CURRENT MICROBIOLOGY Vol. 52 (2006), pp. 158162 DOI: 10.

1007/s00284-005-0266-9

Current Microbiology
An International Journal
Springer Science+Business Media, Inc. 2006

Real-Time PCR Detection of the Effects of Protozoa on Rumen Bacteria in Cattle

Yuhei Ozutsumi,1 Kiyoshi Tajima,2 Akio Takenaka,2 Hisao Itabashi1
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan 2 Department of Animal Physiology and Nutrition, National Institute of Livestock and Grassland Science 2 Ikenodai, Tsukuba-shi, Ibaraki 305-0901, Japan Received: 26 August 2005 / Accepted: 21 September 2005
1

Abstract. A real-time PCR approach was used in this study to clarify the populations of major bacterial species in the rumens of faunated and unfaunated cattle. The sensitivity of this novel real-time PCR assay was evaluated by using 101 to 108 plasmid copies of target bacteria. The numbers of plasmid copies of Ruminococcus albus, Ruminococcus flavefaciens, Prevotella ruminicola, and the CUR-E cluster were higher in the unfaunated than in the faunated rumens. The CUR-E cluster belongs to the Clostridium group. In contrast, Fibrobacter succinogenes was higher in the faunated than in the unfaunated rumens. Although it is well known that an absence of protozoa brings about an increase in the bacterial population, it was clarified here that an absence of protozoa exerted differential effects on the populations of cellulolytic bacteria in cattle rumens (i.e., F. succinogenes, R. albus, and R. flavefaciens). In addition, real-time PCR analysis suggested that the CUR-E cluster was more prevalent in the unfaunated rumens.

Ruminal anaerobic microorganisms include a variety of bacteria, ciliate protozoa, and fungi. These microorganisms constitute the rumen microbial ecosystem. In the analysis of this system, interactions among the microbes are of paramount importance. In particular, protozoa that account for a large biomass in the rumen influence not only rumen volume but also the relative proportions of bacterial species and the overall size of the bacterial population [17]. The presence of ciliate protozoa in the rumen is termed faunated, whereas its absence is termed unfaunated. In a previous study, we compared the bacterial compositions of faunated and unfaunated rumens using 16S rDNA clone libraries, and we determined that Bacteroides and Prevotella were the major groups in the faunated rumens, whereas the Clostridium botulinum group and its relatives (CLUSTER-B and CLUSTER-E) were the major groups in the unfaunated rumens [11]. Analysis with the computer program LIBSHUFF clari-

fied that the presence of ruminal protozoa markedly affected the composition of the rumen bacterial community [11]. However, the investigation of 16S rDNA clone libraries did not lead to the determination of the structure of the rumen bacterial population. Consequently, there was a need for novel, specific methods to monitor and quantify rumen bacteria in faunated and unfaunated rumens. Real-time PCR enabled the direct quantification of dominant and subdominant rumen bacterial populations by using DNA isolated from rumen fluid [13]. In this study, to analyze the relationships between protozoa and bacterial populations, we carried out the detection and quantification of rumen bacteria in faunated and unfaunated rumens using species-specific and group-specific primer sets targeting the 16S rDNA using a real-time PCR approach. Materials and Methods
Reference strains and culture conditions. The following were used as reference strains: Fibrobacter succinogenes (ATCC 19169T), Ruminococcus albus (ATCC 27210T), Ruminococcus flavefaciens

Correspondence to: Hisao Itabashi; email: hita@cc.tuat.ac.jp

Ozutsumi et al.: Real-Time PCR Analysis of Bacteria in Cattle Table 1. Rumen bacteria targeted and primer information Target bacteria or bacteria group Streptococcus bovis Ruminobacter amylophilus Anaerovibrio lipolytica Treponema bryantii Megasphaera elsdenii Succinivibrio dextrinosolvens Fibrobacter succinogenes Ruminococcus albus Ruminococcus flavefaciens Prevotella ruminicola Prevotella bryantii Prevotella albensis CUR-E cluster Primer (forward / reverse) Sb-F/Sb-R Ram-F/Ram-R Al-F/Al-R Tb-F/Tb-R MelsF/MelsR Sd-F/Sd-R Fs-F/Fs-R Ra-F/Ra-R Rf154f/Rf425r Pr-F/Pr-R Pb-F/Pb-R Pa480F/Pa840R OZ660F/OZ780R Annealing temp. (C) 58 58 58 58 60 58 63 62 55 58 68 55 60 Product size (bp) 869 642 597 421 128 854 445 176 295 485 540 400 140 Reference

