Metagenomics
Vol. 1 (2012), Article ID 235571,11pages doi:10.4303/mg/235571
Research Article
The Bacterial Community Composition of the Bovine Rumen Detected
Using Pyrosequencing of 16S rRNA Genes
Sitao Wu,1Ransom L. Baldwin 6th,2Weizhong Li,1Congjun Li,2Erin E. Connor,2and Robert W. Li2
1Center for Research in Biological Systems, University of California, San Diego, CA, USA 2USDA-ARS, Bovine Functional Genomics Laboratory, Beltsville, MD, USA
Address correspondence to Robert W. Li, [email protected]
Received 8 April 2012; Revised 23 April 2012; Accepted 24 April 2012
Abstract The rumen bacterial composition of both pre-ruminant dairy calves and cows and beef steers was surveyed using pyrosequencing of the 16S rRNA gene. Sequences were analyzed using taxonomy-dependent and -independent clustering methods. The core rumen microbiome, regardless of the rumen developmental status or breeds, consisted of 8 phyla, 11 classes, 15 families, and 17 genera. Principal component analysis and clustering demonstrated that the bacterial communities in the rumen of pre-ruminant dairy calves, dairy cows, and beef steers were clearly distinguishable. Approximately 66% of phyla and 41% of Operational Taxonomic Units (OTUs) in a typical rumen bacterial community differed in relative abundance between the developing and mature rumen. Greater abundance of Fibrobacteraceae and Ruminococaceae in the rumen of beef steers likely reflected the need for enhanced fiber-digesting capacity in beef cattle. Our results should facilitate understanding of the structural and functional relationships in the rumen microbial ecosystem.
Keywords 16S rRNA; cattle; hypervariable regions; meta-genomic; rumen; pyrosequencing
1 Introduction
The rumen as a complex microbial ecosystem has played a critical role in sustainable agriculture throughout human civilization. Microorganisms in the rumen, such as bacteria, archaea, protozoa, and fungi perform essential fermentation, including the conversion of plant fiber to small molecules, such as volatile fatty acids (VFA) and vitamins, for the pro-duction of meat and milk for human consumption, thereby influencing the host’s nutrition. In addition, rumen microor-ganisms modulate host immunity and enhance host resis-tance to invading pathogens [23]. Furthermore, certain bac-teria detoxify naturally occurring compounds in the diet that are harmful to the host [13]. Microorganisms in the rumen are highly responsive to changes in diet, host genetics, and physiology, as well as geographical and environmental fac-tors.
platforms has stimulated the research in rumen microbial diversity and rumen ecosystem dynamics [15,19,22,24]. For example, the rumen microbiota of dairy cows harbors 54.5±6.1 genera (mean±sd) and 127.3±4.4 operational taxonomic units (OTUs), respectively [24]. Collectively, 21 prokaryotic phyla have been identified in the rumen of lactating dairy cows. However, the microbial diversity and bacterial population dynamics in the rumen of beef cattle, especially during their critical physiological stages such as adaptation to a high-grain diet in feedlots, have not received sufficient attention. In this study, we examined the ruminal bacterial composition of beef steers using pyrosequencing of 16S rRNA genes. Moreover, we compared the bacterial community composition of the rumen between dairy and beef cattle. The difference in taxonomical profiles of the microbiota between the mature rumen and the developing rumen of dairy cattle was investigated as well.
2 Materials and methods
2.1 Animals and diet
A total of 15 animals were used in this study, including 8 Angus beef steers (∼1 year old), 4 Holstein cows in mid-lactation (∼3 years), and 3 pre-ruminant Holstein bull calves of 42 days old. All animal procedures were conducted according to the protocols approved by the Beltsville Area Animal Care and Use Committee. Eight Angus beef steers (272.5±17.6 kg initial body weight), fitted with ruminal and abomasal infusion catheters, were used in a randomized complete block design. Prior to experimentation steers were dewormed, vaccinated, and acclimated for 30 d. During this time, steers were halter broken and introduced to the tie stall facility. During the acclimation period and throughout the experiment, steers received a pelleted basal diet and were fed to ∼1.5 maintenance metabolizable energy requirements (160 Kcal metabolizable energy/(kg BW0.75·d)) for growing steers of this frame size.
The basal diet consisted of 89.45% timothy grass hay, 5% corn grain, 5% Soybean meal (48% Crude Protein), 0.50% trace mineral-salt and 0.05% vitamin premix on a dry matter basis. Steers received the diet in 12 portions daily at 2-h intervals and were allowed ad libitum access to water. Steers were housed in individual tie stalls (1.1×1.8 m) in a temperature-controlled barn and were exercised for 1 h twice weekly when infusion lines were cleaned. Two post-ruminal treatments were administered per abomasal infusion and included: casein (C; N =4) or starch (D;
N=4) for 42 d. Treatments were administered on an equal energy basis (167.4 kJ Metabolizable Energy/kg metabolic BW). Abomasal infusion treatments were introduced incrementally (25, 50, 75, 100%; every other day) during the first 7 d. Steers were weighed weekly and rations offered were adjusted accordingly on a weekly basis. On day 42, rumen contents (∼200 ml) were collected via rumen fistula.
