presence of free linoleic acid. In total,18 genetically distinct, based on pulsed field gelelectrophoresis, CLA producing strains wereisolated and identified as bifidobacteria based onthe enzyme fructose-6-phosphate phosphoketo-lase, their partial 16S ribosomal DNA or HSP60heat shock protein DNA sequences and by Bifi-dobacterium species specific PCR reactions.The CLA producing bifidobacteria strains wereidentified as either B. breve, B. longum, B. infan-tis, B. dentium or B. catenulatum, with CLA con-version efficiencies varying from 2.6–76%, as

Session II: Microbial metabolism of dietary components S43

determined by gas liquid chromatography. Thepredominant CLA isomer produced by thesebifidobacteria was cis-9, trans-11 C18:2, with themost efficient producers belonging to the spe-cies B. breve and B. longum. Patients infectedwith C. difficile were the best source of CLA pro-ducing bifidobacteria, in terms of both produc-tion efficiency and prevalence. The results showthat 1 in 4 subjects harboured CLA producingbifidobacteria. This diversity and prevalence ofCLA producing bifidobacteria among subjectsof varying ages may be a further potential healthpromoting property of the genus.

P-73 Alternative mechanisms of metaboliccross-feeding between human gut bacteria. A.Belenguer, S.H. Duncan, G. Calder, G. Holtrop,P. Louis, G.E. Lobley, H.J. Flint (RowettResearch Institute, Bucksburn, Aberdeen, AB219SB UK).

Carbohydrates that reach the human colon havea major influence on fermentative metabolismby gut bacteria. Resistant starch and fructo-oli-gosaccharides (FOS) have been considered topromote Bifidobacterium strains, but their influ-ence on metabolism and microbial competitionis likely to be complex. Bifidobacteria form ace-tate and lactate as major fermentation products,but these may be further metabolised by otherspecies via cross-feeding. Furthermore, a widevariety of gut bacteria can be expected to com-pete for starch and FOS breakdown products.The aim of this study was to investigate possiblemechanisms of metabolic cross-feeding, partic-ularly in relation to the formation of butyrate,which plays an important role in gut health. Outof twelve Bifidobacterium strains tested, fourgrew on starch and nine on FOS in pure culture.Metabolic cross-feeding was investigated in co-cultures between strains of Bifidobacteriumadolescentis able to grow on starch or FOS, andstrains of butyrate-producing bacteria that aloneare unable to utilise starch or FOS for growth.Stable isotopically labelled lactate and acetatewere used to study their utilization. 13C-acetateor 13C-lactate labelling confirmed that Eubacte-rium hallii was able to convert acetate and lac-tate into butyrate in the co-cultures. Separately,a non lactate-utilising Roseburia strain, how-ever, also produced butyrate in co-cultures withB. adolescentis, implicating a second mecha-nism of cross-feeding. We conclude that thereare two distinct mechanisms of cross-feeding

between Bifidobacterium and butyrate-produc-ing strains, one due to consumption of lactateand acetate and the other due to cross feeding ofpartial breakdown products from complex car-bohydrates.

P-74 The ability of the rumen ciliate Diplo-plastron affine to digest and use 1,3-β-D-glu-cans. G. Be ecki, R. Miltko, T. Micha owski(The Kielanowski Institute of Animal Physiol-ogy and Nutrition, Polish Academy of Sciences,05-110 Jab onna near Warsaw, Poland).

It is well known that 1,3-β-D-glucans are presentin cereals and in the fungal cell wall. Cereals arecommonly used in ruminant nutrition whilesome species of fungi inhabit the rumen. Rumenciliates engulf and digest small particles of theground cereals and fungal zoospores. However,little is known about the digestion and role of thementioned polysaccharides in nutrition of rumenprotozoa. The aim of the present study was todetermine the ability of the rumen ciliate Diplo-plastron affine to digest and use 1,3-β-D-polysaccharides for growth. In cultivationexperiments the ciliates were grown in vitro.They were fed a diet consisting of powdered hay(0.3 mg·mL–1 culture·d–1), and wheat gluten(0.08 mg·mL·d–1) while experimental cultureswere also supplemented with 1,3-β-D-glucanfrom Euglena gracilis (0.015, 0.03, 0.06 and0.12 mg·mL·d–1). No positive effect of the addedpolysaccharide on the growth of ciliates wasfound as the number of ciliates in the control cul-tures was about 2300 mL–1, while in experimen-tal cultures there were 2400, 2300, 1800 and1200 individuals, respectively. In biochemicalexperiments the degradation rate of 1,3-β-D-glucan (lichenan) and laminarinobiose (1,3-β-D-disaccharide) from Laminarina digitata wasmeasured by crude enzyme preparation of bac-teria-free Diploplastron affine. The degradationrate of lichenan and laminarinobiose was 33.5and 81 µM glucose released mg–1 protein h–1,respectively, while pH optima of these reactionswere 5.0 and 5.5. Non-denaturing polyacryla-mide gel electrophoresis combined with zymo-gram technique revealed the presence of twoprotein bands active against lichenan. End prod-ucts of the digestion of this polysaccharide wereidentified by thin layer chromatography (TLC).Glucose was the main product of substratehydrolysis, however, traces of longer oligosac-charides were also present.

ł ż ł

ł

S44 Session II: Microbial metabolism of dietary components

P-75 Interaction of buckwheat seed extractsand intestinal microflora regarding changesof bacterial profile of intestinal microfloraand antioxidant status of extracts. E. Biedrzy-cka, L. Markiewicz, M. Bielecka, R. Amarowicz(Institute of Animal Reproduction and FoodResearch of the Polish Academy of Sciences,Tuwima 10, 10-747 Olsztyn, Poland).

Buckwheat (Fagopyrum esculentum) contains avariety of valuable antioxidant compounds suchas flavonoids, tocopherols and phenolic acids,but little is known so far about their relation withintestinal microflora (IM). The aim of the studywas, therefore, to define interaction of: (1) themonocultures of pure strains of IM representa-tives, including Bifidobacterium animalis 30,Lactobacillus plantarum IB, Escherichia coli 94and Enterococcus faecium T-208, (2) their co-culture, and (3) human faecal microflora (FM,3 volunteers), and acetone buckwheat seedextracts (whole grain WGBE or shelled SBE;2.5 mg·mL–1 of medium). DPPH and ABTSscavenging, pH and IM quantitative (culturingmethods) and qualitative (PCR-DGGE) profileswere determined in 24-h cultures at 37 °C underanaerobic conditions. The controls were:medium without bacteria and cultures in themedium without extracts. The buckwheatextracts stimulated growth and acidifying activ-ity of B. animalis 30, and slightly decreased thatof L. plantarum IB and E. coli 94 (WGBE)monocultures. In the co-culture, intensified growthof mainly B. animalis 30 and L. plantarum IBwas observed. The growth of FM bacterial pop-ulations was differentiated and reflected hostFM-specific responses (inhibition or stimulationof growth) with lactobacilli reduced to the great-est extent. No significant qualitative alterationswere observed in DGGE profiles of eubacteria,Bifidobacterium, Lactobacillus and Clostridiumcoccoides-group, whereas the respectiveextract-induced quantitative changes were con-firmed. IM generally decreased scavengingactivity of both extracts, similarly for the purestrain co-culture and FM. The share of WGBEsoluble compounds in DPPH scavenging activ-ity (43–50%) lowered to a higher extent (by43%) in the IM co-culture than upon FM (onlyby 2–19%). The high share (64–76%) of solublecompounds in ABTS scavenging activity ofWGBE was significantly reduced by both co-culture and faecal microflora (by 79–89%).

P-76 Accumulation of rumen biohydrogena-tion intermediates through grazing of botan-ically diverse pastures is associated withchanges in the rumen protozoal population.C. Boeckaerta,b, N. Boona, M. Lourençob, W.Verstraetea, V. Fievezb (a Laboratory of Micro-bial Ecology and Technology, Ghent University,Ghent, Belgium, b Laboratory for Animal Nutri-tion and Animal Product Quality, Ghent Univer-sity, Ghent, Belgium).

In an attempt to study the effect of variation inthe botanical composition of pastures on thefatty acid metabolism of grazing ruminants,21 lambs were assigned for 12 weeks to eitheran intensive ryegrass, clover or herbage rich pas-ture and the fatty acid profile of rumen, abo-masum, intramuscular and subcutaneous fat,sampled at slaughter from non-fasted animals,was determined. Concentrations of hydrogena-tion intermediates (C18:1 t10 + t11, C18:2t11c15, CLA c9t11, CLA t10c12) in the rumenof lambs grazing the herbage rich pasture were54% higher compared to lambs grazing inten-sive ryegrass or clover pasture while the abo-masum fatty acid profile showed no significantdifferences in the amount of hydrogenationintermediates between lambs grazing the differ-ent pastures. The disappearance of these signif-icant differences in the abomasum, subcutane-ous and intramuscular fatty acid profile suggestthey might have been associated with rumen pro-tozoa, as the latter mainly sequestrate in therumen. Indeed, molecular fingerprinting of theciliates by PCR-DGGE analysis revealed thatthe protozoal communities evolved differentlyaccording to the grazed pasture, provoking theappearance of an extra DGGE band in rumensamples of herbage rich grazing animals. Iden-tification of the latter, after excising andsequencing, showed 98% similarity to Diplod-inium dentatum. Overall, DGGE patterns ofrumen ciliates in animals grazing the herbagerich pasture mutually clustered and were sepa-rated from those grazing other pastures. In addi-tion, quantitative Real Time-PCR of rumen cil-iates showed a significantly lower amount ofprotozoa in association with the herbage richpasture.

P-77 Rapid screening of bacterial genes cod-ing histidine and tyrosine decarboxylaseusing PCR. R. Burdychovaa, A. Spanovab,B. Rittichb, T. Komprdaa (a Mendel University

Session II: Microbial metabolism of dietary components S45

of Agriculture and Forestry in Brno, Faculty ofAgronomy, Department of Food technology,Czech Republic, b Masaryk University, Facultyof Science, Institute of Experimental Biology,Brno, Czech Republic).

Biogenic amines (BA) are the low-molecularorganic bases which are formed due to the aminoacid decarboxylase activities of microorganismsin fermented foods. These compounds have beenstudied due to the toxicological effects derivedfrom their vasoactive and psychoactive proper-ties The formation of histamine or tyramine infoods depends on the presence of free histamineand/or tyramine in samples and the presence ofmicroorganisms having histidine or tyrosinedecarboxylase activity. The aim of this work wasto use a recently published PCR procedure(Coton et al., 2005, J Microbiol Meth 63: 296–304) for the identification of microorganismswith tyrosine and histidine decarboxylase genes.The microorganisms isolated from cheese(3 strains), lactic acid bacteria (LAB) of thegenus Lactobacillus obtained from the CzechCollection of Microorganisms (CCM, Brno)(19 strains), the Culture Collection of DairyMicroorganisms (CCDM, Tabor, Czech Repub-lic) (7 strains) and newly isolated from humanfaecal samples (20 strains), were used for theanalysis. Altogether 10 ng of genomic DNA iso-lated using a phenol extraction procedure wasused as template in PCR. All DNAs were shownto be amplificable using a primer set targetingthe 16S rRNA gene as an internal PCR control.PCR products were detected using agarose gelelectrophoresis. It was shown that PCR productsof specific length were amplified using a primerset for the tyrosine decarboxylase gene and aprimer set for the histidine decarboxylase genewith DNA of 3 strains isolated from cheese, 1strain collected in CCM, 1 strain collected inCCDM and 1 strain of human origin. The resultspresented in this report show that PCR is a suit-able and quick method for the rapid screening ofbacterial genes encoding histidine and tyrosinedecarboxylase.

Financial supports by IGA Grant of MUAF in BrnoIG460271 and long-term research program MSM151100002 of the Ministry of Education of the CzechRepublic.

P-78 Identification and characterisation ofxylan-degrading bacteria from the human

colon. C. Chassarda, M. Adams-Leclercb, A.Bernalier-Donadillea (a Unité de Microbiologie,INRA, 63122 Saint-Genès-Champanelle, France,b Unité d’Écologie Physiologie et du SystèmeDigestif, INRA, Domaine de Vilvert, 78 332Jouy-en-Josas, France).

In humans, plant cell wall polysaccharides(mainly cellulose and hemicelluloses) representan important source of dietary fibres that aredigested by gut microorganisms. Despite theextensive degradation of xylan in the colon, thepopulation structure and the taxonomy of thepredominant bacteria involved in degradation ofthis polysaccharide have not been extensivelyexplored. Xylanolytic isolates were first obtainedfrom faecal samples, using xylan growthmedium. Phylogenetic analysis of these strainsshowed that they belonged to Roseburia andBacteroides genera. Isolates related to Bacter-oides corresponded to four new species of thisgenus, whereas Roseburia strains could beassigned to the species R. intestinalis. Thexylanolytic Roseburia and Bacteroides speciesdegraded and efficiently fermented xylan fromdifferent botanic origins. End-products of xylanfermentation by Bacteroides isolates weremainly acetate, succinate and propionate, withsome lactate being also produced to a lesserextent, depending on the species considered.Roseburia isolates fermented xylan into butyrate,formate, lactate and H2. The xylanase activity ofBacteroides and Roseburia isolates was quanti-fied and compared with that of xylanolytic bac-teria already isolated from the human gut andclosely related to our strains. Most of our xylano-lytic isolates showed higher xylanase activitythan that of Roseburia intestinalis (L1-82) andBacteroides ovatus (0038). In addition, zymo-gram analyses revealed common characteristicsbetween Roseburia isolates and R. intestinalis,while the endoxylanase system of Bacteroidesisolates appeared specific to each species andvery different from that of B. ovatus. Our resultsprovide new information on the diversity of thexylanolytic flora in the human colon and dem-onstrate that unidentified xylanolytic bacteriaexist in the human gut.

P-79 Molecular quantification of the humangut acetogen, R. hydrogenotrophicus, in faecalsamples using real-time quantitative PCR. C.Del’hommea, E. Delmasa, Y. Leblonda, A.

S46 Session II: Microbial metabolism of dietary components

Bernalier-Donadilleb (a Laboratoires MayolySpindler, 78400 Chatou, France, b Unité deMicrobiologie, INRA, 63122 Saint-Genès-Cham-panelle, France).

Gas production associated with the fermentativeprocess in the human gut can be responsible fordigestive troubles (abdominal pain and bloatingand/or flatus). H2, whose production is higher inpatients with these digestive disorders, must beremoved from the colon in order to prevent gas-associated symptoms. Among the three micro-bial H2-consuming mechanisms existing in thecolon, reductive acetogenesis is of particularinterest, since acetate formed from CO2 reduc-tion by H2 is non gaseous, non toxic and anenergy source for eukaryotic cells. Utilisation ofthe hydrogenotrophic potential of acetogenic bac-teria was thus suggested as new strategy to preventgas-associated troubles through enhancement ofH2 consumption in the gut (patent 0006009). Inthis context, the effect of daily administration ofR. hydrogenotrophicus, an acetogenic speciesisolated from the human gut, to decrease H2 pro-duction was investigated in an animal model. Amethod allowing specific identification andquantification of the added R. hydrogenotrophi-cus in the gastrointestinal (GI) tract was firstdeveloped. Real-time PCR with SYBR Green Iwas used for the detection of the acetogenic spe-cies from faecal DNA, using specific primers.With this molecular approach, the distribution ofthis acetogenic species in the human populationwas found to be close to 12%, including faecalsamples from children. The correlation betweenthe important decrease in H2 excreted by human-flora associated (HFA) rats receiving R. hydro-genotrophicus and the presence of the ace-togenic species in the GI tract of these animalswas further evidenced. Finally, excretion kinet-ics and survival of R. hydrogenotrophicus wasevaluated using real-time-PCR after its inocula-tion to HFA rats.

P-80 Role of ciliate protozoa in the metabo-lism of unsaturated fatty acids in the rumen.E. Devillarda, F.M. McIntosha, C.J. Newboldb,K. Younga, M. Barbiera, R.J. Wallacea (a RowettResearch Institute, Bucksburn, Aberdeen AB219SB, UK, b University of Wales, Aberystwyth,Wales, UK).

Conjugated linoleic acids (CLA) have beenshown to improve human health. CLA aremainly found in milk and beef. They are inter-mediates in the ruminal biohydrogenation ofdietary linoleic acid (LA). It seems that most ofthe biohydrogenation of LA occurring in therumen is carried out by bacteria. The role ofrumen protozoa in this process remains equivo-cal. In this study, analysis of cellular lipids indi-cated that protozoa contain proportionally moreCLA and trans-vaccenic acid (TVA), which isa precursor of CLA in animal tissues, than rumi-nal bacteria. In incubations with different rumencontent fractions (strained rumen fluid SRF,bacterial fraction BAC, protozoal fraction PRO)with LA, the rate of LA metabolism was verysimilar in SRF and BAC and was much higherthan in PRO. The products formed in SRF andBAC were identified as CLA and TVA, and werenot present in the incubations with PRO. A pos-sible alternative route for formation of CLA andTVA was that protozoa could carry out desatu-ration of stearate. Incubations of SRF, BAC orPRO with 14C-stearate showed that desaturationof stearate does not occur in any of the rumencontent fractions. Furthermore, using PCR-based methods, no homologous genes to fattyacid desaturases were found in DNA extractedfrom ruminal protozoa. Thus, although protozoaare rich in CLA and TVA, they do not form thesetwo fatty acids. The most likely explanation forthese high concentrations is that protozoa incor-porate CLA and TVA formed by bacteria. Thiswork also suggests that the flow of unsaturatedfatty acids, including CLA and TVA, from therumen could be higher in protozoa than in bacteria.

P-81 Differential export of two Ruminococcusalbus cellulases by the Tat and Sec transloca-tion pathways of Escherichia coli. J. Esbelin,C. Martin, E. Forano, P. Mosoni (Unité deMicrobiologie, INRA, Clermont-Ferrand-Theix,63122 Saint-Genès-Champanelle, France).

Ruminococcus albus is a Gram-positive bacte-rium that degrades plant cell walls in the rumenof herbivores. In order to ensure this function ofdegradation, R. albus synthesizes a high numberof glycoside hydrolases, which are active afterthey have been exported and, some of them,anchored, at the cell-surface. In bacteria, the pro-teins destined to cross the cytoplasmic mem-brane are synthesized as precursors and possess

Session II: Microbial metabolism of dietary components S47

a signal sequence (SS) directing them to the“Sec” or “Tat” translocation pathways. Thecomposition of the signal sequence of the twomajor cellulases of R. albus strain 20, Cel9D andCel48B, suggests that each enzyme translocatesusing one of the two pathways. In order to con-firm this hypothesis, the translocation of the twoenzymes was studied by expressing SS-GFPfusions in E. coli. The cytoplasmic or periplas-mic localization of the recombinant proteins wasmonitored by Western blot and fluorescencemicroscopy in a wild type E. coli strain and inSec and Tat isogenic mutants. This study showsthat the SS of Cel9D directs the pre-protein to the“Tat” translocation pathway, while the GFPfused to the SS of Cel48B is exported throughthe “Sec” machinery. These observations sug-gest that genes encoding components homolo-gous to the Tat and Sec systems exist in R. albusand should be looked for once the genomesequence of R. albus strain 8 is complete.

P-82 Cross-feeding between Bifidobacteriumlongum BB536 and acetate-converting,butyrate-producing colon bacteria duringgrowth on oligofructose. G. Falony, A. Vlachou,K. Verbrugghe, L. De Vuyst (Research Group ofIndustrial Microbiology and Food Biotechnol-ogy, Department of Applied Biological Sciencesand Engineering, Vrije Universiteit Brussel,Brussels, Belgium).

Cross-feeding between colon bacteria is thoughtto be the link between the bifidogenic and thebutyrogenic effect caused by the addition of oli-gofructose to the diet. Acetate and lactate pro-duced by bifidobacteria may form the substratesfor butyrate-producing colon bacteria that arereported to account for 2–3% of the human gutmicrobiota. In this study, cross-feeding betweenBifidobacterium longum BB536 and acetate-converting colon bacteria was studied through invitro mono- and coculture fermentations.Growth of B. longum BB536 on fructose or oli-gofructose led to the production of acetic acidand, in lesser amounts, formic acid, lactic acid,ethanol, and succinic acid. Anaerostipes caccaeDSM 14662 produced only butyric acid and gas-ses when growing on fructose; no growth on oli-gofructose was observed. In a coculture withB. longum BB536, production of butyric acid byA. caccae DSM 14662 was detected, attributedto growth of the latter strain on the acetic acid,

lactic acid, and fructose formed during the deg-radation of oligofructose by B. longum BB536.Roseburia intestinalis DSM 14610 grew on bothfructose and oligofructose, but showed an abso-lute requirement for acetate. Only butyric acidand gases were formed. A coculture of R. intes-tinalis DSM 14610 with B. longum BB536 onoligofructose showed initial growth and acetateproduction by the Bifidobacterium strain, fol-lowed by oligofructose degradation and acetateconversion by R. intestinalis DSM 14610, lead-ing to the production of butyrate. Two distincttypes of cross-feeding between B. longumBB536 and acetate-converting colon bacteriawere observed, both leading to the production ofbutyrate from oligofructose.

P-83 Bile tolerance mechanisms of Listeriamonocytogenes: implications for survival inthe host GI tract. C.G. Gahan, R.D. Sleator, M.Begley, C. Hill (Department of Microbiologyand Alimentary Pharmabiotic Centre, Univer-sity College Cork, Cork, Ireland).

Listeria monocytogenes is an important food-borne pathogen that is related to the low G/C lac-tic acid bacteria. In order to colonise the host GItract prior to invasion, the pathogen must survivea number of sub-optimal microenvironments,including regions of elevated osmolarity and thepresence of bile salts within the small intestine.Since these same challenges face the coloniza-tion of related probiotic bacteria, we proposethat L. monocytogenes represents a useful,genetically tractable model with which to studymicrobial adaptation to conditions in the GItract. We have used functional genomic analysisof the L. monocytogenes genome to examine anumber of loci responsible for bile tolerance inthis organism. This work demonstrated a signif-icant role for bile salt hydrolase (BSH) and bileacid dehydratase in both bile tolerance and col-onization of the murine GI tract. Furthermore, itwas demonstrated that bsh expression is tightlyregulated by the alternative sigma factor SigmaB and the major regulator of virulence geneexpression, PrfA. Examination of bilE, a locuswith significant homology to osmolyte uptakesystems, demonstrated that this system is essen-tial for bile tolerance in L. monocytogenes. Wedetermined that BilE functions as a bile exclu-sion system that prevents intracellular accumu-lation of bile acids. Mutants in bilE are incapable

S48 Session II: Microbial metabolism of dietary components

of effective colonization of the GI tract and sub-sequent infection in the murine model of disease.Finally, we employed a transposon mutagenesisapproach to determine novel bile tolerance lociin L. monocytogenes. This approach uncovered12 loci that are required for bile tolerance in thisorganism, including zurR, lytB and a homologueof the capA locus required for capsule formationin Bacillus anthracis. Interestingly the majorityof loci are located within a 46-gene region of theL. monocytogenes genome that contains genesresponsible for stress resistance and homeosta-sis. Overall this work represents a characteriza-tion of a number of genetic loci that are essentialfor microbial adaptation to conditions in theupper GI tract.

P-84 Comparison of microbial pellets for esti-mation of microbial protein synthesis in con-tinuous-culture fermenters. A.I.M. Garcíaa,M.J. Ranillab, E.M. Alcaidea, M.D. Carrob

(a Unidad de Nutrición Animal, EstaciónExperimental del Zaidín (CSIC), Camino delJueves s/n, 18100 Armilla, Granada, Spain,b Departamento de Producción Animal I, Uni-versidad de León, 24071 León, Spain).

Measurement of ruminal microbial protein syn-thesis relies on the isolation of a microbial pelletrepresentative of those microbes leaving therumen. The aim of this study was to compare theestimates of microbial protein synthesis in con-tinuous-culture fermenters fed two diets withdifferent forage:concentrate ratio (80:20 [F80]and 20:80 [F20]) obtained by using microbialpellets isolated from effluent (EMP) or from fer-menters content (FMP). A 14-day incubation runwas carried out, each diet was fed to four fer-menters and 15N was used as a microbial marker.On days 12 and 13, total effluent was collectedfor analyses of non-ammonia N and 15N enrich-ment, and for isolation of EMP after incubatingabout 500 g of effluent at 38 °C for 15 min witha saline solution containing 0.1% methylcellu-lose. On the last day, the content of fermenterswas treated with methylcellulose solution beforeisolating FMP. Microbial protein synthesis wasestimated from 15N enrichment in both effluentand microbial pellets. For both diets, FMP pre-sented a greater (P < 0.02) 15N enrichment thanEMP. Compared to FMP, the use of EMP over-estimated microbial protein synthesis by 4.4 and9.2% for diets F80 (365 vs. 381 mg N/d) and F20

(427 vs. 465 mg N/d), respectively, but differ-ences were not significant (P = 0.66 and P =0.18). These results indicate that, under the con-ditions of the present experiment, both FMP andEMP can be used as a reference pellet for esti-mating microbial protein synthesis in continu-ous-culture fermenters.

P-85 Antioxidant activity and total polyphe-nol content studies in selected products usingan in vitro digestion tract model with humanfaecal flora. M. Gumienna, K. Goderska, Z.Czarnecki (Institute of Food Technology ofPlant Origin, Department of Fermentation andBiosynthesis, The August Cieszkowski Agricul-tural University of Pozna , Poland).

Intestinal microflora is the main component ofthe digestion tract and plays an important role indigestive functions. The aim of this research wasto analyse changes in microbial composition andinteractions of the microflora with biologicallyactive compounds. Products from leguminousplants (Red Kidney bean instant flour) and aro-nia (fruit juice) were selected for our investiga-tions. The digestion tract model, which com-prises 3 compartments: stomach, small intestineand large intestine, was used for controlling andregulating the environment in which digestionprocesses occurred. In each compartment of themodel the total amount of phenolic compounds(Folin-Ciocalteu Reagent), antioxidant ABTS•+

(mg Trolox) activity and changes in microbialcomposition were investigated during the diges-tion. Faecal flora was isolated from feces of3 human volunteers. The intestinal model wasinoculated with bacterial flora at a concentrationof 106 cfu·mL–1. Growth of Lactobacillus, Bifi-dobacterium, Enterococcus and Enterobacte-riaceae was enumerated on selective media.Growth of investigated bacteria, especially ofthe Enterococcus species, was affected by thedigested aronia juice. The amount of bacteriaafter digestion in the large intestine was main-tained at 108 cfu·mL–1 and for Enterococcus at106 cfu·mL–1. The instant flour from red Kidneybeans also stimulated bacterial growth. In effect,the amount of bacteria detected in the large intes-tine was 1011 cfu·mL–1. The highest antioxidantactivity as well as amount of phenolic com-pounds after the digestion process was deter-mined for the instant flour, while for aronia juice

ń

Session II: Microbial metabolism of dietary components S49

a decrease in the antioxidant activity and totalamount of phenolic compounds was observed.

P-86 Effect of defaunating the rumen onmicrobial protein synthesis in native Iraniansheep. K.K. Jafaria, M. Rezaeianb, M. Zahedi-farc, A. Mirhadic (a Islamic Azad University,Branch of Savadkooh, Iran, b Faculty of Veteri-nary Medicine, University of Tehran, Iran, cAn-imal Science Institute, Karaj, Iran).

The rumen microbial ecosystem mainly consistsof bacteria, protozoa and anaerobic fungi, whichhave an important function on the rumen fer-mentation process. In this study, the contributionof protozoa on the amount of microbial proteinsynthesis was determined. Four Iranian cas-trated male lambs (breed of shal) in a latin squaredesign were used. Defaunating the rumen wasdone by using an emptying and washing method(Jouany and Senaud, 1979). The amount ofmicrobial protein synthesis was measured usingthe Chen procedure (1992). The basis of thismethod is determining the amount of purinederivatives excreted via urine. The results of thisexperiment indicated that defaunating the rumenof sheep increased microbial nitrogen synthesis(11.8 vs. 7.73 g·day–1), and a significant differ-ence was observed (P < 0.05).

P-87 Strain-specific architecture in cellulos-ome assembly in Ruminococcus flavefaciens.S. Jindoua, I. Borovoka, M. Rinconb, H.J. Flintb,M. Bergc, B.A. Whitec, E.A. Bayerd, R. Lameda

(a Tel Aviv University, Ramat Aviv 69978,Israel, b Rowett Research Institute, Bucksburn,Aberdeen AB21 9SB, UK, c University of Illi-nois, Urbana, IL 61801, USA, d The WeizmannInstitute of Science, Rehovot 76100, Israel).

Ruminococcus flavefaciens FD-1 is a Gram-pos-itive, anaerobic, cellulolytic bacterium, whichcommonly inhabits the digestive tracts of rumi-nants. Shotgun and complementary manualsequencing of the genome has revealed about sixcellulosome-organising proteins (scaffoldins)and circa 190 dockerins, representing at leastfour different phylogenetic types. The architec-ture of the cellulosome system in strain FD-1was assessed by bioinformatic analysis and bystudying the interactions among overexpressed

cellulosomal protein modules. Cellulosomeorganisation in strain FD-1 is generally similarto that of strain 17 but also exhibits some verynovel properties. Surprisingly, the major scaf-foldin, ScaB, comprises two different types ofcohesins in strain FD-1, five of which are relatedto the cohesin sequences of ScaB in strain 17 andfour others are similar to those of ScaA. Thehybrid nature of ScaB scaffoldin is reflected inits ability to bind both ScaA and ScaC dockerinsas well as several dockerin-containing enzymes.Scaffoldin ScaA of strain FD-1 has three cohesinmodules, whose N-terminal cohesin is verydivergent from the others but still appears to rec-ognize weakly the latter type of dockerin. More-over, the cohesin of the FD-1 ScaC adaptor pro-tein binds certain enzyme-borne dockerins, incontrast to the corresponding cohesin of strain17, which apparently fails to recognise the cor-responding dockerins. Finally we also demon-strate that the cohesin of the FD-1 anchoringScaE binds to a novel class of dockerins (e.g.,from ScaB, ScaF and ScaH), but only limitedcross-strain cohesin-dockerin interactions havebeen observed.

P-88 Monitoring of rumen bacteria attachedto ruminally incubated rice straw quantifiedby real-time PCR. S. Koike, H. Yabuki, Y.Kobayashi (Creative Research Initiative Sousei,Hokkaido University, Sapporo 001-0021, Japan).

Real-time PCR assays were developed for therumen bacteria including 12 representative spe-cies and 2 uncultured groups. Untreated- andsodium hydroxide (NaOH) treated rice strawwas prepared to evaluate the effect of differentialdigestibility on the formation of bacterial com-munity. The nylon bags containing respectiverice straws were incubated for 10 min to 96 h inthe rumen of sheep, and the target bacterial spe-cies associated with the straws were quantifiedby real-time PCR. As expected, the digestibilityof NaOH-treated rice straw was higher than thatof untreated straw. Irrespective of the treat-ments, the fiber-associated populations forS. ruminantium, F. succinogenes and unculturedbacterial group U2 were higher than those of theother bacterial species during the initial stage ofincubation (within 6 h), then they maintainedtheir population sizes on the straws. These 3 spe-cies/group may play an important role in ricestraw degradation in the rumen. The kinetics of

S50 Session II: Microbial metabolism of dietary components

bacterial attachment was different between thetreatments, i.e. the higher peak level of attach-ment for fibrolytic species was detected at theinitial stage of incubation on NaOH-treatedstraw than on untreated straw. On the other hand,some of the non-fibrolytic species showed aslower increase on NaOH-treated rice strawcompared to untreated straw. This suggests thatthe higher digestibility of NaOH-treated strawswas achieved under the less synergistic relation-ship between fibrolytic and non-fibrolytic spe-cies. The disappearance of less-digestible frac-tions and soluble substances from the rice strawby NaOH treatment might provide higher acces-sibility of easily digestible fiber for fibrolyticspecies and lower availability of the substratesfor the non-fibrolytic bacteria.

P-89 Molecular identification of carbohy-drate-fermenting bacteria in the humancolon by RNA-stable isotope probing. P.Kovatcheva-Datchary, M. Egert, A.A. De Graaf,N.E.P. Deutz, A. Maathuis, H. Smidt, W.M. DeVos, K. Venema (WCFS, PO Box 557, 6700 ANWageningen, The Netherlands).

Prebiotic carbohydrates are believed to beimportant promoters of human gut health,mainly through their interactions with the micro-bial community in the human colon. However,these interactions are far from being fully under-stood. We used the RNA-stable isotope probing(SIP) technique to identify those microbial pop-ulations that are involved in the fermentation ofdietary relevant carbohydrates in the humancolon. In a pilot study, the microbial communityof an in vitro model of the human colon (TIM-2)was supplemented with 40 mM [U-13C]-glucose.Within 2 h, the glucose was completely fer-mented yielding mostly lactate, acetate andbutyrate. Moreover, 13C-labelled bacterial 16SrRNA was isolated already 1 h after incubationwith [U-13C]-glucose. Terminal-restriction frag-ment length polymorphism (T-RFLP) analysisshowed five dominant microbial species com-prising the bacterial community of the colonmodel. Phylogenetic analysis of 13C-labelled16S rRNA linked the most active bacterial glu-cose-fermenters in the model to Clostridiumperfringens and Streptococcus bovis. In a secondexperiment, 1 g of [U-13C]-starch isolated frompotatoes, grown in the presence of 13CO2, wasadded to the in vitro model. The starch was fer-

mented more slowly than glucose, yieldingmainly acetate, butyrate and propionate. After 4and 8 h of incubation, isotopically labelled RNAcould be isolated. T-RFLP fingerprinting oflabeled 16S rRNA identified an ~ 70 bp T-RF asrepresentative for primary starch-fermentingbacteria. Further phylogenetic analyses areunderway to determine the taxonomic affiliationof these starch consuming bacteria. In conclu-sion, RNA-SIP combined with metabolic anal-yses appears promising to identify bacteria actu-ally involved in colon fermentations and linkthem to metabolic products, not only in thecourse of in vitro but also of in vivo studies.

P-90 Evaluation of the effect of tannin-richplants on rumen microbial fermentation. C.Longoa,b,c, J. Hummelb, J. Liebichc, R.S. Diasa,E.F. Nozellaa, S.L.S. Cabral Filhoa, P. Burauelc,A.L. Abdallaa, K.-H. Südekumb (a Centre forNuclear Energy in Agriculture, SP, Brazil, b Uni-versity of Bonn, Germany, c ForschungszentrumJülich, Germany).

Some plants have been neglected as fodder dueto the presence of anti-nutritional factors, suchas condensed tannins (CT), affecting microbialactivity. Significance of influence of CT can beevaluated via a test for CT, or via in vitro fermen-tation (with and without PEG). Four legumes,Styzolobium aterrimum L (STA), Styzolobiumdeeringianum (STD), Leucaena leucocephala(LEU) and Mimosa caesalpiniaefolia (MIC),and Cynodonx Cynodon grass (CYN) as controlwere tested in the Hohenheim gas test and fortheir CT content. Gas production (GP) wasrecorded at 10 intervals over 96 h of incubation(two runs with three syringes for each plant). GPparameters were determined by an exponentialmodel. CT in the legumes, expressed as leuco-cyanidin-equivalent, ranged from 20 (STA) to205 (MIC) g·kg–1, and 96-h GP ranged from 98(MIC) to 215 (LEU) mL·g–1. LEU, STA and STDpresented similar 96-h GP values, but differed(P < 0.05) from CYN and MIC. There was aPEG effect on 24-h GP for all plants except CYN(P < 0.05), which lasted until 96-h GP only forMIC. Although STA had less CT than STD andLEU, they expressed a similar PEG effect (P >0.05). MIC had a much higher Lag-phase (2.7 h)than the other plants, which ranged only from0.07 to 0.64. PEG largely reduced Lag-phase ofMIC to 0.20 h. Despite the presence of CT, LEU

Session II: Microbial metabolism of dietary components S51

and STD were well fermented, showing differ-ences in biological activity of CT, which can bestbe evaluated by in vitro fermentation tests.

P-91 Butyrate metabolism in human colonicbacteria: characterisation of a novel CoA-transferase. P. Louis, C. Charrier, H.J. Flint(Rowett Research Institute, Gut Health Divi-sion, Microbial Ecology Group, GreenburnRoad, Bucksburn, Aberdeen AB21 9SB, UK).

Low mol% G+C Gram positive bacteria com-prise a major group within the microbial com-munity of the human large intestine. Theyexhibit a fermentative metabolism and for manystrains butyrate is one of the main fermentationacids produced. Butyrate serves an importantrole in maintaining gut health, as it is the pre-ferred energy source for the colonic wall and hasregulatory effects on cell differentiation andapoptosis. We therefore aimed at investigatingthe pathway leading to butyrate formation inrepresentative strains. The final step of butyrateformation can proceed via two alternative path-ways. We have shown previously that the CoA-transferase route is the predominant route forbutyrate formation within human colonic bacte-ria (Louis et al., 2004, J Bacteriol 186: 2099–2106). While all the genes for butyrate formationvia the alternative butyrate kinase route havebeen described in clostridia, the gene for theCoA-transferase reaction remained elusive. Wehave recently identified a novel CoA-transferasegene from the clostridial cluster XIVa strainRoseburia sp. A2-183. Characterization of thegene product showed that butyryl-CoA is thepreferred substrate for this enzyme, making it avery strong candidate for the CoA-transferaseinvolved in butyrate formation. Similar genescould be identified in various other human gutbacteria from clostridial cluster IV and XIVa.The identification of this gene will further ourunderstanding of butyrate formation in humangut anaerobes and enable us to study its regula-tion in response to environmental changes.

P-92 Retention time of substrate affects fer-mentation characteristics in Rusitec ferment-ers. M.E. Martínez, S. Ramos, M.L. Tejido, M.J.Ranilla, L.A. Giraldo, M.D. Carro (Departamento

de Producción Animal I, Universidad de León,24071 León, Spain).

Previous studies have shown that simulation ofrumen fermentation in Rusitec fermenters fedhigh-concentrate diets is usually unsatisfactory.Since rumen retention time of digesta is one ofthe factors affecting rumen fermentation in vivo,the objective of this study was to investigate theeffects of two retention times of concentrate –24 h (T24) and 48 h (T48) – on rumen fermen-tation parameters in Rusitec fermenters. Eightfermenters were fed daily 21 g dry-matter (DM)of concentrate and 9 g DM of alfalfa hay con-tained in separate nylon bags. Forage retentiontime was 48 h in all fermenters. After 10 days ofadaptation, diet degradability and production ofvolatile fatty acid (VFA) and methane weremeasured on four consecutive days. Concentrateretention time did not affect (P > 0.05) the dilu-tion rate (mean value of 3.79% h–1) or the rumenpH (mean value of 6.47). Degradability of bothDM and fibre was lower (P < 0.01) for T24 (66.5and 18.9%, respectively) than for T48 (71.2 and26.6%), but no differences between treatmentswere observed in total VFA production (80.1 vs.81.6 mmol·d–1; P = 0.645). Reducing concen-trate retention time from 48 to 24 h decreasedmethane production (21.7 vs. 18.2 mmol·d–1;P = 0.005) and molar proportions of acetate(52.3 vs. 48.6%; P = 0.008) and butyrate (24.3vs. 22.9%; P = 0.004), but it increased the pro-portion of propionate (14.8 vs. 19.5%; P =0.020). As a consequence of these changes, bothacetate:propionate and methane:VFA ratioswere lower (P < 0.01) for T24 (2.52 and 0.23)than for T48 (3.54 and 0.27). These results indi-cate that changing the substrate retention time isa suitable means to alter rumen fermentationcharacteristics in Rusitec fermenters.

P-93 NMR study of plant fibre degradation byRuminococcus albus and Fibrobacter succino-genes. M. Matulovaa, R. Nouailleb,c, P. Capeka,M. Péand, A.-M. Delortb, E. Foranoc (a Instituteof Chemistry, Slovak Academy of Sciences,Bratislava, 845 38 Slovak Republic, b Labora-toire de Synthèse et Étude de Systèmes à IntérêtBiologique, UMR 6504 Université Blaise Pas-cal-CNRS, 63177 Aubière Cedex, France,c Unité de Microbiologie, INRA, Centre deRecherches de Clermont-Ferrand-Theix, 63122

S52 Session II: Microbial metabolism of dietary components

Saint-Genès-Champanelle, France, d DEVM/GRAP, CEA Cadarache, France).

Cellulose and wheat straw degradation by Rumi-nococcus albus and Fibrobacter succinogeneswas monitored using NMR spectroscopy andchemolytic methods. In situ solid state NMR,13C CP MAS (cross polarisation magic anglespinning) was used to monitor the modificationof the composition and the structure of celluloseand 13C-enriched wheat straw during growth ofthe bacteria on this substrate. There was no pref-erential degradation either of amorphous regionsof cellulose versus crystalline ones, or of cellu-lose versus hemicelluloses in wheat straw, what-ever the bacterial species. Liquid state 2D NMRexperiments and chemolytic methods were usedto analyse in detail the sugars released in the cul-ture medium, and integration of NMR signalsenabled their quantification at various times ofculture. The results showed a different mode ofattack of the plant fibres by the two bacteria: cel-lodextrins were accumulated in the culturemedium of R. albus, but not of F. succinogenes,and sequential activity of the esterases on hemi-celluloses was also different in the two species.This suggests that the two cellulolytic bacteriahave specific roles in plant cell wall degradationin the rumen ecosystem.

P-94 Variations in intermediates and prod-ucts of linoleic acid biohydrogenation by twodifferent human mixed colonic flora. F.M.McIntosh, R.J. Wallace, E. Devillard (RowettResearch Institute, Bucksburn Aberdeen AB219SB, UK).

Conjugated linoleic acids (CLA) are consideredto be beneficial to human health. They are pro-duced in the rumen as intermediates in the bio-hydrogenation of linoleic acid and passed intothe ruminant products. In this study, we showthat CLA can also be produced endogenously inthe human gut from linoleic acid, and that thisprocess varies considerably between humansubjects. Fresh faecal samples were obtainedfrom two healthy female volunteers (V1 andV2). Faecal samples were incubated anaerobi-cally with linoleic acid (LA) and samplesremoved at time intervals. LA was metabolisedat a similar rate in the samples from both volun-teers. However the products of biohydrogena-tion were different between the 2 samples. The

sample from V1 showed low production ofCLA, present as mainly one isomer (C18:2, cis-9, trans-11) and a high production of stearate. Incontrast, the sample from V2 produced highamounts of CLA of several isomers. The relationbetween differences in the ability to produceCLA and microbial diversity was investigated.The biodiversity of the bacterial populations ofthe samples from the two volunteers was shownusing denaturing gradient gel electrophoresisand 16S rDNA libraries. From the analysis of16S rDNA sequences, we observed that the sam-ple from V1 contained a very high proportion ofbacteria close to Butyrivibrio hungatei (ClusterXIVa), whereas the sample from V2 showed alarge population of bacteria related to Eubacte-rium siraeum (Cluster IV). The endogeneousproduction of CLA in the human gut variesbetween subjects, and is probably linked to dif-ferent colonic flora. This is particularly interest-ing since it has been recently shown that specificCLA isomers have potential local effects on thecolon.