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Tajima et al. [13] Tajima et al. [13] Tajima et al. [13] Tajima et al. [13] Ouwerkerk et al. [10] Tajima et al. [13] Tajima et al. [13] Tajima et al. [13] Koike and Kobayashi [7] Tajima et al. [13] Tajima et al. [13] This study This study

(ATCC 19208T), Prevotella ruminicola (ATCC 19189T), Prevotella bryantii (B14T), Prevotella albensis (M384T), Prevotella brevis (ATCC 19188T), Anaerovibrio lipolytica (ATCC 33276T), Streptococcus bovis (JCM 5802T), Succinivibrio dextrinosolvens (ATCC 19716T), Ruminococcus productus (ATCC 27340T), Treponema bryantii (ATCC 33254T), Eubacterium ruminantium (ATCC 17233T), Eubacterium cellulosolvens (ATCC 43171T), Megasphaera elsdenii (JCM 1772T), Clostridium proteoclasticum (ATCC 51982 T), Ruminobacter amylophilus (NIAH3), Selenomonas ruminantium (JCM 6582T), Butyrivibrio fibrisolvens (ATCC 19171T), Butyrivibrio fibrisolvens (OB156), and Butyrivibrio fibrisolvens (OB157). All strains were cultured in medium 10 [2]. Animals and sampling. Seven castrated Holstein cattle (half-siblings, mean body weight 144 kg), reared at the Field Science Center of the Tokyo University of Agriculture and Technology, were divided into two groups: four faunated and three unfaunated. The cattle were isolated from their dams within a few days of birth, and were penned individually in an isolated building. After weaning at 6 weeks of age, four cattle were inoculated with a mixed protozoa population obtained from other adult cattle. The other three cattle were maintained protozoa-free. All seven cattle were fed a diet consisting of 66% (DM) chopped Sudangrass hay and 34% concentrate mixture at a maintenance level for energy and crude protein, twice daily at 09:00 h and 17:00 h in equal amounts. The concentrate contained 41% corn, 23% wheat bran, 8% rapeseed meal, 7% soybean meal, 12% corn byproducts, 5% grains, and 4% minerals and vitamins. Water was continuously available. Samples of ruminal fluid (ca. 300 mL) were obtained before feeding by a suction pump using a flexible polyvinyl chloride stomach-tube. The collected samples were squeezed through two layers of surgical gauze and stored at )20C until analysis. DNA extraction. Total chromosomal DNA from the rumen bacterial strains was extracted as described by Tajima et al. [13]. Total DNA from the rumen fluid was extracted as previously described [11]. To minimize animal-to-animal variations, each sample of faunated DNA (4 samples) and unfaunated DNA (3 samples) was mixed with a same concentration of DNA (final concentration of 15 ng/mL) after DNA extraction. Design of species-specific primers and group-specific primers. The primers used are presented in Table 1. We reported the new operational taxonomic units (OTUs) obtained from the unfaunated

rumen in CLUSTER-E [11]. These primers were designated as characteristic of unfaunated rumens in CLUSTER-E (CUR-E). Species-specific PCR primers used to amplify partial 16S rRNA gene regions were chosen from those described in the literature [7, 10, 13] as well as from the newly designed Prevotella albensis and the group-specific CUR-E cluster. Primers for P. albensis were Pa480F (5-ATG TTA TCC ACG TGT GGA TAT-3) and Pa840R (5-CCA AAT CCA AAA GGA CTC AGA-3). Primers for CUR-E were OZ660F (5-TAA GGT GGG AAT TGC TTT GGA T-3) and OZ780R (5-CAG TTA CAG TCC AGT AAG TC-3). The specificity of the amplifications was confirmed by sequencing and phylogenetic analysis. After detection of species-specific and group-specific PCR amplicons in the total rumen DNA, the PCR products were sequenced with an ABI PRISM 3730 Genetic Analyzer (Applied Biosystems), and phylogenetic analysis was conducted as described previously [11]. The primers and PCR conditions were tested in a qualitative PCR assay to ensure the specificity of the reaction. Conventional PCR. PCR amplification was conducted with an iCycler thermal cycler (Bio-Rad, Hercules, CA) as described previously [11]. The reaction mixtures were treated according to the following protocol: 95C for 3 min, followed by 30 cycles consisting of 95C for 30 sec, various annealing temperatures (described in Table 1) for 30 sec, 72C for 1 min, and a final extension period of 72C for 7 min. Real-time PCR. The targeted bacteria were cellulolytic bacteria (F. succinogenes, R. albus, and R. flavefaciens), Prevotella species (P. ruminicola, P. bryantii, and P. albensis), and a group-specific CUR-E cluster. To establish a quantitative assay, we cloned plasmids containing the amplified target region of each species by using the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA). With the use of the QIAprep kit (Qiagen, Venlo, The Netherlands), the purified plasmids were quantified by spectrophotometry with multiple dilutions. The target DNA used for these experiments possessed an A260/A280 ratio greater than 1.8. The target DNA was quantified by using serial 10-fold dilutions from 101 to 108 plasmid copies of the previously quantified plasmid standards. Real-time PCR amplification and detection were performed in a LightCycler system (Roche, Mannheim, Germany) as described by Tajima et al. [13]. In brief, FastStart DNA Master SYBR Green I was used for PCR amplification. Samples were assayed in duplicate in a 20-lL reaction mixture contained 5 mM MgCl2, 2 lL of