P-95 The existence and composition of dis-tinctive bacterial communities colonisingeach of three insoluble dietary substrates ina model of the human colon. E.C. McWilliamLeitch, A.W. Walker, S.H. Duncan, H.J. Flint(Rowett Research Institute, Bucksburn, Aber-deen AB21 9SB, UK).

The breakdown of insoluble dietary carbohy-drates, such as resistant starch and plant polysac-charides, and of endogenously-produced mucinby gut bacteria, has an important impact upon thehealth of the human colon. While substrate pref-erences have been determined for a few isolatedgut bacteria, the identity and diversity of primarycolonisers of a given insoluble substrate in thecomplete gut community remains largelyunknown. Continuous flow fermentors inocu-lated with faecal samples were used here tomodel bacterial colonisation of starch, bran andmucin. Attached bacteria were identified byPCR-amplified 16S rRNA sequences and fluo-rescent in situ hybridisation. Diversity within theresulting attached communities was low. Therewas evidence for a high degree of substrate-spe-cificity, and 81% of the identified OTU’s wererecovered from only one substrate. The mainstarch-adherent species Ruminococcus bromii

Session II: Microbial metabolism of dietary components S53

and Bifidobacterium adolescentis were onlydetected on starch. Likewise, an unculturedgroup related to Clostridium hathewayi wasfound only on bran, and B. bifidum was detectedonly on mucin. R. lactaris and closely relateduncultured clones were major colonisers ofmucin for samples from three individuals. Inter-individual variation was also apparent. Bifido-bacteria were the predominant species found onstarch for samples from two individuals,whereas Eubacterium rectale and R. bromiiwere the co-dominant colonisers for the othertwo individuals. In conclusion, this study showedthat distinctive bacterial communities form ondifferent insoluble substrates in a model of thehuman colon. In addition, it appears that thedominant primary colonisers of a given substratemay differ between the gut microbiota of differ-ent individuals.

P-96 Isolation of xylanase genes from two newxylan-degrading species from the humancolon, Bacteroides sp. and Roseburia intesti-nalis. C. Mirande, C. Chassard, A. Bernalier-Donadille, E. Forano, C. Béra-Maillet (Unité deMicrobiologie, INRA, CR de Clermont-Ferrand/Theix, 63122 Saint-Genès-Champanelle, France).

Fibrolytic bacteria of the human gut ecosystemplay a major role in the degradation of plant cellwall polysaccharides of the ingested dietaryfibres. However, these microorganisms have notbeen extensively studied. Hemicellulolytic bac-teria were found predominant in this ecosystemcompared to the cellulolytic populations. Ashemicellulolytic activity was found in the pre-dominant colonic microflora and xylans consid-ered as a preferential substrate for the fibrolyticbacteria, we recently isolated bacterial strainswith high xylanolytic activities from the feces ofhealthy volunteers. These isolates were identi-fied as Bacteroides sp. and Roseburia intestina-lis species. Genomic libraries were then con-structed for both Bacteroides sp. and R.intestinalis isolates and xylanase genes weredetected by screening of the xylanolytic activityof the clones. Furthermore, PCR products weregenerated by using specific primers designedfrom the sequence of xylanase genes present inthe databases, comprising other Bacteroidesspecies xylanase genes, and from family 10 and11 xylanase sequences. Xylanolytic equipment

of Bacteroides sp. and R. intestinalis is furtherdiscussed.

P-97 Effect of red beet juice and crisps fromred beet on the growth of intestinal microor-ganisms and antioxidant stability. M.Neumann, W. Grajek (Department of Biotech-nology and Food Microbiology, AgriculturalUniversity, ul.Wojska Polskiego 48, 60-627Pozna , Poland).

One of the most interesting vegetables, rich inantioxidant compounds, is red beet. It containsred betacyanins and yellow betaxanthins. Anti-oxidants, taken up with food into the gastroin-testinal tract, react with many physiologicalsecretions like HCl, hydrolytic enzymes, bilesalts and pancreatic fluids and have contact withintestinal microorganisms. This treatment leadsto chemical changes in antioxidant structure andresults in changes of antioxidant capacity. Theaim of the work was to study the influence ofgastrointestinal digestion and microbial fermen-tation of red beet products on the growth of somegroups of intestinal microflora and on the anti-oxidant capacity. In the experiments, red beetjuice and crisps from red beet were used. Theproduct digestion was performed using an invitro model consisting of a gastric reactor (pep-sine, 1 M HCl, pH 2.0, 2 h) and small intestinereactor (pancreatic extract, bile salt, NaHCO3,pH 7.4, 2.5 h). The culture medium was inocu-lated with a fecal human bacterial suspension at~106 cfu·mL–1. Drawing of samples was doneafter 2 and 21 h of digestion. In samples, the anti-oxidant capacity of red beet products andchanges in the microorganism cell densitieswere determined. Bacterial groups were countedusing the following agar media: McConkey forEscherichia, MRS for Lactobacillus, Garche’sfor Bifidobacterium and Kanamycin EsculinAzide for Enterococcus. To determine the anti-oxidant capacity, the samples were centrifugedand the antioxidant capacity was assessed in thesupernatant on the basis of the scavenging activ-ity of ABTS and DPPH radicals. The results ofour investigation showed a correlation betweenthe type of product digested and the intensity ofintestinal microorganism growth, as well as theeffect of product digestion on its antioxidantcapacity. It was found that crisps from red beetintroduced into the culture medium stimulatedgrowth of the studied microorganisms. The

ń

S54 Session II: Microbial metabolism of dietary components

results also indicated decreases of the antioxi-dant capacity after exposure to the gastrointes-tinal tract.

P-98 Binding and biotransformation of theFusarium mycotoxin zearalenone to α-zearale-nol by lactic acid bacteria. V. Niderkorna,b,H. Boudraa, D.P. Morgavia (a HerbivoreResearch Unit, INRA-Theix Research Centre,France, b Lallemand S.A.S, 31702 Blagnac,France).

Lactic acid bacteria (LAB) are widely used in thepreparation of fermented foods and feeds and asprobiotics in human and animal nutrition. Somestrains of this group of bacteria have the abilityto decrease the bioavailability of mycotoxinspresent in foods and feeds. The objective of thiswork was to select LAB able to detoxify zea-ralenone (ZEN), an estrogenic Fusarium myco-toxin frequently found in maize grain and silage.We screened 197 strains of LAB for their abilityto remove ZEN by either binding or biotransfor-mation in a model representative of maize silage.Bacteria (5 × 108 cfu·mL–1) were incubated at25 °C for 24 h in maize infusion acidified to pH4 and containing 5 µg·mL ZEN. After incuba-tion, the medium was centrifuged and the super-natant was analysed by HPLC. Efficiency ofremoval by binding was genus specific. WhileLactobacillus, Leuconostoc, Lactococcus, andPediococcus bound about 20% of ZEN, the mostactive genera, Streptococcus and Enterococcusbound 40 and 35% of ZEN, respectively (P <0.05). In addition, 8 Lactobacillus and 3 Leucon-ostoc strains were able to biotransform between28 and 59% of ZEN to a single metabolite. Thiscompound was identified by LC-MS as α-zea-ralenol (α-ZOL), which is more estrogenic thanZEN and is thought to be the activated form ofthe toxin in mammals. The ability of somestrains to bind ZEN is a positive technologicalproperty. However, the biotransformation ofZEN to α-ZOL, which was identified for the firsttime in bacteria, cannot be considered as adetoxification.

P-99 Ability of lactic acid bacteria to bind or/and metabolize N-nitrosodimethylamine(NDMA) connected with growth stages of thebacteria. A. Nowak, Z. Libudzisz (Institute ofFermentation, Technology and Microbiology,

Technical University of Lodz, ul. Wolczanska171/173, 90-924 Lodz, Poland).

Lactic acid bacteria have been reported to haveantimutagenic or anticarcinogenic properties invitro and in vivo. The purpose of the presentinvestigation was to study the capacity of lacticacid bacteria to decrease the level of carcinogen– NDMA. Bacterial strains tested were: Lacto-bacillus plantarum WL, Lactobacillus casei DN114 001, Lactobacillus casei/paracasei KNE1,Lactobacillus casei J/III, Lactobacillus caseiBD. The concentration of NDMA examined was10 µg·mL–1 in a special MRS broth with lownitrogen quantity. Samples were inoculated andincubated for 168 h at 37 °C under anaerobicconditions. Using Koch’s plate method, thenumber of cells was determined at 0 h, 4, 8, 12,24, 48, 72 (or 96) and at 168 h incubation. Simul-taneously, with high performance liquid chro-matography (HPLC), the level of NDMA insamples was determined. The addition ofNDMA did not affect the growth of bacteria dur-ing 168 h incubation. There was a relationshipbetween the level of NDMA in the sample andthe stage of growth of bacteria. In all cultures theamount of NDMA was decreased at the begin-ning (between 0 h and 4 h) from 10 µg·mL–1 to8.6 µg·mL–1 (for L. plantarum WL) or to9.5 µg·mL–1 (for L. casei/paracasei KNE1).This could indicate the initial ability of lacticacid bacteria to adsorb the compound. Alongwith reaching the logarithmic stage of growth,the resorption of NDMA from broth wasobserved for all strains. After 24 h incubation(after reaching the stationary phase of growth)the concentration of NDMA was the same as atthe “zero” time for all strains (10 µg·mL–1). Asof 96 h of incubation, the level of NDMA wasdecreasing just along with decreasing number ofliving cells in the samples. The results indicatethat lactic acid bacteria can lower NDMAconcentration in the culture and it could beeither bound or metabolized. The decrease inmutagenic activity of the compound was con-firmed in comet assay.

P-100 Influence of heterocyclic aromaticamines (HCA) on growth and survival ofLactobacillus strains. A. Nowak, Z. Libudzisz(Institute of Fermentation, Technology andMicrobiology, Technical University of Lodz, ul.Wolczanska 171/173, 90-924 Lodz, Poland).

Session II: Microbial metabolism of dietary components S55

The aim of the study was to estimate the abilityof intestinal lactobacilli to grow and survivein the presence of 3 HCA: IQ, MelQx andPhIP. Concentrations selected for the researchwere: 5 µg·mL–1, 25 µg·mL–1; additionally100 µg·mL–1 for IQ and 75 µg·mL–1 for MelQx.Bacterial strains tested were: Lactobacillusplantarum WL, L. casei/paracasei KNE1,L. casei J/III, L. casei BD and L. casei DN 114001. To define the influence of HCA on growthof lactobacilli, the cells were cultivated 24 h inMRS medium at 37 °C under anaerobic condi-tions with the addition of final concentrations ofIQ, MelQx and PhIP. For all HCA tested, therewas no reduction in the number of living bacte-rial cells as compared to the control. To definethe influence of HCA on survival of bacteria, thebiomass achieved after 24 h incubation in MRSbroth was centrifuged, suspended in phosphatebuffer with appropriate concentrations of eachcompound and incubated for 168 h. All the HCAhad some influence on the survival of lactic acidbacteria and it depended on the strain, incubationtime, the compound and its dosage. The mostsignificant impact was observed in case of IQ,when the number of living cells was reduced for:L. casei DN (at 100 µg·mL–1) after 72 h andL. casei J/III (at 100 µg·mL–1) after 120 h,L. casei BD (at 5, 25 and 100 µg·mL–1) after 48 hand L. paracasei/casei KNE1 (at 100 µg·mL–1)after 120 h of incubation. MelQx had someimpact on survival of the bacteria after 144 h forL. casei DN (at 5, 25, 75 µg·mL–1), L. paracasei/casei KNE1 (at 25 and 75 µg·mL–1) andL. plantarum WL (at 75 µg·mL–1). In case ofPhIP, a decreased number of living cells wasnoticed for L. casei DN (at 25 µg·mL–1) after96 h, L. paracasei/casei KNE1 up to 48 h, andL. plantarum WL after 72 h. There was no influ-ence observed from 48 h up to 72 h of incubationcompared with the control. The results indicatethat intestinal lactic acid bacteria can survive inthe presence of all the concentrations of HCAtested up to 72 h with no influence on the numberof living cells during the transit time, which lastsfrom 48 h to 72 h.

P-101 Activation of pro-estrogens from hopsby intestinal bacteria. S. Possemiersa, S.Bolcaa,b, A. Heyerickb, W. Verstraetea (a Labo-ratory of Microbial Ecology and Technology,Ghent University, Belgium, b Laboratory of

Pharmacognosis and Phytochemistry, GhentUniversity, Belgium).

Hops have been used for centuries in beer brew-ing, but in the last few years they have gainedincreasing attention as a source of prenylfla-vonoids, with 8-prenylnaringenin (8-PN) beingthe most potent phytoestrogen identified so far.Typically, isoxanthohumol (IX) is the major pre-nylflavonoid present in beer (≤ 4 mg·L–1) and8-PN concentrations are low (<100 µg·L–1).Therefore, with the current knowledge, nohealth effects due to estrogens in beer were to beexpected through moderate beer consumption.However, we recently showed that, in parallelwith other phytoestrogens, the human intestinalmicrobiota may crucially influence the activityof hop prenylflavonoids. Upon incubation offecal samples, an O-demethylation of IX into8-PN was discovered, leading to increased expo-sure to the strong phytoestrogen. Analysis of51 intestinal communities revealed interindivid-ual differences in the intestinal transformationpotential, separating individuals into high, mod-erate and slow IX activators. Using the SHIME,a five-step simulator of the intestine, we showedthat the production of 8-PN mainly occurs in thedistal colon. In vivo, HFA rats were used and anIX-containing supplement was dosed to 50 post-menopausal women, and a clear correlationbetween intestinal transformations and urinary8-PN excretion was shown. Finally, a bacteriumcapable of this O-demethylation was selected,which produced 8-PN and which could startupIX conversion in the SHIME and in a pig inter-vention trial. Therefore, the activity of the intes-tinal microbiota could easily increase the 8-PNlevels tenfold and the final exposure to 8-PNafter moderate beer consumption does notdepend on the 8-PN concentrations, but rather ona combination of the IX concentrations and theintestinal transformation potential to activate IXinto 8-PN.

P-102 Proteomics-based investigation of majormetabolic pathways of a human gut anaerobe,Fusobacterium varium. J. Potrykusa,b, S.L.Bearnea, R.L. Whiteb (a Department of Bio-chemistry and Molecular Biology, DalhousieUniversity, Halifax, Canada, b Department ofChemistry, Dalhousie University, Halifax, NovaScotia B3H 1X5, Canada).

S56 Session II: Microbial metabolism of dietary components

Bacteria comprising the normal flora are oftenbeneficial to the host, making the investigationof their physiology relevant to our health. Wedescribe a proteomics-based exploration of themetabolism of the butyrate-producing gut anaer-obe Fusobacterium varium. Changes in the pro-teome of F. varium in response to differentgrowth substrates were monitored using two-dimensional polyacrylamide gel electrophore-sis. Cells were grown in a chemically definedminimal medium supplemented with glucose,pyruvate, intermediates of the core acetate-butyrate pathway (acetoacetate or crotonate), oramino acids (L- or D-glutamate, L-histidine, L- orD-lysine, L- or D-serine) as major energy sources.In the selected pH isoelectrofocusing range,pH 4–7, approximately 380 protein spots weredetected that were common to all investigatedgrowth conditions. Pronounced differenceswere observed between the proteomes of bacte-ria thriving on glucose and those fermenting anamino acid as the main energy source. About 90protein spots were chosen for MS/MS analysisand de novo sequencing. The analysis revealedthat some proteins (e.g. specific catabolicenzymes) serve as unique markers for a partic-ular growth substrate, while intracellular con-centrations of other proteins (e.g. translation fac-tor EF-Tu) appear to be independent ofvariations in the identity of the growth substrate.

P-103 Effects of dilution rate on methane pro-duction and fibre degradability in Rusitecfermenters fed a high-concentrate diet. S.Ramos, M.E. Martínez, M.J. Ranilla, M.L.Tejido, L.A. Giraldo, M.D. Carro (Departamentode Producción Animal I, Universidad de León,24071 León, Spain).

Increasing dilution rate in rumen fermentersoften results in an increase in pH, and thereforeeffects of both factors on fermentation charac-teristics are confounded. The aim of this studywas to analyse the effects of two dilution rates –low (LDR, 3.8% h–1) and high (HDR; 5.4% h–1)– on fermentation parameters in Rusitec fer-menters maintained at a similar pH. Eight fer-menters were fed daily 30 g dry-matter (DM) ofa 30:70 alfalfa hay:concentrate diet and eachexperimental treatment was assigned to four fer-menters. After 10 days of adaptation, methaneand volatile fatty acid (VFA) production and dietdegradability after 48 of incubation were meas-ured on four consecutive days. There were no

differences (P > 0.05) between treatments inruminal pH (6.55 and 6.47 for LDR and HDR,respectively) or methane production (21.7 and22.4 mmol·d–1). On the contrary, both produc-tion of total VFA and fibre digestibility aug-mented from 81.6 to 90.5 mmol·d–1 (P = 0.028)and from 29.8 to 36.8% (P < 0.001), respec-tively, by increasing the dilution rate. Molar pro-portions of acetate and butyrate were decreased(P < 0.001) by 4.0 and 10.4%, and those of pro-pionate and other VFA (calculated as the sum ofisobutyrate, isovalerate and valerate) wereincreased by 20.1 and 21.6% when the dilutionrate was changed from 3.8 to 5.4% h–1. As a con-sequence, the acetate:propionate ratio was lowerfor HDR than for LDR (2.83 vs. 3.54; P < 0.001).These results indicate that increasing the dilu-tion rate in Rusitec fermenters can influencerumen fermentation by increasing diet degrada-bility and VFA production and by changing thepattern of VFA production.

P-104 Metabolism of the pyrrolizidine alka-loid senecionine by an enrichment of ovineruminal microbes as detected by high-per-formance liquid chromatography and massspectrometry. R.M. Rattray, J.M. Duringer,A.M. Craig (Oregon State University, Corvallis,Oregon, USA).

Pyrrolizidine alkaloids (PAs) produced by theplant tansy ragwort (Senecio jacobia) have beenshown to act as hepatotoxins when metabolitesof these molecules are created by eukaryoticenzymes. Sheep have been shown to have anextremely high in vivo resistance to PAs whencompared to cattle and horses fed tansy ragwort.Previous work has demonstrated that this resist-ance is conveyed by unique ruminal bacteria. Abacterial consortium capable of degradingmixed PAs from tansy has been enriched fromovine rumen fluid and designated L4M2. Thedisappearance of the PA senecionine fromexperimental cultures of L4M2 was observed byhigh performance liquid chromatography(HPLC) analysis within the first four hours ofincubation. The detection of metabolites as aresult of degradation of senecionine was per-formed by monitoring absorbance at 220 nm aswell as full-spectrum analysis. In addition, massspectrometry was used to determine the putativechemical configuration of senecionine metabo-lites. The appearance of metabolites was

Session II: Microbial metabolism of dietary components S57

detected after about three hours of incubationand persisted through the experimental period.Since animal pathways in the liver have beenshown to convert some non-toxic metabolites ofsenecionine into toxic forms, it is desirable toknow the putative structure of metabolites forthis compound created by L4M2 in vitro. We canthen begin to determine whether the bacterialmetabolites produced in the host animal’s rumensimply pass through the digestive system or areabsorbed, thereby providing the opportunity fortoxic conversion and subsequent liver cirrhosis.

P-105 Novel structural and catalytic elementsin the Ruminococcus flavefaciens cellulosomerevealed by genome analysis. M. Rincona, I.Borovokb, M.E. Bergc, D.A. Antonopoulosc, R.Kimc, L. Liuc, J. Thimmapuramc, S. Jindouc, R.Lamedb, H.J. Flinta, E.A. Bayerd, B.A. Whitec

(a Rowett Research Institute, Bucksburn, Aber-deen AB21 9SB, UK, b Tel Aviv University,Ramat Aviv 69978 Israel, c University of Illi-nois, Urbana, IL 61801, USA, d The WeizmannInstitute of Science, Rehovot 76100, Israel).

Ruminococcus flavefaciens is one of the majorcellulolytic bacteria cultured from the rumen ofherbivores. The bacterium specializes in thebreakdown of complex polysaccharides fromthe plant cell wall thanks to the synthesis of avariety of exocellular hydrolytic enzymes.Recent investigations have established thatR. flavefaciens strain 17 produces a large, mul-ticatalytic, protein complex that is associatedwith the bacterial cell envelope. This complexshares many characteristics with the cellulos-ome reported in Clostridium thermocellum, butalso displays many novel features. Shotgunsequencing of the genome (currently at 4× cov-erage) from a different strain (R. flavefaciensFD1) provided an ideal opportunity to gain abroader perspective on R. flavefaciens cellulos-ome organisation, by extending the functionaland sequence analyses performed with R. flave-faciens 17. In silico analysis revealed the occur-rence of about 180 ORFs which contain adockerin-like motif. Surprisingly, many dockerin-containing sequences contain unusual flankingregions with dissimilar predicted activities, suchas cysteine proteases, serine protease inhibitorsand other regions of unknown function. Pre-dicted glycosyl hydrolase-coding sequencesrepresent at least 36% of all retrieved dockerin-containing ORF’s. Structural proteins and pro-

teases account for 3 and 8% respectively,whereas ORF’s with unknown regions accountfor 53% of the total. In addition to scaffoldin-likehomologs of the previously characterized ScaA,ScaB, ScaC and ScaE of strain 17, newly iden-tified cohesin-like domains were also retrieved.Phylogenetic analysis of dockerin motifs andcohesin-like modules revealed a divergence,which is predicted to be linked to different dock-erin-cohesin specificities in the cellulosomeassembly.

P-106 Polysaccharidases isolated from therumen metagenome. M.T. Rincon, D.Henderson, J.C. Martin, K.P. Scott, H.J. Flint(Rowett Research Institute, Greenburn Road,Bucksburn, Aberdeen AB21 9SB, UK).

Microbes in the rumen are responsible for pre-gastric fermentation of feedstuff, from which thevolatile fatty acid products provide the host ani-mal with major sources of energy and carbon.The ruminant diet is particularly rich in ligno-cellulosic material. In order to fully degrade therecalcitrant matrix of complex polysaccharidesin the plant cell wall, microbes adapted to livein the rumen ecosystem have evolved a greatvariety of enzymes that cleave the wide varietyof linkages present in ligno-cellulose. The smallnumber of cultured cellulolytic rumen bacteriathat is available may not, however, reflect thefull range of cellulose degraders that are presentin the rumen ecosystem. Metagenome approachestherefore offer the prospect of recovering novelgenes and activities. In this work, carried outthrough the EU FPV GEMINI project, bacterialDNA was extracted from total rumen contents,and libraries were constructed using a bacteri-ophage lambda vector (average insert size of ~5.5 kb). Functional screening of the phagelibrary for polysaccharidase activities was car-ried out using plate overlays, followed by Congored staining. Insert DNA was recovered after cir-cularization by in vivo excision, and subjectedto sequence analysis. Enzymes belonging to gly-cosyl hydrolases family 5 (http://afmb.cnrs-mrs.fr/CAZY/acc.html) were the most abundantamong those retrieved after screening with car-boxymethyl cellulose. However, novel glycosylhydrolases were retrieved after screening withthe substrates xyloglucan and lichenan. Phylo-genetic relationships suggested by the sequencesindicate that that cloned inserts originate from

S58 Session II: Microbial metabolism of dietary components

relatives of a wide range of anaerobic bacterialgenera, including Prevotella, Fibrobacter,Bacteroides, Clostridium and Selenomonas.

P-107 Transcriptome analysis of the clusterXIVa human colonic anaerobe Roseburiainulinivorans. K.P. Scott, J. Martin, J. Potrykus,G. Campbell, M.T. Rincon, G. Rucklidge, C.-D.Mayer, H.J. Flint (Rowett Research Institute,Aberdeen, UK).

Roseburia species together with Eubacteriumrectale represent one of the main groups ofbutyrate producers in the human colon, and cancomprise up to 10% of the total bacterial popu-lation in human faeces. Roseburia inulinivoransA2-194 is able to utilize a particularly widerange of growth substrates, including inulin,FOS, resistant starch and fucose for growth. Dif-ferential gene expression of R. inulinivoransduring growth on different host-derived and die-tary substrates was investigated using a combi-nation of genomic microarray screening andproteomics. Genes induced on inulin encoded asucrose hydrolase, fructose kinase and PTStransport system proteins specific for fructoseuptake. Genes induced on starch included anα-amylase functionally similar to one expressedby the rumen anaerobe Butyrivibrio fibrisolvens,an α-glucanotransferase and various ABC trans-porters. Comparison of 2D gels of the proteomeof the bacteria grown on the same substratesindicated fourteen differentially expressed pro-teins, and further analysis indicated that one ofthem was identical to the sucrose-6-phosphatehydrolase identified through microarray analy-sis. A complete switch in metabolism wasrequired to enable the bacterium to grow onfucose and resulted in the formation of propion-ate and propanol. The genes induced on fucosewere primarily directly involved in fucose andpropanediol utilisation.

P-108 Localization of cellulolytic rumen bac-teria on the plant fibrous materials deter-mined by a newly developed fluorescent insitu hybridization protocol. T. Shinkai, Y.Kobayashi (Graduate School of Agriculture,Hokkaido University, Japan).

In this study, we established a new fluorescentin situ hybridization (FISH) protocol to mini-

mize the self-fluorescence of orchardgrass hayas a representative grass material. By using thisprotocol, we have successfully detected fibro-lytic rumen bacteria attaching to stem and leafsheath sections of orchardgrass hay under a flu-orescent microscope. Real-time PCR assayswere also employed to quantitatively monitorrepresentative fibrolytic rumen bacteria on thematerial. Fibrobacter succinogenes was esti-mated to account for 6.9–8.1% of total bacteriaboth in the stem or the leaf sheath in terms of 16SrDNA copy number. F. succinogenes waslocated as firmly attaching not only to cut edgesbut also to undamaged plant surfaces. AlthoughRuminococcus flavefaciens was present at only0.34–0.59%, this bacterium was detected to agreater extent in the leaf sheath than in the stem(according to real-time PCR assay values andfrequency of FISH signals). Furthermore, R. fla-vefaciens created a lot of pits on the sheath andmany cells of this species were located alongedges of the pits. F. succinogenes was consid-ered to play the central role in fiber digestion oforchardgrass hay stem, since this bacterium isclearly detectable by FISH and assayable as thelargest in population size in the less degradablestem section. Further use of this FISH protocolin combination with quantitative PCR assaysmay clarify the whole scheme of fiber digestionby a bacterial consortium comprised of the rep-resentative fibrolytic bacteria and even function-ally unexplored bacteria.

P-109 Detection and quantification of chiti-nases in the human fecal chitinolytic bacte-rium Clostridium paraputrificum J4. J.Šim neka, G. Tishchenkob, H. Barto ováa

(a Institute of Animal Physiology and Genetics,Czech Academy of Sciences, Víde ská, 1083,140 00 Prague 4, Czech Republic, b Institute ofMacromolecular Chemistry, Academy of Sci-ences of the Czech Republic, Heyrovský Sq. 2,162 06 Prague 6, Czech Republic).

A strictly anaerobic, mesophilic and chitinolyticbacterium, Clostridium paraputrificum J4, wasisolated from human faces. In response to vari-ous types of chitin used as growth substrates, thebacterium produced a complete array of chitino-lytic enzymes: endochitinase, exochitinase,N-acetylglucosaminidase, chitosanase and chitindeacetylase. The high activity of endochitinase(256.4 pkat·mL–1), exochitinase (346.5 pkat·mL–1)

ů ň

ň

Session II: Microbial metabolism of dietary components S59

and N-acetylglucosaminidase (154.4 pkat·mL–1)was induced in the presence of colloidal chitin.The chitinolytic enzymes from the culture fil-trate were fractionated by an ultrafiltration proc-ess using polymer membranes (Millipore, USA)with cut-off 100, 50, 30 and 10 kDa. The frac-tions were characterized by enzymatic activities,SDS-PAGE and concentration of proteins. Thechitinase variability was confirmed on zymo-grams of renaturated SDS-PAGE. The enzymeswere visualized as fluorescent bands by using4-methylumbelliferyl derivatives of N-acetyl-β-D-glucosaminide, β-D-N,N’-diacetylchitobio-side, or β-D-N,N’,N’’-triacetylchitotriose for N-acetyl-β-glucosaminidase, chitobiosidase, orendochitinase, resp. The stability of the chitino-lytic enzymes within the pH (3–8) and temper-ature (10–60 °C) range as well as the tempera-ture optimum of the enzyme activity weredetermined. In the second step the chitinolyticenzymes of Cl. paraputrificum J4 were sepa-rated from a culture filtrate by an ion exchangechromatography on the carboxylic POLY-GRAN-27 sorbent. The adsorbed enzymes wereeluted under a stepwise pH gradient (pH 5; 5.5;6; 6.5; 7; 7.5 and 8) in 0.1 M phosphate buffer.The protein and enzyme recovery reached 90%.

The project was supported by The Grant Agency ofCzech Republic (grant No. 525/05/2584).

P-110 Fermentation activity of intestinalLactobacillus strains and end products inthe presence of fructooligosaccharides. K.li ewska (Institute of Fermentation, Technol-

ogy and Microbiology, Technical University ofLodz, ul. Wolczanska 171/173, 90-924 Lodz,Poland).

Twenty-six intestinal Lactobacillus bacteria,belonging to the species: L. acidophilus, L. casei,L. crispatus, L. gasseri, L. paracasei/casei andL. rhamnosus, were grown in MRS medium con-taining inulin (Sigma, Raftiline®HP and ST) andoligofructose (Raftilose®P95), as well as mon-osaccharides (glucose, fructose, galactose) anddisaccharides (lactose, sucrose), as a source ofcarbon. In media containing inulin the biomasswas low and ranged from 0.2 to 1.1 g·L–1 (aver-age 0.4 g·L–1). In media with oligofructose thebiomass ranged from 0.2 to 1.4 g·L–1 (0.6 g·L–1).In control media, containing mono- or disaccha-rides, the biomass was much higher and reached

from 0.7 to 2.3 g·L–1 (average 1.2 g·L–1). Thefermentation of inulin led to the production ofacetic acid as a predominant metabolite. Theamount of this acid ranged from 2.3 to 6.5 g·L–1

(average 4.0 g·L–1), while that of lactic acidranged from 0.1 to 3.9 g·L–1 (average 1.0 g·L–1).As a result of oligofructose fermentation, lacticacid was a predominant metabolite for L. caseiand L. paracasei/casei, while acetic acid was apredominant metabolite for L. acidophilus,L. rhamnosus, L. gasseri and L. crispatus. Theamount of lactic acid ranged from 1.7 to10.9 g·L–1 (average 5.2 g·L–1), while acetic acidfrom 3.0 to 5.7 g·L–1 (4.1 g·L–1). As a result ofmonosaccharide and disaccharide fermentationlactic acid was a predominant metabolite. Theamount of this acid ranged from 6.0 to 20.3 g·L–1

(9.2 g·L–1), while acetic acid ranged from 2.8 to6.7 g·L–1 (4.2 g·L–1). Moreover, it was foundthat in the group of the strains tested, a variedamount of succinic acid, acetaldehyde, diacetyland ethanol were produced, irrespective of thecarbon source applied.

P-111 The influence of non-enzymatically gly-cosylated potato proteins on the survival andmetabolic activity of chosen bacteria.D. wi teckaa, K. Marciniak-Darmochwa a, H.Kostyraa, A. wi teckib (a Institute of AnimalReproduction and Food Research of the PolishAcademy of Sciences, Olsztyn, Poland, b Fac-ulty of Biology, Department of Microbiology,Warmia and Mazury University, Olsztyn,Poland).

The non-enzymatic glycosylation of proteins isconsidered to be a very important process occur-ring in living organisms and food, and modifiesthe structure and properties of proteins. Thus, itbrings about decreases in the nutritional value offood. Additionally, such modified proteins arelikely to become the source of biologicallyactive peptides as well as allergens. Conse-quently, it greatly affects human and prokaryoticorganisms, causing changes in their functioning.Regrettably, little is known about the influenceof such modified proteins on the intestinalmicroflora and ensuing health consequences forthe host. Hence, the aim of this study was toestablish the dependences between potato pro-teins modified by non-enzymatic glycosylationand bacterial proliferation rate, survival andmetabolic activity. The application of various

Ś ż

Ś ą ł Ś ą

S60 Session II: Microbial metabolism of dietary components

fluorescent techniques was carried out to assessthe mentioned impact exerted on Enterococcusfaecalis, Escherichia coli and Bacillus subtilis.The rate of bacterial proliferation in the presenceof examined proteins was estimated directlywith DAPI, whereas bacterial dehydrogenaseactivity was indicated with the fluorogenic sub-strate-tetrazolium chloride (CTC). Subse-quently, bacterial survival in the presence ofnon-enzymatically glycosylated proteins wasestablished with the fluorescent probe (Live/Dead® BacLight™ Bacterial Viability Kit).Glycosylated potato proteins were preferablyutilized as a substrate and energy source by pro-biotic bacteria to unmodified ones, and wereused likewise by pathogenic bacteria during thelater period of cultivation. The opposing impactswere exerted on the opportunistic bacteria bytwo different fractions of glycosylated potatoproteins. Obtained results confirm the highlysignificant influence of glycosylated proteins onbacterial physiological and metabolic status.

P-112 Metabolism of the food associated carcin-ogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) by human intestinal micro-biota. L. Vanhaecke, T. Van de Wiele, W. Ver-straete (Laboratory of Microbial Ecology andTechnology, Ghent University, Coupure Links653, 9000 Ghent, Belgium).

2-Amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine(PhIP) is a mutagenic, carcinogenic heterocyclicamine formed from meat and fish duringcooking. Although the formation of hazardousPhIP metabolites by mammalian enzymes iswell documented, nothing is known about thePhIP transformation potency of human intestinalbacteria. In this study, the in vitro metabolism ofPhIP by human faecal samples was investigated.Following anaerobic incubation of PhIP withstools freshly collected from 6 healthy volunteers,we found that PhIP was extensively transformedby the human intestinal bacteria. HPLC analysisshowed that the 6 human faecal microbiotatransformed PhIP with efficiencies from 55 to95% after 72 h incubation, resulting in one majorderivative. ESI-MS/MS, 1D (1H, 13C, DEPT)and 2D (gCOSY, gTOCSY, gHMBC, gHSQC)NMR and IC analysis elucidated the completechemical identity of the microbial PhIP metabolite,being 7-hydroxy-5-methyl-3-phenyl-6,7,8,9-tetra-hydropyrido[3’,2’:4,5]imidazo[1,2-a]pyrimi-

din-5-iumchloride. At present, no information isavailable about the biological activity of thisnewly discovered bacterial PhIP metabolite. Ourfindings however suggest that bacteria derivedfrom the human intestine play a key role in theactivation or detoxification of PhIP, a digestivefate ignored so far in risk assessments. Moreo-ver, the variation in transformation efficiencybetween the 6 human microbiota indicates inter-individual differences in the ability to convertPhIP. This may predict individual susceptibilityto carcinogenic risk from this suspected dietarycarcinogen.

P-113 Determination of breakdown pathwayof tryptophan and indole acetic acid to skatoleby Clostridium scatologenes and swinemanure slurry. T.R. Whitehead, N. Price, M.A.Cotta (USDA-ARS-National Center for Agri-cultural Utilization Research, Peoria, IL, 61604USA).

Production of the odorant chemical skatole (3-methyl-indole) is a problem both within swine(boar taint) and during storage of swine manure,where skatole can contribute to odor productionfrom large-scale swine facilities. Previousresearch has suggested that skatole is producedfrom the bacterial breakdown of tryptophan, butdefinitive proof has not been published.Clostridium scatologenes is one of the few iso-lated bacterial strains reported to produce ska-tole. Skatole production was initially deter-mined from medium containing increased levelsof tryptophan or indole acetic acid (IAA) usinggas chromatography-mass spectrometry (GC-MS) following growth of C. scatologenes. Inorder to conclusively determine that skatole wasbeing produced from these substrates, deute-rium-labeled tryptophan or IAA were added tothe medium and deuterated products were iden-tified by GC-MS. Deuterated skatole was foundto be synthesized from both tryptophan and IAA,and deuterated IAA was also formed from tryp-tophan. When the same deuterium-labeled sub-strates were incubated with fresh swine manureslurry, deuterated IAA and skatole were againidentified, as well as a number of oxidation prod-ucts. These results demonstrate conclusivelythat skatole can be derived from tryptophan and/or IAA by C. scatologenes, and bacteria arepresent in stored swine manure that can produceskatole and other products from these substrates.

S63Reprod. Nutr. Dev. 46 (2006) S63–S77© INRA, EDP Sciences, 2006

O-15 Comparison of direct-fed microbial andantibiotic supplementation on innate andadaptive immune characteristics of weanlingpigs. M.E. Davisa, D.C. Brownb, M.S. Dirainb,H.D. Dawsonc, C. Maxwellb, T. Rehbergera

(a Agtech Products, Inc., Waukesha, Wisconsin,USA, b University of Arkansas, Fayetteville,Arkansas, USA, c USDA/ARS Beltsville HumanNutrition Research Center, Beltsville, MD, USA).

The commensal microflora within the gastroin-testinal tract have a profound effect on immunesystem development. Therefore, strategicallyselected direct-fed microbials may guide immunesystem development toward effective, appropri-ate responses to encountered challenges. Sup-plementation with Lactobacillus brevis to pigsbefore weaning decreased the expression of sev-eral genes within the jejunum involved in thetoll-like receptor pathway, specifically TLR4,TLR9, TRAF6, SARM, and TIRP, indicatingthe influence of this direct-fed microbial on thedeveloping innate immune system of the porcinegastrointestinal tract. Lactobacillus brevis incombination with a Bacillus-based direct-fedmicrobial administered after weaning resulted ina decrease in the percentage of phagocyticmonocytes during the post-weaning period sim-ilar to that observed with antibiotic (Carbadox)supplementation. Furthermore, supplementationwith Lactobacillus brevis, the Bacillus-baseddirect-fed microbial, or Carbadox resulted inincreased proportions of specific T cell popula-tions determined flow cytometrically within theperipheral blood during the post-weaning periodthat distinctly differed from proportions inunsupplemented pigs. These differing popula-tions expressed cell surface molecules indicativeof activated and memory populations of T cells,including major histocompatability complex-II,cells double-positive for CD4 and CD8, the γδT cell receptor, and the interleukin-2 receptor(CD25). These data demonstrate the effect ofdirect-fed microbial supplementation on theyoung pig’s developing immune system. Inaddition, the alteration of the gastrointestinalmicrobial population by direct-fed microbialsupplementation seems to elicit anti-inflamma-tory effects and immune characteristics similarto those observed with antibiotic administration.

O-16 Intestinal microbial composition affectsthe ratio of digestive ileal brush border

enzyme activity to gene expression in thegnotobiotic pig. B.P. Willing, A.G. Van Kessel(Department of Animal and Poultry Science,University of Saskatchewan, Saskatoon, Sas-katchewan, Canada).

In early life, colonization of autochthonousmicrobes induces major morphological andphysiological changes in the gastrointestinaltract of the host, including nutrient digestion andabsorption. To study microbial influence onintestinal development, two separate gnotobi-otic experiments were performed, each with16 piglets allocated to 4 treatment groups: germfree (GF), mono-association with Escherichiacoli (EC), mono-association with Lactobacillusfermentum (LF) or conventionalisation with sowfeces (CV). At day 14 isolated epithelial cells(IEC) and whole tissue were collected at 75% ofthe small intestinal length. Enzyme activity andgene expression of lactase-phlorizin hydrolase(LPH) and aminopeptidase N (APN) were meas-ured in homogenized ileal tissue and IEC usingspecific substrates and quantitative PCR (qPCR),respectively. Similar results were obtained forboth whole tissue and IEC. CV pigs had reducedAPN activity, but had increased gene expressionrelative to GF, making the specific activity/mRNA (A:G) ratio dramatically lower (P <0.05). Similarly, LPH A:G ratio was signifi-cantly reduced (P < 0.05) in CV pigs as com-pared to GF, although gene expression was notup-regulated. LF pigs had A:G ratios that weresimilar to GF, while the EC pigs were more sim-ilar to CV. Co-incubation of LF, EC and fecalbacteria with APN indicate a direct relationshipbetween enzyme inactivation and specific activ-ity in ileal tissue. We hypothesize that enterocyteup-regulation of brush border enzyme expres-sion occurs as either a direct response to micro-bial colonization or as a feedback mechanism inresponse to reduced enzyme activity throughmicrobial degradation. This mechanism mayplay a role in ensuring effective competition ofthe host with the intestinal microbiota for avail-able nutrients.

O-17 Interactions between the mucin-degraderAkkermansia muciniphila and host cells in theGI tract. M. Derriena, E.G. Zoetendala, E.Norinb, W.M. De Vosa (a Laboratory of Micro-biology, Wageningen University, Hesselink van

S64 Session III: Microbial interactions with the host

Suchtelenweg 4, 6703 CT Wageningen, TheNetherlands, b Microbiology and Tumour Biol-ogy Center, Karolinska Institute, Nobels väg 16,171 77 Stockholm, Sweden).

The first level of interactions between host andbacteria starts in the mucus layer that protectsepithelial cells from the luminal content. Whilethe mucins (mucus glycoproteins) present in thislayer have a protective role, they are also knownto serve as carbon and nitrogen source for someintestinal microbes. However, the exact interac-tion between mucin-degrading microbes and thehost is largely unknown. In the current study, wefocus on the interaction between the mucin-degrading bacteria and the host using both cul-tivation and high throughput molecular approaches.In a previous study, Akkermansia muciniphila(CIP 107961T, ATTC BAA-835 T), a novelGram-negative anaerobic organism, which iscapable of utilizing mucin as sole carbon andnitrogen source, was isolated from a healthy vol-unteer. Fluorescent In situ Hybridization (FISH)combined with flow cytometry using an Akker-mansia-specific 16S rRNA-targeted probe wasused to quantify Akkermansia-like bacteria in15 faecal samples and revealed it to account forapproximately 1% of the total faecal cells. Inorder to determine the in vivo impact ofA. muciniphila on its host, mono-association ofgerm-free mice was performed with A. mucini-phila, and L. plantarum WCFS1, which servesas control for a non mucin-degrading bacterium.Biopsy samples were collected from differentareas of the intestine 7 days after the inoculation,and subsequently used for transcriptomic anal-ysis using Affymetrix Genechip Mouse genome430A arrays. The data from this study will bepresented and discussed with specific referenceto the expression of mucin genes.