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CURRENT MICROBIOLOGY Vol. 52 (2006)

Fig. 1. PCR products resulting from the use of 11 species specific and 1 species cluster primer sets in faunated and unfaunated rumens. Lane M, molecular weight marker, with bands of 1,000, 750, 500, and 100 bp depicted. Lane F, faunated rumens; lane U, unfaunated rumens. 10 Mastermix (including FastStart enzyme, FastStart Taq DNA polymerase, reaction buffer, dNTP mixture, MgCl2, and SYBR Green I dye), 30 ng of template DNA, and 0.5 lM of each primer. All PCRs were performed in duplicate. Table 2. Sequence analysis of clone libraries generated by speciesspecific primers from total rumen DNA samples No. of clones sequenced Fibrobacter succinogenes Ruminococcus albus Ruminococcus flavefaciens Prevotella ruminicola Prevotella bryantii Prevotella albensis CUR-E cluster 27 21 32 32 20 20 40 % Similarity (mean SD) 98.2 99.9 96.9 99.6 99.0 98.0 97.5 1.0 0.3 1.5 0.5 0.5 0.0 0.7

Results and Discussion

Detection of rumen bacteria. Sets of specific primers (Table 1) were used to detect the presence of these bacterial species in the faunated and unfaunated rumens (Fig. 1). In the faunated rumens, all targeted species were detected with specific primers, except for M. elsdenii. In the unfaunated rumens, PCR signals of R. amylophilus, P. bryantii, and P. albensis were not detected. The signals of R. albus, T. bryantii, and S. dextrinosolvens in the faunated rumens were markedly weaker than the signals in the unfaunated rumens (Fig. 1). Specificity of primers. The primer pairs of F. succinogenes, R. albus, R. flavefaciens, P. ruminicola, P. bryantii, and the newly designed P. albensis did not amplify any of the 20 other rumen bacterial species tested (data not shown), indicating that the primer pairs were highly specific. The primer pairs of the group-specific CUR-E primers did not amplify any of the 21 other rumen bacterial species tested (data not shown). To evaluate the specificity of the primers, specificprimer PCR assays were applied to DNA extracted from rumen samples. Mini-libraries were constructed from each amplification reaction, and 20 to 40 randomly chosen clones from each of the seven libraries were sequenced. The similarities were calculated from the specific library sequences (Table 2), and the values

within the specific libraries were well within the species definition range proposed on the basis of an analysis of 16S rRNA similarity [12]. All clones of the group-specific CUR-E primer sequences were positioned at target regions in the phylogenetic analysis (data not shown). Each specific primer set gave positive PCR results for the corresponding target bacteria. Quantification of bacteria in the faunated and unfaunated rumens. The quantification results are shown in Table 3. The number of 16S rRNA gene copies of F. succinogenes was higher in the faunated than in the unfaunated rumens. The number of F. succinogenes in the faunated rumens was 2.4 times that of the unfaunated rumens. In contrast, the numbers of R. albus, R. flavefaciens, and P. ruminicola, as well as the frequency of the CUR-E cluster, were higher in the unfaunated than in the faunated rumens. The number of R. albus in the unfaunated rumens was 3.5 times that in the faunated ones. The number of R. flavefaciens in the

Ozutsumi et al.: Real-Time PCR Analysis of Bacteria in Cattle Table 3. Quantification of rumen bacteria by real-time PCR Log10 No. of 16S rRNA gene copies/mLa Bacteria targeted Fibrobacter succinogenes Ruminococcus albus Ruminococcus flavefaciens Prevotella ruminicola Prevotella albensis Prevotella bryantii CUR-E cluster Faunated 8.1 7.7 8.4 8.9 6.0 6.1 9.6 6.4 6.6 6.6 7.6 5.0 5.3 7.3 Unfaunated 7.7 8.3 8.8 9.3 ND ND 10.3 6.8 6.3 7.9 7.9

161 species in the rumen [5]. The present results revealed that the numbers of R. albus (log10 8.3 6.3 per mL) and R. flavefaciens (log10 8.8 7.9 per mL) were higher in the unfaunated rumens than in the unfaunated rumens (log10 7.7 6.6 per mL and log10 8.4 6.6 per mL, respectively). However, the number of F. succinogenes (log10 7.7 6.8 per mL) was lower in the unfaunated than in the faunated rumens (log10 8.1 6.4 per mL) (Table 3). Although the reason for this is not clear, the absence of protozoa did not necessarily lead to an increase in the population of cellulolytic bacteria (i.e., F. succinogenes, R. albus, and R. flavefaciens). The number of 16S rRNA gene copies of CUR-E clusters was log10 10.3 8.6 per mL rumen fluid in the unfaunated and log10 9.6 7.3 per mL rumen fluid in the faunated rumens (Table 3). Real-time PCR suggested the CUR-E cluster was more prevalent in the unfaunated rumens, in which this cluster may, therefore, play an important role. As the function of this CUR-E cluster remains unknown, the characteristics of the cultured representatives of this cluster are of considerable interest and merit further study. In conclusion, we have demonstrated the applicability of real-time PCR for the quantification of bacterial species in the complex microbial ecosystems of faunated and unfaunated rumens. Although it is well known that an absence of protozoa brings about an increase in the bacterial population, the present study clarified that an absence of protozoa exerted differential effects on the populations of cellulolytic bacteria in cattle rumen (i.e., F. succinogenes, R. albus, and R. flavefaciens). In addition, real-time PCR analysis suggested that the CUR-E cluster was more prevalent in the unfaunated rumen.
ACKNOWLEDGMENTS We would like to thank the staff and students at the Field Science Center of the Tokyo University of Agriculture and Technology for their help with the animal care and sampling.

8.6

ND, not detected. a Results are shown as logarithms of 16S rRNA gene copy numbers per 1 ml of rumen fluid standard deviation. The values were calculated with total DNA purified from 1 ml of rumen fluid. The detection limit of the quantitative PCR was log10 6.0 copies per ml of rumen fluid.

unfaunated rumens was 2.0 times that in the faunated ones. The number of P. ruminicola in the unfaunated rumens was 2.6 times that in the faunated ones. The number of CUR-E clusters in the unfaunated rumens was 4.5-fold that in the faunated ones. P. bryantii and P. albensis were detected in the faunated rumens only, though in small numbers (Table 3). Prevotella species are some of the most numerous among rumen bacteria [16]. P. ruminicola is one of the principal species among the amylolytic bacteria [3, 8]. All entodiniomorphid protozoa are known to engulf starch grains [6]. Amylolytic bacteria were shown to attach to starch grains, and the ingestion of starch was found to be accompanied by selective predation [15]. These findings may account for the higher number of P. ruminicola in the unfaunated than in the faunated rumens (Table 3). Rumens that did not contain protozoa had enhanced populations of amylolytic bacteria; this finding is in agreement with previous results [1, 15]. Logarithms of 16S rRNA gene copy numbers per 1 mL of rumen fluid were calculated. The number of 16S rRNA gene copies of P. ruminicola was log10 9.3 7.9 per mL rumen fluid in the unfaunated rumens and log10 8.9 7.6 per ml rumen fluid in the faunated ones (Table 3). Approximately one-quarter to one-third of the fiber degradation in the rumen is protozoal [4, 9]. Takenaka et al. [14] employed molecular techniques to demonstrate that ruminal protozoa possess fibrolytic enzymes. When the protozoa were removed from the rumen, the total viable counts and cellulolytic bacteria increased [1, 15], and the digestion of fiber in the rumen was reduced [17]. At present, F. succinogenes, R. albus, and R. flavefaciens are considered the representative cellulolytic

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