O-18 Engineering of the human commensalBacteroides ovatus for the controlled in situdelivery of immunomodulatory proteins.Z.Z.R. Hamadya, M.D. Farrara, T. Whiteheadb,K.T. Hollanda, J.P. Lodgea, S.R. Cardinga

(a Institute of Molecular and Cellular Biologyand Department of Surgery, University ofLeeds, Leeds, UK, b Fermentation Biotechnol-ogy Research, National Center for AgriculturalUtilization Research, Peoria, Illinois, USA).

Soluble growth factors that promote epithelialrepair and can suppress inflammation are of clin-ical interest as therapeutic agents for chronicintestinal inflammation that is characteristic ofinflammatory bowel disease. However, whenadministered orally as recombinant proteinsthey are unstable and systemic administrationincreases the risk of unwanted side effects.Alternative means of delivery have been consid-ered of which delivery via live micro organismshas shown real promise. The aim of this workwas to genetically engineer the human commen-sal colonic bacterium Bacteroides ovatus to pro-duce and secrete mammalian cytokines underthe control of the xylanase promoter. The xyla-nase promoter was cloned and sequenced usingan inverse-PCR approach. The coding sequenceof the mature human cytokines TGF-β or KGFwas PCR amplified from cDNA and cloneddownstream of the xylanase promoter in the E.coli-Bacteroides suicide vector pBT2 that wasintroduced into B. ovatus by conjugation.Resulting transconjugants were tested for cytok-ine gene expression by reverse transcription-PCR and protein production by enzyme-linkedimmunosorbent assay (ELISA) and bioassay.Recombinant strains of B. ovatus secreted highlevels of human TGF-β or KGF in a xylan-dependent manner. Cytokine expression wasminimal in the absence of xylan. Studies toassess the efficacy of these strains in the treat-ment and prevention of chemically induced coli-tis in mice are ongoing.

O-19 Probiotic bacteria impede Listeriamonocytogenes infection and modulate themucosal immune response. S.C. Corra,b, C.Hilla,b, C.G.M. Gahana,b (a Department ofMicrobiology, University College Cork, Ire-land, b Alimentary Pharmabiotic Centre, Bio-sciences Research Institute, University CollegeCork, Ireland).

Invasion of the gastrointestinal epithelium,translocation across this epithelial barrier andsurvival of the host immune response are criticalto the pathogenesis of Listeria monocytogenes.However, despite a thorough understanding ofthe intracellular pathogenesis of this organism,little is known about how the pathogen interactswith the host microflora at the mucosal surface.This study utilises in vitro culture of the C2bbe1intestinal cell line to examine the effect of

Session III: Microbial interactions with the host S65

pre-treating epithelial monolayers with probi-otic bacteria on Listerial invasion. We found thatprobiotic bacteria significantly reduce L. mono-cytogenes invasion of epithelial cells in vitro.We determined that this inhibition is due to asecreted factor directly affecting the epithelialmonolayer, thus inhibiting invasion. Further-more, Enzyme-Linked Immunosorbent assays(ELISA) demonstrated a significant reduction ofpro-inflammatory cytokine (IL-8) and increasedanti-inflammatory cytokine (IL-10) followingprobiotic treatment of monolayers and subse-quent Listerial challenge. Finally, a novel luci-ferase reporter system was used to analyseorgan-specific infection by L. monocytogenes inthe A/J mouse model following oral dosing ofmice with Lactobacillus salivarius UCC118.We have shown for the first time that a probioticbacterium can significantly reduce L. monocy-togenes infection in vivo. We observed a dra-matic reduction of Listerial infection of liversand spleens following probiotic dosing of micecompared to a placebo control. This studyassesses the ability of various well-characterisedprobiotic bacteria to inhibit the initial step ofL. monocytogenes host cell infection and theinflammatory response. As not all probiotic bac-teria have the same therapeutic effects, under-standing the mechanism of action of some ofthese probiotics will aid selection of strains use-ful for therapeutic application and treatment ofinfections.

O-20 Enterohaemorrhagic Escherichia coliinhibit nitric oxide synthesis by activatedhuman intestinal epithelial cells. M. Vareille,T. De Sablet, A. Durand, C. Martin, A.P. Gobert(Unité de Microbiologie, INRA, Centre deTheix, 63122 Saint-Genès-Champanelle, France).

Enterohaemorrhagic Escherichia coli (EHEC)is a food borne pathogen causing human diseasesranging from uncomplicated diarrhea to haem-orrhagic colitis and life-threatening complica-tions, such as haemolytic-uremic syndrome(HUS) or thrombotic thrombocytopaenic pur-pura. The majority of EHEC carry a pathogenic-ity island called Locus of enterocyte effacement(LEE) which encodes virulence proteins and canalter activated mucosal intestinal cells, the firstline of defense against these pathogens. There-fore, the capacity of EHEC to modulate theinnate immune response in infected colon may

be a critical step in pathogenesis. Inducible nitricoxide synthase (iNOS)-derived nitric oxide(NO) is a free radical synthesized by cytokine-stimulated cells, which possesses numerousinflammatory and immunological functions. Todetermine the effect of EHEC on NO productionby human intestinal epithelial cells we used thehuman epithelial cell lines HcT-8 and Caco-2which were stimulated for 6 h with cytokines, inthe presence or absence of different strains ofEHEC. Total RNA was purified and iNOSmRNA was analysed by RT-PCR and real-timePCR. After washing, cells were cultured for 24 hin a fresh medium containing antibiotics;nitrates, the stable products of NO in culture,were then measured in the supernatants. Theseresults showed that iNOS mRNA expression andNO production were induced in cytokine-acti-vated cells when compared to unstimulatedcells. These effects were inhibited by addingEHEC to the cultures. The effect on iNOS induc-tion was observed when LEE-positive and LEE-negative strains were used and did not requiredirect contact with bacteria. Moreover, this inhi-bition was not observed when a non-pathogenicE. coli was used.

IL-1 Resident intestinal bacteria directexpression of a bactericidal lectin. H.L. Cash,C.V. Whitham, C.L. Behrendt, L.V. Hooper(Center for Immunology, The University ofTexas Southwestern Medical Center at Dallas,Dallas, TX, USA).

The human gut harbors a vast consortium of res-ident bacteria that make important contributionsto human nutrient metabolism and intestinaldevelopment. Maintaining the mutually benefi-cial nature of this host-microbial relationshiprequires strict corralling of gut bacteria in theintestinal lumen, as microbial incursions acrossepithelial layers can elicit damaging inflamma-tory responses. Members of the Reg family ofC-type lectins are expressed in intestinal epithe-lial cells and show increased expression in themucosa of inflammatory bowel disease patients.We have found that intestinal bacteria inducePaneth cell RegIIIγ expression in mice, that theprotein is secreted into the gut lumen, and thatit binds preferentially to the surfaces of grampositive bacteria. Further studies have revealedthat both RegIIIγ and its human counterpart HIP/PAP bind to bacterial peptidoglycan via its

S66 Session III: Microbial interactions with the host

carbohydrate backbone. Consistent with thisbinding, both RegIIIγ and HIP/PAP exhibitdirect bactericidal activity against a variety ofgram positive bacteria. This activity can beinhibited by soluble carbohydrates that mimicthe sugar structures found in peptidoglycan, sug-gesting that peptidoglycan binding is requiredfor microbial killing. Thus, RegIIIγ and HIP/PAP represent a new family of bactericidal pro-teins that seek out their microbial targets viainteractions with bacterial cell wall carbohydrates.These proteins likely play an important role inpreventing epithelial penetration by resident gutbacteria, thus preserving a commensal host-microbial relationship. Furthermore, our resultssuggest that human Reg proteins exhibit increasedexpression in inflammatory bowel disease mucosaas a compensatory mechanism designed to limitbacterial invasion of mucosal surfaces.

P-114 Characterisation of the effects of lacto-bacilli on the integrity of intestinal epithelialcells. R.C. Andersona, A.L. Cooksona, R.D.Hursta,b,c, L.A. Cassidyb, W.C. McNabba, N.C.Roya (a Metabolism and Microbial GenomicsSection, Food and Health Group, AgResearchLimited, Grasslands Research Centre, Palmer-ston North, New Zealand, b Meat Quality andSafety Section, Food and Health Group, AgRe-search Limited, Ruakura Research Centre, Ham-ilton, New Zealand, c Current address: HumanHealth and Performance Group, HortResearchLimited, Hamilton, New Zealand).

The gastrointestinal barrier (GIB) is compro-mised in conditions such as inflammatory boweldiseases. It is hypothesised that commensal bac-teria, such as lactobacilli, play a critical role inmaintaining the integrity of the GIB. Our objec-tive was to determine the effect of two Lactoba-cillus sp. (L. plantarum and L. acidophilus) onthe integrity of the GIB. The lactobacilli wereisolated from a commercial probiotic productand identified using 16S rDNA sequencing. Theeffects of these bacteria and the probiotic prod-uct were tested in an in vitro bioassay that mim-ics the GIB. This assay consisted of a monolayerof intestinal epithelial cells (Caco-2) grown ona semi-permeable membrane. The trans-epithe-lial electrical resistance (TEER) across the mon-olayer was measured using a voltohmmeter todetermine the strength of the tight junctionsbetween the individual epithelial cells. The addi-

tion of the whole probiotic product increased theTEER by 30%. L. plantarum alone, isolatedfrom the product, increased the TEER by 50%compared to the controls (i.e. media with no bac-teria added). This is in agreement with thereported literature. Conversely, the L. acido-philus strain isolated from the same productdecreased the TEER by 30% compared to thecontrols. These results indicate that L. planta-rum has the potential to improve gastrointestinalhealth and wellness and may be a useful organ-ism to evaluate further as a potential additive fornovel health-promoting foods.

P-115 A tool for studying adherence of intes-tinal pathogens on intestinal mucus – oppor-tunities for screening products for pathogeninhibition in various hosts. J. Apajalahti, O.Siikanen, L. Purkamo, M. Lauraeus (AlimetricsLtd, Höyläämötie 14, 00380, Helsinki, Finland).

The pathogenesis of many enteric pathogens isinitiated by adherence of the bacteria on theintestinal epithelium lining. Through adherence,bacteria can resist washout with digesta passageand get into the vicinity of the host cells, the ulti-mate targets of toxins and intracellular patho-gens. Prevention of pathogen adherence on themucus overlying epithelial cells would removea significant mediator of pathogenesis and makethe host less susceptible for the respectiveenteric diseases. In the method presented here,intestinal mucus covering the epithelium wasisolated from an animal species, breed, age, die-tary group and intestinal section relevant for theapplication and the problem of interest. Mucuswas then washed and immobilised onto polysty-rene wells and used for studying adherence ofradiolabeled pathogens representing differentbacterial species, serotypes and strains isolatedfrom animal hosts, and even from specific farmswith recurrent pathogen outbreaks. The methodprovides a means to study the mechanisms ofadherence and to classify bacterial strainsaccording to their binding potential and specifi-city. Furthermore, this in vitro method can beapplied to the development of health productsaimed at prevention of pathogen colonisation inthe animal and human intestine. We have foundspecific representatives of product categoriessuch as direct fed microbes (probiotics) andcomplex carbohydrates that are especially effec-tive in preventing the adherence of pathogenic

Session III: Microbial interactions with the host S67

Escherichia coli and Salmonella enterica strains.Some products have to be present prior to path-ogen introduction to prevent adherence, whereassome actually detach pathogens already adhered.The former case characterises compounds suit-able for preventive treatment, whereas the lattercould work also in the therapy of pathogenesis.

P-116 Effect of inoculation with intestinalbacteria on bodyweight and intestinal inflam-mation in the interleukin-10 knockout mouse.M.P.G. Barnetta, S.T. Zhub, A. Cooksona, R.Broadhurstc, B. Knocha, W.C. McNabba, N.C.Roya (a Metabolism and Microbial GenomicsSection, Food and Health Group, AgResearchLimited, Palmerston North, New Zealand,b Faculty of Medical and Health Sciences, TheUniversity of Auckland, Auckland, New Zea-land, c National Resources Group, AgResearchLimited, Hamilton, New Zealand).

The interleukin-10 (IL-10) knockout (KO)mouse develops Crohn’s disease-like colitiswhen raised under conventional conditions. Thiscolitis is caused in part by an inappropriateresponse to normal intestinal bacteria. Our aimwas to produce a more reliable model of inflam-mation by orally inoculating IL-10 KO micewith 12 Enterococcus strains and complex intes-tinal flora collected from C57BL/6 mice raisedunder conventional conditions (EF+CIF). Bod-yweight was recorded throughout the trial. At12 weeks of age, intact intestinal sections wereassigned a histological score (HIS) based oninflammatory cell infiltrates and tissue destruc-tion. Bodyweight of IL-10 KO mice was lower(P < 0.05) than controls, while the HIS (partic-ularly colon) of IL-10 KO mice inoculated withEF+ CIF was higher (P < 0.05) than that of con-trol mice. Inoculation also gave more consistentHIS compared to IL-10 mice which were notinoculated. These results show that IL-10 KO miceinoculated with intestinal bacteria are an improvedmodel of intestinal inflammation. This modelwill now be used to test how specific dietarycomponents affect gene expression relevant tothe development of Crohn’s disease-like colitis.

Nutrigenomics New Zealand is a collaborationbetween AgResearch, Crop & Food Research, Hor-tResearch and The University of Auckland and islargely funded by the Foundation of Research, Scienceand Technology (FRST). MPG Barnett is funded by aFRST Postdoctoral Fellowship.

P-117 Effect of selected strains of lactic acidbacteria on non-specific cellular and humoraldefence mechanisms of healthy and Salmo-nella-infected chickens. M. Bieleckaa, A.K.Siwickib, E. Biedrzyckaa, R. Wójcikb, W.Smoragiewiczc, A. Or owskia, A. Majkowskaa

(a Institute of Animal Reproduction and FoodResearch of the Polish Academy of Sciences,Tuwima 10, 10-747 Olsztyn, Poland, b Univer-sity of Warmia and Mazury, Olsztyn, Poland,c University of Québec, Montreal, Canada).

The ability to enhance the immune system is oneof the most important probiotic features. Theaim of the study was to determine the influenceof potentially probiotic strains of Lactobacillusand Bifidobacterium on selected non-specificcellular and humoral defence mechanisms andprotection against salmonellosis in chickens.The experiment was performed on 240 chickens(Ross 308) randomly allocated into 6 groups. Allchickens were fed the standard diet (includingthe control group), those in one group receivedfeed supplemented with the antibiotic avilamy-cin, and those in four groups were administeredwith the selected, well-defined single strains ofLactobacillus salivarius AWH, L. acidophilusBS, Bifidobacterium longum KNA1 and B. ani-malis 30 as active cultures in the amount of 109–1010 cells per chicken per day. On day 14 beforethe end of the experiment, half of the chickensin each group were separated and infected withSalmonella (~ 106 cells per chicken every thirdday). Among the strains tested, L. acidophilusBS and B. animalis 30 were found to be the mosteffective. The strains increased phagocytic abil-ities of blood leukocytes, stimulated lysozymeactivity (LSM) and increased the level ofgamma-globulin in healthy chickens. In thechickens infected experimentally with Salmo-nella, the selected strains (BS, 30) significantly(P < 0.05) activated phagocytic ability of leuko-cytes and proliferative response of lymphocytes,and increased activity of LSM, the level ofgamma-globulin and Ig Y in blood serum incomparison to non-infected chickens. In thegroups of chickens receiving avilamycin, immu-nosupression was observed. The results indi-cated the possibility to use the carefully selectedprobiotic strains as one of the alternatives forantibiotics for chicken rearing.

ł

S68 Session III: Microbial interactions with the host

P-118 Probiotic bacteria in early defenceagainst viral infection. T. Boti a, M. Iveca,S. Korenb, M. Jakobsenc, H. Weingartld,A. Cenci a,e (a University of Maribor, Faculty ofAgriculture, Vrbanska c.30, 2000 Maribor,Slovenia, b University of Ljubljana, MedicalFaculty, 1000 Ljubljana, Slovenia, c The RoyalVeterinary and Agricultural University, Dept. ofDairy and Food Science, Rolighedsvej 30, 1958Frederiksberg C, Denmark, d Canadian FoodInspection Agency, National Centre for ForeignAnimal Disease, 1015, Arlington Street, Winni-peg, Manitoba, R3E 3M4, Canada, e Universityof Maribor, Medical Faculty, Slomskov trg 15,2000 Maribor, Slovenia).

Probiotics are undoubtedly important in sup-porting a functional yet balanced immune sys-tem. A further application of immunomodula-tory bacteria in health care is in the control ofmicrobial pathogens, including various viruses.We investigated if probiotic bacteria can preventor reduce viral infection. The in vitro systemused was composed of the pig small intestinalcell line IPEC-J2 (A. Blikslager, USA), pig mac-rophage cell line 3D4/21, and Vesicular Stoma-titis Virus –VSV, as a model virus. L. paracaseiF19, L. paracasei/rhamnosus Q85 and B. longumQ46 were used in the experiment as the probioticstrains. Cell survival and viral inhibition weredetermined by antiviral assay, NO productionand the activity of mitochondrial enzymes ininfected versus non-infected cells. Pre-incuba-tion of cell monolayers with probiotic bacteriareduced viral infectivity up to 60%, in the intes-tinal epithelia and in the macrophages. The high-est accumulation of NO in the culture superna-tants was observed in macrophages aftertreatment with L. paracasei F19. The MTT-testshowed that L. paracasei F19 has a positiveeffect on cell viability in the virus infected andnon-infected cells. The results of our study, in acell culture model, showed that probiotic bacte-ria exhibit antiviral activity by direct interac-tions with the virus, triggering the intestinal epi-thelia and activation of macrophages.

P-119 Adaptation of adhesion test on modelepithelial cells for anaerobic bacteria. T.

epeljnik, M. Narat, B. Lah, B. Dolenec, R.Marinšek-Logar (Zootechnical Department,

Biotech. Fac., University of Ljubljana, Groblje3, 1230 Domžale, Slovenia).

Caco-2 cells are often used as model epithelialcells in studies of bacterial adhesion. So far onlyaerobic or facultatively anaerobic bacteria wereused in this kind of assays. In our study theadhesion test was adapted in such a manner thatit was suitable for anaerobic bacteria such as e.g.Pseudobutyrivibrio xylanivorans Mz5T, origi-nating from the rumen of a Holstein-Friesiancow. This bacterium has some attributes thatmake it suitable as probiotic strain (very activehydrolases, bacteriocin and CLA production).One of the criteria for a probiotic candidate isalso the ability of the bacterial strain to adhereto epithelial cells. In our case the adhesion testwas performed in an anaerobic chamber instandard 96-well plates. A thousand bacterialcells were added per Caco-2 cell at neutral pHfor 30 min. During the test optimization it wasdetermined that the best method for separatingadhered bacteria from Caco-2 cells was homog-enization with an automatic pipette. The nextstep was the development of a reliable quantifi-cation of the adhered bacteria, that are usuallytoo few in number to be determined by micros-copy. Therefore our work focused on viablecounting methods. After the adhesion test hadbeen carried out the adhered bacterial cells wereenumerated by a pour plate method and by anewly developed method, i.e. measurement oftheir lag phase duration in growth medium M330in parallel. We have confirmed the adhesioncapability of P. xylanivorans Mz5T: 1.04 bacte-rial cells from the late logarithmic growth phaseadhered per Caco-2 cell under the selected assayconditions. We have shown that the adaptedadhesion test could be suitable for anaerobicbacteria, too. Further optimisation of the methodis planned.

P-120 Microbial extinctions and modern dis-eases. M.G. Domínguez-Bello (Department ofBiology, University of Puerto Rico, Rio Piedras,San Juan, Puerto Rico, USA).

Animals evolved in a microbial world. Thedynamics of co-evolution of animals with theirindigenous microbes – eukaryotic, prokaryoticand viral – has shaped animal’s immunity.Selectivity of the immune function – combating

č

č

Č

Session III: Microbial interactions with the host S69

specifically pathogens – is important for thehost-microbial balance. This dynamic equilib-rium that evolved through millions of years, issuddenly altered in modern humans and theirdomesticated animals by cultural and technolog-ical antimicrobial practices (hygiene, antibiot-ics), leading to modern diseases involvingautoimmune disorders. This paper discusseshost microbial function in health, from an exam-ple of true digestive mutualism in the rumen, toindigenous microbes in indigenous peoples andmicrobial related diseases of modernity.

P-121 Probiotic bacteria alter intestinalabsorption and metabolism of aflatoxin B1 invitro. S. Gratza,b, H. Mykkänena, H. El-Nezamib(a Department of Public Health and ClinicalNutrition, University of Kuopio, Finland, b Foodand Health Research Centre, University of Kuo-pio, Finland).

Probiotic bacteria are known to have numerousbeneficial effects, especially on gut health.Recently, probiotics have been shown to bindseveral food carcinogens, including aflatoxins,to their surface. Until now, extensive in vitrowork has identified potential candidate strainssuch as Lactobacillus rhamnosus strain GG(LGG), and there is some evidence that aflatoxinbinding by LGG also occurs in vivo in animalsand humans. Nevertheless, little is known aboutthe interactions between bacteria, aflatoxin andthe intestinal tract. Aflatoxin B1 (AFB1) is a lowmolecular weight lipophilic compound, effi-ciently absorbed in the intestinal tract by passivediffusion and metabolized into free metabolites,such as aflatoxin M1 (AFM1), aflatoxin Q1(AFQ1) and aflatoxicol (AFL), or to a highlyreactive epoxide which then forms adducts withDNA and proteins. The metabolism and toxicityof AFB1 have been studied in human cellularsystems derived from liver or respiratory tract,but the impact of the intestinal metabolism is stillto be investigated. We therefore wanted to studythe uptake and metabolism of AFB1 by Caco-2intestinal cells and the impact of LGG on theseprocesses. AFB1 was mixed into the cell culturemedium and AFB1 and its metabolites weremeasured by HPLC. Furthermore, LGG wassuspended in culture medium and added to cellsbefore adding AFB1 and metabolites were meas-ured as above. Preliminary results show thatCaco-2 cells were able to metabolize AFB1 into

AFM1 and AFL and production increased withincubation time. In the presence of LGG, lessAFM1 and AFL were detected in culturemedium. These results indicate that LGG mightreduce the absorption and metabolism of AFB1in intestinal epithelium cells.

P-122 Inhibition of human atopic dermatitislike skin lesion by the administration ofLactobacillus johnsonii NCC533 (La1) duringweaning period in a model mouse, NC/Nga –the predicted inhibitory mechanism of lesionby La1 from gene expression analyses. R.Inouea, M. Otsukaa, A. Nishioa, Y. Nakamuraa,Y. Fukushimab, K. Ushidaa (a Kyoto PrefecturalUniversity, Laboratory of Animal Science,b Nestlé, Japan).

We previously found that the oral administrationof probiotic Lactobacillus johnsonii NCC533(La1) during a specific time of weaning periodsignificantly inhibited the development ofhuman atopic dermatitis (AD) like skin lesion ina model mouse, NC/Nga. In this study, we pro-pose the predicted inhibitory mechanism of skinlesion by La1 from gene expression analyses.Skin, Peyer’s patch (PP) and mesenteric lymphnodes (MLN) were collected from the mice ineither control or La1 administrated group afterinduction of AD and total RNA was extractedfrom these organs. The expression of variousgenes, proinflammatory cytokines for instance,was examined by real time RT-PCR. The geneexpression of proinflammatory cytokines suchas IL-8 was significantly down-regulated in allexamined organs of the La1 administrated groupcompared to those of the control group. Interest-ingly, significant down-regulation of co-stimu-latory molecules, CD80 and CD86, wasobserved in skin and PP of the La1 administratedgroup. CD80 and CD86 were known to beexpressed in activated APCs and promote T cellactivation through its receptor, CD28. Thus thisresult suggests that both mucosal and systemicAPCs in AD condition may be unnecessarilyactivated by antigens and that they induceimmune responses such as inflammation andinfiltration of mast cells. The administration ofLa1 in weaning period seems to work on APCsand eventually inhibit redundant activation ofthem both mucosally and systemically. As a con-sequence of this, development of AD-like skinlesion may be inhibited by La1.

S70 Session III: Microbial interactions with the host

P-123 Rose hip and lactobacilli used in anischemia-reperfusion model in mice to sup-press the intestinal injury. M. Jakesevica,Å. Håkanssona, D. Adavib, S. Ahrnéa, B.Jeppssonb, G. Molina (a Food Hygiene, Depart-ment of Food Technology, Engineering andNutrition, Lund University, PO Box 124, 221 00Lund, Sweden, b Department of surgery, MalmöUniversity Hospital, Lund University, Malmö,Sweden).

Ischemia-reperfusion (I/R) in the intestines is aninflammatory condition which results in the lossof mucosal barrier function, increased translo-cation of pathogenic bacteria and elevation ofsystemic cytokines. During I/R there is activa-tion of leukocytes and reactive oxygen species(ROS) which leads to lipid peroxidation andDNA damage. Polyphenols, such as flavonoidsand tannins, act as antioxidants and may preventlipid peroxidation. Rose hip has a high contentof antioxidants and is thought to be effective inprevention of lipid peroxidation. Bioavailabilityand absorption of polyphenols in the bodydepend on their metabolism in the small intes-tine. The non-absorbed fractions of the polyphe-nols are degraded by colonic microflora to moresimple compounds, such as phenolic acids. Tan-nins form strong complexes with protein and are,in that way, nutritionally undesirable, but theymight have positive effects by being anticarci-nogenic and antimutagenic. Tannins are quiteresistant to microbial attack, but some bacteria,such as L. plantarum, L. pentosus and L. para-plantarum, are capable of degrading tannins tosimpler and non-toxic constituents like phenolicacids, which can further be spliced into derivatesof phenylpropionic or phenylacetic acids. In thisstudy, nine strains belonging to L. plantarum, L.pentosus and L. paraplantarum together withtwo different species of rose hip were used in anI/R model in mice. The aim of the study was toinvestigate, if administration of Lactobacillusstrains and/or rose hips can suppress or preventI/R-induced injury of the intestinal tract.

P-124 Serological and genetic markers inCrohn’s disease patients and their healthyrelatives. M. Joossens, S. Joossens, N. VanSchuerbeek, K. Claes, P. Rutgeerts, S. Vermeire(Division of Gastroenterology, University Hos-pital Gasthuisberg, Leuven, Belgium).

Crohn’s disease (CD) is a chronic inflammatorydisease of the gut of unknown aetiology. Bothgenetic (e.g. CARD15) and microbial (e.g.ASCA for Anti-Saccharomyces cerevisiae Anti-body) markers have been associated with CD.An exceptionally high frequency of familial CDhas been reported in Northern France and Bel-gium. We hypothesize that a high prevalence ofgenetic and/or environmental risk factors mightexplain high disease prevalence in these fami-lies. Therefore 21 families (74 patients;87 healthy relatives, HR) with at least three CDpatients per family and 10 matched healthy con-trol families (HC, 58 persons) were studied.ASCA IgG/A/M (Sendid B et al., 1996) and gen-otypes for CARD8 (C10X), CARD15 (R702W,G908R, 1007fs), TLR4 (A299G) and DLG5(R30Q) were determined, as well as 4 novelserological markers: anti-outer membrane porin(OMP IgG) (INOVA, USA), anti-mannan(gASCA), anti-chitobioside and anti-laminar-ibioside carbohydrate (ACCA IgA, ALCA IgG)(all Glycominds, Israel). In the CD families, thegenetic variants were equally found in patientsand their relatives. The prevalence was higherthan previously observed in sporadic CD andHC. The prevalence of ASCA IgG/A/M (71%;38%; 4%), gASCA (60%; 12%; 6%) and ALCAIgG (44%; 13%; 13%), but not of ACCA IgA(40%; 32%; 24%) and OMP IgG (36%; 27%;14%) was significantly higher in CD patientscompared with their first-degree HR and withHC, respectively. Whereas HR of CD patientsshowed a higher prevalence of ASCA IgG/A/Mcompared with HC (P = 0.001), no significantdifference was found between HR and HC forgASCA and ALCA IgG. In multiple affected CDfamilies, an exceptionally high prevalence ofgenetic and microbial markers was found. Cor-relations with faecal microbiota are currentlyinvestigated using DGGE.

P-125 Study of pathogenesis of Helicobacterpylori in Iran. P. Khaki, S.M. Bidhendi, H.Eliasi, M. Alimoradi (Razi Vaccine andResearch Institute, Karaj, Iran).

H. pylori is a causative agent of chronic activegastritis, gastric and duodenal ulcers, and gastricadenocarcinoma. The organism is one of themost common human bacterial pathogens, esti-mated to be infecting at least half of the world’s

Session III: Microbial interactions with the host S71

population. The goal of our study was to evaluatethe pathogenesis of H. pylori in vitro and in vivo.250 H. pylori strains were isolated from330 patients who attended at the Gastroenterol-ogy Department of Taleghani hospital, Tehran,during May 2003 to August 2005. Three celllines (BK, Hela and BHK), were used for eval-uation of the cytopathic effects of urease andcytotoxicity of H. pylori isolates. For study ofthe pathogenesis of H. pylori in vivo: (1) His-topathology slides were prepared from 50 patientswith dyspeptic complaints. (2) The pathogenesisof the bacteria was studied in rhesus monkeys.Orally we administered 106 cfu·mL–1 of theH. pylori to 2 monkeys, and one served as neg-ative control. The H. pylori isolates were iso-lated from 144 (97.3%) out of 148 gastritispatients, 43 (86%) out of 50 duodenal ulcers, 10(50%) out of 20 gastric ulcers, 6 (60%) out of10 gastric adenocarcinomas, and 48 (47%) outof 102 asymptomatic attenders. We found thatH. pylori can induce vacuolation, deformationand finally lysis of epithelial cells in vitro andperhaps in vivo, either by direct activity of cyto-toxin or by its strong urease activity. The resultsshowed that the cell lines of BK and Hela (epi-thelial cells) were susceptible to cytotoxin andurease of H. pylori isolates. 63.64% of the iso-lates showed cytotoxin activity of which 98%were from duodenal ulcers, 66.7% from gastritispatients and 33.3% from asymptomatic attend-ers. The bacterium was isolated from experi-mentally infected rhesus monkeys 2 monthsafter oral injection. Also the histopathologicslides showed vaculation, presence of polymor-phonuclears and inflammation in their stomach,while the control was negative. H. pylori infec-tion is extraordinarily common in Iran. Theresults showed that H. pylori is implicated as akey risk factor in gastritis, duodenal ulcer andperhaps gastric adenocarcinoma. This studysuggests that the cytotoxicity and urease activityof H. pylori are two important virulent factors ofthe organism. The in vivo observations demon-strate that H. pylori infection in rhesus monkeymay serve as a model for human infection.

P-126 Comparison of the influence of propolisand virginiamycin on broiler performanceand immune response to ND vaccine. S.M.M.Kiaeia, M. Modirsaneia, H. Bozorgmeherifardb,B. Mansooria, B. Gholamianb, A. Ghalyanchib,M. Rabanib (a Departement of Animal and Poul-

try Health and Nutrition, Faculty of VeterinaryMedicine, University of Tehran, Tehran, Iran,b Faculty of Veterinary Medicine, University ofTehran, Tehran, Iran).

This study was carried out to compare the effectsof propolis and virginiamycine on the perform-ance parameters and on the immune response ofbroiler chicks to the ND vaccine. Three hundredand sixty male day-old Ross308 broiler chickswere randomly assigned to four treatments(three replicates of 30 chicks). Chicks were fedone of the four dietary treatments: (1) a corn–soymeal as control diet, (2) Control diet with3000 ppm propolis for starter and 2000 ppmpropolis for grower and finisher diets, (3) Con-trol diet with 6000 ppm propolis for starter and4000 ppm propolis for grower and finisher diets,(4) Control diet with 100 ppm virginiamycineduring the whole experimental period. Bodyweight gain (BWG), feed intake (FI), feed con-servation ratio (FCR), survivability and effi-ciency productive index (EPI) were recorded at21 and 42 days of age. Prior to administration ofND vaccine (day 17) and 10 days after the vac-cination, blood samples were taken from10 chicks of each treatments for HI test. Datawere analysed using ANOVA. Scheffe test wasalso used to compare means showing signifi-cance (P < 0.05). Chicks on treatment 4 showedhigher BWG, FI and EPI compared with theother treatments, although treatment 2 showedlower FCR. Virginiamycine improved theimmune response of the chicks against ND vac-cine while no effect was observed by Propolis.It seems that propolis could not have any immu-nostimulant effects but appeared to be promisingas a potential growth promoter in chicks.

P-127 Time-course of the progression of intes-tinal inflammation in interleukin-10 knock-out mice inoculated with intestinal bacteria.B. Knocha,d, M.P.G. Barnetta, S.T. Zhub, R.Broadhurstc, A. Cooksona, W.C. McNabba, S.O.Knowlesa, C.E. Ugarted, N.C. Roya (a Metabo-lism and Microbial Genomics Section, Food andHealth Group, AgResearch Grasslands, Palmer-ston North, New Zealand, b Faculty of Medicaland Health Sciences, The University of Auck-land, Auckland, New Zealand, c NationalResources Group, AgResearch Ruakura, Ham-ilton, New Zealand, d Institute of Food, Nutrition

S72 Session III: Microbial interactions with the host

and Human Health, Massey University, Palmer-ston North, New Zealand).

Between 5 and 12 weeks of age the interleukin-10 (IL-10) knockout (KO) mouse develops aCrohn’s Disease-like colitis when raised underconventional conditions of housing and feed.Inoculation at 5 weeks with intestinal bacteriamay hasten the onset and progression of thisintestinal inflammation. Our aim was to deter-mine the optimal tissue sampling time for theIL-10 KO and C57BL/6J (control) mice wheninoculated orally with 12 Enterococcus strainsplus complex intestinal flora collected from othercontrol mice. At 7, 8.5, 10, 12 and 14 weeks,intact intestinal sections were assigned a histo-logical score (HIS) based on inflammatory cellinfiltrates and tissue destruction. The HIS of theintestinal tissues in the IL-10 KO mice increased(P < 0.05) until 12 weeks with the colon beingthe most affected tissue. While bodyweight ofthe control mice continued to increase up to14 weeks, IL-10 KO mice failed to gain weightbeyond 11 weeks. These results show that by11 weeks IL-10 KO mice inoculated with intes-tinal bacteria reliably develop sufficient inflam-mation to be a suitable model of InflammatoryBowel Disease. Nutrigenomics New Zealand isa collaboration between AgResearch Limited,Crop and Food Research, HortResearch and TheUniversity of Auckland and is largely funded bythe Foundation of Research, Science and Tech-nology (FRST). MPG Barnett is funded by aFRST Postdoctoral Fellowship.

P-128 Isolation and partial purification ofchicken intestinal mucus for use in the studyof attachment of Lactobacilli to the poultryGI tract. K. McGreeghan-Crosbya, J.K.Thompsonb, C. Nicolsonb, M.A. Collinsa,b

(a Department of Food Science, Queen’s Uni-versity of Belfast, UK, b Agriculture, Food andEnvironmental Science Division, Department ofAgriculture and Rural Development for N. Ire-land, Newforge Lane, Belfast, BT9 5PX, North-ern Ireland, UK).

Intestinal mucus is considered to be the primarysite of attachment for probiotic microorganisms.In order to construct a simple yet realistic model,chicken intestinal mucus was extracted and par-tially purified. The product proved to be a glyc-

oprotein with a carbohydrate content of > 20%.A poultry GI tract strain of Lactobacillus ther-motolerans was observed to flocculate in thepresence of both chicken mucus and commer-cially produced porcine mucin which may beassociated with cell adhesion to the glycoproteinmatrix. This model system will be used to studyadhesion associated changes in gene expressionin Lb. thermotolerans.

P-129 Cloning of the murC gene in order tostudy its putative role in attachment of Lacto-bacilli to poultry GI tract epithelium. K.McGreeghan-Crosbya, J.K. Thompsonb, M.A.Collinsa,b (a Department of Food Science,Queen’s University of Belfast, Agriculture,Food and Environmental Science Division, Bel-fast, Northern Ireland, UK, b Department ofAgriculture and Rural Development for N. Ire-land, Newforge Lane, Belfast, BT9 5PX, North-ern Ireland, UK).

Differential Display experiments have sug-gested that the murC gene in Lactobacilliappears to be upregulated in the presence ofmucin. As this glycoprotein gel is normallyfound as a protective layer coating the GI tractepithelium, this could indicate that murC playsa role in the attachment of Lactobacilli to theintestinal epithelium. In order to confirm thishypothesis, the murC gene from a poultry GItract strain of Lactobacillus thermotolerans wascloned and its expression studied. Degenerateprimers based on known murC DNA sequenceswere used to generate a 700 bp murC amplicon.DNA sequencing revealed an identity with amurC fragment previously identified by differ-ential display as being upregulated by mucin.Northern analysis, using a model system basedon MRS broth containing increasing concentra-tions of porcine mucin and chicken intestinalmucus, was used to confirm the Differential Dis-play data. A transcript of ~ 3 000 nucleotides inlength was observed suggesting the murC genemay be part of a polycistron. An attempt wasmade to identify the flanking regions of themurC gene using inverse PCR. Sequence datawere consistent with murC forming part of apolycistron. Experiments using Differential Dis-play to study changes in gene expression in thepresence of intestinal mucin have generated sev-eral other amplicons of interest, including pro-teins with mucus binding motifs.

Session III: Microbial interactions with the host S73

P-130 The aerobic/anaerobic cell culturemodel for studies on bacterial adhesion. A.Olejnik, M. Lewandowska, W. Grajek(Department of Biotechnology and Food Micro-biology, August Cieszkowski Agricultural Uni-versity of Pozna, ul. Wojska Polskiego 48, 60-627 Pozna , Poland).

Adhesion to the intestinal tract has become gen-erally accepted as an important colonization fac-tor used for the selection of probiotic cultures.Studying bacterial adhesion in vivo is difficultand in vitro models with intestinal cell lines arewidely accepted methods for this assessment.The most common model used is the enterocyte-like cell line Caco-2, obtained from an adeno-carcinoma of the human colon and showing sim-ilarity to the epithelial cells of the gastrointesti-nal tract. This model is recommended to studyprobiotic adhesion, invasion of pathogenic bac-teria, trans-epithelial transport of nutrients,immune stimulation, and others. In the conven-tional Caco-2 culture system, the Caco-2 cellsare attached to the surface of T-flasks or to aporous membrane as a cell monolayer. The cellsare usually fed with the Dulbecco’s modifiedEagle medium from the apical side of the mon-olayer. In the new culture system, we used a two-chamber reactor, divided in the middle with aporous polycarbonate membrane. The uppersurface of the membrane was colonized withCaco-2 cells and fed with non-aerated, low-nutrient medium to simulate intestinal, anaero-bic conditions. The bottom chamber was fedwith the Dulbecco’s modified Eagle medium,rich in nutrients and well aerated to create aero-bic condition. We studied some physiologicalaspects in the new model of Caco-2 culture,especially the influence of initial inocula andserum addition on the Caco-2 cell differentiationand growth kinetics. Well-developed monolay-ers of intestinal cells were obtained after 21 dayswhen the initial concentration of Caco-2 cellswas 1 × 104·cm–2 and at foetal bovine serum sup-plementation at 20%. In the adhesion study,three probiotic strains were used: Lactobacilluscasei (ATCC 39539), Lactobacillus rhamnosusGG (ATCC 53103) and Lactobacillus acido-philus LC1 (ATCC). The yield of bacterial adhe-sion was strain dependent, and Lactobacillusrhamnosus GG exhibited the best adhesionability.

P-131 Intestinal epithelial cell responses toLactobacillus plantarum. S. Pavana,b, J.J.M. Vande Sandtb, K. Venemaa,b, M. Kleerebezema,c

(a Wageningen Centre for Food Sciences,Wageningen, The Netherlands, b TNO Qualityof Life, Zeist, The Netherlands, c NIZO foodresearch, Ede, The Netherlands).

Intestinal epithelial cells (IEC) constitute theinterface between the host and the gut luminalenvironment and are the first cells sensing thepresence of intestinal bacteria, which results ina variety of immunological and physiologicalresponses. The global transcriptional responseof differentiated IEC monolayers (Caco-2, HT29-MTX) to Lactobacillus plantarum WCFS1, amodel of a commensal as well as probioticmicroorganism, was studied using combined invitro and in silico approaches. IEC were culti-vated alone or in the presence of human bloodmonocyte-derived dendritic cells. The mono- orco-cultures were stimulated with L. plantarum,using either standard laboratory cultures or bac-terial suspensions subjected to passage througha dynamic in vitro model of stomach and smallintestine. Statistical analyses of the humancDNA microarray data revealed a clear anddose-dependent transcriptional response of IECto L. plantarum, involving modulation of cellcycle and cell signaling functions. Althougheach of the two IEC lines used in these experi-ments displayed distinct native gene expressionprofiles, both specific and common responses tobacterial stimulation were observed. In co-cul-ture experiments, the presence of dendritic cellstriggered strong transcriptional responses inCaco-2 cells, while the IEC response toL. plantarum stimulation remained limitedunder these conditions. These results show thatIEC are able to sense and react to the presenceof harmless gut bacteria and that responses gen-erated after exposure to physiological bacterialdoses include modulations of IEC signaling.

P-132 Daily oral supplementation withL. paracasei CNCM I-2116 leads to a down-modulation of milk protein hypersensitivityin mice. S. Pecquet, S. Chibani-Chennoufi, F.Rochat, A. Mercenier (Nutrition and HealthDepartment, Nestlé Research Center, Vers chezles Blanc, Nestec Ltd, 1000 Lausanne, Switzer-land).

ń

S74 Session III: Microbial interactions with the host

Cow’s milk allergy (CMA) is one of the leadingcauses of food allergy in children. Sensitisationto milk proteins occurs early in life and leads toatopic symptoms. Some clinical studies showedthat probiotic treatment of “at risk” infants mightprevent them from allergic symptoms, in partic-ular from atopic dermatitis. In this study, weused a milk hypersensitivity mouse model toevaluate the beneficial effect of the probioticL. paracasei CNCM I-2116 on the onset ofatopic symptoms. C3H/HeJ mice were sensi-tised 4 times at weekly intervals with whey pro-teins in combination with cholera toxin. Allalong the trial, mice received either L. paracaseiCNCM I-2116 (5 × 108cfu·mL–1) or maltodex-trine (matrix control) in drinking water. Oneweek after the last sensitisation, mice wereorally challenged with β-lactoglobulin andsymptoms (scratching, puffiness and activityloss) were followed for 45 min. At sacrifice,blood and faeces were collected for seric IgE,IgG1, MMCP1, histamine and faecal IgA. Fae-cal probiotic counts were recurrently controlledduring the trial. Mice treated with maltodextrineshowed a high symptom score (4.1 ± 1.8) com-pared to L. paracasei CNCM I-2116 treatedmice (1.3 ± 0.9). This significant differencecould be correlated to a similar trend observedwith MMCP1, total IgE and specific IgG1 levels.This preliminary data firstly indicates that thisexperimental model is suitable to evaluate thepreventive effect of probiotics in allergy. Sec-ondly, this study provides the evidence that pro-biotics can modulate food hypersensitisation inmice. Indeed, we demonstrate that daily admin-istration of L. paracasei CNCM I-2116 protectsmice from cow’s milk protein hypersensitivity.

P-133 An immunoblot for the determinationof antibodies against Lactobacilli. A-L. Prangli,M. Utt, R. Uibo (Department of Immunology,University of Tartu, Estonia).

The development of specific adaptive immuneresponses to indigenous Lactobacillus spp. hasbeen insufficiently characterised among humans.Partly, this is connected to the fact that no goodtools are available for antibody measurementagainst these microbes. In the present study wehave developed an immunoblot assay for thedetermination of serum antibodies against dif-ferent lactobacilli. Different lactobacilli were

obtained from the culture collection of lactoba-cilli from the Department of Microbiology, Uni-versity of Tartu. Our results showed IgG typeantibodies in a group of 55 healthy children (agerange 1–17 years) most commonly againstL. plantarum 20 kDa, 28 kDa, 29 kDa, 31 kDa,47 kDa, and 68 kDa proteins, and against L. fer-mentum and L. acidophilus proteins of 38 kDaand 31 kDa, respectively. When results in dif-ferent patient groups (64 children either withceliac diseases, type I diabetes or Helicobacterpylori infection with abdominal complaints; agerange 0.7–18) were compared to the healthychildren group, we revealed a characteristic pat-tern of reactivity in a celiac disease group withL. fermentum and L. acidophilus antigens. Chil-dren with type I diabetes and with H. pyloriinfection did not differ from the control group inIgG type antibody frequency. A higher fre-quency of IgA type antibodies against L. fermen-tum proteins was observed in the celiac diseaseand H. pylori infection groups. Our studyshowed that immunoblot is a reliable method forthe determination of serum antibodies of IgGand IgA types against Lactobacillus spp. in chil-dren. Whether the revealed reactivity is charac-teristic for the tested lactobacilli strains needs tobe controlled in further studies. However, in thepresent configuration our assay is usable for thedetermination and comparison of antibody pres-ence in different clinical associations.

P-134 Mannan induced changes in cytokineexpression and growth of enteropathogenicE. coli-challenged broilers. P. Singboottraa,F.W. Edensa, A. Kocherb (a North Carolina StateUniversity, Department of Poultry Science,Raleigh, NC 27695-7635 USA, b Alltech Bio-technology Centre, Sarney Summerhill Rd,Dunboyne, Co. Meath, Ireland).

Mannan oligosaccharides (MOS) isolated fromthe outer cell wall of a specific strain of Saccha-romyces cerevisiae (Bio-Mos®, Alltech Inc.) isan alternative to the prophylactic use of antibi-otics in poultry feeds and has been used to pro-mote enteric health. The mechanism of action ofMOS is agglutination of enterobacteria withtype-1 fimbriae preventing them from coloniz-ing the GIT. We have shown in vitro that mac-rophages given a mannan-rich fraction (MRF)found in the commercial product Bio-Mos have

Session III: Microbial interactions with the host S75

altered abilities too express inflammatorycytokines such as IL-6 and nitric oxide. Thealtered cytokine expression was mediatedthrough a transitory decrease in the expressionof the toll-like receptor 4 (TLR-4), which bindsto pathogen-associated molecular patterns stim-ulating NF-κB translocation from the cytoplasmto the nucleus where it activates genes encodingthe inflammatory cytokines. MRF was fed tobroiler chickens in four treatments: (1) basaldiet-non-challenged, (2) MRF-non-challenged,(3) basal-enteropathogenic E. coli-challenged(EPEC), and (4) MRF-EPEC. EPEC (106 cfu)was administered by gavage to day old chicks.At three weeks of age, MRF-fed chicks had sig-nificantly increased body weights even withEPEC. Abdominal exudate macrophages, col-lected after Sephadex G50 stimulation, weredivided into primary and LPS-stimulated groupsand assayed for IL-6 and nitric oxide expression.Both primary and LPS-stimulated macrophagesfrom basal-fed-EPEC and non-challenged chickshad elevated IL-6 activity and nitric oxide levelsas compared with MRF-fed-EPEC and non-challenged chicks. These data confirm in vitroobservations and suggest that mannan can regu-late macrophage activity associated with intes-tinal health.

P-135 Interleukin-18 levels in the small bowelafter oral infection with Salmonella. A.Splichalovaa, I. Trebichavskya, I. Rychlikb, Y.Munetac, Y. Moric, I. Splichala (a Institute ofMicrobiology, Academy of Sciences of theCzech Republic, Novy Hradek, Czech Republic,b Veterinary Research Institute, Brno, CzechRepublic, c National Institute of Animal Health,Tsukuba, Japan).

IL-18 is a pleiotropic cytokine playing a criticalrole in intracellular infections as an IFN-γinducer. It was shown recently that IFN-γ is themajor protective factor in protection againstSalmonella. The effect of IL-18 in Salmonellainfections is, however, not so unequivocal. Wehave therefore used a microbiologically definedgerm-free model and compared it with a conven-tional one. One-week-old germ-free (GF) pigs orconventional pigs (CV) from the pigsty wereorally challenged with 108 cfu of Salmonellaenterica serotype Typhimurium for 24 h: eitherwith a virulent LT2 strain (causing similar dis-

ease in both humans and pigs but fatal forGF pigs) or with a non-pathogenic LT2 aroAdeletion mutant. IL-18 levels were measured byELISA in ileal washes (IL-18 was not detectedin plasma even after the infection). GF pigs didnot contain IL-18 in intestinal washes, whereasCV pigs contained 146 pg·mL–1. LT2 infectioncaused an increase of IL-18 levels, whereas thearoA– mutant did not cause any significantchange. These findings show that IL-18 issecreted in the gut in response to the infection.Sterile GF pigs have no IL-18 in the lumen of thesmall bowel, whereas endogeneous microflorastimulate its production in CV counterparts fromthe pigsty. The vaccinal mutant did not induceIL-18 contrary to the virulent Salmonella. Thus,IL-18 levels in the small bowel correlated withthe belligerence of the associated microbe.

This work was supported by the grant No. 524/05/2248of the Czech Science Foundation.

P-136 The preventive effect of Enterococcusfaecalis cell preparation (EC-12) against theexperimental infection of porcine edema dis-ease. T. Tsukaharaa,b, N. Nakanishic, C.Kameuea, K. Nakayamab, N. Matsubarad, M.Hamasakie, K. Ushidaa (a Laboratory of AnimalScience, Kyoto Prefectural University, Shimog-amo, Kyoto 606-8522, Japan, b Kyoto Instituteof Nutrition and Pathology, Kyoto 610-0231,Japan, c Kyodoken Institute, Kyoto 612-8073,Japan, d Combi Corporation, Saitama 338-0832,Japan, e Kanematsu Corporation, Tokyo 105-8005, Japan).

Porcine edema disease (ED) is caused by Shigatoxin 2e-producing Escherichia coli (STEC).ED has become frequent in pig farms and the useof antimicrobials has resulted in the develop-ment of antimicrobial-resistant STEC. Accord-ingly, the use of materials other than antimicro-bials is requested for the prevention of ED. Oraladministration of the cell preparation of Entero-coccus faecalis strain EC-12 (EC-12) to wean-ing piglets decreased their mortality in a STEC-contaminated farm. Experimentally STEC-infected piglets were used as a model for ED todetermine the appropriate dose level of EC-12 toprevent the ED. Fifteen 21 days old pigs weredivided into five groups; STEC challenge withthe control diet, STEC challenge with dietaryEC-12 either at 0.005, 0.01 or 0.05% (w/w), and

S76 Session III: Microbial interactions with the host

no STEC challenge with the control diet. Chal-lenge was carried out at 25, 26 and 27 days oldusing STEC contained in capsules resistantagainst gastric digestion. All pigs were eutha-nized at 32 days old. Daily weight gain, feed effi-ciency and the palpebral edema was improvedby supplementation of 0.05% EC-12. The eosi-nophil infiltration in the lamina propria of jeju-num of the same piglets reduced to 1/2 level ofthose observed in STEC challenge with the con-trol diet group. Accordingly, 0.05% dietary EC-12 appeared to be effective to improve clinicalsymptoms in the weaned piglets infected bySTEC.

P-137 Modulating mucin dynamics usingfunctional carbohydrates. Z. Uni, A. Smirnov(Department of Animal Science, Faculty ofAgriculture, Hebrew University, Rehovot, Israel76100).

The epithelium of the gastrointestinal tract iscovered by a mucus layer that acts as a mediumfor protection, lubrication and transport betweenthe luminal contents and the epithelial cells. Themucus layer is composed predominantly ofmucin glycoproteins produced by goblet cellswhich are believed to be capable of aggregatingseveral bacterial species and preventing theattachment of pathogenic bacteria. The mucuslayer is also a component of the innate hostresponse that is regulated in response to inflam-mation and infection. A number of protectivemucus-layer-associated proteins are either co-secreted with mucins or interact with the mucousenvironment to perform their protective func-tion. Recently it has been proposed that nutri-tional and microbial factors may affect bothmucin biosynthesis and secretion in the smallintestine. The use of “functional carbohydrate”such as mannan oligosaccharides (MOS) derivedfrom a specific strain of yeast can change mucindynamics. It can interact with Galectins, a familyof animal lectins. This lectin can regulate cellgrowth and survival, by interacting with cyto-plasmic and nuclear proteins, thereby affectingintracellular signaling pathways. Its presence inthe intestine may also lead to changes in the gutmicroflora by competitive exclusion of selectivebacterial species and therefore change themicroenvironment of intestinal epithelial cells,including mucin-producing cells. Results from a

recent study showed that adding MOS supple-mentation (Bio-Mos® Alltech Inc. 2 g·kg–1) tobroiler diets led to increased expression of thechicken intestinal mucin gene, accumulation ofthe mucin in the goblet cells and thicker mucusadherent layer. These changes may contribute togut health and may influence gut function byaffecting nutrient uptake. The mucin layer is anunstirred layer which contains mucus, IgA, andprobably digestible nutrients (dipeptides and di-tri-carbohydrated and fat micelles), it may serveas a medium which incorporates these nutrientsand makes them more available for the entero-cytes.

P-138 Oral dose of Lactobacillus plantarumand Megasphaera elsdenii increased intesti-nal IgA secretion, colonic butyrate andgrowth of colonic mucosa in piglets. K.Ushidaa, R. Inouea, N. Shimojoa, S. Takahashia,C. Kameuea, T. Tsukaharab (a Kyoto PrefecturalUniversity, Shimogamo, Kyoto 606-8522,Japan, b Kyoto Institute of Nutrition and Pathol-ogy, Kyoto 610-0231, Japan).

Piglets are often vulnerable after weaning toentero-pathogens due to the incomplete immunedefense. This is particularly true when they areraised without dietary antimicrobials. Stimula-tion of IgA, which is the primary immunologicaldefense line in the mucosal immune system, mayhelp the situation above. Lactobacilli may stim-ulate IgA secretion when orally dosed. The pig-lets also suffer from non-pathogenic diarrheadue to maladaptation to the diet. Colonicbutyrate may help the situation in supporting thegrowth and function of the colonic mucosa.Megasphaera elsdenii, a lactate-utilizing butyrateproducer, may help butyrate production partic-ularly when combined with lactobacilli. Weanedpiglets (21 days old) were orally dosed once aday either (L) 1010 L. plantarum LQ80, or (LM)1010 LQ80 with 109 M. elsdenii pig isolate.LQ80 was contained in capsules resistant to gas-tric digestion. M. elsdenii was contained in cap-sules resistant to gastric and intestinal digestion.An untreated control (C) was also prepared. Dur-ing 2 weeks of test period, fecal IgA was higherin L and LM than C. At the end of the test period,jejunal and ileal IgA tended to be higher in L andLM than in C. Colonic butyrate was higher inLM than the others. Thickness of the colonic

Session III: Microbial interactions with the host S77

mucosa was greater in LM than the others. As aresult, fecal score was improved in LM and dailyweight gain during the test period was improvedin both LM and L. This study was supported bySecure and healthy livestock farming project ofNILGS, Japan.

P-139 Adhesion of Bifidobacterium strains tointestinal mucus and its influence on compet-itive exclusion of Escherichia coli from theintestine. E. Wasilewska, M. Bielecka (Instituteof Animal Reproduction and Food Research ofthe Polish Academy of Sciences, 10-747 Olsz-tyn, Tuwima 10, Poland).

Adherence or attachment of some bacteria toeukaryotic cells or tissue surfaces and blockingof epithelial receptors may represent one of themechanisms for prevention of pathogenic micro-flora invasion. The aim of the studies was toinvestigate the ability of live, dead or disruptedbifidobacterial cells to competitively excludeadherent enterohemorrhagic and enterotoxi-genic Escherichia coli from intestinal mucosa.Commercial preparations (Sigma), as well asmucus freshly isolated from human and labora-tory rats, was used in the studies. The resultsrevealed a great diversity of bifidobacteria bind-ing to glycoproteins included in the mucus.Depending on the strain, 100-800000 live bifi-dobacterial cells attached to 1 mm2 surface of themicroplate covered by the mucus. The source ofmucus isolation had no significant influence onthe Bifidobacterium adherence; however, the

strains of human origin adhered slightly better tomucus isolated from humans. Such preferenceswere not observed in the well-adhering strains ofB. animalis and B. animalis/lactis species – iso-lated from rats and fermented milks, respec-tively. The tested bifidobacterial strains exhib-ited a diverse influence on the adhesion ofenterohemorrhagic/enterotoxigenic Escherichiacoli. Depending on the strain or its physiologicalstate, a significant decrease (0.5–50%) orincrease of the number of E. coli cells attached(in some cases over twofold) was observed. Livebifidobacterial cells revealed the strongestinhibiting effect towards enterohemorrhagicE. coli 94 (serotype O157:H7), which was char-acterized by the strongest adhesion to the mucus.Blocking the adherence of the remaining strainstested, i.e. E. coli DSM 10973 (serotype 06),DSM 30083 (serotype O1:K1:H7) and s20 strainfreshly isolated from rat, was somewhat lower.Testing of lactic acid bacteria adhesion to mucusand careful selection of the strains with profita-ble adherence abilities seems to be very impor-tant for a probiotic effect in the intestine.

P-140 Disappearance of purified plasma IgGfrom the rumen. Y.J. Williams, S. Popovski, S.Rea, A.D.G. Wright (CSIRO Livestock Indus-tries, Centre for Environment and Life Sciences,Private Bag 5, Wembley Western Australia6913).

Abstract withdrawn.

S81Reprod. Nutr. Dev. 46 (2006) S81–S123© INRA, EDP Sciences, 2006

O-21 Effects of mannan-oligosaccharide-based yeast cell wall preparations on theprevalence of antibiotic resistant bacteria inaxenic culture of intestinal bacteria: arethere strategies for decreasing the prevalenceof antibiotic resistant bacteria in the intesti-nal tract? S.M. Scheuren-Portocarrero, M.M.Newman, K.A. Dawson, C.A. Moran (Depart-ment of Animal and Food Sciences, Universityof Kentucky, Lexington, KY 40546, USA, All-tech Biosciences Center, Nicholasville, KY40356 , USA).

A series of studies have examined the effects ofvarious yeast cell wall components from thecommercial product Bio-Mos on the preva-lence of multi-drug resistant Salmonella and E.coli in in vitro culture systems. While long-term(24 h) exposure to low levels of mannan-enriched yeast cell wall preparations (0.3%) didnot affect the growth rate of any of the bacterialstrains studied, the relative proportion of Salmo-nella monterido in a multi-resistant populationthat were resistant to streptomycin and ampicil-lin and E. coli XL1-blue that were resistant tolincomycin and tetracycline decreased overtime. Decreased resistance to streptomycin wasassociated with the loss of a 2.5 kb plasmid inthe s. enteritidis strain and the loss of 6 plasmids(1 kb, 1.5 kb, 2 kb, 2.5 kb, 3.5 kb and 7 kb) fromthe multi-resistant strain of S. monterido. How-ever, similar plasmid losses were not seen ingenetically engineered strains of E. coli. Expo-sure to the yeast cell wall preparations delayedthe formation of plasmid-containing transcon-jugates when antibiotic-resistant strains ofE. coli were incubated with antibiotic-sensitiverecipient cells in a defined laboratory media andin cultures containing mixed populations ofswine fecal bacteria. The results suggest thatfractionated yeast cell wall preparations may beuseful in strategies for decreasing the preva-lence of antibiotic resistant bacteria in the gas-trointestinal tract.

O-22 SAFEWASTES’ by-products as poten-tial preventives of intestinal adhesion of bac-teria. P.M. Beckera, S. Gallettib, J. Van derMeulena (a Animal Sciences Group, Wagenin-gen UR, PO Box 65, 8200 AB Lelystad, TheNetherlands, b Faculty of Veterinary Medicine,University of Milan, Via Celoria 10, 20133Milan, Italy).

The recent EU-wide ban on the use of antibioticsas growth promoters in animal feed hasprompted a huge demand for substitutes. Withinthe EU-project SAFEWASTES, specific func-tional plants and their post-processing deriva-tives are to be screened both in vitro and in vivowith regard to their potential as alternatives toantibiotics. In general, antimicrobial strategiestake advantage of the Achilles’ heels or weakspots of bacteria, like their susceptibility todamaging agents, or their ability to attach toalternative adhesion sites. By impeding the col-onization of the intestine by enteropathogenicbacteria, not only animal pathogens can bewarded off, but also the risk of transmission ofhuman pathogens via the food-chain can bereduced. The SAFEWASTES' products arederived from artichoke pomace, carrot pomace,coneflower, larch sawdust, linseed, mango peel,pumpkin fruit, sunflower, thyme, and willow,amongst other plants, and are provided as rawmaterials and as water, ethanol, and heptaneextracts. To test the adhesion of enteropatho-genic bacteria to complex substrates, a newmicrotitration plate-based assay was developed.First results with different raw materials indi-cate that artichoke pomace, pumpkin fruit, lin-seed, sunflower, and coneflower have a bindingcapability for E. coli ATCC 25922, which hasan affinity to mannose due to its type 1 fimbriae.The impact of different water soluble plantextracts on the adhesion of E. coli K88ac(ETEC, type 4 fimbriae) to brush-border cells ofpiglets was evaluated microscopically. Of allthe products tested, mango inhibited the mostefficient brush-border adhesion. The test resultsindicate that some of the SAFEWASTES' by-products that are at present regarded as wastematerials might gain an added value in animalnutrition in the near future.

O-23 Effect of diet and protozoa on the com-munity composition of feed-adherent bacte-ria in the rumen. D.P. Morgavia, D. Gravioua,M.J. Ranillab, C. Martina (a Herbivore ResearchUnit, INRA-Theix, 63122 Saint-Genès-Cham-panelle, France, b Departamento de ProducciónAnimal I, Universidad de León, 24071 León,Spain).

The microbial population attached to feed par-ticles is essential for the efficient degradation ofcomplex plant substrates in the rumen. The aim

S82 Session IV: Nutritional manipulation

of this work was to investigate the changesinduced by contrasting feeding regimes and pro-tozoa on the structure of the rumen feed-adher-ent bacterial population. Sheep (n = 6) were sep-arated into two groups that received an alfalfahay or a wheat concentrate:alfalfa hay diet(60:40) in a cross over design with two blocks:faunated and defaunated. The bacterial commu-nity was studied by PCR-DGGE on five sub-strates, 2 cereals and 3 forages that were incu-bated in sacco for 24 h. Feed degradation wasevaluated in sacco (alfalfa hay) and in vitro (con-centrate:hay diet, wheat, alfalfa hay, and cornsilage). Diversity of the bacterial population forall substrates as measured by Shannon’s index(H) was higher for the forage than for the con-centrate-rich diet (P < 0.01) and when protozoawere absent (P < 0.01). This greater diversitywas associated with a more efficient in vitro drymatter degradation of forage-fed (67 vs. 59%,mean of all substrates, P < 0.01) and protozoa-free (71 vs. 56%, P < 0.01) inocula. Degradationof alfalfa hay in sacco showed similar results(P < 0.05). An animal variation was observed,variation that was translated into dissimilarDGGE profiles. However, PCA analysis arrangedby substrate showed that while most samplesfrom forage diets grouped rather closely, sam-ples from concentrate-rich diets were more dis-persed, possibly indicating the ecosystem’s insta-bility. Diversity of rumen feed-adherent bacteriawas a trait positively associated to substrate deg-radation and it was affected by a cereal-rich dietand the presence of protozoa.

O-24 Inulin and Lactobacillus amylovorussupplemented to human gut microbiota lowerthe microbial bioactivation of dietary aro-matic contaminants to estrogenic metabo-lites. T. Van de Wielea, L. Vanhaeckea, H.Jacobsb, W. Verstraetea (a Laboratory MicrobialEcology and Technology, Ghent University,9000 Gent, Belgium, b COSUCRA, 7740 War-coing, Belgium).

One of the lesser studied mechanisms by whichpre- and probiotics exert their health-promotingeffects in the gut is through the detoxification ofpotential toxicants, termed chemopreventiveactivity. Using a Simulator of the Human Intes-tinal Microbial Ecosystem (SHIME), we inves-tigated how administration of inulin and Lacto-bacillus amylovorus to an intestinal microbial

suspension affected the bioactivation of dietaryaromatic contaminants to metabolites with estro-genic properties. The compounds of interestwere polycyclic aromatic hydrocarbons (PAH),polybrominated diphenyl ethers (PBDE) andzearalenone. Estrogen bioassay data on PAH-incubated SHIME colon samples revealed, thatinulin addition significantly decreased the estro-genicity in the ascending colon (39%) and trans-verse colon (14%), whereas no chemopreven-tive effects were seen in the descending colon.Yet, the estrogenic response from descendingcolon samples, incubated with PAHs, decreasedby 20% when Lactobacillus amylovorus wassupplemented. Interestingly, the colon-specific-ity of the inulin chemopreventive effect corre-sponded to the colon-specificity of its prebioticeffects. Conventional culture-based techniquesand PCR-DGGE analysis on the SHIME colonsuspension revealed the selective effect of inulintreatment towards stimulation of lactic acid bac-teria in the ascending colon compartment. More-over, realtime PCR showed a significant increasein Bifidobacteria, with more than 1 log cells·mL–1

from the proximal to distal colon. Our data showthat the prebiotic effects of inulin and the probi-otic activity of Lactobacillus amylovorus mayalso inhibit the bioactivation of dietary contam-inants. Additionally, we infer that the specificconditions in the proximal colon region are mostsuited to bring about the chemopreventiveeffects from inulin.

O-25 Quantification of faecal Bifidobacteriumand Lactobacillus species in infants receivinga prebiotic infant formula. M. Haarmana,B. Stahlb, G. Boehmb, J. Knola (a NumicoResearch B.V., Wageningen, The Netherlands,b Friedrichsdorf, Germany).

A healthy intestinal microbiota, like the micro-biota of breast-fed infants, is considered to beimportant for priming of the infants’ mucosaland systemic immune system. Generally Bifido-bacterium and Lactobacillus predominate themicrobiota of breast-fed infants. To study theBifidobacterium and Lactobacillus compositionof infants in more detail, duplex 5’ nucleaseassays were designed for Bifidobacterium ado-lescentis, Bifidobacterium angulatum, Bifido-bacterium bifidum, Bifidobacterium breve,Bifidobacterium catenulatum, Bifidobacterium

Session IV: Nutritional manipulation S83

dentium, Bifidobacterium infantis, Bifidobacte-rium longum, Lactobacillus acidophilus, Lacto-bacillus casei, Lactobacillus delbrueckii, Lacto-bacillus fermentum, Lactobacillus paracasei,Lactobacillus plantarum, Lactobacillus reuteriand Lactobacillus rhamnosus. These assayswere used to determine the relative amounts ofBifidobacterium and Lactobacillus species infaecal samples of infants, aged 28 to 90 days,receiving a standard formula (SF; n = 10) or astandard formula with 0.8 g/100 mL galacto- andlong chain fructo-oligosaccharides (9:1) (OSF;n = 10). A breast-fed group (BF; n = 10) wasstudied in parallel as reference. After 6-weeksintervention a significant increase in bifidobac-teria and lactobacilli was shown in the OSF-group as well as in the BF-group but not in theSF-group. At the end of the study the Bifidobac-terium sp. and Lactobacillus sp. composition ofthe OSF-group looked rather similar to that ofthe BF-group with relatively high levels ofB. infantis, B. longum, B. breve, L. acidophilus,L. paracasei and L. casei. Faeces of the SF groupcontained more B. adolescentis, B. catenulatumand L. delbrueckii and less L. paracasei com-pared to the BF- and OSF-group. In conclusion,the distribution of the different Bifidobacteriumand Lactobacillus species in infants receivingthe prebiotic formula mimics that of breast-fedinfants whereas that of infants receiving thestandard formula does not.

O-26 PCR-DGGE and real-time PCR analy-ses of total bacteria, Bifidobacteria andLactobacilli populations in human subjectsconsuming calcium gluconate. K. Andersona,J. Chena, Z. Yua, J. Jenkinsb, P. Courtneyb, M.Morrisona (a Department of Animal Sciences,Ohio State University, Columbus, USA, b Depart-ment of Food Science and Technology, OhioState University, Columbus, OH 43210, USA).

There is a continued interest in the identificationand development of prebiotics that selectivelyincrease the abundance of Bifidobacterium andLactobacillus in the human gastrointestinaltract. Much attention has been directed towardsthe use of oligosaccharides of fructose or galac-tose, but there are also monosaccharides thatappear to have similar effects. One of these isgluconic acid, which has been shown in animaltrials to be associated with increased numbers oflactic acid bacteria and increased butyrate con-

centrations; a single human trial showed anincreased number of bifidobacteria in responseto gluconate supplements. Gluconic acid (or itssalts) is widely used as a food additive and it alsoexists naturally in rice, wine, beer, grape juice,honey, vinegar, and other fermented products.The objective of this study was to examine theimpact of a calcium gluconate supplement on theintestinal microbiota of healthy humans, withspecific emphasis on bifidobacteria and lactoba-cilli. Twelve healthy adults consumed a dailycalcium carbonate supplement for the first2 weeks (control period), followed by 2.4 g ofcalcium gluconate daily for 3 weeks (testperiod), then reverted back to a daily calciumcarbonate supplement for two more weeks (post-test period). Stool samples were collected fromeach subject once a week and microbiome DNAwas extracted from these stool samples and ana-lyzed by V3-based PCR-DGGE and real-timePCR assays. The gross PCR-DGGE profiles foreach subject appeared to be unaffected by thetreatment schedule. Genus-specific PCR-DGGEprofiles also showed that the bifidobacterialpopulations present in 9 of the 12 subjects wereunaffected throughout the study. Conversely,the lactobacilli-specific PCR-DGGE profilesdid vary in most of the subjects during the studyperiod. Real-time PCR assays showed there wasan appreciable increase in bifidobacterial abun-dance (0.43 to 1.48 logs) while the calcium glu-conate supplement was consumed in only 4 ofthe 12 subjects, and this stimulatory effectdiminished within the two weeks following thetermination of calcium gluconate supplementa-tion. Similar observations were also made for thelactobacilli, but they were less pronounced, andcollectively, these findings paralleled thoseobtained from cultivation-based analyses of thesame samples. We conclude that gluconate canpositively impact bifidobacteria and lactobacillipopulations in the human gastrointestinal tract,but the consumption of 2.4 g·day–1 is not suffi-cient in most cases to confer a significant preb-iotic effect on these bacteria. Different dosesmight be required to achieve certain desirableprebiotic effects in different individuals.

P-141 Effect of antibiotics in diets and level offeeding on caecal microbial biodiversity inlactating does as estimated by DGGE. L.Abeciaa, N.R. McEwanb, J. Balcellsa, G.E.Lobleyc, M. Fondevilaa (a Departamento de

S84 Session IV: Nutritional manipulation

Producción Animal y Ciencia de los Alimentos,Universidad de Zaragoza, Spain, b Institute ofRural Sciences, University of Wales, Aberyst-wyth, Wales, UK, c Rowett Research Institute,Aberdeen, UK).

Studies in growing rabbits highlighted a side-effect of medicated diets on the symbiotic diges-tive population. Besides, the feeding level influ-ences digestive microbiota. The effect of dietaryantibiotics to modify caecal population, and thefeeding level on bacterial caecal biodiversity oflactating does was studied. Thirty-six lactatingdoes (6 per treatment) were given a commercialdiet alone (no antibiotics, NA) or with zinc baci-tracin (BAC) or tiamulin (TIA). Intake was mod-ified by manipulating the litter size at birth to 9(LS9, high feeding level) or 5 (LS5, low level)pups per doe. Does were slaughtered at day 26of lactation and their caecal content sampled.DNA was extracted and amplified for DGGEanalyses. DNA profiles within a gel were com-pared using similarity trees from Hamming Dis-tances and presented in pictorial form usingUPGMA analysis. The effect of the feeding levelwas clearly manifested for either NA or BACdiets, whose resulting trees were divided intotwo different areas for LS5 and LS9. However,this effect was not clear for TIA. When NA andBAC were compared, the antibiotic effect didnot manifest and the major factor on diversitywas litter size. In contrast, comparison of TIAwith NA gave a tree firstly split by antibiotic andthen by litter size. Tiamulin has a marked effecton biodiversity, that prevails over that of feedinglevel, whereas without antibiotics or with thosewith a low effect on biodiversity, samples clustermainly according to the feeding level.

P-142 Microbial biodiversity in the caecum of lit-ters from lactating does given antibiotics in thediet to manipulate their digestive population. L.Abeciaa, N.R. McEwanb, J. Balcellsa, E. Solanasa,G.E. Lobleyc, M. Fondevilaa (a Departamento deProducción Animal y Ciencia de los Alimentos,Universidad de Zaragoza, Spain, b Institute ofRural Sciences, University of Wales, Aberyst-wyth, Wales, UK, c Rowett Research Institute,Aberdeen, UK).

Since the microbial digestive population of theoffspring is significantly influenced by that ofthe mother, changes in caecal biodiversity of lac-

tating does induced by antibiotic dietary supplyaffects the instauration of digestive microbiotain their litters. From parturition, 3 groups of3 lactating does were given a diet with no anti-biotics (NA) or supplemented either with zincbacitracin (BAC) or tiamulin (TIA). Throughoutlactation access of the litter to solid food wasavoided to ensure the pups were only milk-fed.After 26 days of lactation, two pups from eachlitter were slaughtered and their caecal contentsampled. After DNA isolation, fragments of 16SrDNA genes were amplified and analysed byDGGE. DNA profiles were compared by pair-wise Hamming Distance analysis and presentedin pictorial form using UPGMA analysis. Theresulting tree of pups from does given either NAor BAC diets was split by litter, with no majoreffect from the antibiotic. However, the DGGEgel comparing pups belonging to NA and TIAgroups primarily showed a split by antibiotic inthe diet fed to the mother and then by litter.Accordingly, the tree from bacterial DGGE pro-files of pups from the therapeutic diets (BAC andTIA) showed two distinct areas according to theantibiotic used in the maternal diet. It is shownthat the bacterial population in the offspring islargely dependent on the microbial biodiversityof their mothers, primarily when this is affectedby the dietary inclusion of antibiotics.

P-143 Rumen microbial population charac-teristics and detection of transgenic DNA inrumen bacteria from sheep fed Bt176 maizefor three years. G. Acutia, M. Trabalza-Marinuccia, E. Foranob, P. Mosonib, L. Mughettia,S. Costarellic, C. Rondinic (a Dipartimento diPatologia, Diagnostica e Clinica Veterinaria,Università degli Studi di Perugia, 06126 Peru-gia, Italy, b INRA, Centre de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France,c Istituto Zooprofilattico dell’Umbria e delleMarche, 06126 Perugia, Italy).

The use of genetically modified feeds and thelevel of public concern are increasing world-wide. One concern is the gene transfer to therumen bacteria, but there is a lack of studies onthis matter. Forty-four Bergamasca × Appenni-nica ewes were used in this 3-year study. Ani-mals were divided into two groups and receivedhay ad libitum and either nontransgenic or Bt176maize. Twelve, 20 and 12 ewes during the first,

Session IV: Nutritional manipulation S85

second and third year of the trial, respectively,were slaughtered 90 d after lambing and approx-imately 12 h after feeding. Rumen contents werecollected and microbial populations were numer-ated. The presence of endogenous and transgenicDNA was searched for in DNA extracted frombacterial sub-populations grown for 7 (total andamylolytic) or 14 d (cellulolytic) in vitro. No dif-ference was found between control and Bt176-fed ewes as far as bacterial numbers and proto-zoal genera distribution were concerned. Norwas the 211 bp fragment from the CryIA(b)transgene or the 226 bp fragment of maize inver-tase gene detected by PCR in the bacterial cultures.As expected, the rrs bacterial gene (1485 bp),used as a control, was found in all samples.These results show that a long-time exposure totransgenic feed does not affect the rumen micro-bial populations tested and that the conditions ofthe rumen environment are likely to prevent bac-teria from integrating recombinant DNA.

P-144 Detection of recombinant maize DNAin rumen contents of dairy cows and in mixedruminal cultures. G. Acutia, P. Mosonib, M.Trabalza-Marinuccia, C. Antoninia, E. Foranob

(a Dipartimento di Patologia, Diagnostica e Cli-nica Veterinaria, Università degli Studi di Peru-gia, 06126 Perugia, Italy, b INRA, Centre deClermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France).

The fate of transgenic plant DNA fragments inthe gastro-intestinal tract of mammals is notcompletely known. This experiment wasdesigned to compare the persistence of trans-genic maize DNA in the bovine rumen and invitro mixed ruminal cultures. The persistence ofmaize invertase gene fragment (226 bp) and ofCryIA(b) transgene fragment (211 bp) was mon-itored by PCR in 2 rumen cannulated cows. Ani-mals were fed for 2 weeks a diet containingBt176 maize (10% of dry matter intake). Sam-ples of whole rumen contents were collected at0, 30 min, 2, 4, 6, 8, 10, 12, 16, 24, 36, 48 and72 h after removing the transgenic maize fromthe diet. An in vitro experiment was performedusing rumen fluid from the same cows. Fourteenml of inoculum (mixed 1:4 with buffer) wereadded to 175 mg of ground Bt176 maize andincubated at 39 °C in triplicate vials for eachsampling time according to the in vivo protocol.

Bacterial DNA was detected in all in vivo andin vitro samples. The invertase fragment wasdetected at all incubation times in the in vitrostudy and in most in vivo samples. The CryIA(b)fragment was detected up to 2 h in the in vitrotrial and found in rumen contents in one caseonly (at 2 h in one cow). Detection of single-copy genes originating from transgenic feed isdifficult in the complex rumen ecosystem. Cau-tion is needed when extrapolating results fromin vitro to in vivo studies.

P-145 A new promising promoter for the pro-duction of prebiotic polysaccharides fromsucrose or maltose using recombinant bacte-ria and yeasts. T. Alamäe, K. Tammus, J.Lashmanova, T. Visnapuu (Institute of Molecu-lar and Cell Biology, University of Tartu, Riia23, 51010 Tartu, Estonia).

We have isolated the maltase gene promoter ofthe yeast Hansenula polymorpha. The promoterworks perfectly in both, bacteria and yeasts. InEscherichia coli expression from the promoteris strong and constitutive while in H. polymor-pha it is induced by maltose and sucrose. Nota-bly, in yeast the promoter is bidirectional,enabling coordinated expression of two pro-teins. Properties of the promoter suggested itspotential application in expression cassettes forthe production of prebiotic polysaccharidesfrom either sucrose or maltose in bacteria andyeasts. To test the assumption, two levan sucrasegenes were amplified from the genome of apseudomonad and cloned under the control ofthe maltase gene promoter in a E. coli/H. poly-morpha shuttle vector. Bacterial levan sucrasespolymerize fructose residues from sucrose intoa levan polymer, and some bacterial levans orlevan-type polysaccharides have positive healtheffects as immune stimulators or prebiotics.E. coli transformants expressing the levansu-crase genes formed slimy colonies on sucrose-containing medium and gained the ability toassimilate sucrose in minimal medium. The bestlevan producers were selected, respective plas-mids were isolated and partially sequenced. Thecloned levan sucrases were also functional inyeast, because H. polymorpha maltase-disrup-tion mutant transformed with levan sucrasegenes regained the ability to metabolize sucrose.Expression conditions of levansucrases in

S86 Session IV: Nutritional manipulation

H. polymorpha will be further optimized toachieve sufficient expression level. Recombi-nant E. coli clones will be used to produce levansucrase proteins for the study of their biochem-ical properties. Levan produced from sucrose byrecombinant bacteria will be purified and furtherstudied with the final aim to examine its poten-tial prebiotic effect.

P-146 Effect of oral nitroethane administra-tion on ruminal nitroethane reduction andmethane production in cattle. R.C. Andersona,G.E. Carstensb, E.G. Brownb, J.L. McReynoldsa,L.J. Slayb, T.R. Callawaya, D.J. Nisbeta(a USDA/ARS, Food and Feed Safety ResearchUnit, Texas A&M University, USA, b Depart-ment of Animal Science, Texas A&M Univer-sity, College Station, TX, USA).

Interventions are sought to reduce economic andenvironmental costs of ruminant methane emis-sions. To test the effects of nitroethane on rumi-nal nitroethane reduction and methane produc-tion, we orally administered 0, 1, 2 or 4 gnitroethane·kg–1 forage diet·day–1 (0, 1×, 2× or4×, respectively) to Holstein steers (319 ± 6.5 kg).Treatments were administered twice daily andthe experiment was replicated twice (3 steers/treatment replicate–1). In the first replicate, invitro incubation of ruminal fluid collectedbefore and on day 8 of treatment revealed thatruminal nitroethane-reducing activity increased(P < 0.05) from 0.022 ± .04 to 0.185 ± 0.12 µmolnitroethane/mL h–1 suggesting an in vivo enrich-ment of nitroethane-reducing microbes. Incuba-tion of ruminal fluid collected before and onday 2, 4 and 8 of treatment revealed methane-producing activity was 25% lower (P < 0.05) in2×- and 4×-treated steers than those measured influid from 0×- and 1×-treated steers (7.87 ± 2.14and 8.00 ± 1.82 µmol CH4/mL·h–1). In the sec-ond replicate, which immediately followed thefirst, pretreatment ruminal nitroethane-reducingactivity (0.408 ± 0.04 µmol nitroethane/mL·h–1)was higher (P < 0.05) and methane-producingactivity (3.21 ± 0.81 µmol CH4/mL·h–1) lower(P < 0.05) than in the first replicate (0.022 ±0.04 µmol nitroethane/mL·h–1 and 8.60 ±1.59 µmol CH4/mL·h–1, respectively). Meth-ane-producing activities measured in ruminalfluid from 1×- and 2×-treated steers were 22 and26% lower (P < 0.05), respectively, compared to0×-treated steers (3.71 ± 0.67 µmol CH4/mL·h–1).

Methane-producing activity from the 4×-treatedsteers was 19% lower, but not significantly, thanthat of the 0×-treated controls. Results demon-strate that nitroethane treatment effectively mit-igates methane production for up to 8 days.

P-147 The microbiological contamination ofgrains used in the production of probiotics forbroiler chickens. J. Biernasiak, M. Piotrowska,K. li ewska, Z. Libudzisz (Institute of Fermen-tation Technology and Microbiology TechnicalUniversity of Lodz, 171/173 Wólcza ska Street,90 – 924 Lodz, Poland).

Fodder is prone to contamination with parasitesor pests as well as pathogenic bacteria andmoulds, with the products of their metabolism –mycotoxins, contributing to changes in nutritionvalues and influence on animal health. The aimof this study was to determine the level of micro-biological contamination and of aflatoxins andochratoxin A in grains. Additionally, the influ-ence of the fermentation process of probioticbacteria and yeast (Lactobacillus paracasei/casei LOCK 0920, L. brevis LOCK 0944,L. plantarum LOCK 0945, Saccharomyces cer-evisiae LOCK 0142) on reduction of pathogenicmicroflora and mycotoxin concentration wasevaluated. Three kinds of grains from differentregions of Poland were used in the experiment:barley, wheat and corn. The work included iden-tification of selected microorganisms (totalnumber of bacteria, Enterobacteriaceae, Esche-richia coli, Salmonella, aerobic and anaerobicbacterial spores, Staphylococcus, lactic acidbacteria, yeast and moulds) in the grain. For esti-mating the level of aflatoxins and ochratoxin Athe ELISA test was used. The fermentation wasconducted in previously optimized conditions(at 37 °C, during 24 h) for the production of pro-biotic preparations. The fermentation mediumwas composed of barley (50%), wheat (45%)and corn (5%) grain mixed with water in 1:1.5proportions. The most frequently detected micro-organisms in grains appeared to be coliforms(present at the range of 102–104 cfu·g–1), meso-philic aerobic spores (present at the range of105–106 cfu·g–1) and moulds (present at therange of 102–103 cfu·g–1) of which the most fre-quent were Aspergillus, Penicillium, Cladospo-rium, Alternaria and Fusarium. Salmonella waspresent in 25 g of three samples. E. coli andanaerobic bacterial spores were not detected. In

Ś ż

ń

Session IV: Nutritional manipulation S87

all samples of grain aflatoxins were present and89% of them were above the UE limit, which is4 µg·kg–1. Ochratoxin A was detected in one bar-ley grain and its value was 5 times higher thanthe UE limit, which is 5 µg·kg–1. After the fer-mentation, a decrease in the level of aflatoxins(at about 61%) and ochratoxin A (at about 57%)was observed. Mesophilic aerobic spores andcoliforms were not detected in 0.1 g of the sam-ple and Salmonella in 25 g of the sample.

P-148 Survival of probiotics in a dynamic arti-ficial gastrointestinal system. S. Blanqueta, S.Denisa, V. Rousseaub, F. Paulb, S. Holowaczc,G. Garraita, G. Hébrarda, E. Beyssaca, M. Alrica

(a ERT CIDAM, Faculty of Pharmacy, Univer-sity of Auvergne, 63000 Clermont-Ferrand,France, b GENIBIO, 09190 Lorp Sentarail,France, c PiLeGe, 75738 Paris, France).

Probiotic bacteria and yeasts have been sus-pected to have beneficial effects on health. Thesurvival of ingested microorganisms influencesthe efficacy of probiotics. Validated models arerequired to study the mechanisms influencingthe behaviour of microorganisms in digestiveconditions. Here we present an artificial gas-trointestinal system as a powerful tool to predictthe survival of probiotics in the stomach andsmall intestine of human. This dynamic, compu-ter-controlled system has been designed toaccept parameters and data obtained from invivo studies on human volunteers. The mainparameters of digestion, such as pH, tempera-ture, peristaltic mixing and transport, digestivesecretions and passive absorption of small mol-ecules and water are reproduced as accurately aspossible. Gastrointestinal passage and succes-sive conditions are controlled to mimic param-eters in humans at different life stages, differentfood intakes and physiological or pathologicalconditions. This model offers reproducibility,easy manipulation and sample collection at anylevel of the digestive tract and at any time duringdigestion. Survival rates varied from 0.5 to 95%depending on the strains (Saccharomyces, Lac-tobacillus, Bifidobacterium, Lactococcus andStreptococcus spp.) and the simulated condi-tions (infant or adult conditions, different foodintakes). In vitro results were consistent with invivo data, showing the predictive value of thesystem.

P-149 Bioavailability of phytoestrogens fromsoy and hops. S. Bolcaa,b, S. Possemiersa,A. Heyerickb, D. De Keukeleireb, H. Depyperec,M. Bracked, I. Huybrechtse, S. De Henauwe,W. Verstraetea (a Laboratory of Microbial Ecol-ogy and Technology, Faculty of BioscienceEngineering, Ghent University, Coupure Links653, 9000 Ghent, Belgium, b Laboratory of Phar-macognosy and Phytochemistry, Faculty ofPharmaceutical Sciences, Ghent University,Harelbekestraat 72, 9000 Ghent, Belgium,c Department of Gynaecological Oncology,Ghent University Hospital, Belgium, d Departe-ment of Radiotherapy and Clinical Oncology,Ghent University Hospital, Belgium, e Departe-ment of Public Health, Ghent University Hospi-tal, De Pintelaan 185, 9000 Ghent, Belgium).

Phytoestrogens are plant constituents whichpossess pro- and antiestrogenic properties andare therefore considered to play a beneficial rolein the prevention of hormone related disorders.Soy is the major dietary source of phytoestro-gens and hops contain the most potent phytoes-trogen. The microbial metabolism is importantfor the bioactivity of the ingested phytoestro-gens. There is a large interindividual variation inthe metabolism partially due to the interindivid-ual differences in activity and composition of thegut microbiota. The factors influencing themicrobial metabolism are still largely unknown.A randomized intervention study with 150 healthymenopausal women was undertaken. Exclusioncriteria were hormone replacement therapy,allergy to soy products and the use of antibioticsup to 3 month before the study. After a 4-daywashout period, all participants delivered a fecalsample for incubation purposes and microbio-logical characterization, a control urine sample,and filled a gas collection bag for methane anal-ysis. Participants ingested a soy or hop extractor 250 mL soymilk 3 times a day for 5 consec-utive days. A 24-h urine sample of the fifth daywas collected. Subjects consumed their habitualdiets but were asked to avoid all products basedon soy or hops during the study. A food fre-quency questionnaire was used to elicit the usualfat and fiber consumption.

P-150 Effects of a gluco-oligosaccharide onperformance and gut microflora in weanedpiglets. M.L. Callegaria, S. Soldia, F. Rossia, M.Morlacchinia, P. Gattib, L. Morellia (a Università

S88 Session IV: Nutritional manipulation

Cattolica del Sacro Cuore, Piacenza, Italy,b Roquette Italia, Alessandria, Italy).

One hundred twenty-eight weaned Duroc piglets(7.2 ± 1.04 kg) were fed for 77 days alternativelywith: basal diet (CTR); basal diet supplementedwith 2% Nutriose® (GOS); basal diet supple-mented with chlortetracycline and spiramycine(1000 and 400 mg·kg–1 of feed, respectively),during the first 14 days of trial and then fed withbasal diet (CTRM) or GOS diet (GOSM) untilthe end of the study. At day 0, 14, 35, 56 and77 animals were weighed and feed conversionratio (FCR) was determined. Nutriose® reducedthe average daily gain in the first 35 d, but from35th day animals fed GOSM grew faster thananimals fed medicated diet. No differences weredetected on FCR. The central aim of this studywas to evaluate the impact of Nutriose®on theintestinal bacterial community of piglets usingmicrobiological plate counts and a cultivation-independent molecular technique. No effect wasdetected on bifidobacteria and lactobacillicounts. The PCR-DGGE profiles, obtained withHDA1GC and HDA2 primers, of all piglets werecompared and the differences of bacterial com-munity fingerprints were evaluated based on theDice coefficient. Analysis of profiles consider-ing the presence or absence of bands or their rel-ative intensity demonstrated that there were sta-tistically significant differences between thefour groups of animals. The most significantmodification in the microflora composition con-cerned Escherichia coli, in fact we detected aquantitative reduction, confirmed by Real timePCR, of this organism in animals fed with preb-iotic supplemented diet. These results indicatedthat a Nutriose® supplementation was able toreduce potential enteropathogenic bacteria.

P-151 A combination of inulin and type 3resistant starch alters bacterial fermentationcharacteristics in the rat cecum and colon.D.J. Campbella, P.R. Heavya, F. Brounsb, A.Adamb, S. Silvic, C. Coudrayd, R.M. Hughesa,I.R. Rowlanda (a Northern Ireland Centre forfood and Health, Centre for Molecular Bio-sciences, School of Biomedical Sciences, North-ern Ireland, b Cerestar-Cargill Vilvoorde R&DCentre, Havenstraat 84, 1800 Vilvoorde, Bel-gium, c Dipartimento di Scienze Morfologiche eBiochimiche Comparate, Università degli Studidi Camerino, Camerino, Italy, d Centre de

Recherche INRA Clermont-Ferrand/Theix, Saint-Genès-Champanelle, France).

Certain dietary resistant starches (RS) and otherindigestible carbohydrates, such as fructans, arethought to benefit gut health by acting as sub-strates for bacterial fermentation leading to theproduction of short chain fatty acids (SCFA) inthe large intestine. These SCFA, particularlybutyrate, play an important physiological role ingut epithelial cell energy metabolism and arebelieved to act as chemoprotectants by suppress-ing the genesis and growth of tumour cells, inaddition to maintaining gut barrier integrity. Todate few studies have examined the effects ofcombining non-digestible carbohydrates onSCFA production in vivo. The purpose of thecurrent study was to elucidate the interactiveeffects, of a fructan source, inulin (FibrulineInstant Cosucra) and a type 3 RS, based on ret-rograded maltodextrins, (Actistar RM) onSCFA profiles and associated parameters of guthealth in the rat caecum and colon. Wistar rats(n = 48) were randomly assigned to one of fourdietary regimes for a period of 90 days; a basalmaintenance diet, inulin; RS3 or an inulin/ RS3combination. The experimental diets resulted inhigher total SCFA pools in both the rat caecumand colon compared with the control. Caecal pHwas lower on the test diets and faecal and caecalbulk and wall weights were higher. Combiningthe Actistar RS with inulin appeared to have asynergistic effect on butyrate production in thecolon. In animals fed the combination diet,molar proportions of faecal butyrate were com-parable with those found in the caecum, the pri-mary site of bacterial fermentation in the rat.These findings suggest that the addition of RS3to an inulin containing functional food, or Inulinto a diet high in RS, increases the amount of fer-mentation by butyrate producing bacteria in thecolon and may have a beneficial effect on guthealth.

P-152 Fermented liquid feed and fermentedcereal grains – microbial composition andeffect on the gastrointestinal ecology of pig-lets. N. Canibe, O. Højberg, B.B. Jensen (DanishInstitute of Agricultural Sciences, Departmentof Animal Health, Welfare and Nutrition,Research Centre Foulum, PO Box 50, 8830Tjele, Denmark).

Session IV: Nutritional manipulation S89

Feeding fermented liquid feed and fermentedcereal grains are strategies used to improve thegastrointestinal health of pigs. Fermented liquidfeed decreases the counts of coliform bacteriaand Salmonella along the gastrointestinal tract(GIT), decreases the incidence of swine dysen-tery, and delays the excretion of L. intracellu-laris in challenged pigs. Lactic acid bacteria arethe dominating microorganisms in FLF and fer-mented grain, but knowledge on microbial com-position to genus or species level is scarce. Threedietary treatments were designed: a weaner dietfed on as-is basis (DRY), fermented liquid cerealgrain feed (FLG), and fermented liquid feed(FLF). The FLF diet was prepared by storing thefeed and water in a closed tank at 20 °C, and theFLG diet by fermenting only the cereals and add-ing the remaining dietary ingredients immedi-ately before feeding. On day 14 post-weaning,one piglet from each treatment was killed andsamples collected from the GIT. Terminal restric-tion fragment length polymorphism (T-RFLP)analysis of the fermented diets showed a cleardominance of 598 bp fragments, representinge.g. Lactobacillus pentosus, L. plantarum,L. paraplantarum, Lactococcus sp., 604–605 bpfragments representing e.g. L. sakei, and 606–608 bp fragments representing Pediococcuspentosaceus. T-RFLP profiles of digesta fromvarious segments of the GIT indicated that thelactic acid bacteria detected in the diets were alsopresent in the GIT. Further differences betweendietary groups in the T-RFLP profiles along theGIT were observed. These results are presentedand discussed together with parameters describ-ing the ecology of the GIT of the three dietarygroups

P-153 In vivo and in vitro characterisation ofantibiotic safety and competitive exclusionproperties of Lactobacillus salivarius andEnterococcus faecium. A.J. Cartera,b,d, R.M. LaRagionea, V. Perretonc, M. Adamsb, M.J.Woodwarda (a Department of Food and Environ-mental Safety, Veterinary Laboratories Agency(Weybridge), New Haw, Addlestone, Surrey,KT15 3NB, UK, b School of Biomedical andMolecular Science, Food Safety ResearchGroup, University of Surrey, Guildford Surrey,GU2 7XH, UK, c Institute of Veterinary Bacte-riology, University of Berne, Langass-Strasse122, Postfach, 3001 Bern, Switzerland, d Probi-otics International Ltd, Stoke-sub-Hamdon,Somerset, TA14 6QE, UK).

Probiotic bacteria are thought to act as compet-itive exclusion agents against food borne patho-gens such as Salmonella enteritidis. However,the exact mechanisms of action are still to be elu-cidated. Here we report phenotypic and geno-typic characterisation of antibiotic resistance ofLactobacillus salivarius using minimum inhib-itory concentrations and microarray analysis. Inaddition preliminary studies on the competitiveexclusion of Salmonella enteritidis in chickensby a mono-culture of Lactobacillus salivarius ora mixed culture of Enterococcus faecium andLactobacillus salivarius are discussed. Mini-mum inhibitory concentrations were used todetermine the antibiotic resistance of Lactoba-cillus salivarius to a selection of 18 antibiotics.Lactobacillus salivarius was found to be resist-ant to vancomycin. However, previous reportshave indicated that resistance to this antibiotic isintrinsic to Lactobacillus salivarius by a nontransferable, chromosomal D-Ala:D-Lac ligase.The genome of Lactobacillus salivarius wasalso screened for transferable resistance genesusing a Gram positive antibiotic microarray chipcontaining 90 antibiotic resistance genes. Lacto-bacillus salivarius did not harbour any of the 90antibiotic resistance genes screened. To test thecompetitive exclusion properties of the probiotictest organisms, day old White Leghorn SPF(SPAFAS) chicks were inoculated by oral gav-age with either ~ 5 × 108 Lactobacillus salivar-ius or ~ 3 × 108 of Lactobacillus salivarius and~ 3 × 108 of Enterococcus faecium. On day 2 thebirds were challenged with ~ 5 × 105 of Salmo-nella enteritidis by oral gavage. Salmonellaenteritidis colonisation of the caeca, ileum andcolon was evaluated by direct bacterial counts.Interestingly, in the group treated with the dualprobiotic culture, colonisation of the caeca,ileum and colon by Salmonella enteritidis wasreduced by approximately two logs on day 44,compared to the control group. Here we havedemonstrated the lack of transferable antibioticresistance and competitive exclusion propertiesof Lactobacillus salivarius.

P-154 Fungal colonisation of alkali treatedwheat straw in the rumen of sheep. A.S.Chaudhry (School of Agriculture, Food andRural Development, University of Newcastle-upon-Tyne, NE1 7RU, UK).

S90 Session IV: Nutritional manipulation

While anaerobic rumen fungi facilitate degrada-tion of fibrous foods in ruminant animals, theirefficacy is limited by the presence of cellulose-lignin bonds in foods such as wheat straw.Therefore, it would help if such bonds are mod-ified in order to enhance the efficacy of rumenfungi in degrading and hence utilising wheatstraw in sheep. This in sacco study examined theeffect of alkali treatments (calcium oxide = CaO,sodium hydroxide = NaOH and alkaline hydro-gen peroxide = AHP) on the fungal colonisationand hence utilisation of wheat straw when incu-bated in the rumen of sheep. Comparisons weremade between treated and untreated straws toobserve colonization microscopically using atrypan blue lactophenol staining technique toreveal the fungal thalli and rhizoids. Structuraldisintegration of un-incubated and rumen incu-bated straw particles following alkali treatmentswas also observed. AHP showed the greatest fra-gility to straw particles followed by NaOH andCaO treatments. The untreated straw revealedrelatively less fungal colonization than thetreated straws, which were heavily colonized byvariable amounts of rhizoids and thalli. AHPtreated straw showed most extensive coloniza-tion with the largest network of fungi containingbranched and multi-directional rhizoids pene-trating deep into the straw tissues, particularlythe bundle sheath cells. The thalli of this studyresembled mono- and poly-centric genera ofanaerobic Chytridiomycete fungi. The extentand pattern of fungal colonization comparedwell with the previous degradability data ontreated straws, demonstrating the importance ofalkali treatments to improve fibre degradationby rumen micro-organisms, particularly fungi.The efficacy of rumen fungi to utilise fibrousfoods can be enhanced if their cellulose-ligninbonds are modified by using alkali pre-treatments.

P-155 The effect of two different types of ara-binoxylanoligosaccharides on the level ofhuman faecal bifidobacteria determinedusing real-time PCR. L. Cloetensa, V. DePretera, K. Swennenb, T. Van de Wielec, W.Verstraetec, W.F. Broekaertb, C.M. Courtinb,J.A. Delcourb, P. Rutgeertsa, K. Verbekea

(a Gastrointestinal Research, University Hospi-tal Gasthuisberg, KU Leuven, Leuven, Belgium,b Laboratory of Food Chemistry, KU Leuven,Leuven, Belgium, c Laboratory Microbial Ecol-ogy and Technology, Ghent University, Gent,Belgium).

Arabinoxylanoligosaccharides (AXOS) can beobtained by enzymatic hydrolysis of arabinoxy-lan and have been shown to exert prebioticpotential in animals. However, their physiolog-ical properties in humans have not yet beendescribed. In this study, AXOS with an averagedegree of polymerisation (avDP) of 15 and anaverage degree of substitution (avDS) of 0.27(AXOS-15-0.27), and AXOS with an avDP of58 and an avDS of 0.58 (AXOS-58-0.58) werecompared to investigate their impact on the levelof bifidobacteria in human faecal samples. In arandomized cross-over study, 9 healthy volun-teers were administered daily either AXOS-15-0.27 or AXOS-58-0.58 in a dose correspondingto 4.88 g arabinoxylan during 2 weeks andswitched to the opposite treatment after a wash-out period of 2 weeks. Faecal samples were col-lected on the day before (baseline) and the dayimmediately after each intake period. Theamount of bifidobacteria in the faecal sampleswas measured using real-time PCR (Van deWiele T et al., 2004, FEMS Microbiol Ecol 51:143–153). Results were expressed as log bifido-bacteria/g faeces. The level of faecal bifidobac-teria significantly increased (P = 0.021) after a2-week intake period of AXOS-15-0.27 (7.55 ±0.35) as compared to baseline (6.86 ± 0.63). Incontrast, no differences were observed betweenbaseline (6.94 ± 0.71) and a 2-week intakeperiod of AXOS-58-0.58 (6.95 ± 1.05). Theseresults demonstrate the favourable effects ofAXOS-15-0.27 administration on bifidobacteriaand indicate its prebiotic potential in humans,whereas AXOS-58-0.58 did not stimulate thebifidobacteria. The results further suggest thatthe structural conformation of the AXOS mole-cules influences their prebiotic potential.

P-156 Molecular and functional characterisa-tion of new Lactobacillus isolates from Bul-garian dairy products. S.T. Danovaa, R.N.Aleksandrovaa, I.N. Ilievb (a Institute of Micro-biology, Bulgarian Academy of Sciences, 26Acad. G. Bontchev str, 1113 Sofia, Bulgaria,b Plovdiv University, Tzar Assen str, 4000 Plov-div, Bulgaria).

The new consumer’s demand for more naturaland minimally processed food has stimulatedconsiderable interest for fermented products,containing living bacteria. Thus, intensive stud-ies on the health promoting properties of Lactic

Session IV: Nutritional manipulation S91

acid bacteria (LAB) have been established. Wecharacterised LAB microflora of artisanal dairyproducts from different rural regions of Bul-garia. As a pre-selection procedure 39 strains,determined as mesophilic homo- and hetero-fer-mentative lactobacilli, were examined for anti-microbial activity. Despite the high percentageof activity against Listeria, no positive signalwas obtained in Dot hybridization assays with aDIG-labelled DNA probe recognising the anti-Listeria motif for bacteriocins of class IIa.Selected active strains did not produce H2O2 andwere moderate acidifiers in skim milk and MRSbroth. They expressed different technologicallyimportant characteristics such as resistance tohigh concentrations of NaCl and to Nisin, alongwith some probiotic properties and long termviability during cold storage. Molecular charac-terisation of selected isolates was done accord-ing to polyphasic taxonomy. The species affili-ation was confirmed by PCR using species-specific primers and the first discrimination wasperformed by RAPD analysis. Box and rep PCRwere used as DNA fingerprinting techniques forthe identification of active isolates at strain level.Selection and characterisation of new techno-logically relevant Lactobacillus strains, withpotential to guarantee the quality and safety offood, is a necessary step in the development ofprobiotic foods with impact on consumeracceptance.

P-157 Effect of dietary intervention with dif-ferent pre- and probiotics on intestinal bacte-rial enzyme activities. V. De Preter, L. Cloetens,E. Houben, P. Rutgeerts, K. Verbeke (Departmentof Gastrointestinal Research, University Hospi-tal Gasthuisberg, KU Leuven, Herestraat 49,3000 Leuven, Belgium).

One of the claimed beneficial effects of pre- andprobiotics for the human host is, that these sub-strates are able to reduce the production of toxicand carcinogenic metabolites by suppressingspecific enzyme activities in the colon. In thepresent study, the influence of long-term admin-istration of different pre- and probiotics on thefaecal β-glucuronidase and β-glucosidase activ-ity was investigated. The effect was evaluated ina randomized, cross-over study in 50 healthyvolunteers. At the start of the study and at the endof each 4-week treatment period the volunteers

collected faeces during 72-h. Lactulose and oli-gofructose-enriched inulin (OF-IN) were cho-sen as prebiotic substrates, whereas Lactobacil-lus casei Shirota, Bifidobacterium breve andSaccharomyces boulardii were selected as pro-biotic strains. Two synbiotic combinations wereevaluated as well (Lactobacillus casei Shirota +OF-IN and Saccharomyces boulardii + lactu-lose). The enzyme activity was assessed spec-trophotometrically and the results wereexpressed as mg of substrate released/g faeces/h. Lactulose and OF-IN significantly decreasedβ-glucuronidase activity (P = 0.001 and P <0.001, respectively), whereas no effect wasobserved on β-glucosidase activity. The addi-tion of L. casei Shirota and B. breve to the dietresulted in a tendency to a decreased faecal bac-terial β-glucuronidase activity. To the contrary,B. breve significantly increased β-glucosidaselevels in the faeces (P = 0.02). Supplementationwith the synbiotic did not appear to be more ben-eficial than either compound alone. No influenceof S. boulardii on bacterial enzyme activity wasnoted. In conclusion, administration of lactu-lose, OF-IN, L. casei Shirota or B. breve resultedin a decrease in β-glucuronidase activity, whichis considered beneficial for the host, because thisenzyme can produce carcinogenic end-products.

P-158 Effect of short- and long-term dietaryintake of oligofructose-enriched inulin on thecolonic fate of NH3 in healthy volunteers. V.De Preter, L. Cloetens, P. Rutgeerts, K. Verbeke(Department of Gastrointestinal Research, Uni-versity Hospital Gasthuisberg, KU Leuven,Herestraat 49, 3000 Leuven, Belgium).

Oligofructose-enriched inulin (OF-IN) is a preb-iotic which is able to modify the balance of theintestinal flora, thereby stimulating the growthand/or activity of beneficial bacteria such as bifi-dobacteria. In the present study, the influence ofOF-IN on the absorption of NH3 was evaluated.The biomarker lactose-[15N, 15N]-ureide (LU)was used as a source of 15NH3 to study its met-abolic fate (De Preter et al., 2004, Br J Nutr 92:439–446). Before the start of the study and at thebeginning and end of a 4-week treatment period,19 healthy volunteers consumed a test meallabelled with 75 mg LU. During the studyperiod, they received 10 g OF-IN (b.i.d.). Aftereach test meal, urine (48 h) and faeces (72 h)

S92 Session IV: Nutritional manipulation

were collected and analysed for 15N-content bycombustion-IRMS. Results were expressed as %of administered dose. Short-term OF-IN intakeresulted in a significant decrease of the urinary15N content (P < 0.001) and a concomitantincrease in faecal 15N as compared to baseline(P < 0.001). This was probably due to a stimu-lation of bacterial metabolism, since a signifi-cant increase in 15N content in the bacterial frac-tion was found (P = 0.001). After long-termadministration of OF-IN, a statistically signifi-cant reduction of the urinary 15N content wasfound (P < 0.001) without a significant increasein the faecal 15N content, resulting in anincreased retention of the label in the colon. Thismight be explained by the hypothesis that OF-IN primarily stimulates the growth of mucosa-associated bacteria which might use the 15NH3for their growth or metabolism.

P-159 Effect of short- and long-term admin-istration of Lactobacillus casei Shirota, oligof-ructose-enriched inulin and their synbioticcombination on the colonic fate of ammonia.V. De Preter, L. Cloetens, P. Rutgeerts, K. Verbeke(Department of Gastrointestinal Research, Uni-versity Hospital Gasthuisberg, KU Leuven,Herestraat 49, 3000 Leuven, Belgium).

In the present study the effect of short- and long-term administration of Lactobacillus casei Shi-rota (LacS), oligofructose-enriched inulin (OF-IN) and their synbiotic combination on thecolonic NH3 metabolism was investigated bymeans of the biomarker lactose-[15N, 15N]-ure-ide (LU). A reduced urinary 15N-excretion afterintake of LU has been shown to reflect animproved removal of the potentially toxic NH3from the colonic lumen by stimulation of thebacterial metabolism (De Preter et al. 2004, BrJ Nutr 92: 439–446). Therefore, 9 healthy vol-unteers received consecutively for 1 month 6.5 ×109 LacS (2×/d), 10 g OF-IN (2×/d) and the com-bination of both. Consecutive intake periodswere separated by 2 weeks wash-out. Before thestudy, on the first day of each intake period(short-term effect) and after each period (long-term), the volunteers consumed a test meallabelled with LU and performed a fractionated48-h urine collection. The 15N-content of theurine was determined by combustion-IRMS.Both short- and long-term administration of OF-

IN resulted in a significantly decreased urinary15N-excretion: from 51.75 (IQR 41.56–63.12) to28.24 (IQR 24.01–30.97) (P = 0.008) for short-term and to 28.24 (IQR 24.01–30.97) (P = 0.011)for long-term intake. For LacS, only short-termadministration resulted in a significant effectcompared to baseline (51.75 (IQR 41.56–63.12)vs. 47.41 (IQR 27.00–56.05) (P = 0.038)),whereas a tendency to a decrease was observedafter long-term intake. The synbiotic combina-tion LacS + OF-IN resulted in a significantreduction of the cumulative excretion of thelabel compared to baseline with P = 0.008 forshort-term and long-term synbiotic administra-tion. In conclusion, dietary addition of LacS,OF-IN and their synbiotic combination results ina favourable effect on the colonic NH3 metabo-lism.

P-160 Effect of crude linseeds, extruded lin-seeds or linseed oil on digestion and fatty acidmetabolism in dry cows. M. Doreaua, E.Aurousseaua, S. Gachona, F. Glassera, J.Normandb, G. Chesneauc, C. Martina (a INRATheix, 63122 Saint-Genès-Champanelle France,b Institut de l’Élevage, 5 rue Hermann Frenkel,69364 Lyon Cedex 07, France, c Valorex 35210Combourtillé France).

Effects of fatty acids (FA) from linseeds onruminal digestion and fatty acid metabolismwere studied in dry cows fitted with ruminal andduodenal cannulas in a 4×4 Latin square design.Four diets based on maize silage (65%), hay(10%) and concentrates (25%) were compared:control diet (C); diet supplied with 7.5% cruderolled linseeds (RL), diet supplied with 7.5%extruded linseeds (EL); diet supplied with 2.6%linseed oil and 4.9% linseed meal (LO). Thesefour diets did not differ in total OM and fibredigestibility, in OM ruminal and intestinaldigestibility, and in duodenal N flow. MicrobialN duodenal flow was lower for RL than for Cdiet. Volatile fatty acid concentration and pat-tern, and protozoa concentration in the rumendid not vary among diets. Ruminal biohydrogen-ation of linolenic acid did not significantly differamong diets (average 96.2%) whereas biohydro-genation of linoleic acid was higher for RL(94.5%) than for C (90.7%), EL (91.5%) and LO(92.6%) being intermediate. Trans 18:1 FA duo-denal flows were higher for LO and EL than forRL and C diets (93.3, 82.4, 56.1 and 32.1 g·d–1,

Session IV: Nutritional manipulation S93

respectively), this difference being due to t10and/or t11 (coeluted) flows: 59.3, 51.6, 24.0,18.2 g·d–1. The same result was observed forother intermediates of biohydrogenation: c9t11CLA (0.6, 0.6, 0.2 and 0.2 g·d–1) and t11c15 18:2(14.0, 12.5, 4.7, 1.5 g·d–1). No differencebetween RL, EL and LO diets was observed forcis 18:1 FA and for stearic acid.

P-161 Effect of essential oils on degradationof starch-rich substrates in a rumen simulat-ing fermenter (Rusitec). S.M. Duvala, R.J.Wallaceb, C.J. Newbolda (a Institute of RuralSciences, University of Wales, Aberystwyth,SY23AL, UK, b Rowett Research Institute,Bucksburn, Aberdeen AB21 9SB, UK).

We have previously shown that a commercialblend of essential oil (EO) compounds was ableto decrease the rate of degradation of starch-richsupplements in the rumen. Here we furtherinvestigate this phenomenon in the rumen sim-ulating fermenter (Rusitec). Four vessels wereused as controls fed daily with 14 g of a basal diet(80% hay, 10% soyabean meal, 10% molasses),3 g of cut maize and 3 g of rolled barley and4 were supplemented with 15 mg of EO (CRINARUMINANTS®) equivalent to 0.02 mg·mL–1.To investigate the effect of EO on bacterialattachment, DNA was extracted from barley andmaize incubated in the fermentor for 3, 6 and15 h and used as a template for 16S rDNA PCR-DGGE. EO supplementation had no effect on thenumber of total, cellulolytic or amylolytic bac-teria that could be cultured from the fermentor,nor did it affect VFA production or the degrada-tion of the basal diet. However, pH tended to behigher in vessels supplemented with EO andammonia production was significantly decreased.Degradation of barley (but not maize), after 48 hincubation, was reduced in the presence of EOand methane production was significantlydecreased. Cluster analysis of the DGGE bandprofile showed that the EO had no detectableeffect on attached populations with either sub-strate. Instead both incubation time and substrateappeared to be major factors affecting the band-ing pattern. These results support the idea thatwhile EO can have dramatic effects on microbialactivity in the rumen, the bacterial groupsaffected by EO may not be represented by themajor bacterial groups as enumerated throughculture counts or visualized by DGGE.

Study sponsored by CRINA S.A., 15, rue de la Combe,Gland, CH-1196, Switzerland

P-162 Fermented feed for laying hens. R.M.Engberg, N.F. Johansen, B.B. Jensen (DanishInstitute of Agricultural Sciences, ResearchCentre Foulum, PO Box 50, 8830 Tjele, Den-mark).

The influence of fermented liquid feed on theactivity and composition of the gastrointestinalmicroflora was studied in an experiment with480 layers (Babcock, 16 weeks old) housed infloor pens (30 animals/pen). From week 16 toweek 38 during the laying period, the birdsreceived an organic feed, which was either fedas dry mash (experimental group 1) or as wetfeed (feed:water ratio, 1:1.4) following naturalfermentation (experimental group 2). The fer-mented feed was characterised by a high lacticacid concentration (≈ 260 mmol·kg–1 feed) andmoderate amounts of acetic acid (20–30 mmol·kg–1 feed), high numbers of lactic acidbacteria (109–1010/g feed) and a pH of about 4.5.At 38 weeks of age, 5 birds of each floor penwere killed and the contents of crop, gizzard,ileum and caeca were separately collected andpooled. In crop contents, the pH was about 0.2units lower in birds receiving fermented feed ascompared to hens receiving dry feed (4.51 vs.4.73). In the contents of ileum and caeca, the pHwas higher (pH ileum, 7.4 vs. 7.2; pH caeca, 6.5vs. 6.8), indicating a reduced microbial fermen-tation in the lower digestive tract followingintake of fermented feed. In crop contents, fer-mented feed increased the number of lactic acidbacteria and reduced the numbers of entero-cocci. The counts of coliform bacteria werelower in the contents of crop, gizzard and ileumof hens fed with fermented feed. Consideringcoliform bacteria as indicator organisms for e.g.Salmonella, it can be concluded that fermentedfeed improves gastrointestinal health due toacidification of the upper digestive tract.

P-163 Effect of a Saccharomyces cerevisiaesupplementation on microbial profiles andfermentation patterns in faeces of horsesunder transportation. C. Faubladiera, F.Chaucheyras-Durandb,c, L. Veigaa,b, V. Jullianda

(a ENESAD, Dijon, France, b Lallemand Animal

S94 Session IV: Nutritional manipulation

Nutrition, Blagnac, France, c Unit of Microbiol-ogy, INRA, Saint-Genès-Champanelle, France).

Transport is a stressful procedure for horses,which can disturb intestinal microbial commu-nities as well as their environmental parametersand finally provoke digestive disorders likecolic. Several studies on Saccharomyces cerevi-siae have reported that a yeast supplementationcould limit the imbalance of the microbial com-munities in horses, which would provide apotential interest in stressful situations. The aimof our work was to evaluate the effect of a yeastsupplementation on the microbial communitiesand fermentation parameters in faeces of horsesunder transportation. Four mature geldingsreceived daily a morning meal providing a min-imum of 200 g/100 kg BW of starch without orwith a supplementation of S. cerevisiae (2 ×1010 units), in a Latin square design. After four-teen days of adaptation to the diet, horses weretransported at d0. Faecal content was collectedat d –1, d0 (after transportation) and at d +3, 4 hafter the morning meal, in order to count totalanaerobic, cellulolytic, and lactic acid-utilizingbacteria, lactobacilli, and streptococci. Lacticacid, volatile fatty acids and pH were measuredon faecal fluid samples collected at the sametime. Microbial concentrations were statisticallyanalysed using the GLM procedure of SASv8.The transport had an immediate impact on theconcentration of the total anaerobic bacteria,which depended on the supplementation of theyeast. When S. cerevisiae was supplemented,the concentration of streptococci increased afterthe transport and the concentration of lactoba-cilli at d3 was changed. Further analyses areunder process and more data will be reported.

P-164 Capric acid concomitantly affectsrumen methanogenesis and biohydrogena-tion of linoleic and linolenic acid. V. Fieveza,C. Boeckaerta, G. Bruggemanb, K. Deschepperb

(a Laboratory of Animal Nutrition and AnimalProduct Quality, Ghent University, Belgium,b Nutrition Sciences n.v., Belgium).

Medium-chain fatty acids (MCFA) are efficientantimicrobial compounds, with lauric and myr-istic acid being effective in suppressing rumenmethane production. Few studies using capricacid (CA), suggest several rumen bacteria also

to be susceptible to inhibition by CA. During24 h batch in vitro incubations with 50 mL buff-ered rumen fluid, three CA concentrations (0, 20and 30 mg) were tested in combination withstandard dairy concentrate (0.5 g), sunflower(10 mg) and linseed oil (10 mg). Both CA dosesinhibited rumen CH4 production by 85 and 28%,respectively, and equally suppressed rumenshort-chain fatty acid (SCFA) production by23%. Shifts in the rumen SCFA proportionswere observed, with 30 mg CA mainly stimulat-ing propionate (+64%) and suppressing butyrate(–50%) proportions, whereas 20 mg CA pro-voked a significant increase in butyrate (+35%)and decrease in acetate (–9%) proportions. CAsignificantly reduced apparent biohydrogena-tion of linoleic and linolenic acid (84.5, 76.6 and73.8%, SEM 1.0%) and both CA doses equallyinhibited further hydrogenation, provokingaccumulation of C18:2 t11c15 (+391%) andC18:1t11+t10 (+142%) and reducing C18:0(–29%). This study further illustrates that sup-plements inhibiting rumen methanogenesis con-comitantly affect hydrogenating bacteria, as hasbeen suggested earlier for fish oil, micro algaeand monensin.

P-165 Monitoring the bacterial populationchanges in the rumen of the camel associatedwith change in diet by real-time PCR. M.B.Ghalia, P.T. Scottb, G.A. Alhadramic, R.A.M. AlJassima (a School of Animal Studies, The Uni-versity of Queensland, Gatton, QLD 4343, Aus-tralia, b ARC Centre of Excellence for Integra-tive Legume Research, The University ofQueensland, St Lucia, QLD 4072, Australia,c UAE University, Al-Ain, UAE).

Rumen studies in the past focused on the diver-sity and characteristics of the different microbesinhabiting the rumen, mainly of cattle and sheep,while very few studies dealt with other rumi-nants such as camels. In an earlier report weestablished the presence of common rumen spe-cies including Streptococcus bovis, Lachnospirapectinoschiza, Butyrivibrio fibrisolvens, Prevo-tella ruminicola and Selenomonas ruminantium.In this study changes in the population of thesebacterial species following changes of diet weremonitored by real time PCR. Four rumen fistu-lated camels were fed Rhodes grass hay alone(R) or with barley grain (R+G, 40:60). The

Session IV: Nutritional manipulation S95

experimental schedule involved 3 consecutivefeeding periods each of 3 weeks duration. Dur-ing the pre-treatment and post-treatment periodsall camels were fed the R diet, while during thetreatment period they were fed the R+G diet.Genomic DNA was extracted from rumen sam-ples collected during day 8, 11, 18 and 21 at 0,8 and 16 h after feeding of each period. All thebacterial species, except S. ruminantium,decreased in number (P < 0.01) following die-tary change from R to R+G. S. bovis, L. pectin-oschiza, B. fibrisolvens and P. ruminicoladecreased 3, 2.1, 1.9 and 1.7 fold, respectively.In contrast, the population of S. ruminantiumwas increased by 4.2 fold during the sameperiod. The bacterial population decreased fur-ther when the camels were shifted back to the Rdiet. Results indicate that S. ruminantium, whichis a lactate utiliser, is the only bacterium toincrease in number during the grain supplementperiod. The residual effect of the grain supple-ment needs further investigation.

P-166 Post-weaning maturation of rabbit cae-cal microbial communities: impact of liveyeast intake. T. Gidennea, N. Bennegadi-Laurenta,V. Monteilsb, G. Fontyc (a INRA Toulouse,SRC, BP 52627, 31326 Castanet-Tolosan,France, b ENSA Toulouse, BP 32607, 31326Castanet-Tolosan, France, c INRA, C.R. Cler-mont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France).

The balance of some caecal microbial popula-tions was assessed in the young healthy rabbit,at weaning (28 d) and two weeks after (42 d), bydot-blot hybridization with phylogenetically tar-geted 16S rRNA oligonucleotide probes. Ani-mals (doe and litters) were fed ad-libitum a con-trol pelleted diet (group C) or the same dietcontaining 106 cfu of live yeast (Levucell SB®,Lallemand, group SB), from parturition to 70 dof age for youngs. Whatever the age or group,no response was observed with probes targetingrRNA of E. coli, anaerobic fungi, Fibrobacterintestinalis and Fibrobacter succinogenes (detec-tion limit = 25 ng·µL–1 hybridised rRNA, i.e.≈ 106 bact·g–1). The total quantity of rRNAdecreased from 28 to 42 d of age (394 vs. 241 µg,P < 0.001), originating from a decrease in bac-terial rRNA (324 vs. 216 µg, P < 0.001) and inarchaeal rRNA (62 vs. 24 µg, P < 0.001), without

effect or interaction with dietary treatment. Iden-tified bacteria represented about 50% of the totalbacterial community. From 28 to 42 d, bacterialrRNA proportions increased from 84 to 90%(P < 0.001) while that of archaea decreased (15vs. 10%), without an impact of the group effect.The Flexibacter-Cytophaga-Bacteroides groupwas prevalent and remained steady with age(41% of bacterial rRNA), as well as that of R. fla-vefaciens (0.8%), without an effect of yeast addi-tion. The fact that, after weaning, the rRNA pro-portions of R. albus increased more in the SBgroup (0.3%) than in the C group (0.1%; P <0.01) suggests the existence of a positive inter-action between the yeast and this fibrolytic bac-terial species.

P-167 Digestive fate of Saccharomyces cerevi-siae CBS 493 94, fed at 3 different concentra-tions to horses. J. Goberta, G. Bertinb, V.Jullianda (a ENESAD, 26 bd Dr Petitjean, BP87 999, 21079 Dijon Cedex, France, b AlltechRegulatory Department, 14 place Marie-JeanneBassot, 92300 Levallois-Perret, France).

The digestive fate of live yeast, used as microbialadditives in herbivores, has been studied inlambs but not in horses. The current recommen-dation is to supply live yeast each day to horses.This study was undertaken to evaluate and com-pare the appearance and disappearance kineticsof Saccharomyces cerevisiae CBS 493 94 (Sc)(Yea-Sacc 1026®) in the right ventral colon andin the faeces of horses. Eight adult geldings,allotted into homogeneous pairs based onweight, were fed a high fibre diet (35% straw/65% pellets) supplemented daily with Sc at 0 –4.5 × 108 –9 × 108 or 4.5 × 109 cfu·kg–1 DMintake, according to a 4 × 4 Latin square design.Colonic contents and faeces were collected fromhorses at 0, 3, 6, 9, 12, 24, 36, 48, and 72 h afterthe first and the last meal supplemented with Scand yeast concentrations were determined. Scwas detected 3 h and 9 h after the first supple-mented meal, in the colon and in faeces, respec-tively. Peak concentrations were obtainedbetween 3 h and 12 h in the colon (from 5.7 ×103 to 190 × 103 cfu·mL–1) and at 24 h in the fae-ces (from 8 × 102 to 780 × 102 cfu·g–1). First datafor the disappearance kinetics confirm that Sc donot colonize the digestive tract, being no longerdetected after 48 h in the colon and 72 h in the

S96 Session IV: Nutritional manipulation

faeces. Knowing the digestive fate of Sc in thecolon will explain more precisely their action onthe microbial communities. Furthermore, it willcontribute to assessing the optimal daily doseof Saccharomyces cerevisiae CBS 493.94 forhorses.

P-168 Shifts of rumen microbial populationdetected by real-time PCR when methano-gens are inhibited. Y.Q. Guoa, J.X. Liua, W.Y.Zhub (a Institute of Dairy Science, Zhejiang Uni-versity, Hangzhou 310029, PR, China, b Labo-ratory of Gastrointestinal Microbiology, Nan-jing Agricultural University, Nanjing 210095,PR China).

Ruminal microbiologists have long sought todevelop strategies for the enteric methanogene-sis especially within the rumen ecosystem. How-ever, most methane-inhibiting strategies per-formed different in vivo and in vitro, and theunderlying influence on the microbial flora isunclear as well. Real-time PCR was conductedto quantify the rumen microbes including meth-anogens, total bacteria, Fibrobacter succino-genes, Ruminococcus flavefaciens and fungi inthe rumen inoculum in vitro when methane syn-thesis was inhibited. The primer pairs of thosemicrobes and PCR conditions referred to Den-man and McSweeney’s work. Bromoethanesul-fonic acid (BES), a known inhibitor of metha-nogenesis, was selected as agent. The sodiumsalt of 2-BES added at a concentration of 5 mMinhibited methane production by 85.7% after24 h incubation (P < 0.01). Total VFA concen-tration and molar proportions of acetate, propi-onate and butyrate were significantly affected byBES. Compared with the control, the molar pro-portion of acetate and the acetate-to-propionateratio were reduced to 91.8% and 76.1% (P <0.01), and the proportions of propionate andbutyrate were increased by 20.9% and 15.0%(P < 0.01), respectively. With BES addition,production, utilization and recovery of hydrogenwere significantly reduced (P < 0.01). The quan-tity of methanogens in relation to bacteria inBES-added fluid reduced to 10.4% of the valueof control fluid (P < 0.01). When methanogenswere inhibited, the rumen microbial fauna estab-lished a new balance to utilize hydrogen. After24 h incubation, Fibrobacter succinogenes, oneof the major bacteria for fiber degradation, was50.0% higher in BES-treated fluid than control

(P < 0.01). The growth of fungi relative to bac-terial growth fell by 61.9% (P < 0.01) in com-parison with the control. However, the BES didnot significantly influence the total number ofbacteria and the growth of Ruminococcus flave-faciens. In summary, addition of BES couldchange the rumen microflora and have a differ-ent influence on different microbes by inhibitingthe methanogens.

P-169 Effects of urea and controlled releasenon-protein nitrogen on ruminal fermenta-tion and microbial protein synthesis inrumen-simulating cultures. G.A. Harrison,J.M. Tricarico, K.A. Dawson (Alltech, Inc.,Nicholasville, KY, USA).

Twelve rumen-simulating cultures were used toinvestigate the effects of two concentrations andtwo sources of non-protein nitrogen (NPN) onruminal fermentation and microbial protein syn-thesis. Sources of NPN were either urea or Opti-genII® (Alltech Inc., Nicholasville, KY, USA)and the concentrations tested were equivalent to50 or 125 g NPN·day–1 at a dry matter intake of22.7 kg·day–1. Cultures were fed a 50:50 for-age:concentrate diet twice daily for six days. Alldiets were isonitrogenous and treatments wereapplied in premixes. Samples were collectedprior to morning feeding during the last 3 daysof the experiment for pH, ammonia, VFA, DMdisappearance, and microbial protein synthesisdeterminations. Data were analyzed as a com-pletely randomized design and orthogonal con-trasts used to test the effects of NPN source,NPN concentration, and their interaction. Flowrate differed (P < 0.05) among treatments andwas used as a covariate for all analyses. Culturefluid pH, ammonia, and DM digestibility (appar-ent or true) were not affected by treatment. Theconcentration of NPN did not affect bacterialyield or efficiency of microbial protein synthe-sis. However, feeding OptigenII® increased (P <0.10) bacterial yield (0.213 vs. 0.274 g for 50g·d–1 urea or OptigenII®; and 0.149 vs. 0.239 gfor 125 g·d–1 urea or OptigenII®) and improved(P < 0.10) the efficiency of microbial protein syn-thesis (17.2 vs. 20.1 g bacterial N·kg–1 DMTD for50 g·d–1 urea or OptigenII®; and 13.0 vs. 18.2 gbacterial N·kg–1 DMTD for 125 g·d–1 urea orOptigenII®). Compared to urea, cultures feddiets containing OptigenII® had an average of

Session IV: Nutritional manipulation S97

42% greater bacterial yield and 27% improvedefficiency of microbial protein synthesis.

P-170 Effect of allicin on fermentation andmicrobial populations in the rumen simulat-ing fermentor Rusitec. K.J. Hart, S.E. Girdwood,S. Taylor, D.R. Yáñez-Ruiz, C.J. Newbold (TheInstitute of Rural Sciences, University of WalesAberystwyth, Aberystwyth, SY23 3AL, UK).

Garlic (Allium sativum) has been used as amedicinal herb in human and animal feeds formany years. However, there is a lack of detailedinformation on the effects of garlic and its com-ponents in such systems. Here an experiment isreported in which the effects of a commercialallicin extract (Neem Biotech), prepared fromgarlic, on fermentation in the rumen simulationtechnique (Rusitec) was investigated. Allicinwas introduced to each vessel at 0, 3 or 30 mg·d–1

over a period of 17 d. Samples were taken foranalysis over the last 5 d. There was no signifi-cant effect of allicin concentration on daily ace-tate, propionate, butyrate or ammonia productionwith mean values of 18, 7, 8 and 44 mmol·d–1,respectively. However, there was a significant(P < 0.05) effect on daily methane productionwith mean values of 4.1, 3.0 and 0.2 mmol·d–1

(s.e.d. = 1.16) for 0, 3 and 30 mg allicin·d–1,respectively. Microbial DNA was extractedfrom residual feed and vessel liquor homoge-nates, and amplified by qPCR using bacterialuniversal and methanogen specific primers.There was no significant difference of allicinconcentration on the number of cycles to thresh-old (CT) for total bacteria with a mean value of14.3 CT. However, there was a significant (P <0.05) difference in CT for methanogenic archaea,with mean values of 22.1, 23.4 and 24.6 CT(s.e.d. = 0.70) for 0, 3 and 30 mg allicin·d–1

respectively. Preliminary results from this exper-iment have shown that allicin had no effect onfermentation parameters with the exception ofmethane production. It is concluded that allicinhad a selective effect on reducing the number ofmethanogens but had no effect on the total bac-terial population.

P-171 Recovery and phylogenetic analysis ofthe fumarate reductase gene from the bovinerumen. K. Hattori, H. Matsui (Faculty of Biore-sources, Mie University, Tsu 514-8507, Japan).

Supplementation of fumarate is one of the waysto reduce methane emission from the rumen.Fumarate reducing bacteria utilize hydrogen toreduce fumarate and compete for hydrogen withmethanogens in the rumen. Therefore, it isimportant to gain knowledge about fumaratereducing bacteria to enhance fumarate reduc-tion. However, knowledge on the ecology offumarate reducing bacteria in the rumen is lim-ited. The fumarate reductase gene (frdA) can bea marker gene for fumarate reducing bacteria. Inthis study, we newly developed a set of specificprimers that amplify the frdA fragment andexamined the diversity of frdA in the rumen. Aspecific primer set for the fumarate reductasegene (frdA) was newly developed from selectedfrdA sequences. By using the primer set, a clonelibrary was constructed from DNA extractedfrom cow rumen and phylogenetically analyzed.Thirty-one clones were obtained and analyzedfor DNA sequences. Deduced amino acidsequences were subjected to homology searchand phylogenetic analysis. Twenty sequencesshowed the highest homology to frdA of Pas-teurella multocida at 78% identity and weregrouped into a cluster on a phylogenetic tree.Seven sequences showed the highest homologyto Proteus vulgaris frdA at 85% identity andwere grouped into a cluster. The rest of thesequences showed homology to succinate dehy-drogenase (sdh) and were clustered with sdhsequences of various bacteria. All frdA-likesequences obtained were distantly related tofrdA sequences from known bacterial species.These results suggest the presence of unculturedfumarate reducing bacteria in the rumen.

P-172 Bellis perennis tested as new antiproto-zoal feed additive in vivo achieved a persistentpartial defaunation in sheep. E.M. Hoffmanna,N. Seljea, R.J. Wallaceb, K. Beckera (a Univer-sity of Hohenheim, 70599 Stuttgart, Germany,b Rowett Research Institute, Bucksburn, Aber-deen AB21 9SB, UK).

The EU project Rumen-up aimed to identify nat-ural plant additives which may replace growthpromoting antibiotics in ruminant nutrition. Inthis context an antiprotozoal activity was dem-onstrated in vitro for Bellis perennis (daisy). Tovalidate this observation in vivo, eight fistulatedsheep were fed a control and a treatment diet

S98 Session IV: Nutritional manipulation

with 5% inclusion of grass meal or Bellis peren-nis, respectively, in a cross-over design over atotal period of 60 days. Rumen fluid sampleswere withdrawn regularly and analysed for gen-eral fermentation parameters and protozoacounts. Effects observed in response to the addi-tive were an increase in branched SCFA concen-tration (1.40 vs. 1.50 mM, P = 0.024) and a trendto a higher concentration of soluble protein (0.35vs. 0.40 mg·mL–1, P = 0.098). Total SCFA con-centration (97.1 vs. 94.9 mM) and ammoniumconcentration (14.3 vs. 15.2 mM) were not dif-ferent (P > 0.1). Protozoal numbers showed adaily fluctuation pattern in response to feedingand high variation within and between the ani-mals. Nevertheless, Bellis perennis significantlyreduced the numbers of protozoa (2.20 vs. 1.76 ×106·mL–1, P = 0.031), irrespective of thesequence in which the diets were fed. This cor-responds to a partial defaunation of 20%. Asdaisy was shown to contain saponins, these arelikely to be the defaunating agent. In contrast toother published reports, where antiprotozoaleffects of plant derived saponins were only tran-sitory, the daisy effect persisted in vivo for morethan 8 weeks.

P-173 Coarse structured feed stimulatesmembers of the genera Lactobacillus and Mit-suokella as well as propionate and butyrateproducers in the pig stomach. O. Højberg, L.L.Mikkelsen, B.B. Jensen (Danish Institute ofAgricultural Sciences, Research Centre Foulum,8830 Tjele, Denmark).

Feeding coarse structured diets has been shownto stimulate bacterial production of lactate,butyrate and propionate in the pig stomach andthereby reinforce the stomach as a barrier againstspreading of pathogens and zoonoses to distalparts of the gastrointestinal tract. However,detailed information on the influence on themicrobial community is still scarce. TerminalRestriction Fragment Length Polymorphism(T-RFLP) analysis involving multiple restric-tion enzymes was used to investigate the effectof diet on bacterial community structure in gas-trointestinal samples collected from twenty-fourcrossbreed Danish Landrace × Yorkshire pigsfed either a fine non-pelleted (FNP), fine pel-leted (FP), coarse non-pelleted (CNP) or coarsepelleted (CP) diet. A strong dietary influence on

bacterial community structure was observed inthe pig stomachs, whereas the effect was lesspronounced in the ileum, cecum and colon. Pigsfed the CNP diet had the highest microbial diver-sity in the stomach as revealed by a highernumber of terminal-restriction fragments (T-RFs).By comparison to 16S rRNA gene sequencesobtained from a pig bacteria culture collectionand GenBank, the phylogenetic inference of theT-RFLP analysis suggested that phylotypes ten-tatively identified as Lactobacillus delbrueckii,L. mucosae, L. reuteri, L. amylovorus, Mitsuokellajalaludinii, Megasphera elsdenii (butyrate pro-ducer) and Selenomonas ruminantium (propion-ate producer) were stimulated in the stomach ofpigs fed the CNP diet. T-RFLP analysis of iso-lates from the pig bacteria culture collectionshowed good agreement between predicted andobserved T-RFs. These results further verifiedthe tentative identifications of the T-RFs fromstomach and demonstrated that dominatingmembers of the microbiota could be distin-guished by using multiple restriction enzymes.

P-174 Fatty acid composition of rumen pro-tozoa: a link with chloroplast ingestion? S.A.Huws, E.J. Kim, M.R.F. Lee, A.K. Kingston-Smith, N.D. Scollan (Institute of Grassland andEnvironmental Research (IGER), Plas Gogerd-dan, Aberystwyth SY23 3EB, UK).

Recent data show that rumen protozoa are richin beneficial polyunsaturated fatty acids (PUFA)and monounsaturated fatty acids (MUFA), com-pared to rumen bacteria. Evidence suggests thatcertain rumen protozoa are apt at ingestingPUFA-rich plant chloroplasts, and we hypothe-sise that this may contribute towards their higherPUFA content. To evaluate this hypothesissteers fitted with rumen cannulae were offeredeither hay (low chloroplast content) or freshgrass (high chloroplast content) for 2 week peri-ods, before collecting liquid and solid associatedbacteria (LAB and SAB, respectively) and pro-tozoal (LAP) fractions at various time intervalspre- and post-feeding. Fatty acid data revealedthat the protozoa contained c. × 2–3 more C18:0(stearic acid), C18:3 n-3 (α-linolenic acid), andC18:1 trans-11 (TVA) than LAB and SAB pop-ulations at all time intervals and under both feed-ing regimes. The protozoa also contained c. × 2–3 more α-linolenic acid at all time intervals whenfed fresh grass as opposed to hay. Protozoal

Session IV: Nutritional manipulation S99

chlorophyll content was also c. × 2–3 higher, atall time intervals, on the grass compared to haydiet. Fresh grass is known to contain ca. 55% oftotal fatty acid in the form of α-linolenic acid,thus it is unsurprising that the main increases inprotozoal PUFA content under a fresh grass dietoccurred with α-linolenic acid. The increases inα-linolenic acid along with concomitant increasesin protozoal chlorophyll content provides evi-dence that rumen protozoa may be rich in PUFAdue to the ingestion of chloroplasts. Confocalmicroscopy also revealed that Epidinium spp.were saturated with seemingly intact, intracellu-lar chloroplasts as opposed to the other specieswhich contained very few (< 5). This evidencesupports the hypothesis that ingestion of chloro-plasts by protozoa increases their PUFA content.The co-interactions of the rumen ciliates withchloroplast are now the focus of further investi-gation.

P-175 Effect of forage type and level of fish oilinclusion on bacterial diversity in the rumen.S.A. Huwsa, M.R.F. Leea, S. Muetzelb, R.J.Wallaceb, N.D. Scollana (a Institute of Grasslandand Environmental Research (IGER), Plas Gog-erddan, Aberystwyth SY23 3EB, UK, b RowettResearch Institute, Bucksburn, Aberdeen AB219SB, UK).

The fatty acid composition of ruminant productsis of particular concern due to the link betweenfatty acid composition of dietary fat and cardio-vascular disease. Ruminant products such asmilk and meat are often criticised for high pro-portions of saturated fatty acids (SFA) comparedto polyunsaturated fatty acids (PUFA). Thislargely reflects the extensive biohydrogenationof dietary PUFA in the rumen by specific bacte-ria. Much attention is now focused on enhancingour understanding of the microbial speciesinvolved in biohydrogenation, with a view toestablishing methods of reducing this process.Feeding red clover and fish oil in the ruminantdiet has been demonstrated to decrease biohy-drogenation in the rumen. It is uncertain if thisrelates to changes in the bacterial community.Eight steers prepared with ruminal and duodenalcannulae were offered either red clover or grasssilage with 0, 1, 2, and 3% fish oil in a Latinsquare 3-period changeover design. Following2 weeks on each diet, ruminal digesta sampleswere collected and processed into liquid and

solid associated fractions (LAB and SAB,respectively). Bacterial PCR-DGGE was thenperformed on 16S rRNA sequences amplifiedfrom these samples. Dendograms revealed thatthe profiles for both LAB and SAB clustered dis-tinctly due to the type of silage fed, and that,within these diet-dependent clusters, sub-clus-ters were evident according to the fish oil con-centration. It is evident that feeding red clovercaused differences in the rumen bacterial popu-lations in comparison to feeding grass silage.Fish oil inclusion also caused a shift in the rumenbacterial diversity. We now intend to quantifythe bacteria that we currently know are involvedin the biohydrogenation pathway using qPCR.

P-176 Monitoring of bioactive peptides iso-lated from artisanal Balkan yogurts. I. Ivanovaa,I. Tzvetkovaa, N. Tzekovaa, M. Dalgalarrondob,T. Haertleb (a Biological Faculty, Sofia Univer-sity, 8 Dr Tzankov, bd 1164 Sofia, Bulgaria,b INRA-LEIMA, 44316 Nantes Cedex 3,France).

The bioactivities of peptides encrypted in majormilk proteins are latent until released and acti-vated by enzymatic proteolysis, e.g. during gas-trointestinal digestion or food processing.21 LAB strains isolated from the three differenttypes of Balkan artisanal yogurts cow, sheep andbuffalo, were used. Strains isolated from arti-sanal fermented dairy products represent partic-ular interest as a source of new potential startersfor application in the food industry. The presentwork quantifies the various products of LAB fer-mentations relative to the inhibition of St. aureusand E. coli and evaluated the interaction betweenthe isolated LAB and food pathogens in spoiledmilk products. The proteolytic activity was eval-uated for α-caseins and whey proteins by reversephase chromatography, PAGE and anti-micro-bial assays. A food derived bioactive peptideobtained upon the action of an artisanal strain ofLb. delbrueckii on α-casein is claimed to be ahealth-enhancing component which could beused for functional foods and pharmaceuticalpreparations.

P-177 Characterization and possible probi-otic influence of Bacillus smithii strainLTN074 in a murine model. E. Jõgi, I. Suitso,E. Talpsep, P. Naaber, A. Nurk (Institute of

S100 Session IV: Nutritional manipulation

Technology University of Tartu Nooruse St 1,50411 Tartu, Estonia, United Laboratories ofTartu, University Clinics, Puusepa 1a, 50406,Tartu, Estonia).

Bacillus sp. spores based probiotics are wide-spread, especially in South East Asia, but theprophylactic effect of this kind of probiotics isstill not very clear. The purpose of the presentstudy was to test survival of Bacillus smithiistrain LTN074 in the gastrointestinal environ-ment of mice and hamsters as well as its influ-ence on infection caused by invasive Salmonellaenterica serotype Enteritidis and Clostridiumdifficile. Our strain Bacillus smithii LTN074 wasisolated from human faeces. Typical Bacillussmithii strains are thermophilic lactic acid bac-teria usually found as contaminants in the foodindustry. Due to its flexible metabolism (ther-mophilic and aerotolerant growth ability) it isquite easy to detect and differentiate this strainfrom the other gut habitants. We have tested theinfluence of this strain against two enteropatho-gens, Clostridium difficile and Salmonellaenterica serotype Enteritidis, in a murine model.We have also combined antibiotics and othermetabolically active substances to test the pro-biotic influence of our strain. We used the mouseand hamster model to determine the colonisationrate of B. smithii and possible prevention ofpseudomembranous colitis and salmonellosis.B. smithii LTN074 was shown to persist in thegastrointestinal tract of mouse and hamster.

P-178 Changes in rumen pH and populationdensity of Streptococcus bovis in sheep duringdietary transition. F.M. Jonesa, N.D. Costaa,K.R. Guestb, P.E. Vercoeb, R.H. Jacobc, J.M.Snowdenc, A-D.G. Wrightd (a School of Veter-inary and Biomedical Sciences, Murdoch Uni-versity, Murdoch, WA 6150, Australia, b TheUniversity of Western Australia, Nedlands, WA,6009, Australia, c Department of Agriculture,South Perth, WA 6151, Australia, d CSIRO Live-stock Industries, Floreat, WA 6014, Australia).

Changes in pH and the relative population den-sity of lactic acid producing bacteria, such asStreptococcus bovis, have not been well docu-mented for ruminants during transition ontograin-based diets. Real time quantitative PCR(RT qPCR) was used to quantify the relative pro-

portion of S. bovis in rumen samples from sheepfitted with rumen cannulas. These sheep werefed diets based entirely on either lucerne hay(Medicago sativa), yellow lupins (Lupinusluteus), soy beans (Glycine hispida) or whitelupins (Lupinus angustifolius) at maintenance(M) ME or at 3 × M. The change in proportionof S. bovis (as cells·mL–1) after the first 24 h onfeed was 3900-fold in sheep fed 3 × M, 1300-fold in sheep fed soybeans, but only 50-fold onthe lucerne diet. At these times, rumen pHdecreased to 6.05 in sheep fed 3 × M whitelupins, and to 6.3 on the lucerne diet, but rumenpH in the sheep fed soybeans barely changed to7.02. Thus the changes in the proportion ofS. bovis were not related to the pH in the rumenenvironment. Moreover, lupins contain β-glu-cans, and are thought to decrease the predispo-sition to acidosis associated with the rapidly fer-mentable starch in cereal grains. These findingscall into question the sequence of events leadingto large increases in S. bovis, and the precise rela-tionship between increases in S. bovis andchanges in pH.

P-179 Determination of the effective dose ofSaccharomyces cerevisiae CBS 493.94 used asmicrobial additive for horses. J.-P. Jouanya,B. Medinab,c, V. Julliandb, G. Bertinc (a INRA,Centre de Clermont-Ferrand-Theix, 63122Saint-Genès-Champanelle, France, b ENESAD,26 bd Dr Petijean, 21000 Dijon, France, cAlltechRegulatory Department, 14 place Marie-JeanneBassot, 92300 Levallois-Perret, France).

Live yeasts, which are actually recommended asmicrobial additive for herbivores such as rumi-nants and horses, do not grow in the digestivetract and have to be supplied continuously to thehost. The objective of the present work was toassess the optimal daily dose of Saccharomycescerevisiae CBS 493.94 (Sc) (Yea-Sacc 1026)for horses. Eight adult geldings weighing 383 ±9 kg were fed twice a day 670 g DM 100 kg–1

BW of a pelleted diet of dehydrated lucerne(55%), wheat bran (25.5%), barley (13%),molasses (2.5%) and a mineral-vitamin mixture(3.5%). Long wheat straw was given on a dailybasis of 340 g DM 100 kg–1 BW in one meal.Animals, allotted into homogeneous pairs basedon weight, were assigned to a 4 × 4 Latin squaredesign, fed twice a day with 4 doses of Sc (0;

Session IV: Nutritional manipulation S101

4.1 × 109; 8.3 × 109; 1.65 × 1010 cfu·kg–1 feedDM) top-dressed on the diet. The experimentlasted 170 days planned over 4 periods of adap-tation (21 days), measurements (7 days) andwash out period (15 days). Digestibility of DM,OM and fibre was determined based on a totalfeces collection. The supplementation of Scregardless of the dose had no significant effect(P > 0.10) on feed intake, animal weight or fecesdry matter. Positive and significant effects wereobserved on digestibilities of dry (+5%) andorganic matter (+3.5%) (P < 0.10), and ondigestibilities of NDF (+8%) and ADF (+12%)(P < 0.05) for the two doses of 8.3 × 109 and1.65 × 1010 cfu·kg–1 feed DM, which were notdifferent. No effect was noted when Sc were fedat the dose 4.1 × 109 cfu·kg–1 feed DM. Theseresults indicate that the minimum effective doseof Saccharomyces cerevisiae CBS 493.94 forhorses weighing less than 400 kg is 8.3 ×109 cfu·kg–1 feed DM. Doubling of the minimaldose had no effect on the measured digestiveparameters.

P-180 Dietary essential oil supplementationenhanced intestinal immunocompetence inyoung broiler chicks. H. Kettunena, A. Ouwe-handa, H. Schulzeb, N. Rautonena (a Enteromix®

Research, Danisco Innovation, Kantvik, Fin-land, b Danisco Animal Nutrition, Leiden, TheNetherlands).

Essential oils (EOs) may act as antimicrobials,antioxidants, and immune enhancers in animals.Thus, EOs may support gut health of farm ani-mals in antibiotic-free nutrition programs. Theconcentration of immunoglobulin A (IgA) in thegut lumen can be used as a measure of the intes-tinal immunocompetence. We studied the effectof EOs on the performance and intestinal IgA inmale Ross 508 broiler chicks. Newly hatchedchicks were allocated to six feeding treatments(12 pens per treatment; 30 chicks per pen).Wheat-based mash diet without antibiotics, coc-cidiostats, or enzymes was supplemented withfive different EO blends. Chick performancewas measured for days 1–21 and 21–42. On days21 and 42, we sampled the ileal and caecal con-tents (six replicate pools of two chicks per diet).Ileal and caecal IgA was measured from the con-trol and two EO blend treatments (“low EOlevel” and “high EO level”) by ELISA. Com-pared with the control group, the “low EO level”

diet significantly improved weight gain andFCR, but did not affect the ileal or caecal IgA ateither time point. The “high EO level” dietincreased the IgA concentration on day 21 (P =0.009 for caecum, P = 0.051 for ileum), andshowed a non-significant trend for improvedperformance at both time points. The IgA con-centrations increased from day 21 to 42. On day42, all the dietary treatments showed similar IgAconcentrations. The results suggest that dietaryessential oil supplementation may improveintestinal immunocompetence and performanceof young broiler chicks, and that the effect islikely dependent on the quality and quantity ofessential oils.

P-181 Probiotics in treatment of patients withCrohn’s disease and ulcerative colitis. P.Kohouta, D. Tuèekb, V. Zboøilc, Z. Beneša

(a IInd Department Internal Medicine, TeachingThomayer’s Hospital, Prague, Czech Republic,b Department of Metabolic Care, Charles Uni-versity Hospital, Hradec Králové, Czech Repub-lic, c IIIth Department Internal Medicine, Teach-ing Hospital Brno, Czech Republic).

Abstract withdrawn.

P-182 The influence of dairy season on thesafety of sheep milk. M. Ladyková (SlovakUniversity of Agriculture, Faculty of Biotech-nology and Food Science, Slovak Republic).

The aim of this research was to determine theinfluence of the dairy season on sheep milksafety. Bulk samples of sheep milk were col-lected from May to September 2004 from themiddle part of Slovak Republic. The analysisinvolved eight samples from each factory in var-ious stages of the dairy season. Milk analysesincluded determination of composition, techno-logical quality, hygienic and microbial quality.The average value of somatic cell counts (SCC)was ranging from 67 750 mL–1 at the beginningof the dairy season to 416 125 mL–1 at the endof the dairy season, and the difference was sig-nificant (P < 0.01). The total bacteria count(TBC) in milk samples ranged from 1.47 ×106 cfu·mL–1 (beginning of dairy season) to9.55 × 105 cfu·mL–1 (end of dairy season). A

S102 Session IV: Nutritional manipulation

similar tendency of lower numbers was foundfor coliform bacterial counts, with an averagevalue at the beginning of dairy season of 1.01 ×106 cfu·mL–1 and at the end of dairy season1.72 × 104 cfu·mL–1. The average value for psy-chrotrophic bacteria was from 9.18 ×105 cfu·mL–1 (beginning of dairy season) to8.23 × 106 cfu·mL–1 (end of dairy season).

P-183 Diet modulates resilience of the gut eco-system in human microbiota associated rats.F. Levenez, V. Rochet, M. Sutren, S. Droineau,A. Foussié, P. Guillaume, S. Rabot, M. Leclerc,J. Doré (INRA-UEPSD, 78350 Jouy-en-Josas,France).

Human gut microbiota appears essentiallyunique, specific to the host and stable over timeat the level of dominant species. It is resilientupon antibiotic treatment in human volunteers.In the present study we investigated the impactof minor perturbations and diet on resilience ofthe gut ecosystem. Sixteen human microbiotaassociated rats received a stable diet (basal orinulin 10%) throughout the study (84 d) or a dietwith minor perturbations (basal versus inulindiet alternated every 2 d) followed by stable dietafter the major perturbation (single dose of200 mg·kg–1 amoxicillin and clavulanic acid,administered to each animal on day 32). Domi-nant diversity of microbiota was followed usingTTGE and its composition using FISH. Themicrobiota was affected by antibiotic treatmentwithin one day and tended to return to its initialstructure within 1–2 d (dominant diversity) to upto 24 d (composition). Enterobacteria were dra-matically increased one day after antibiotictreatment, and the increase was statistically sig-nificant for 3 d. Firmicutes (C. coccoides group)were markedly decreased for 10 d while Bacter-oides were initially decreased and thereafterincreased until day 24. In the absence of minorperturbations, resilience was not affected by thediet. With minor perturbations before antibiotictreatment, resilience was affected by the dietsuch that inulin promoted a significantly fasterreturn to initial dominant diversity (1 d) and toEnterobacteria initial levels. Effects of dietaryperturbations observed on microbiota composi-tion using FISH depended on diet and microbialgroup. Our results indicate that inulin favorsresilience of the gut microbiota after antibiotictreatment. Functional foods should be further

investigated in their ability to modulate resil-ience of the gut ecosystem upon stress.

P-184 Dietary carbohydrate source deter-mines molecular fingerprints of the rat fecalmicrobiota. T.R. Licht, M. Hansen, M. Poulsen,L.O. Dragsted (Danish Institute for Food andVeterinary Research, Mørkhøj Bygade 19, 2860Søborg, Denmark).

A study was designed to elucidate effects ofselected carbohydrates with different digestibil-ity on the intestinal physiology and microbiota.Five groups of eight rats were fed a western typediet containing cornstarch (reference group),sucrose, potato starch, Raftiline® (a long-chained fructan) or Raftilose® (a short-chainedfructan). The animals fed the diets containingpotato starch, Raftiline and Raftilose had a sig-nificantly higher caecal weight and lower caecalpH when compared to the reference group, indi-cating increased fermentation. Selective cultiva-tion from faeces revealed a higher amount ofLactobacillus spp. in these animals. Addition-ally, the fructan-fed groups had a lower amountof coliform bacteria in faeces. In the Raftiline-and Raftilose-fed groups, higher levels ofbutyrate and propionate, respectively, weremeasured. Principal Component Analysis ofprofiles of the fecal microbiota obtained byDenaturing Gradient Gel Electrophoresis (DGGE)of PCR amplified bacterial 16S rRNA genes aswell as of Reverse Transcriptase-PCR amplifiedbacterial rRNA revealed a different microbiotain each of the five animal groups. Comparisonof DNA-based and RNA-based profiles revealedthat 2 species within the phylum Bacteroideteshad a particularly high ribosome content in theanimals fed with Raftiline, indicating thatgrowth of these species was specifically stimu-lated by this particular fructan.

P-185 Effect of gas pressure and buffer com-position on in vitro ruminal fermentation.D. Macheboeuf, B. Lassalas, R. Bergeault,D.P. Morgavi (INRA CR de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France).

In vitro fermentation is a rapid and efficient wayto evaluate ruminant’s feedstuffs. However,comparison among published works is difficultbecause of variations in the methodology. The

Session IV: Nutritional manipulation S103

most common variations are buffer compositionand whether the produced gas is vented or not.The aim of this study was to assess the effect ofa buffer developed for protozoa and the effect ofgas pressure build-up in an in vitro batch system.The buffers tested were: (protozoa) Simplexmedium (SIM) (carbonate/phosphate ratio1.52 mol·mol–1) from Coleman and Goering-Van Soest medium (GVS, carbonate/phosphateratio 5.45 mol·mol–1). Fermentation vials con-tained 25 mL buffer, 15 mL rumen fluid and400 mg of substrate composed of corn, soja beanmeal, and alfalfa hay. Incubation was for 16 h at39 °C under N2 gas. At 5h-incubation, aftermeasuring gas production, half of the bottleswere depressurized to atmospheric pressure.Depressurization had no significant effect onfermentation parameters and there was no inter-action between pressure and media, althoughhydrogen utilization was slightly higher withhigh pressure. GVS incubations produced moregas (10.7 vs. 9.7 mmol·g–1 DM, P < 0.01) andmethane (3.0 vs. 2.78 mmol·g–1 DM, P < 0.05)than SIM, while no differences were observedon fatty acid production. In addition, the chem-ical CO2 production and hydrogen utilizationwere lower (P < 0.01) with SIM. These differ-ences could be explained by a more efficienthydrogen fixation in SIM due to its higher phos-phate concentration. The dry matter digestibilitywas higher with GVS (+3.5 points, P = 0.011)and the pH lower (6.18 vs. 6.30, P < 0.01). GVSbuffer seems more appropriate than SIM for rou-tine evaluation of feedstuffs for ruminants.

P-186 Dose-response effect of diallyl disulfideon ruminal fermentation and methane pro-duction in vitro. D. Macheboeufa, B. Lassalasa,M.J. Ranillab, M.D. Carrob, D.P. Morgavia(a INRA-Theix, 63122 Saint-Genès-Champan-elle, France, b Producción Animal I, Universi-dad de León, 24071 León, Spain).

Ruminal methanogenesis is an inefficient pro-cess resulting in losses of energy intake. The aimof this study was to assess in vitro the dose effectof diallyl disulfide, a compound present in garlicessential oil, on ruminal fermentation, with anemphasis on methane production. Ruminal con-tents collected from sheep fed concentrate:alfalfahay (40:60) diet were incubated at 39 °C inbuffer for 16 h with a mixed substrate. Diallylwas added at concentrations of 0, 0.5, 1, 1.5, 2,

2.5, and 4 mM in the batch mixture. Treatmentswere in triplicate. Diallyl, at the lower dose used,drastically decreased the amount of methaneproduced by 90% as compared to 0 control (P <0.001). Methane proportion of the total gas pro-duced fell from 28% in control to 3.4% with adose of 0.5 mM. This was associated with anincrease in the hydrogen ratio in the gas phasefrom 0 to 15.3% (P < 0.001). Total gas produc-tion, although also significantly reduced, wasless affected by the additive (from –18 to –31%compared to control). Total VFA productionwas also linearly reduced from –33 down to–55% for the lower and higher dose, respectively(P < 0.001). Acetate was the most affected, witha fall of 50% at 0.5 mM, while butyratedecreased from –8 up to –17% for the 0.5 and2.5 mM dose, respectively. In contrast, propi-onate production was not different from the con-trol up to the 2.5 mM concentration. Interest-ingly, dry matter digestibility was not affectedwith 0.5 mM and then decreased with dose from7 to 12% (P < 0.01). Diallyl ability to reducemethane production merits further studies.

Supported by Acción Integrada HF2004-0026 (MECof Spain) and Action integrée Picasso (EGIDE ofFrance).

P-187 Combination of feed additives to meetproduction and environmental targets in rumi-nants – in vitro optimization. D. Macheboeufa,M.J. Ranillab, M.D. Carrob, M.L. Tejidob,B. Lassalasa, D.P. Morgavia (a INRA-Theix,63122 Saint-Genès-Champanelle, France, b Pro-ducción Animal I, Universidad de León, 24071León, Spain).

Feed additives for ruminants, normally used toimprove production, could also serve to reducethe emission of environmental pollutants likemethane and ammonia. However, the mode ofaction of individual additives often limits theireffect to a particular objective. The aim of thisstudy was to optimize a mixture of additives thatact concurrently on several fermentation param-eters. Three essential oils (diallyl disulfide (DIA),cinnamaldehyde (CIN), carvacrol (CAR)) and2 organic acids (disodium malate (MAL) andfumarate (FUM)) were tested alone or in com-bination using a simplex centroid experimentaldesign. The vertices of simplex were one com-pound alone (coded as i) at the maximum dose(coded as 1) and the other points in the design

S104 Session IV: Nutritional manipulation

were blends of compounds (1 < i ≤ 5, 0 < dose(i)< 1, Σdose(i) = 1). The additive mixtures wereadded to buffered rumen fluid from sheep con-taining a 50:50 forage:concentrate substrate andincubated in vitro for 16 h with 3 repetitions intime. Fermentation variables were compared tocontrol without additives and fitted to quadraticor special cubic models; R2 for methane, ammo-nia, and volatile fatty acids (VFA) were 0.91,0.54, and 0.58, respectively. When specifica-tions were targeted to one parameter, like reduc-ing methane, ammonia, or maximizing VFAproduction, the major compound of the blendwas DIA, CIN, and MAL-FUM, respectively.When specifications were to keep dry matterdigestibility and VFA production at control lev-els, but reducing methane and ammonia, themodel suggested a mixture (0.60 MAL,0.32 CIN, 0.02 CAR, 0.07 DIA) that decreasedmethane (–20%), ammonia (–15%) and the C2/C3-ratio (–30%). Selected combinations ofadditives can be tailored to a specific type of pro-duction or ration as well as to reduce pollutantemissions.

Supported by Acción Integrada HF2004-0026 (MEC ofSpain) and Action integrée Picasso (EGIDE of France)

P-188 The influence of chestnut tannins ongrowth and enzymatic activities of selectedrumen bacteria. R. Marinšek-Logar, T.

epeljnik (University of Ljubljana, Biotechni-cal Faculty, Zootechnical Department, Dom-zale, Slovenia).

During the last years there has been a generalinterest recognised in tannin use in animal nutri-tion, including ruminants. Tannins can improvethe general performance of ruminants throughtheir influence on rumen microflora. They inter-act with rumen bacteria in different ways: theyinhibit bacterial growth, enzymatic activitiesand attachment to substrates, and inhibit certainpathogenic bacteria and methanogenesis in therumen. As tannins are a broad group of chemi-cally diverse polyphenolic plant compounds,and their influences are strongly dependent onthe nature of the compound and the nature ofbacterial carbohydrates and proteins in interac-tion with it, research is needed on defined bac-terial species and defined tannin products. Theaim of this study was to follow the effects ofchestnut hydrolysable tannins of the commercialproduct Farmatan® on four selected rumen bac-

terial species: highly active fiber degrader Pseu-dobutyrivibrio xylanivorans Mz5 and veryabundant starch and protein degrading rumenbacteria Prevotella bryantii B14, Streptococcusbovis DSMO 4551 and Selenomonas ruminan-tium DSMO 2150 – the two bacteria that areinvolved in rumen acidosis. The effects of dif-ferent concentrations of Farmatan® in the anaer-obic bacterial growth medium (0.00, 0.05, 0.25and 1.00 g·L–1) on bacterial growth and enzy-matic activities (xylanolytic, amilolytic, cellulo-lytic and proteolytic) compared to negative con-trols were followed after 48 h incubation at37 °C. Statistically significant effects of chest-nut tannins were detected at concentrationshigher than 0.25 g tannins·L–1, which is the con-centration of tannins in the cow rumen whenapplied at recommended doses. The growth ofall four investigated bacteria is slightly inhibitedat concentrations higher than 0.25. The mostprofound effect of tannins was found on theamylolytic activity in P. bryantii and S. bovis.The results suggest that the application of chest-nut tannins does not effect the normal ruminalbacteria too much, but it does hinder the fermen-tation of starch in S. bovis, which may be usefulin mitigation or prevention of rumen acidosis.

P-189 Effect of probiotics, prebiotics and syn-biotics as functional food supplements on gutmicroflora of rats. L. Markiewicz, A.Majkowska, M. Bielecka (Institute of AnimalReproduction and Food Research of the PolishAcademy of Sciences, ul. Tuwima 10, 10-747Olsztyn, Poland).

The impact of functional food supplements onthe complex intestinal microecosystem is stillnot enough recognised. The aim of the study wasto assess quantitative and qualitative changes inthe selected groups of colonic microflora of ratsadministered with probiotics, prebiotic and syn-biotics. The research was carried out on Wistarrats (8 groups, 8 rats in each) administered with:potentially probiotic strains of Lactobacillusacidophilus BS and/or Bifidobacterium anima-lis 30, commercial preparation of Raftilose Syn-ergy 1 (OF) (ORAFTI, Belgium) as a prebiotic,and their combinations as synbiotics. The activebacterial cultures were given per os ~ 109 cells/rat·day–1; 5% OF was added to the diet. Thecontrol group was fed with standard diet.After 35 days, selected groups of bacteria were

Č

Session IV: Nutritional manipulation S105

examined quantitatively and qualitatively usingselective cultivation and PCR-DGGE methods,respectively. In the groups of rats receivingLactobacillus and/or Bifidobacterium strains,population counts of these genera increased. Therespective DGGE patterns showed the presenceof both administered strains in the rat colon, withB. animalis 30 as a dominant strain. Probioticsneither affected the eubacteria and Clostridiumcoccoides group DGGE patterns nor the popu-lation numbers of other colonic bacteria deter-mined. The microflora of animals administeredwith prebiotic and synbiotics was characterisedby significantly higher counts and a more diversepattern of the Bifidobacterium population,whilst the Lactobacillus count was stable in spiteof inhibition of some of its subpopulation. More-over, as DGGE profiles showed, prebiotic andsynbiotics stimulated some subgroups of eubac-teria and C. coccoides group, simultaneouslyinhibiting other subgroups of C. coccoides. Theimpact of synbiotics on the Lactobacillus popu-lation was rather individual-dependent. The fur-ther studies on combined supplements will becontinued, as their impact on the microflora maybe a sum of their separate effects.

P-190 Effects of non-antibiotic additives onthe microbial equilibrium of broiler chickenintestine. B. Massiasa, M. Arturo-Schaanb,A.-M. Eliea, K. Bebinb, V. Hocdec, M.Denayrollesa, M.C. Urdacia (a Enitab, LMBA 1,Cours du Général de Gaulle, 33170 Gradignan,France, b CCPA, Z.A. Nord Est du Bois deTeillay, 35150 Janzé, France, c Deltavit, France).

Sub-therapeutic doses of antibiotics have beenused systematically in the poultry industry inorder to promote growth and fight avian patho-gens, thus improving productivity. A rise in bac-terial resistance has constrained Europeanauthorities to ban this practice. The search forsubstitutes has, therefore, become a priority,because animal health and development isstrictly dependent on gastrointestinal micro-flora. The influence of three feed additives onbroiler chicken intestinal microbiota were inves-tigated: Fructooligosaccharidic (sc FOS) prebi-otic (additive I) and two plant aromatic essential-oil extracts (additives II and III). Thirty subjectsper treatment were sacrificed at 21 and 35 daysand samples were collected from intestinal andcaecal contents. Samples were cultured by clas-

sical methods in MRS medium for LAB isola-tion and colonies were subsequently sequencedfor identification of 16S rDNA. In parallel, aDGGE fingerprinting method was applied tocharacterize the predominant bacterial popula-tions. Electrophoresis of amplified fragments of16S rDNA using Lactobacillus genus-specificor universal eubacterial primers produced feedadditive-specific patterns. Dendrometric analy-ses of gel profiles and sequencing of DGGEbands permitted identification of bacteria whosepresence was modulated by the different addi-tives. Approximately twenty different bacteriain four principal genera were identified: Bacter-oides, Clostridium, Lactobacillus, and Rumino-coccus. Variations in bacteria were observedaccording to age and intestinal compartment.Additives I and II gave the greatest variationsespecially within the Lactobacillus population.Compared to FOS, the tested essential-oil extractsalso modulated the gut microflora and so may beinteresting as growth promoters in poultry.

P-191 Role of protozoa and antiprotozoalagents on CLA biohydrogenation in mixedruminal microorganisms. N. McKain, R.J.Wallace (Rowett Research Institute, Bucksburn,Aberdeen AB21 9SB, UK).

Conjugated linoleic acids (CLA) are health-pro-moting 18:2 fatty acids which are formed asintermediates during the biohydrogenation ofdietary linoleic acid (C18:2, cis-9, cis-12) tostearic acid (C18:0) in the rumen. A range ofmaterials was investigated for their ability toinhibit CLA breakdown and thereby enhance theflow of CLA from the rumen and into ruminantproducts, including meat and dairy products.Foliage from Sesbania sesban, a leguminoussubtropical forage tree, was the most potentinhibitor of CLA breakdown. As S. sesban hadbeen previously shown to be antiprotozoal byvirtue of its saponin content, other antiprotozoalplants and saponins were investigated for theirability to inhibit CLA biohydrogenation. Lon-icera japonica (Japanese honeysuckle) decreasedthe rate of CLA metabolism by 50% in a 3-hincubation with strained ruminal digesta. Theinhibition fell to only 20% if the protozoa wereremoved by slow speed centrifugation.L. japonica had little effect on CLA metabolismwhen the experiments were repeated withdigesta from defaunated sheep. The experiments

S106 Session IV: Nutritional manipulation

were repeated with the saponin, glycyrrhizicacid (GA), and its corresponding sapogenin, gly-cyrrhetinic acid (GTA). In faunated sheep, GAdecreased the rate of CLA metabolism by 50%in a 3-h incubation with strained ruminal digesta,whereas the GTA, which as a sapogenin has noantiprotozoal activity, had no effect. There wasno effect of GA or GTA if the protozoa wereremoved by centrifugation. No significant inhi-bition occurred in digesta from defaunatedsheep. Thus, the interaction between antiproto-zoal agents and ciliate protozoa leads to an inhi-bition of biohydrogenation of CLA in ruminaldigesta, possibly via materials released fromprotozoa by lysis.

P-192 Survivability of L. delbrueckii ssp. bul-garicus and S. thermophilus in the human gas-trointestinal tract. M.L. Michaylova, Zh.P.Dimitrov, L. Vlachkova, P. Petrova, (LB Bul-garicum R&D Center, 12A Malashevska Str.,Sofia 1202, Bulgaria).

Twelve healthy volunteers consumed over tendays their usual diet supplemented with 250 gbuffalo’s yogurt. The yogurt was administratedtwice a day (2 × 125 g). L. bulgaricus and S. ther-mophilus had been isolated from Crepis biennislinei, showing high β-galactosidase activity anddemonstrating a positive influence on the fecalmicroflora and metabolites in above human vol-unteer’s tests. Survivability of L. bulgaricus(5.9 ± 0.7 log cfu·g–1 fecal samples) was regis-tered. The strain identity was confirmed bystrain specific oligonucleotide DNA marker.Survivability of S. thermophilus was not regis-tered. This result supports our observations frompreliminary studies to determine the effect ofmilk base (sheep’s or buffalo’s or cow’s) and thestrain specificity for the survivability of yogurtbacteria in the gastrointestinal tract.

P-193 Effects of probiotics on body weightgain, body height, diarrhea occurrence andhealth condition of newborn calves. M.R.Mokhber-Dezfouli, P. Tajik (Department ofClinical Sciences, Faculty of Veterinary Medi-cine, University of Tehran, PO Box 14155-6453,Tehran, Iran).

The effects of probiotic administration werestudied on 120 newborn calves. Sixty calves

were randomly assigned as treatment group andthe probiotic was added in their daily milkintake. Each received 0.25 g probiotics until 90 dof age. After the first week, all calves (includingcontrol group) received starter ration containing21.0% crude protein and 3.0% crude fat. Bodyweight gain, body height and general health con-dition of all calves were observed at d 30, 60 and90. The condition of feces was also examineddaily and the occurrence of diarrhea wasrecorded throughout the experiment. Mean val-ues of weight gain during three successivemonths for treatment and control groups were57.52 and 50.58 kg, respectively. Body weightgained was not significantly different for the firstand second month between treatment and con-trol groups (16.9 and 33.87 vs. 14.49 and 33.07for first and second month in treatment and con-trol groups, respectively). However, these val-ues were significantly different (P < 0.001)between treatment (57.52) and control (50.58)groups at three months of age. Diarrhea wasobserved in 35 calves of the control group, whichwas higher than 11 cases in calves treated withprobiotic (P < 0.001). The body height values ofcontrol and treatment groups in three successivemonths were 5.49, 10.82 and 15.00 cm for thecontrol and 5.44, 9.25 and 15.75 cm for the treat-ment group in the first, second and third monthrespectively, which showed no significant dif-ference between the two groups during thisstudy. The results of this study indicate that theprobiotic compound has beneficial effects, espe-cially on the third month of age in rearing calves.

P-194 Behaviour of live yeast Biosaf® Sc 47during digestive transit in dairy cows. V.Monteilsa, L. Cauquilb, E. Auclairc, C. Bayourthea

(a ENSAT Ave de l’Agrobiopole, BP 32067,31326 Castanet Tolosan cedex, France, b INRAToulouse, SRC, BP 52627, 31326 Castanet-Tolosan, France, c LFA, 1 rue du Haut Touquet,59520 Marquette-Lez-Lille, France).

The knowledge of the behaviour of live yeasts(LY) in the ruminant digestive tract (DT) wasassessed by counting the number of viable LYin the rumen, the ileum and the faeces of fistu-lated dry cows after a single dose (20 g contain-ing 8 × 109 cfu·g–1) along with their morningmeal or after repeated daily doses along with themeal for 3 consecutive days. The transit of LY

Session IV: Nutritional manipulation S107

through the DT was monitored for 4 and 5 days,respectively. The number of viable LY in digestawas determined by the method for counting thecfu on YM agar containing 1% chlorampheni-col. LY appeared at a high level of 1.4 × 105 cfuper mL rumen fluid, 5.3 × 105 cfu per mL ilealcontent and 1.4 × 106 cfu per g in faeces at 2, 4and 16 h after administration of a single dose, i.e.82, 91 and 98% of the dose initially distributed.LY were then gradually eliminated. The numberdecreased more slowly in the faeces than in therumen to a negligible amount of 102 cells per gafter 96 h. When supplementation was repeateddaily, LY persisted over 24 h in the rumen at alevel equal to 68% of the dose administered. Inthe faeces, a concentration of 12 × 102 cfu per gwas found after 128 h. The results indicate thatingested Biosaf® Sc 47 cannot colonize therumen and are not entirely killed under the spe-cific conditions of the DT.

P-195 Quantification by real-time PCR of cel-lulolytic bacteria in the rumen of sheep aftersupplementation of a forage diet with readilyfermentable carbohydrates. P. Mosoni, F.Chaucheyras-Durand, C. Béra-Maillet, E. Forano(Unité de Microbiologie, INRA Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle,France).

The supplementation of forage diets with readilyfermentable carbohydrates is known to depressruminal fiber degradation. The most likelyexplanation for this observation is a decrease inthe number of the cellulolytic flora and/or oftheir fibrolytic activity. The aim of this studywas to examine the effect of concentrate supple-mentation to a forage diet on the number of threerumen cellulolytic bacterial species. Rumencontents were collected from six sheep one hourbefore feeding with hay and one hour before andthree hours after feeding with a hay plus concen-trate (50/50) diet. The pH of the rumen contentswas monitored on both diets. Fibrobacter suc-cinogenes, Ruminococcus albus and Ruminoco-cus flavefaciens were quantified by real-timePCR (SYBR Green, LightCycler) from totalDNA extracted from the rumen contents usingspecies-specific primers. External standardcurves were performed with the 16S rDNA geneof each species (PCR amplified and serialdiluted from 102 to 108 copies) and used for

absolute quantification. The addition of 50%concentrate in the diet of sheep resulted in adecrease (1 Log) of the three cellulolytic speciesjust after feeding concommittent with rumenacidification, but this effect was erased at thepre-prandial state with rumen contents close toneutral pH and a number of cellulolytic bacteriasimilar to that observed on a hay diet.

P-196 Molecular analysis of the intestinalmicrobiota of weaning piglets fed with dietssupplemented with xylo-oligosaccharides.P. Mouraa, F. Simõesa, S. Marquesa, L. Alvesa,F. Gírioa, J.P.B. Freireb, M.P. Estevesa (a INETI,Department Biotechnology, Estrada Paço Lumiar,22, 1649-038 Lisboa, Portugal, b ISA, Depart-ment Agricultural and Animal Production,Tapada da Ajuda, 1349-017 Lisboa, Portugal).

The effects of xylo-oligosaccharides (XOS)obtained by autohydrolysis of corn cobs, on themodulation of piglet intestinal microbiota wereinvestigated. Experimental diets supplementedwith 2% of XOS-rich hydrolysates, with a com-mercial probiotic, or with both XOS and probi-otic, were fed for 4 weeks to 24 weaning piglets(Exp. I) and for 2 weeks to 24 piglets submittedto ileo-rectal anastomosis (Exp. II). Samplesfrom the ileum, caecum and distal colon (Exp. I)and jejunum and ileum (Exp. II) were used forcharacterisation of the intestinal microbiota byERIC PCR fingerprinting and analysis of theLactobacillus population using group-specificprimers. ERIC PCR profiles from Experiment Iand from piglets after weaning (t0) were com-pared with ERIC PCR fingerprints of referencestrains. The dendrogram obtained revealed amajor cluster grouping all the samples from t0,bifidobacteria, lactobacilli, and most of the sam-ples from RB+XOS and RB+XOS+probiotic. Inpartial comparisons from each experimentaldiet, the similarity level between the cluster ofbifidobacteria and lactobacilli and the clustergrouping most of the samples from RB,RB+XOS, RB+probiotic and RB+XOS+probi-otic was 28, 71, 43 and 54%, respectively. WithLactobacillus group-specific primers, the high-est detection limits were obtained with samplesfrom the colon of animals fed with XOS. InExperiment II, the diets including XOS pro-duced an increased positive PCR signal in sam-ples from the ileum, as determined by the use of

S108 Session IV: Nutritional manipulation

Lactobacillus group-specific primers and serialdilutions of total DNA. ERIC PCR profiles fromExperiment II originated three main clusters, butonly one sample from the ileum of a RB+XOSfed piglet was clustered within the group of bifi-dobacteria and lactobacilli.

The authors are grateful to the Commission of theEuropean Communities and the FEDER Funds forfinancial support of this work (Project POCTI/CVT/37545/2002).

P-197 Evaluation of a natural dietary strategyfor controlling the severity of pathogenic pro-tozoal infections in the gastrointestinal tract.L. Nollet, R. Murphy (Alltech European Bio-sciences Centre, Dunboyne, Ireland).

Recent legislation in Europe has led torestrictions on the use of certain pharmaceuticalproducts for the control of protozoal diseases inpoultry feeds. As a result, there is an increasedemphasis on new nutritional strategies that canaddress the production problems associated withsuch diseases. A series of studies evaluated theeffects of new feeding strategies that use a nat-ural blended yeast feed ingredient, NatustatTM ,on two protozoal diseases in poultry. In Eimeriachallenge studies in broilers, the weight gain andfeed conversion in birds fed NatustatTM

(1.925 kg·T–1) was greater (4% and 5%,respectively) than that of the birds receiving anunsupplemented control diet. This performancewas comparable to that of birds receiving aSalinomycin (66 ppm) supplemented diet. Birdsreceiving NatustatTM also had less severelesions in upper and caecal regions. A trialcomparing benefits of NatustatTM supplementationon the improved efficacy of a coccidiosisvaccination (Coccivac B) demonstrated thatbirds fed NatustatTM had significantly better FCRand lower gut lesion scores compared to thechallenged control. In turkey trials using a mildhistomoniasis (“Blackhead”) challenge, thebenefits of NatustatTM at 1.925 kg·T–1 wascompared to an unchallenged control group anda group of birds receiving the antiprotozoal drug,Histostat® (0.1875 kg·T–1). The results indicatethat turkey poults receiving NatustatTM hadweight gains and feed conversion ratios whichwere not significantly different from theunchallenged control birds while caecal andliver lesion were the least severe of all challenged

birds. The results of these studies suggest thatNatustatTM may be useful as part of a non-pharmaceutical strategy to reduce the risk andseverity of intestinal protozoal diseases in poultry.

P-198 DNA stability and transformability offeed derived DNA in the gastrointestinal tractof rats. L. Nordgårda,b, T. Traavika, K.M.Nielsena,b (a Norwegian Institute of Gene Ecol-ogy, Science Park, N9294 Tromsø, Norway,b Department of Pharmacy, Faculty of Medi-cine, University of Tromsø, N9037 Tromsø,Norway).

Considerable amounts of foreign DNA are intro-duced into the gastrointestinal (GI) tract withfood ingestion. Studies have shown that minorproportions of this DNA survive the passagethrough the GI tract and that it can be absorbedto reach white blood cells, the spleen and theliver, or be present in feces (Schubbert et al.,1994). Furthermore, several bacterial speciesthat are present in the GI tract are known to becompetent for natural transformation (Lorenzand Wackernagel, 1994), suggesting that somebacteria can take up food-ingested DNA throughnatural transformation. The use of antibioticresistance genes as marker genes in geneticallymodified organisms has put forward the ques-tion whether such antibiotic resistance genes cantransform bacteria present in the GI tract in vivo.The aims of this study were to describe the deg-radation dynamics of food-ingested DNA (chro-mosomal DNA and DNA present in bacteriallysates) in the GI tract of rats and to determineif this ingested DNA still remains able to trans-form the naturally competent bacterium Aci-netobacter baylyi strain BD413 (AcBD413). Wedetected that the rat feed given does not degradeDNA completely, and that this DNA stillremains biologically active after being exposedto the rat feed for up to 72 h. We are currentlycharacterizing the degradation dynamics andbiological activity of foreign DNA through theGI tract of rats using molecular methods such asPCR, hybridizations and natural transformationassays and the possibility of in vivo transforma-tion of AcBD413 to occur in gnotobiotic mice.

P-199 The effect of L. plantarum and L. fer-mentum on the microflora in the digestive sys-tem of calves. V. Oberauskas, A. Sederevicius,

Session IV: Nutritional manipulation S109

R. Zelvyte, R. Sutkeviciene, I. Monkeviciene(Lithuanian Veterinary Academy, Departmentof Anatomy and Physiology, Tilzes 18, 47181Kaunas, Lithuania).

High productivity of animals greatly depends oncalves‘ digestive system and its developmentduring the first month after birth. Neonate calvesduring the period of micro-ecological systemformation tend to be especially sensitive to var-ious factors. Therefore, it seems especiallyimportant to control the microbiological compo-sition in the calves. Probiotic preparations seemto be a promising means in this case. The pre-ventive dose of a lyophilized preparation con-taining L. plantarum U-14 no less than 1 ×109 cfu·g–1, and L. fermentum U-5 – no less than1 × 108 cfu·g–1, for calves is 4 g daily when givenwith colostrum or milk. The preventive dose oflyophilized preparation increased the dailyweight gain of calves by 16.2% and decreasedthe signs of diarrhea by 50%. According to theresults of one-way analysis of variance the lyo-philized preparation effected the total count ofLactobacillus in calves’ faeces by 7%, by 13.2%for L. plantarum and by 11.4% for L. fermentum,but had no effect on the number of L. salivariusand L. acidophilus. The lyophilized preparationhad no effect on the total count of enterobacteria.The probiotic preparation was produced fromlocal Lactobacillus plantarum U-14 and Lacto-bacillus fermentum U-5 strains and it is intendedfor the prevention of the digestive diseases ofneonate calves, correction of the digestive sys-tem microflora and calves weight gain.

P-200 Ruminal acetogens in dairy cows: die-tary effects and quantification of E. limosum.K. Olsson, P. Evans, K.N. Joblin (Rumen Bio-technology, Grasslands Research Institute,AgResearch, PB 11008, Palmerston North, NewZealand).

The ruminal acetogenic bacteria with theircapacity to convert H2 and CO2 into acetate havethe potential to act as H2 sinks in the rumen.Management of H2 is a key to controlling rumi-nant methane emissions. To determine whetherruminal acetogens in grazing livestock can beinfluenced by diet or diet change, rumen con-tents from rumen-fistulated dairy cows on threediets were collected and acetogen populationsquantified. The successive diet changes involved

grazing ryegrass/clover pasture, feeding lucerne(Medicago sativa) silage, feeding silage contain-ing 25% grain, and a return to grazing ryegrass/clover pasture. Acetogen population densities infreshly-collected samples were measured byanaerobic culturing under a H2/CO2 headspacein a rumen-fluid medium containing BES toinhibit methanogens. Acetogen-positive cul-tures were identified as those showing H2 utili-sation. The mean ruminal acetogen density foundin grazing animals did not change significantlywhen the diet changed to silage. However, ace-togen density increased markedly (3-fold) whenthe diet contained grain. To obtain further infor-mation from samples, a real-time PCR methodfor quantifying Eubacterium limosum, a com-mon ruminal acetogen, was developed. Themethod is based upon the 16S rRNA gene ofE. limosum and a self–quenched fluorogenic(LUXTM) primer set. LUX primers weredesigned, PCR optimised and primers success-fully tested for specificity against genomic DNAprepared from 3 strains of E. limosum, 5 speciesof non-target ruminal acetogens, and a range ofruminal bacteria, methanogens, and fungi. At thetime of writing, the method has been tested onone of the enumerated samples and showed thatE. limosum is a predominant acetogen.

P-201 The effect of yeast on Listeria innocuasurvival in the digestive tract of sheep.A.M. Olvera-Ramíreza, N.R. McEwana, F.M.McIntoshb, C.J. Newbolda (a Institute of RuralSciences, University of Wales, LlanbadarnCampus, Aberystwyth SY23 3AL UK, b RowettResearch Institute, Bucksburn, Aberdeen AB219SB, UK).

The use of yeast to stimulate ruminant produc-tion is well established, with increases in pro-ductivity reported in both growing and lactatinganimals. Previously, based on in vitro experi-ments, we have suggested yeast might also helpcontrol pathogenic bacteria within the digestivetract of ruminants. Now we present results show-ing in sheep supplemented with 5 g·d–1 of Pro-creatin 7 and challenged with the pathogen Lis-teria innocua, that the flow of pathogen from therumen decreased by 55% (4.0 vs. 1.7 × 108/d).This decreased flow of pathogens was associ-ated with more than doubling of the total bacte-rial population (8.2 vs. 20.8 × 108·mL–1) andan even greater increase in the numbers of

S110 Session IV: Nutritional manipulation

cellulolytic bacteria (1.5 vs. 9.5 × 107·mL–1) thatcould be recovered from the rumen of sheep fedyeast. The distribution of bacterial species asindicated by 16S rDNA PCR-DGGE also shiftedin response to yeast addition. However, despitea decrease in the flow of pathogens leaving therumen, yeast had no effect on pathogen sheddingin faeces (1.5 v 2.4 × 107/d).

Study sponsored by Lesaffre Feed Additives, Km. 57.5Carretera Mexico-Toluca, CP 50200 Toluca, Edo. deMexico, Mexico.

P-202 In vitro activity of essential oils towardsintestinal microbes. A.C. Ouwehanda, H.Kettunena, H. Schulzeb, N. Rautonena (a Ente-romix® Research, Danisco Innovation, 02460Kantvik, Finland, b Danisco Animal Nutrition,2300 AE Leiden, NL).

Essential oils have long been known to exertantimicrobial activity and have been suggestedto be a reason why certain spices contribute tothe preservation of foods. Hence, their effect onfood pathogens has been widely investigated.However, the influence essential oils have onmembers of the intestinal microbiota hasreceived much less attention. We investigatedthe effect of 10 nature-identical essential oils and3 natural extracts on the in vitro growth of mem-bers of the intestinal microbiota. The essentialoils were tested at three different concentrations;5, 50 and 500 µg·mL–1. Growth was followedunder anaerobic conditions at 37 ºC by auto-mated measurement of absorbance at 600 nm.The effects of nature-identical essential oils oninhibiting growth of E. coli, Clostridium perf-ringens or Salmonella varied with source andinclusion level. Beneficial microbes such aslactobacilli and bifidobacteria were found to beless sensitive towards nature identical essentialoils; inhibition occurred only at levels of500 µg·mL–1. The studied natural extracts werefound to be highly antimicrobial. Salmonella,E. coli and C. perfringens were sensitive at lev-els of 5–50 µg·mL–1, while lactobacilli andbifidobacteria were less sensitive at levels of 50–500 µg·mL–1. All tested strains, except Salmo-nella, were sensitive to the tested antibiotic,avilomycin. The observations indicate thatselected nature-identical essential oils and natu-ral extracts can exert a strong antimicrobialeffect against potential pathogenic members of

the intestinal microbiota. Due to their lower sen-sitivity, beneficial members of the microbiotawill be spared, giving an opportunity to improvethe composition of the intestinal microbiota.This would be a major benefit over antibioticsthat often negatively affect the beneficial mem-bers of the intestinal microbiota.

P-203 Functionality of the pig gastrointestinalmicrobiota in response to alternatives for in-feed antimicrobials. O. Pérez, W. Pellikaan, M.Verstegen, H. Smidt, W.M. De Vos (Laboratoryof Microbiology, Wageningen University, Hes-selink van Suchtelenweg 4, 6703 CT, Wagenin-gen, The Netherlands).

Throughout Europe it is normal commercialpractice that pigs are weaned at an earlier agethan under natural conditions. This causes gas-trointestinal disturbances and an increased sus-ceptibility to infection, resulting in economiclosses to the pig industry. Currently applied con-trol measures mostly rely on prophylactic anti-biotics in feeds. This practice, however, is goingto be either banned or significantly reduced dueto the risk of antibiotic resistance for both animaland human health. As a result, interest is focus-ing on alternative ways to stimulate and stabilizethe autochthonous gastro-intestinal microbiota.In the framework of the EU-funded project“FeedForPigHealth”, the current study aims todetect, localize and quantify key microbial spe-cies, their metabolic activity and interaction withthe host, as affected by PENS (plant extracts andother natural substances) supplemented to diets,in health and disease. To this end, integrated cul-tivation-based and direct molecular approaches,targeting phylogenetic as well as functional cat-abolic marker sequences, are used, and com-bined with analyses of the digesta for microbialmetabolites. Faecal and ileal digesta samplesfrom piglets receiving a standardized referencediet were evaluated by molecular fingerprintingof the overall bacterial and Lactobacillus com-munities, focusing on the effect of a large varietyof plant-derived additives on communitydynamics during in vitro fermentation assays. Ingeneral, incubation with the different substrates(i.e. SBP, chyme and starch) lead to substrate-specific shifts in community profiles, while mostof the non-fermentable additives had no signif-icant effect on microbiota composition. Furtherstudies are currently underway to assess the

Session IV: Nutritional manipulation S111

effect of selected PENS on microbiota compo-sition and activity in vivo.

P-204 Yeast cell wall preparations preventthe attachment of enteropathogenic Escheri-chia coli on broiler gut mucus. S. Peuranena,A. Kocherb, K.A. Dawsonb (a Alimetrics Ltd,Höyläämötie 14, FIN-00380 Helsinki, Finland,b Alltech Biotechnology Centre, Sarney, Sum-merhill Road, Dunboyne, Co. Meath, Ireland).

The association of E. coli with mucus is consid-ered to initiate bacterial overgrowth in the intes-tine, leading in the worst case to intestinal oreven systemic infection. The specific adherencereaction is mediated by adhesins on the surfaceof E. coli and receptors on the mucus surface ofthe host intestine. In theory, the adherence canthen be prevented by providing adhesin or recep-tor analogues in feed. An in vitro attachmentmodel with isolated broiler mucus was used tostudy the efficacy of a commercial yeast cellwall preparation Bio-Mos® (BM, Alltech Inc.)to prevent bacterial attachment. Three differententeropathogenic isolates of Escherichia coliwere allowed to attach to broiler mucus, and theattachment with and without BM (1 or 2 g·kg–1)was quantitatively measured after labelling thebacterial cells with radioactive thymidine. Theincubation order of bacteria and BM in themucus-coated microtiter wells was varied toillustrate the interactions between mucus, bacte-ria and yeast cell walls. Despite the fact that birdage and gut section affected the efficacy of BM,and that the three bacterial strains had differentabsolute attachment efficiency to broiler mucus,Bio-Mos showed consistent effect against theattachment of E. coli. Its efficacy was as goodas the positive control, mannose (1 g·kg–1). Inmost cases the higher inclusion level of BMresulted in better attachment inhibition. At thehigher inclusion level, BM was capable ofdetaching some of the previously attached bac-teria from mucus. These observations substanti-ate further studies on the mode of action of yeastcell wall carbohydrates in the interactionbetween bacteria and gut mucins.

P-205 Effect of human milk on the growth ofbifidobacteria: stimulation of Bifidobacte-rium bifidum, inhibition of Bifidobacterium

animalis. V. Rada, J. Nevoral, E. Vlková, I.Trojanová, J. Killer Ji í (Department of Micro-biology, Nutrition and Dietetics, Czech Univer-sity of Agriculture Prague, Pediatric Clinic ofCharles University, Prague – Motol, CzechRepublic).

The growth of four strains of bifidobacteria infive different samples of human milk was tested.Two strains of B. bifidum were isolated frominfant faeces. Two strains of B. animalis wereisolated from fermented milk products. Allstrains were identified using biochemical testsfollowed by species-specific PCR. The faecalflora of infants of milk donors was also analysedusing cultivation methods and FISH. Threeinfants had high numbers of bifidobacteria(>10 log cfu·g–1) in their faeces. The remainingtwo infants did not contain detectable amountsof bifidobacteria in their faecal samples. Goodgrowth of B. bifidum (both strains) in humanmilk was accompanied with a decrease of pH (upto 4.4) and production of lactic acid (up to 2 g·L–1).On the other hand, numbers of viable cells ofB. animalis decreased from 6 log cfu·mL–1 to3 log cfu·mL–1 after incubation in human milk.There were significant differences (P < 0.05)between bacterial counts of B. bifidum and B.animalis in all human milk samples tested. Ourresults suggest that B. bifidum is a suitable spe-cies for probiotic treatment of fully breast-fedinfants, which are free of bifidobacteria. On theother hand, B. animalis seems not to be an effec-tive probiotic bacterium for infants.

P-206 The effects of organic acids and essen-tial oils on methane production in vitro. M.J.Ranillaa, M.D. Carroa, M.L. Tejidoa, D.Macheboeufb, B. Lassalasc, D.P. Morgavib(a Departamento de Producción Animal I,Universidad de León, 24071 León, Spain,b INRA, Clermont-Ferrand-Theix ResearchCentre, Herbivore Research Unit, 63122 Saint-Genès-Champanelle, France).

Organic acids and essentials oils have recentlyattracted much attention as possible manipula-tors of ruminal fermentation, but whilst thesesubstances may have some beneficial effect onanimal production, their mechanisms of actionvary. The effects of two organic acids (disodiummalate (MAL) and fumarate (FUM); 8 mM) and

ř

S112 Session IV: Nutritional manipulation

three essential oils (cinnamaldehyde, 4 mM(CIN); carvacrol, 2 mM (CAR); and diallyl sul-phide, 0.5 mM (DIA)) on ruminal methane pro-duction and fermentation characteristics wereevaluated in vitro. Samples (400 mg) of a sub-strate consisting of concentrate and alfalfa hay(75:25) were incubated with 40 mL of bufferedrumen fluid from sheep for 16 h at 39 °C, withone of the 5 treatments or without additive (con-trol; CON). Methane production was measuredat 5 and 16 h and the production of VFA wasdetermined at the end of incubation. At 5 h, themethane produced was similar (P > 0.05) for alltreatments. However, total methane productionover the 16 h period was lower (P < 0.05) forDIA, CIN and CAR (872, 1039 and 1086 µmol,respectively) compared to CON (1289 µmol),while total VFA production was similar (P >0.05) for all treatments. FUM treatment pro-duced a lower (P < 0.05) acetate:propionate ratio(2.63 vs. 3.77, FUM vs. CON, respectively). Theresults show that under the conditions testedessential oils inhibited methane production invitro without negatively affecting ruminal fer-mentation.

Supported by Acción Integrada HF2004-0026 (MECof Spain) and Action integrée Picasso (Egide ofFrance).

P-207 Antiprotozoal properties of someplants from Iran. M. Rezaeiana, R. Ningratb,R.J. Wallacec (a Faculty of Veterinary Medicine,University of Tehran, Tehran, Iran, b Faculty ofAnimal Husbandry, Andalas University, LimauManis, Padang 25163, West Sumatra, Indonesia,c Rowett Research Institute, Greenburn Road,Bucksburn, Aberdeen AB21 9SB, UK).

The aim of this study was to investigate five dif-ferent plant samples from Iran for their effectson rumen protozoal activity in vitro. Plant sam-ples were Artemisia siberi, Acroptilon repens,Rubia tinctorum, Crocus sativa and Pegamolaharmula. Samples were harvested in April, driedat 100 °C and milled to pass through a 1-mmscreen. Selenomonas ruminantium was grown inthe presence of L-[14C]leucine in medium M2for 24 h. The incorporated bacteria were addedto six Hungate culture tubes (3 sheep in dupli-cate), each containing 7 mL strained ruminalfluid, 22.5 mg plant sample and 5 mM unlabelledL-leucine, after 1 h of incubation under anaero-bic conditions at 39 °C in a shaking water bath.

Samples (1 mL) were taken from each culturetube at 0, 1, 2, and 3 h and prepared for the meas-urement of trichloroacetic acid-soluble radioac-tivity by liquid scintillation spectrometry. Theextent of antiprotozoal activity was determinedfrom the net release of 14C into acid-solublematerial. All tested plants showed antiprotozoalcharacteristics compared with the control sam-ples that were without plant samples. The lowestinhibition was obtained with Rubia tinctorumand the highest value with Artemisia. Furtherstudies are necessary to find whether their prop-erties will be the same in vivo and to identify theprobable chemical component(s) of each plant,which may be responsible for the suppression ofthe bacteriolytic activity of ruminal protozoa.

P-208 Growth of Butyrivibrio fibrisolvensJW11 and Fusocillus P-18 in the presence offish oil, fatty acids and their derivatives. A.J.Richardson, R.J. Wallace (Rowett ResearchInstitute, Bucksburn, Aberdeen AB21 9SB,UK).

Biohydrogenation of dietary unsaturated fattyacids in the rumen is a major factor which affectsthe quantities of health-promoting polyunsatu-rated fatty acids in ruminant meats and dairyproducts. Two bacteria involved in this processare Butyrivibrio fibrisolvens and ‘Fusocillus’.Both species biohydrogenate linoleic acid (LA,cis-9,cis-12-18:2) producing conjugated lino-leic acid (CLA; cis-9,trans-11-18:2) as an inter-mediate and, in the case of Fusocillus, stearicacid (18:0) as an end product. In dairy cattle, theaddition of fish oil to the diet results in anincrease in the CLA content of milk. The aim ofthe experiments reported here was to determinethe influence of fish oil and its components onbiohydrogenation by B. fibrisolvens JW11 andFusocillus P-18. Fish oil, the two main fatty acidsof fish oil, eicosapentaenoic acid (EPA,C20:5(n-3)), docosahexaenoic acid (DHA,C22:6(n-3)),linoleic acid, linolenic acid (LNA), linoleyl andoleyl methyl esters, trilinolein, linoleyl, linole-nyl and oleyl alcohols were added to an anaero-bic, ruminal fluid-containing medium and thegrowth of the two bacterial strains was followedturbidimetrically. Fish oil, up to a concentrationof 10 mg·mL–1, had no effect on the growth ofeither B. fibrisolvens JW11 or Fusocillus P-18,but EPA and DHA inhibited the growth of both

Session IV: Nutritional manipulation S113

strains at 50 µg·mL–1. LA caused a 12-h lagphase with B. fibrisolvens JW11 and in combi-nation with fish oil the effect was exactly thesame. Growth of P-18 was inhibited completelyby LA and LNA. JW11 was also unable to growin the presence of LNA. Esterified fatty acidsand their alcohols were much less toxic than freefatty acids. It was concluded that the effects offish oil on biohydrogenation require the releaseof free EPA and DHA by lipolysis.

P-209 Recovery of Lactobacillus casei DN-114 001Rif in the intestinal tract of healthyhumans after consumption of fermentedmilk. V. Rocheta, L. Rigottier-Goisa, F. Leveneza,J. Cadioua, A. Mogenetb, J-L. Bressonb, N. Gou-pil-Feuilleratc, J. Doréa (a UEPSD, INRA, 78352Jouy-en-Josas, France, b CIC, Hôpital Necker,75015 Paris, France, c Danone Vitapole, 91767Palaiseau Cedex, France).

Milk fermented with Lactobacillus casei DN-114 001 has health benefits in humans possiblydue to its effects on the intestinal microbiota. Westudied the recovery of L. casei DN-114 001Rif

in the terminal ileum and in faeces of 10 healthyhumans after ingestion as a fermented milk. Therecovery of L. casei DN-114 001Rif was ana-lysed in the terminal ileum for the 8 h followingingestion of 300 mL of the test product, and inthe faeces after 300 mL daily consumption of theproduct for 8 days. Real time PCR was per-formed using primers specific for the L. casei/paracasei group. PCR-TTGE was done for anal-ysis of Lactobacillus spp. dynamics. FISH usingribosomal RNA targeted probes was used tomeasure relative proportions of dominant bacte-ria in faecal samples. Following consumption ofL. casei DN-114 001Rif, increases in bacteriaequivalents of the L. casei/paracasei group wereobserved in all samples. These numbers revertedto initial levels after the end of the intake. PCR-TTGE profiles showed a band characteristic ofL. casei DN-114 001Rif during consumption ofthe test product for all individuals. This band dis-appeared after the end of consumption. FISHshowed no significant differences in the propor-tions of the phylogenetic groups analysedbetween the different steps of the study. Con-sumption of a fermented milk containingL. casei DN-114 001Rif induced a marked andtransient increase of this probiotic in intestinalsamples of healthy subjects. This is associated

with stability in the composition of dominantphylogenetic groups of the gut microbiota.

P-210 In vitro growth inhibition of Helico-bacter pylori and Campylobacter jejuni byLactobacillus salivarius. K.A. Ryan, Y. Li,P.W. O’Toole (Department of Microbiology andAlimentary Pharmabiotic Centre, UniversityCollege Cork, Cork, Ireland).

H. pylori is one of the most common infectionsworldwide. It colonises the human gastricmucosa and is a causative agent of gastritis, pep-tic ulcer disease and gastric adenocarcinoma.The related organism C. jejuni is regarded as acommensal in poultry but has emerged as theleading cause of human acute enteritis in theWestern world. Guillain Barré syndrome hasbeen implicated as a possible sequela to C. jejuniinfection. With increased antibiotic resistancebecoming a problem, we investigated the abilityof twenty-eight strains of Lactobacillus salivar-ius from different sources (five human intestine,five human saliva, four human blood, threehuman other, two pig intestine, one cat, onecheese, five avian and two unknown) to inhibitgrowth of H. pylori and C. jejuni. Seven otherspecies of Lactobacilli were included as con-trols. Lawns of H. pylori or C. jejuni were spreadonto blood agar plates. Either Lactobacilluswhole cells or cell free supernatants were thendotted onto paper disks which had been placedon the agar plates. Plates were incubated for48 h, after which time growth inhibition aroundthe disks was measured. Whole cells from 7/28strains and cell free supernatants from four ofthese seven strains inhibited growth of C. jejuni,while whole cells from 6/28 strains and cell freesupernatants from four of these six strains inhib-ited growth of H. pylori. None of the seventeenstrains of human origin inhibited either H. pylorior C. jejuni. In contrast, 4/5 strains of avian ori-gin inhibited growth of both pathogens. The pHof all Lactobacillus spent cultures was 4.5 ± 0.5,so the killing effect observed was not simply dueto the production of acid by these strains. Thesedata suggest that strains of Lactobacillus sali-varius of avian origin are a good source for effec-tive probiotics against H. pylori and C. jejuni.

P-211 Composition and temporal stabilityof oral and intestinal microbiota during

S114 Session IV: Nutritional manipulation

probiotic ingestion. M. Saarela, J. Maukonen,M.-L. Suihko, J. Mättö (VTT, Biotechnology,Espoo, Finland).

The oral cavity and the colon harbour dense anddiverse indigenous microbiota consisting of spe-cies representing the same phylogenetic clus-ters. The aim of the study was to compare thediversity and temporal stability of oral and intes-tinal bacterial populations during probioticingestion. Saliva and faecal samples were col-lected from ten healthy adult volunteers on threesampling occasions (baseline and after one andtwo weeks ingestion of probiotic capsules) forassessment of the predominant bacteria, bifido-bacteria, lactobacilli and the Eubacterium rec-tale-Clostridium coccoides (Erec) -group byPCR-DGGE and, except for the Erec-group, byculturing. Numbers of culturable anaerobeswere log 8.4 ± 0.6 cfu·mL–1 in saliva and 10.5 ±0.2 cfu·g–1 in faeces, and the corresponding fig-ures for aerobes were 8.0 ± 0.5 and 7.2 ± 0.8. Thepredominant bacterial population was morediverse in faeces (mean 34.9 amplicons detectedby PCR-DGGE) than in saliva (mean 23.6 ampli-cons), and the dominant population was gener-ally stable in both sampling sites (average sim-ilarities in samples obtained from the sameindividual were 83.3% in faeces and 92.2% insaliva). Similarly, the Erec, Lactobacillus andBifidobacterium populations were more diversein faeces than in saliva, and with the exceptionof the Lactobacillus population in faeces, theirpopulations were generally stable. Only fewsimilarly migrating amplicons were detected inthe PCR-DGGE profiles from faecal and salivasamples, which reflects differences in the spe-cies composition between the two sites. In con-clusion, the microbiota of the oral cavity andintestines was generally stable during short termconsumption of probiotic capsules. The micro-biota in faeces was more diverse than in saliva,and the species composition varied between thetwo sampling sites.

P-212 Cultured milk replacer with prophy-lactic influence on intestinal diseases. A.Sarkinas (Food Institute of Kaunas University ofTechnology, Taikos av. 92, LT 51180 Kaunas,Lithuania).

The purpose of the investigation was to producean antagonistic activity strain, possessing the

property of surviving in the intestinal tract. Thestrain of Lactobacillus acidophilus L16-4-18-500B was selected with moderate activity in lac-tic acid production in milk and milk products,capable of surviving in the intestinal tract, inhib-iting the growth of coliforms in the product andin the intestinal tract. Lactobacillus acidophilusL16-4-18-500B can be applied for production ofmilk replacers. By means of the strain described,feeds for young calves can be produced. Feedingto calves resulted in an increase of calves’ aver-age daily liveweight gain when compared withmilk replacer produced according to the sameprescription but devoid of Lactobacillus acido-philus. The described cultured milk replacerdecreases the content of conditionally patho-genic microflora in the intestinal tract andreduces calves’ morbidity. Investigations wereconducted to examine the influence of Lactoba-cillus acidophilus cell survival and titrationacidity dynamics on keeping the condensed milkreplacer and emulsion. The optimum storageregime of the product was determined based onthe exploratory findings. Studies have been car-ried out on the influence of Lactobacillus acido-philus L16-4-18-500B on the development ofEscherichia coli in cultured milk replacer andemulsion. Storing of samples at 30, 25, 20, 15and 8 °C temperatures made it possible to esti-mate the steadiness of the product. It has beenestablished that the development of Escherichiacoli was inhibited by Lactobacillus acidophilusintroduced into the product with starter. In thecase of Lactobacillus acidophilus contamina-tion of milk should not increase significantlyduring storage.

P-213 Dietary essential oil supplementationcan affect broiler performance and digestamicrobial community. H. Schulzea, H.Kettunenb, A.C. Ouwehandb and N. Rautonenb

(a Danisco Animal Nutrition, 2300 AE Leiden,NL, Finland, b Enteromix® Research, DaniscoInnovation, 02460 Kantvik, Finland).

For many centuries, essential oils (EO) have tra-ditionally been used by man for the pleasantodour of the essence, its flavour or its antisepticand/or preservative properties. The antibacterialeffect of EO is well established in vitro. How-ever, information about the effects of EO in live-stock animals is scarce. To assess the effect ofdifferent blends and dietary inclusion levels of

Session IV: Nutritional manipulation S115

EO on performance parameters and digestamicrobial community, a 42-day broiler studywas conducted. The study was conducted with2160 1-day old male Ross broiler chickens, allo-cated to 6 treatment groups (12 replicates pertreatment, 30 birds per replicate). The birds werefed mash wheat based diets, free of antibioticgrowth promoter and coccidiostat, containing21% and 19% crude protein and 11.7 and12.6 MJ ME·kg–1 DM for the starter (day 1–21)and grower (day 21–42) phase, respectively.Five blends of EO, varying in composition andinclusion level, were studied. Bird performanceswere measured for the starter (1–21 days),grower (21–42 days) and overall experimentalperiod. On days 21 and 42, ileal and caecaldigesta were collected for microbial analyses.Compared with the control group, the dietaryaddition of EO improved weight gain and feedefficiency of the birds (statistically significantfor one of the EO treatment groups). Dietaryaddition of EO reduced ileal microbial numbers,in particular at day 42. In caecal digesta, bio-genic amines were reduced and volatile fattyacids increased by the essential oil supplemen-tation of the diet. In addition, for some of the die-tary EO treatments changes of the microbial pro-file in caecal digesta were observed. The resultssuggest that dietary EO can improve broiler per-formance parameters, reduce the microbial pop-ulation measured in ileal digesta and change cae-cal microbial activity.

P-214 Continuous pH measurements in therumen of sheep to test the anti-acidotic effi-cacy of nettles (Urtica dioica). N. Selje, E.M.Hoffmann, P. Lawrence, K. Becker (Institute forAnimal Production in the Tropics and Subtrop-ics, University of Hohenheim, 70599 Stuttgart,Germany).

U. dioica had proven to exhibit an anti-acidoticeffect in vitro (Kliem et al., 2005). This studyaimed at validating this impact on rumen pH invivo. In a crossover feeding trial six cannulatedlambs (BW 54 kg) were fed 900 g concentrateand 400 g per day. At a time, three lambsreceived a control concentrate N0A (5% grasshay) and three sheep were fed concentrate N5A(5% U. dioica). After 3 weeks of adaptation, pHwas recorded during three consecutive days in15 min intervals by probes permanently insertedinto the rumen. Daily samples were taken

throughout the experiment to analyse rumenparameters. Neither ruminal concentrations oflactate, short chain fatty acids, and ammonium,nor body weight gain were significantly differ-ent for the diets. With one exception, all sheepexhibited higher H+ ion concentrations when fedN5A (ave 3.1e–6 M) than with N0A (ave2.3e–6 M), depending significantly on diet andon individual animal (P < 0.0001), irrespectiveof which diet was fed first. These results are con-trary to those obtained in the in vitro systemswith bovine rumen fluid. However, in the in vitroexperiments either 100% ground wheat or a mix-ture of 30% wheat and 70% maize silage servedas substrates, whereas in the in vivo trial the dietwas based on various grains (16% wheat, 44%barley, 12% maize). Thus, it cannot be excludedthat the efficacy of the plant additive is highlydependent on the composition of the diet and/orthe animal species investigated.

P-215 Microbial composition and metaboliteconcentration in fermented liquid diets forpigs. A. Shlimon, N. Canibe, O. Højberg, B.B.Jensen (Danish Institute of Agricultural Sci-ences, Department of Animal Health, Welfareand Nutrition, Research Centre Foulum, PO Box50, 8830 Tjele, Denmark).

Feeding with fermented liquid feed reduces thenumber of enteric pathogens along the gastroin-testinal tract of pigs. However, negative effectson growth performance have been observed,probably due to microbial degradation of dietarylysine. Fermenting only the cereal grains isbeing considered as an alternative to fermentingthe entire diet. But a fast and substantial pH dropin fermented grain affects the microbial prolif-eration in the mixture, resulting in too low lacticacid concentrations, thus reducing the bacteri-cidal effect against pathogens compared to fer-mented liquid feed. Four experimental treat-ments were designed: fermented liquid feed(FLF), fermented liquid grain (GRAIN), fer-mented liquid grain amended with soybean(SOY), and fermented liquid grain amendedwith calcium carbonate and calcium phosphate(CAP). Feed components and water were incu-bated at 20 °C and samples taken at various timepoints during incubation. The pH drop wasreduced in the SOY and CAP compared to theGRAIN diet. At time 180, the lactic acid con-centration was 151, 55, 87, and 101 mmol·kg–1

S116 Session IV: Nutritional manipulation

in the FLF, GRAIN, SOY, and CAP diet, respec-tively. Preliminary results of partial 16S rDNAsequences of bacteria isolated from de Man,Rogosa and Sharp agar revealed that membersof the genus Weissella dominated during the ini-tial hours of incubation, while at 180 h, speciesof the Pediococcus and Lactobacillus weremainly represented. The results suggest that fer-mentation of grain cereals amended with soy-bean or with calcium carbonate and calciumphosphate could be a way of avoiding microbialdegradation of lysine in the mixture while keep-ing relatively high concentrations of lactic acid,and thereby the bactericidal effect.

P-216 Ultrasonic modification of alginatematrices for the develop of probiotic deliverysystems. A.I. Sidorova, O.V. Manaenkova, E.M.Sulmana, L.E. Smirnovab, V.F. Vinogradovb

(a Tver Technical University, A. Nikitin str., 22,Tver, 170026, Russia, b Tver Medical Academy,Sovetskaya str., 4, Tver, 170642, Russia).

In recent years the practical interest to deliverysystems on the basis of polymer matrices hasincreased. They are close to real food objects,have a prolonged effect, and are capable of theselective release of the encapsulate in differentparts of the intestine. Thus, controlling the rateof the matrix dissolving, it is possible to controlthe rate of the probiotic delivery to the organism.The experiments showed that the most effectiveand simple method to influence the rate of algi-nate matrix dissolving is ultrasound. Ultrasoundcauses destruction of sodium alginate macro-molecules, the degree of which depends on thetime of treatment and intensity of ultrasound.Three groups of gel beads on the basis of sodiumalginate with different molecular weight (MW)were prepared. The first group of beads was pre-pared from the raw sodium alginate solution.The time of ultrasonic treatment of the solutionfor the second group of beads was 10 min at theintensity of 92 W/sm2, and for the third group20 min at 460 W/sm2. Thus, the MW of alginatemacromolecules is 1.5 × 106; 0.938 × 106 and0.565 × 106, accordingly. The examination ofthe bead-dissolving process was carried out inenvironments simulating stomach and intestineconditions. Full dissolving of beads on alginatebasis with the molecular weight of 1.5 × 106

occurs by the 50th min, with the molecular

weight of 0.938 × 106 – by the 30th min, and withthe molecular weight of 0.565 × 106 – by the 20thmin. On the basis of the obtained alginate sam-ples with known MW it is possible to developdrug delivery systems with the set properties,able to be dissolved on narrow sites of a gastro-enteric path.

P-217 Influence of Bifidobacterium bifidumon mineral release from bread in the processof enzymatic digestion in vitro. K.A.Skibniewska, B. Nalepa, A. Babuchowski(University of Warmia and Mazury in Olsztyn,Pl. Cieszynski 1, 10-726 Olsztyn, Poland).

Nutrient bioavailability is a key factor whendesigning appropriate diets. Many factors, suchas diet composition, influence the amount ofnutrients to be utilized by the human organism.Enzymatic digestion in vitro is used as a first stepin the assessment of the part of diet to be utilizedby the organism. Bread of wheat, varietyTonacja, was baked with white meal and withaddition of 15%, 30% and 45% of bran. Concen-tration of Ca, Mg, Mn, Cu, and Fe was deter-mined by atomic absorption spectrometry.Breads were next digested in vitro in a processsimulating digestion in the gastrointestinal tractwithout (control) and with addition of Bifido-bacterium bifidum inoculum at 106 cfu·cm–3

(experimental). Bifidobacteria influenced theminerals in different ways. Filtrate after experi-mental white bread digestion contained lessminerals than the control one, especially Ca andFe, probably because of mineral utilization bybacteria. Increase of mineral release wasobserved in solution after experimental breadwith bran addition digestion vs. the same breadswithout bacteria.

P-218 Effect of organic acids and monolaurinon Escherichia coli, Salmonella spp. andClostridium perfringens. E. Skrivanovaa, M.Marouneka,b, G. Dlouhaa (a Research Institute ofAnimal Production, 10401 Prague-Uhrineves,Czech Republic, b Institute of Animal Physiol-ogy and Genetics, Czech Academy of Sciences,14220 Prague, Czech Republic).

Antimicrobial activity of fatty acids, monolau-rin, citric, succinic, fumaric, malic and lactic

Session IV: Nutritional manipulation S117

acid against strains of Escherichia coli, Salmo-nella spp. and Clostridium perfringens was inves-tigated. Antimicrobial activity was expressed asthe minimum inhibitory concentration (MIC)that prevented growth and glucose utilization.Caprylic acid was the only acid inhibiting glu-cose utilization in all cultures. Its MIC variedfrom 1 to 3 mg·mL–1. E. coli strains were inhib-ited also by capric acid at 5 mg·mL–1. Cl. perf-ringens strains were inhibited by medium-chainfatty acids (C8–C14), oleic acid and one strainalso by linoleic acid. The lowest MIC were thoseof lauric and myristic acid (0.1–0.2 mg·mL–1).Growth of clostridia was inhibited by monolau-rin at 3 mg·mL–1 and citric acid at 4 mg·mL–1.Inhibitory effects of other acids were notobserved. In cultures of E. coli treated withcaprylic acid, and in cultures of Cl. perfringenstreated with lauric acid and monolaurin, trans-mission electron microscopy revealed damageof cytoplasmic structures. Bacteria maintainedthe integrity of the outer cell membrane. Toassess the effect of fatty acids on inner mem-brane permeability, K+ efflux from cells ofE. coli and Cl. perfringens treated with caprylicand lauric acid, respectively, was measured bymeans of a K+ ion-selective electrode. Cellstreated with cetyltrimethylammonium bromide(CTAB) served as a control. Potassium ionswere released by action of CTAB, however, noK+ efflux from cells treated with caprylic andcapric acid occurred. Medium-chain fatty acids,thus, do not increase the K+ permeability of thecytoplasmic membrane, which usually leads todissipation of membrane potential.

Supported by Ministry of Agriculture of the CzechRepublic (project MZe0002701403).

P-219 The influence of probiotics on growthof turkeys. L. Sol ianska, D. Vladárová (SlovakUniversity of Agriculture, Faculty of Agrobiol-ogy and Food Resources, Slovak Republic).

The aim of our work was to monitor and evaluatethe influence of probiotic preparations Bio-Mos,Mycosorb, Lactiferm L-5 and LactifermL-200+Se on growth indicators, chosen bodymeasures and slaughter quality of turkeys LargeWhite to 84 days old. One day turkeys weredivided into six groups. In the first group were11 turkeys and we used it as a control group. Inthe second group were 10 turkeys which werefed with feedstuff enriched with 0.2% of probi-

otic preparation Bio-Mos. In the third groupwere 11 turkeys which were fed with feedstuffenriched with 0.1% of probiotic preparation Bio-Mos. In the fourth group were 11 turkeys whichwere fed with feedstuff enriched with 0.1% ofprobiotic preparations Bio-Mos and Mycosorb.In the fifth group were 10 turkeys which were fedwith feedstuff enriched with 0.03% of probioticpreparation Lactiferm L-5. In the sixth groupwere 10 turkeys and we added probiotic prepa-ration Lactiferm L-200 + Se to drinking water.At the end of feeder at the age of 84 days wefound out the highest average live weight in thesixth group with probiotic preparation LactifermL-200 + Se and the lowest average live weightin the second group with 0.2% of probiotic prep-aration Bio-Mos. The highest average bodylength was found in the sixth group with probi-otic preparation Lactiferm L-200 + Se and thehighest average breast half-diameter was foundalso in the sixth group. We found the highestaverage length of carina in the fourth group with0.1% of probiotic preparations Bio-Mos andMycosorb and the highest average leg lengthalso in the fourth group. We found the highestaverage breast meat yield and the highest slaugh-ter-house yield in the fourth group with 0.1% ofprobiotic preparations Bio-Mos and Mycosorb.The highest average share of legs was found inthe sixth group with probiotic preparation Lac-tiferm L-200 + Se.

P-220 Effect of feeding processed karanj(Pongamia glabra) meal on the microbialprotein synthesis and immune response ofgrowing lambs. N.M. Soren, V.R.B. Sastry,T.K. Goswami, S.K. Saha (Indian VeterinaryResearch Institute, Izatnagar 243122, UttarPradesh, India).

Abstract withdrawn.

P-221 Bacterial community change and Strep-tococcus suis disappearance in piglet gut afteroral administration of probiotic S1. Y. Su,W.Y. Zhu, W. Yao (Laboratory of Gastrointes-tinal Microbiology, Nanjing Agricultural Uni-versity, 210095, RP China).

In China, piglets are commonly infected withdiseases such as diarrhoea. In 2005, Streptococ-cus suis-2, a pathogen that can infect both pigs

č

S118 Session IV: Nutritional manipulation

and humans, caused a number of human infec-tions and deaths. Commensal gut bacteria playan important role for the host health, but alsocontain potentially harmful bacteria. The effectsof probiotic S1 on the gut bacterial communityin piglets was investigated. Six litters of neonatalpiglets were divided randomly into controlgroup and treatment group. At 7, 9 and 11 daysof age, piglets in the treatment group orallyreceived probiotic S1 preparation. At 7, 14,21(weaning), 24 and 35 days of age, one pigletfrom each litter was slaughtered. Gut sampleswere collected and total DNA was extracted.The V6-V8 region of 16S rDNA was amplifiedand analyzed by denaturing gradient gel electro-phoresis (DGGE). DGGE profiles showed thathigh G+C% bacteria in the hindgut of piglets dis-appeared after weaning and came back graduallyon d 35. Sequencing analysis showed that mostof these high G+C% bacteria belonged to Lacto-bacillus. Enumeration by plate counting showedsimilar changes in the number of lactobacilli. S1had no marked effect on the gut bacterial diver-sity. However, a special band appeared on d 14for the treatment group, with its correspondingsequence 95% similar to Clostridium disporium.On d 35, a special band appeared in the controlgroup, with its sequence 99% similar to Strep-tococcus suis. In summary, weaning could causedisappearance of high G+C% bacteria (mainlylactobacilli) in the piglet hindgut. Streptococcussuis may be commonly present in pig farms inChina and probiotic S1 has a potential role inpromoting beneficial bacteria and inhibitingpotential pathogens.

P-222 Dynamic single fermenter model forstudy of survival and growth of probiotics inthe human upper gastrointestinal tract. I.Sumeria,b, L. Arikea,b, S. Adambergb, K.Adamberga,b, L. Lappa,b, T-M. Lahta,b, T.Paalmea,b (a Competence Center of Food andFermentation Technologies Akadeemia tee 15,12618 Tallinn, Estonia, b Tallinn University ofTechnologies Ehitajate tee 5, 19086 Tallinn,Estonia).

A dynamic in vitro model to simulate physio-logical conditions characteristic to the humanupper gastrointestinal (GI) tract was developedusing a single fermenter system. Two hundred mLof model food containing probiotic microorgan-isms (106–8 cfu·mL–1) was added to 100 mL

0.1 M HCl. Then 1 M HCl was added at the max-imum rate of 20 mmol·h–1 until pH = 3.0 wasobtained, followed by neutralization of the fer-menter with 1 M NaHCO3 until pH = 6.5 wasreached. Then a 4% solution of bile salts wasadded during 10 min to achieve a 0.4% bile saltconcentration in the fermenter. After 20 min,feeding with a diluted MRS solution was startedat dilution rate D = 0.42 h–1 for 5.5 h. During thistime the pH was kept at 6.5 and bile salts werediluted down to 0.04%. During the whole proc-ess the number of cells were determined by:(1) plating out on MRS; (2) counting the cellnumber in a counting chamber; (3) staining thecells with SYTO9 and propidium iodide andcounting green (live) and red (dead) cells usinga fluorescence microscope. The model was eval-uated using the commercial probiotic Lactoba-cillus acidophilus La-5 (Chr. Hansen A/S,Denmark). It was demonstrated that survival ofLa-5 depends on both the physiological state ofthe microorganism (exponential vs. stationaryphase) as well as on the food matrix. Resultswere compared with behaviour of cells in a staticin vitro model.

P-223 Effects of exogenous fibrolytic enzymeson in vitro ruminal fermentation of grass hayand its cell wall. M.L. Tejidoa, L.A. Giraldoa,M.D. Carroa, J.M. Tricaricob, M.J. Ranillaa

(a Departamento de Producción Animal I,Universidad de León, 24071 León, Spain,b Alltech Inc., 3031 Catnip Hill Pike, Nicholas-ville, KY 40356, USA).

Batch cultures of mixed rumen microorganismswere used to study the effects of a fibrolyticenzyme preparation (FibrozymeTM, Alltech Inc.,Nicholasville, USA) on the in vitro fermentationof grass hay and its isolated cell walls (neutral-detergent fibre, NDF). Samples (500 mg) ofeach substrate were incubated in 120-mL bottleswith 50 mL of buffered rumen fluid from sheepfor 24 h at 39 °C. Enzyme was added directly intothe bottles at the beginning of the incubation atthree levels: 0 (control; CON), 15 (ENZ15) and30 (ENZ30) xylanase units·g–1 substrate DM.Compared to CON, the addition of ENZ30 tograss hay increased (P < 0.05) gas productionand VFA production after 5, 10 and 24 h of incu-bation, without affecting (P > 0.05) the ace-tate:propionate ratio. For grass hay NDF, bothENZ15 and ENZ30 treatments increased

Session IV: Nutritional manipulation S119

(P < 0.05) gas and VFA productions after 5 and10 h of incubation, compared to CON, withhigher values (P < 0.05) for ENZ30 than forENZ15; after 24 h, however, only ENZ30increased (P < 0.05) these parameters. Dry mat-ter disappearance, NH3-N concentration and pHvalues at 24 h of incubation were not affected(P > 0.05) by the addition of enzymes for anysubstrate. The results indicate that both levels ofenzyme addition stimulated the in vitro fermen-tation of grass hay NDF at short incubation times(5 and 10 h), but only the higher level of enzymeaddition (ENZ30) stimulated the fermentation ofgrass hay.

P-224 Effects of dietary nitrogen source andamino acid composition on intestinal micro-biota. M. Tokuraa, Y. Jojimaa, M. Moria,T. Kobayashia, Y. Hongohb, T. Inoueb, M.Ohkumab and R. Fudoa (a Ajinomoto co., Inc.,Suzuki-cho 1-1, Kawasaki-ku, Kawasaki-shi,210-8681 Japan, b RIKEN, Hirosawa 2-1,Wako-shi, Saitama, 351-0198 Japan).

Effects of dietary amino acid (AA) compositionon intestinal microbiota were analyzed in adultrats. Experimental groups were preparedaccording to the dietary protein source: (1) FreeAA mixture based on casein AA composition(group AA: as control); (2) Casein (group C);(3) AA mixture lacking either arginine, cystine,phenylalanine, or tryptophan (group AMOArgetc). T-RFLP profiles of intestinal and fecalmicrobiota among groups were compared byseveral statistical approaches. Cluster and prin-cipal component analyses indicated that themicrobial profile of group AA was more stabi-lized than that of group C. Fixation index (Fst)and Fst-P values indicated that intestinal andfecal microbiota of group AA were significantlydifferent from groups C, AMOPhe and AMOTrp.According to the Wilk's Lambda of discriminantanalysis, relative importance of T-RFs was eval-uated, and the most important two T-RFs wereidentified as Lactobacillus murinus and Turici-bacter sanguinis based on comparison with indi-vidual 16S rRNA gene clones. Real time PCRdemonstrated that Lactobacilli in intestinalmicrobiota were severely decreased in groupsAMOPhe and AMOTrp, though the total bacterialnumber was unchanged. These results suggestthat difference in the dietary nitrogen source

causes changes in intestinal microbiota and thatdeficiency in essential amino acids such as Pheand Trp reduces the intestinal Lactobacilluspopulation.

P-225 Relationships between gut microbialspecies and energy metabolism in broilerchickens. V.A. Toroka, K. Ophel-Kellera, R.J.Hughesb (a SARDI, Field Crops Pathology,GPO Box 397 Adelaide, SA 5001 Australia,b SARDI, Pig and Poultry Production Institute,Roseworthy, SA 5371, Australia).

The role of gut microflora in animal health hasbecome increasingly important, with the use ofantibiotics in animal feeds to promote growthbeing limited due to legislation and consumerpressure. We have developed terminal restric-tion fragment length polymorphism analysis(T-RFLP) to examine chicken intestinal micro-flora based on high-throughput, high resolutionfingerprinting of bacterial gene regions. Thistool is capable of providing a “snap-shot” of thecomplex bacterial population at any particulartime, and combined with advanced multivariatestatistical analysis has enabled relationshipsbetween gut microflora and bird performance tobe investigated for the first time. Changes inmicrobial communities along the chicken gut inresponse to addition of a non-starch polysaccha-ride degrading enzyme product to a barley-baseddiet were investigated. We found significant dif-ferences in the overall microbial communitiesbetween dietary treatments within the ileum andcaecum, as well as between the ileum and cae-cum. Several indicator bacterial species contrib-uting to the diet-induced differences in the over-all gut microbial communities were identifiedand found to be different between the ileum andcaecum. Classical growth/performance analysisshowed chickens fed the barley plus enzyme diethad a significantly higher apparent metabolisa-ble energy (AME) than chickens on the controlbarley diet. Correlations were observed betweenchanges in gut microflora of chicken fed barleyversus barley plus enzyme diets and an increasedAME for individual chickens on the barley plusenzyme diet. The presence of specific beneficialbacterial species and/or the absence of specificdetrimental bacterial species may be indicatorsof improved energy metabolism in these chick-ens.

S120 Session IV: Nutritional manipulation

P-226 Effects of live yeasts on the fatty acidbiohydrogenation by ruminal microflora. A.Troegelera, J.-P. Mardenb, C. Bayourtheb, R.Moncoulonb, F. Enjalberta (a École NationaleVétérinaire de Toulouse, Département Élevageet Produits, Laboratoire d’Alimentation, BP87614, 31076 Toulouse Cedex 03, France.b École Nationale Agronomique de Toulouse,Laboratoire de Zootechnie, 31076 ToulouseCedex 03, France).

Addition of live yeasts in high concentrate dietsfor ruminants has been shown to help maintain-ing the ruminal pH above 6, which couldenhance the microbial biohydrogenation ofunsaturated dietary fatty acids. Moreover, yeastsimprove the growth of Megasphera elsdenii, abacterium which favors the trans-10 pathway ofbiohydrogenation. So the objective of this studywas to investigate the effects of live yeasts (Sac-charomyces cerevisiae) on the biohydrogena-tion in the rumen of dairy cows receiving a highconcentrate diet without added fat. Three rumi-nally fistulated lactating dairy cows were giventhree diets based on corn silage (control, controlplus 0.5 g·d–1 or control plus 5.0 g·d–1 of Sac-charomyces cerevisiae NCYC SC47), accordingto a Latin square design. Ruminal contents weresampled and liquid and solid phases were sepa-rated with a 0.25 mm metal sieve. Fatty acidsprofiles were obtained by gas chromatography.The two doses of yeast resulted in similar effects.Live yeast significantly decreased myristic andstearic acid proportions, and significantlyincreased oleic and linoleic acid proportions by16 and 32% in the liquid and the solid phases,respectively. No significant effect was observedfor other biohydrogenation intermediates, butthe cis9,trans11-C18:2 tended (P = 0.154) toincrease with the addition of yeasts, whereastrans10-C18:1 numerically decreased (P = 0.225).These results suggest that live yeasts affectmicrobial activity, lowering the extent of biohy-drogenation without shifting toward the trans-10 isomers pathway.

P-227 The trial of fermented carrot juice tech-nology development. M. Trzaskowska, D.Kolozyn-Krajewska (Warsaw Agriculture Uni-versity, Faculty of Human Nutrition and Con-sumer Science, ul. Nowoursynowska 159C, 02-776 Warszawa, Poland).

Prebiotics are defined as nondigestible foodingredients that may beneficially affect the hostby selectively stimulating the growth and/or theactivity of a limited number of bacteria in thecolon. Inulin is recognized as a prebiotic. Con-sumption of food with this prebiotic can be ben-eficial for human health. The objective of thestudy was to work out the technology of fer-mented carrot juice with inulin addition, as ahealth additive. The strain of Lactobacillus aci-dophilus CH-2 was applied for fermentation.The criterion of optimization were results of sen-sory assessment, as the sensory attractivenesshas a significant effect on consumer motivationin food and drink choosing. Carrot juice withaddition of 3% inulin and 5% saccharose wasevaluated as the best. The worst was the juicewith lower addition of saccharose (3%) (P <0.05). It confirmed the importance of sweetnessin food acceptance. Juices with inulin (3%) andsaccharose (5%) addition and with only saccha-rose (5%) addition had the same sensoric quality(P > 0.05). It can be concluded that the inulindoes not influence sensory quality of carrotjuice. The number of bacteria directly after fer-mentation was at the level of about 9.04 logcfu·mL–1. After storage (at 10 °C) the number ofbacteria was 9.09 log cfu·mL–1. Sensory qualityof the juice just after fermentation was evaluatedat the level 6.3 + 1.5 (at the scale from 0 to 10).After 32 days of storage at 10 °C, the sensoryquality decreased to the level 3.0 + 1.4 (P < 0.05).

P-228 New generation probiotics: potentialenhancers of rumen function. N.D. Walkera,A. Ameilbonnea, E. Foranob, F. Chaucheyras-Durandc, B. Dulld (a Dansci-Beneflor Inc., Franceb INRA, Clermont-Ferrand-Theix, France, cLal-lemand Inc., Toulouse, France, dNutriscienceTechnologies Inc, USA).

Previous research has demonstrated that probi-otic yeasts may be used to positively enhancedifferent aspects of ruminal fermentation, ani-mal productivity and health. This is due toimproved ruminal pH stabilization, fibre diges-tion and anaerobiosis, which is reflected byincreased dry matter intake, milk yield and liveweight gain for the host. However, these positiveeffects can be strain and diet dependent, with dif-ferent yeast strains exhibiting different stimula-tory effects, depending on diet composition. In

Session IV: Nutritional manipulation S121

the frame of a new joint venture, Dansci-Bene-flor Inc, formed between Lallemand and Nutris-cience Technologies, a program has been under-taken to screen many different strains for theirpotential as new generation probiotics whichwill target specific areas of ruminal fermenta-tion, health and welfare. Different yeast strains(169) were selected on the basis that they wereGRAS organisms, grew rapidly and could beindustrially produced. All were from the genusSaccharomyces and in the first screening stepwere tested for their potential to enhance thegrowth and metabolic activity of Fibrobactersuccinogenes, an important ruminal fibre-degrader, already known to be positively affectedwhen grown in the presence of probiotic yeasts.The majority of yeasts tested had a positiveeffect on the growth and metabolic activity of F.succinogenes; in some instances causing almosta 2-fold increase in the rate of fibre-breakdownand ammonia utilization when compared withcontrol incubations. It can be concluded that theaddition of live yeast cells can stimulate thegrowth and activity of a fibre-degrading organ-ism like F. succinogenes, although the degree ofstimulation can be highly strain dependent.These highly-stimulatory yeast strains will beused in further screening tests.

P-229 Germination and conjugation of Bacil-lus thuringiensis in the gut of gnotobiotic rats.A. Wilcksa, L. Smidtb, L. Andrupb, M. Bahlc,B.M. Hansend, N.B. Hendriksend, T.R. Lichta

(a Danish Institute for Food and VeterinaryResearch, Søborg, Denmark, b National Instituteof Occupational Health, Copenhagen, Denmark,c University of Copenhagen, Copenhagen, Den-mark, d National Environmental Research Insti-tute, Roskilde, Denmark).

Bacillus thuringiensis (Bt) is an entomopatho-genic Gram-positive spore forming bacteriumused worldwide in the combat of insect pests. Inaddition to production of insect-specific toxins,Bt produces enterotoxins that may cause diar-rhoea in humans. Since plant protection agentsbased on Bt are widely used on e.g. tomatoes andcucumbers there is a risk that humans ingestingthose vegetables may also ingest Bt spores thatcould germinate in the gut and express entero-toxins causing disease. We used germfree ani-mals to study whether Bt spores are able to ger-

minate, express enterotoxins and transferplasmids in a mammalian gut. To study germi-nation, germfree rats were fed spores of a Btstrain harbouring a plasmid encoding Green Flu-orescent Protein enabling us to detect germina-tion by flow cytometry. In vivo conjugation wasstudied by associating germfree rats with a donorstrain harbouring the conjugative plasmid pXO16and an isogenic recipient strain. Both strainswere given as spores and transfer was observedfrom the donor to the recipient strain. Besides thefact that this is the first time that conjugationbetween Bt has been shown in a mammaliantract, this also confirms that the strain is able togerminate in vivo, since conjugation only canhappen between vegetative cells. In vivo enter-otoxin production was however only detected inone out of six animals, and in this animal onlyin the ileal sample.

P-230 Encapsulated fumaric acid as a meansof decreasing ruminal methane emissions.T.A. Wooda, R. Wallacea, A. Roweb, J. Pricec,D.R. Yáñez-Ruizc, S.P. Williamsc, C.J. New-boldc (a Rowett Research Institute, Bucksburn,Aberdeen, AB21 9SB, UK, b Rowett ResearchServices, Bucksburn, Aberdeen, AB21 9SB,UK, c The Institute of Rural Science, Universityof Wales, Aberystwyth, SY23 3AL, UK).

A decrease in methanogenesis in ruminantswould be advantageous to the agricultural indus-try in terms of energy loss to the animals as wellas being beneficial for the environment. Fumaricacid used as a feed supplement has the potentialto decrease methane production but the quantityfed has to be restricted because of the risk of aci-dosis and decrease in fibre breakdown as well asfeed intake that may result from a drop in pH.The objective of this study was to determine ifencapsulated fumaric acid (EFA) could decreasemethane formation in vitro and to assess its use-fulness in a lamb feeding trial. Fumaric acidencapsulated in partially hydrogenated vegeta-ble oil (PHVO) did not negatively affect pH orpropionate production when added to ruminalfluid in vitro, but retained its suppression ofmethane formation. Growing lambs on a controldiet with ad libitum straw produced 24.6 L·d–1

of methane whereas a 10% addition of fumaricacid (FA) or EFA decreased methane productionto 9.6 L·d–1 and 5.8 L·d–1 respectively (P < 0.001).

S122 Session IV: Nutritional manipulation

Live weight gain over 43 d was 182, 168 and 202g·d–1 while feed conversion ratios were 108, 119and 132 g gain·kg–1 feed intake for the control,FA and EFA groups respectively. The 75%decrease in methane described in this study is thelargest reported in the literature to date and aswell as having an impact on the environmentwhereby greenhouse gas emissions are abated,there are significant implications for the farmingindustry with increased efficiency of feed con-version.

P-231 Physiological and microbial status ofrats fed a diet containing grapefruit flavo-noids and inulin. M. Wróblewska, E.Biedrzycka (Institute of Animal Reproductionand Food Research of the Polish Academy ofSciences, Division of Food Science, 10 TuwimaStr., 10-747 Olsztyn, Poland).

Phenolic compounds and non digestible saccha-rides are potential functional supplements ofdiet. As both substances are intensively metab-olised in the large intestine, their potential phys-iological interaction is possible. The aim of thiswork was to characterise the physiological effectand caecal bacterial composition of the separateor joint supplementation of rat diet with grape-fruit flavonoids and inulin. The studies wereconducted on 32 male young Wistar rats. All ani-mals were fed everyday a fresh diet ad libitumwith permanent access to distilled water. After4 weeks of treatment rats were anaesthetised andserum and caecum content were collected. Addi-tion of both tested preparations caused enlarge-ment of caecal tissue and digesta, compared tothe control group. The presence of grapefruit fla-vonoids (0.3%) in the diet caused an increase ofdigesta pH, and a decrease of dry matter of cae-cal content, caecal activity of β-glucosidase andβ-glucuronidase, total short chain fatty acids andparticularly butyric acid concentration. Dietaryflavonoids also significantly affected the caecalpopulation of Bifidobacterium and Escherichiacoli. When 5% inulin was added to the diet, theactivity of α- and β-galactosidase and the con-centration of butyric and propionic acidincreased and the pH of the caecum decreased.Inulin addition to the diet reduced the caecalpopulation of Escherichia coli and enlarged theBifidobacterium population. The dietary combi-nation of both tested preparations had a greaterimpact on the activity of galactosidases and

butyric acid content and caused more beneficialchanges in a bacterial population.

P-232 Real-time PCR and PCR-DGGE anal-yses of stool microbiomes from infants fedhuman breast milk, infant formula, or for-mula with added fructo-oligosaccharides.Q. Xia, Z. Yu, T. Premaraj, T. Williams,M. Morrison (Department of Animal Sciences,The Ohio State University, Columbus, OH43210, USA).

The colonization and development of microbi-omes throughout the infant gastrointestinal tractare affected by the mode of post-natal nutrition(human milk vs. infant formulas). There arehypotheses that these differences may havelong-term impact on health and development.Oligosaccharides present in human milk arebelieved to be the major components in humanmilk that stimulate the growth of bifidobacteriaand lactobacilli. For these reasons, we evaluatedinfant formulas without and with an added pre-biotic, fructooligosacharide (FOS), for its impacton fecal flora relative to a reference group ofhuman milk fed infants. We obtained samples ofinfant stools to assess the microbiota approxi-mately 30 days after birth and after 27 days offeeding on a milk-based infant formula (FF), FFsupplemented with FOS at 2.4 and 3.4 g·L–1

(FF-L and FF-H, respectively), or human milk(HM). Sixty-five samples (17 HM, 20 FF-H, 14FF-L, and 14 FF) were analyzed. The abundanceof total bacteria, bifidobacteria, lactobacilli,Clostridium difficile, and E. coli were estimatedby real-time PCR assays. We used PCR-DGGEto assess the impact on total bacteria, as well asintrageneric diversity of bifidobacteria andlactobacilli. The HM group had significantlylower total bacterial abundance than the FOSsupplemented formula-fed groups (HM vs.FF-H, P < 0.02; HM vs. FF-L, P < 0.02, HM vs.FF P > 0.07). Though statistically significant,the differences were rather small. The real-timePCR results found that the least square mean val-ues for total bifidobacteria were also not greatlydifferent among the four feeding groups. Theleast square mean values for the FF-H and FFgroups were slightly higher (by less than 0.9 log)than that for the HM and FF-L groups (not sta-tistically significant). In conclusion, FOS sup-plementation at the described levels did not

Session IV: Nutritional manipulation S123

appreciably affect the total number of bifidobac-teria or lactobacilli in infant stool samples. How-ever, the PCR-DGGE and DNA sequence anal-yses suggest subtle shifts in the species ofbifidobacteria and lactobacilli in infants con-suming infant formula when compared to humanmilk, irrespective of the addition of FOS.

P-233 Microbial numbers and diversity in therumen of sheep supplemented with fumaricacid. D.R. Yáñez-Ruiza, G. Be eckib, R.J.Wallacec

, C.J. Newbolda (a Institute of RuralSciences, University of Wales, Aberystwyth,SY23 3AL, UK, b Kielanowski Institute of Ani-mal Physiology and Nutrition Polish Academyof Sciences, 05–110 Jab onna, Poland, c RowettResearch Institute, Bucksburn, Aberdeen AB219SB, UK).

Organic acids, including malic and fumaric acid,have been shown to decrease methane formationin the rumen, however, their acidic propertiesrestrict the quantity which can be fed. In a recentexperiment we have found that dietary fumaricand encapsulated fumaric acid (as a means ofavoiding the acidic effect) decreased methaneformation by sheep by 50 and 75%, respectively.The present study was designed to determine theinfluence of adding encapsulated and freefumaric acid, compared to a control diet, on bac-terial and protozoal diversities and on the num-bers of bacteria, protozoa and methanogens inthe rumen. Microbial diversity was assessed byPCR-DGGE using universal primers thatamplify specific regions of the 16S and 18SrDNA, respectively, for bacteria and protozoa.Microbial DNA quantification was assessed byreal-time PCR using specific primers designedfor bacteria, protozoa and ruminal methanogens.Cluster analysis of the banding profiles showeda tendency of samples treated with fumaric acidand encapsulated fumaric acid to be clusteredtogether for bacterial DGGE, while no clear pat-tern was observed for rumen protozoa. Real-time PCR revealed no differences in numbers ofbacterial, protozoa or methanogens betweengroups, and a large variability within groups interms of protozoa numbers. Our preliminaryresults suggest that fumaric acid caused a shiftin the type of bacteria in the rumen, but had no

effect on protozoal diversity nor on microbialnumbers.

P-234 Daidzein affects the composition of gas-trointestinal bacterial community of piglets.Z.-T. Yu, W.Y. Zhu, W. Yao (Laboratory ofGastrointestinal Microbiology, Nanjing Agri-cultural University, 210095, PR China).

Daidzein is one of the estrogenic isoflavoniccompounds. Researches have demonstrated thatdaidzein could affect growth hormones, immu-nity, and growth performance in animals. Ourrecent study showed that daidzein could alsoincrease the number of lactobacilli in pigletdigesta after in vitro fermentation. Effects ofdaidzein on the intestinal bacterial communityof piglets were investigated using 16S rDNA-based approaches. Six litters of neonatal pigletswere allocated into control group and treatmentgroup. The piglets in the treatment orallyreceived 1 mL, 2 mL and 3 mL daidzein(10 mg·L–1) on day 7, 9 and 11, respectively. Allpiglets were weaned on day 21. On day 14, 21,24 and 35, one piglet of each group, respectively,was slaughtered. The gut digesta were collectedfor DNA extraction and analysed by PCR/DGGE analysis. Shannon’s index showed noapparent difference in microbial diversitybetween treatment and control groups beforeweaning. However, after weaning, piglets fedwith daidzein showed a higher bacterial diver-sity in colon and caecum compared with thosein the control. This effect was apparent when thepiglets were 35 d old. Three DGGE bands exist-ing in the large intestine of the control wereabsent in the treatment, and their matchingclones had 16S rDNA sequences closest toClostridium thermocellum, Lactobacillus pontisand Streptococcus sp., respectively. Anotherthree bands were present only in treatment pig-lets, and their corresponding clones hadsequences closest to butyrate-producing bacte-rium SL7/1, Clostridium butyricum and Rumi-nococcus obeum, respectively. In summary,daidzein increased the bacterial diversity of thepiglet large intestine, but this effect was notapparent until after weaning, two weeks afterdaidzein treatment.

ł ż

ł

S127Reprod. Nutr. Dev. 46 (2006) S127–S129© INRA, EDP Sciences, 2006

IL-2 Diversity and function of the human gutmicrobiota. H.J.M. Harmsen, L.C.M. Wildeboer-Veloo, G.C. Raangs, R.H.J. Tonk, S.P. VanTongeren, J.E. Degener, G.W. Welling (Depart-ment of Medical Microbiology, UniversityMedical Center Groningen, University of Gro-ningen, Hanzeplein 1, 9713 GZ Groningen, TheNetherlands).

The large number of bacteria present in theintestinal tract of a human is the essential partof an ecosystem that is strongly interacting withthe host and plays an important role in health anddisease. To investigate the diversity we haveused fluorescent in situ hybridisation (FISH) fordetermination of the number and identity ofintestinal bacteria and compared it to the diver-sity detected by clone library analysis. Further-more, the FISH methodology was used to study:(i) what is the diversity and composition of theintestinal microbiota and is it related to the ageof the human host (ii) and what is the functionof all these microorganisms in relation to healthand diseases. Health and disease plays an impor-tant role in elderly. Frailty parameters can beused to describe the vulnerability to disease anddecline of elderly. In our present studies we tryto establish a relation between faecal microbiotacomposition and frailty in elderly (age> 75 years). The results show significant differ-ences between the bacterial composition of fae-cal samples from high frail and low frail elderly.Culture-independent methods have becomeavailable to detect and to quantify bacteria.Analysis of clone libraries from several studieshave revealed the diversity in the human gut.However, it is not fully known what the relationof this diversity is with the health of the host orwhether this diversity is related to age and/or lifestyle. We aim at studying the function of somehard-to-culture bacteria in this microbial diver-sity. Probes were designed for groups of bacteriathat are detected by clone library analysis, butof which the relevance in terms of quantity andfunction in the gut is unknown. Applying theseprobes on faeces of volunteers and the elderlymentioned before showed the presence ofClostridium viride group bacteria, C. propioni-cum, Eggerthella lenta and the Roseburia genusin relation with ageing. Some of these changesmay be related to a changing diet with age, forinstance the intake of dietary and insoluble fiber.These fibers are believed to play a beneficialrole in gut health. Bacterial degradation of these

fibers may increase the production of shortchain fatty acids such as butyrate. The involve-ment of different bacterial groups in fiber deg-radation was demonstrated using FISH on fibersisolated from faeces. We detected the involve-ment of Ruminococcus species, Eubacteriumrectale/C. coccoides, Roseburia and Faecali-bacterium species that were associated with thefiber structures. In all of the above-mentionedstudies large individual differences wereobserved. Our general conclusion is that the bac-terial composition of the intestinal microbiota isunique in each individual.

IL-3 From phylogenetics to metagenomics –insight in gut microbial functionality. J. Doré(INRA, Unité d’Écologie et Physiologie du Sys-tème Digestif, 78352 Jouy-en-Josas Cedex,France).

During the past century, recognition of anaero-biosis and the development of anaerobic culturetechniques allowed the isolation and character-ization of a large bacterial diversity from thedominant human fecal microbiota. It is com-monly reported that over 400 species composethis microbiota. Nevertheless, a large fraction ofthe dominant gut microbes remains uncultura-ble and most recent evaluations convergetowards 70% uncultured cells from fecal dilu-tions. Today, the long awaited culture independ-ent tools that dramatically developed during thepast decades have been allowing a completereassessment of human gut microbial diversity.Molecular tools have their own biases and lim-itations, yet they allow the recognition of a myr-iad of species bearing no cultured representativein currently available strain collections. Com-parison of 16S rDNA libraries obtained by clon-ing has proven especially adapted. We willreview the information derived from this phyl-ogenetic approach that sets the stage for futureperspectives of functional genomics. On a molec-ular basis, 80% of the phylotypes observed in thefecal microbiota of healthy young adults belongto four phylogenetic groups : the Gram negativeBacteroides-Porphyromonas-Prevotella cluster,low-GC Gram positives of the phylum Firmi-cutes, belonging to the Eubacterium rectale-Clostridium coccoides (cluster XIV) phylogeneticgroup and to the Fusobacterium prausnitzii-Clostridium leptum (cluster IV) and high-GCGram positives of the Bifidobacterium and Col-linsella-Atopobium groups. Other approaches

S128 Yakult Symposium

such as fluorescent in situ hybridization haveconfirmed this observation. Further, 80% ofthe phylotypes (60% of the cloned rDNAsequences) derive from microorganisms thathave no culturable representative. This is espe-cially true for the Firmicutes. The proportion ofyet un-recognized species (present in an individ-ual’s gut microbiota) increases from birth to theold age. Accordingly, specificities of the gutmicrobiota associated with ageing or intestinaldisorders such as inflammatory diseases can beoutlined. Comparative analysis of several 16SrDNA based molecular inventories indicatesthat the dominant fecal microbiota is essentiallyspecific to its host at the species level. Highthroughput methods also inform us of its resist-ance to modification and its marked resiliencefollowing stress. Considering the expected func-tional homogeneity of the human intestinal eco-system, it may be speculated that a true func-tional redundancy will be evidenced betweenindividuals. Yet it is not clear at this stagewhether this is to be found at the level of the gutmicrobiota genome, proteome or metabolites.We will review the preliminary results derivedfrom the investigation of the metagenome of thehuman faecal microbiota. This has beenaddressed using a comprehensive metagenomicapproach to investigate the full range of intesti-nal microbial diversity and potential functional-ities. Two libraries of genomic DNA isolateddirectly from fecal samples of six healthy donorsand six patients with Crohn’s disease (CD) wereconstructed to this end, each composed of25 000 clones. Characterization of 16S rDNAswithin metagenomic clones identified 125 non-redundant ribotypes mainly represented by thephyla Bacteroidetes and Firmicutes. Speciesdiversity within Firmicutes was lower in CDpatients than in healthy subjects (13 vs. 43 dis-tinct ribotypes; P < 0.025). This was confirmedby FISH using fecal samples analysed individu-ally (n = 12; P < 0.02). Metagenomic librarieswere also investigated by functional screening.This allows investigation of yet totally unex-plored genomic resources from the gut environ-ment, some of which will prove highly relevantto our understanding of the hots bacteria dia-logue. The metagenomic approach appearspromising to identify most redundant genomictraits of the human intestinal microbiota andthereby identify the functional balance of thisorgan. It will further contribute to substantiatethe concept of a functional core within the intes-tinal microbiome.

IL-4 The gut microflora and colorectal cancerrisk. I. Rowland (Northern Ireland Centre forFood and Health, University of Ulster, Cole-raine, BT52 1SA, Ireland).

Colorectal cancer is one of the major causes ofdeath from malignant disease in Western Europe,USA, and Australia. There is considerable evi-dence from laboratory animals and human stud-ies that the colonic microflora is involved in theaetiology of colon cancer. For example, germ-free rats treated with the carcinogen 1,2-dimeth-ylhydrazine have a lower incidence of colontumours than similarly treated rats having a nor-mal microflora. We have demonstrated potentDNA-damaging activity in faecal extracts froma proportion of healthy human subjects. Further-more, intestinal bacteria possess a range of xeno-biotic metabolizing enzymes that enable them toproduce, from dietary components, substanceswith genotoxic, carcinogenic and tumour-pro-moting activity. They can synthesize carcino-gens such as N-nitroso compounds, activate car-cinogens to reactive DNA-damaging metabolitesand deconjugate detoxified carcinogens in thecolon releasing the active carcinogen. In addi-tion they can produce tumour promoters such asammonia and bile acids. It follows from theabove that dietary regimens that modify the gutmicroflora may alter colon cancer risk. Probiot-ics and non-digestible oligosaccharides (prebi-otics), which significantly increase bifidobacte-ria and lactobacilli in the gut, have considerablepotential in this regard and there is considerableevidence from in vitro studies and animal exper-iments that they inhibit DNA damage and sup-press pre-cancerous lesions and tumours in thecolon. Evidence from epidemiological studiesfor anticancer effects of probiotics is limited, butrecent dietary intervention studies in healthysubjects and in polyp and cancer patients haveyielded promising results on the basis of biomar-kers of cancer risk. Potential mechanisms for theanticarcinogenic activity of probiotics includebinding of carcinogens, induction of apoptosis,stimulation of Phase II enzymes, and modulationof carcinogen formation by gut bacteria.

IL-5 Microbially-induced inflammation andcolon cancer. E.M. El-Omar (Department ofMedicine and Therapeutics, Aberdeen Univer-sity, Aberdeen AB25 2ZD, Scotland).

Yakult Symposium S129

Impressive epidemiologic evidence shows aclear association between chronic inflammatoryconditions and subsequent malignant transfor-mation in the inflamed tissue, particularly of theGI tract. The stimulus for the inflammation isoften an infective agent such as viruses or para-sites (e.g. Hepatitis B/C and Schistosomiasis inhepatocellular carcinoma), and bacteria (e.g.Helicobacter pylori -associated gastric cancer).Chronic inflammation is also very relevant to therisk of colorectal cancer (CRC). Inflammatorybowel disease is known to substantially increasethe risk of CRC and anti-inflammatory medica-tions (e.g. aspirin) are known to reduce it. Therole of gut bacteria in CRC has recently receivedconsiderable attention. There is now definitiveevidence that the commensal gut microbiotaplays a crucial role in maintaining the healthymucosal barrier. Any disruption of this normalhomeostasis is associated with colonic inflam-mation and increased risk of CRC. Our recentwork has focussed on assessing the degree andphenotype of inflammation within normal and

neoplastic colonic tissue. We have establishedthat colorectal polyps have a significantlyincreased complement of acute and chronicinflammatory cells compared to their immedi-ately adjacent normal mucosa. Furthermore,there is a progressive increase in chronic inflam-matory cells and mediators that match the pro-gressive increase in the size of polyps and theirgrade of dysplastic change. This suggests thatchronic inflammation is the fundamental patho-physiological mechanism underlying CRC. Wehave proceeded to investigate the aetiology ofthe inflammation seen in the polyps and our pre-liminary data indicate that a large proportion ofthese polyps have a significantly differentmucosal microbiota compared to their immedi-ately adjacent normal mucosa. This differencemay reflect a disrupted homeostasis within pol-yps caused by acquisition or loss of crucial bac-terial species that are relevant to inflammationand cancer risk. This field is expanding rapidlyand the future promises to be very excitingindeed.