Four ruminally-cannulated Holstein cows used in this study were described previously [24]. These multiparous cows were fed ad libitum standard lactation rations as a Total Mixed Ration (TMR; 50% corn silage and 50% concentrate at a dry matter basis). Diets were formulated to provide or exceed NRC dietary recommendations for lactation. Complete dietary chemical composition was determined weekly on composited daily samples of TMR composited weekly. Cows were moved to a tie stall barn for adaptation and acclimation at least five days prior to the collection of rumen contents. Similarly, the rumen contents of pre-ruminant calves fed milk replacer (Cornerstone 22:20; Purina Mills, Gray Summit, MO, USA) were collected as described [22]. The rumen liquor pH was measured using a standard pH meter. The liquid fraction of rumen contents was passed through a 300-µM metal sieve and centrifuged at 16,000×g for 3 min at 4 °C. The supernatant was decanted and the remaining solid materials were snap frozen in liquid nitrogen and stored in−80 °C until DNA extraction.
2.2 DNA extraction, amplicon library preparation, and pyrosequencing
a GS FLX Titanium System (Roche) following the manu-facturer’s protocol. A total of 15 samples, from the rumen of 8 beef steers in 2 treatment groups, abomasal infusion of casein and starch, 4 dairy cows, and 3 calves were sequenced. The number of raw sequences reads per sample was 37,004.6±6,612.4 (mean±sd) at∼456 bp/read.
2.3 Sequence analysis
Raw reads were first decoded based on 8-bp bar codes; their quality was checked and artifacts were removed as previ-ously described [2]. Sequences were filtered to remove low-quality reads and initially analyzed using BLAST against the GreenGene database (http://greengenes.lbl.gov/). The sequences of non-16S rRNA gene origin, such as host contamination, were removed. Resultant quality sequence reads were then analyzed using RDP Classi-fier [41] from the Ribosomal Database Project (release 10; http://rdp.cme.msu.edu/) at a 80% confidence threshold for taxonomic classification. Raw read counts were normalized. A square root transformation was applied to the relative abundance data. Bray-Curtis similarity matrix was then calculated using PRIMER v6 software (Plymouth Marine Laboratory, Plymounth, UK). Principal component analysis (PCA) was performed using the ade4 package in R as described elsewhere [43].
The 16S rRNA gene sequences also were analyzed using taxonomy-independent methods, such as CD-HIT-OTU [44] (http://weizhong-lab.ucsd.edu/cd-hit-otu/), ESPRIT-tree [4], and CROP [14] for taxonomic inference at the OTU level. The CD-HIT-OTU algorithm uses a greedy incremental clustering process to identify OTUs from 16S rRNA gene sequences, which involves 3 major steps: raw read filtering and trimming, selection of error-free reads, and clustering selected representative reads into individual OTUs at a user-specific cutoff (at both 0.03 and 0.05 distance levels). The program avoids over estimation of OTUs, a common prob-lem for many existing programs, and results in a rapid and more accurate estimation of microbial diversity in complex microbial ecosystems. The consensus sequences of clusters identified using CD-HIT-OTU were then annotated using FR-HIT [29] against the GreenGene database. Unadjusted
P values were calculated based on a modified t-test.
3 Results
3.1 Taxonomical profiles of the bovine rumen microbiota The bacterial community composition in the rumen of pre-ruminant calves and mature dairy and beef cattle was estimated from the 16S rRNA gene sequences generated by Roche/454 pyrosequencing. The mean number of 16S sequences generated per sample was 37,004.6±6,612.4 (mean±sd;N=15). Rarefaction analysis was performed at the family, genus, and OTU levels and suggested that the sequencing depth at this scale was adequate (Figure1).
(a)
(b)
(c)
Table 1: The number of taxa identified in the bovine rumen using pyrosequencing of the 16S rRNA genes. The ruminal taxonomical profiles at the phylum to genus levels were derived from RDP Classifier. The operational taxonomic units (OTUs) at the 0.03 distance level (bacterial species) was identified using greedy heuristic clustering-based CD-HIT-OTU.
∗Shared by all samples (may represent the core rumen microbiome).∗∗Collectively identified in this study, including both
the developing and mature rumen of dairy and beef cattle.
Phylum Class Family Genus OTU
Developing rumen (core∗) 10 18 35 53 59
mean (±sd) 12.3 (±1.2) 20.0 (±1.4) 49.0 (±6.5) 97.0 (±11.9) 133.3 (±10.3)
Mature rumen (core) 10 14 18 30 102
Dairy cows
core 14 16 26 50 368
mean (±sd) 17.3 (±0.8) 22.8 (±1.1) 41.0 (±4.1) 73.8 (±5.5) 537.5 (±30.5) Beef steers
core 11 16 22 37 175
mean (±sd) 15.6 (±1.2) 22.3 (±2.3) 39.4 (±4.6) 76.5 (±12.7) 499.6 (±34.1)
Total∗∗ 21 31 93 219 1,079
Several quality control procedures were applied to raw reads, including removal of putative artifacts. The RDP classifier was able to assign∼96% of input 16S sequences to a given phylum at an 80% confidence threshold. The RDP classifier identified 21 phyla, 31 classes, 93 families, and 219 genera in the study, from both the developing and mature rumen, regardless of breed (Table 1). In the developing rumen of pre-ruminant calves fed milk replacer, the mean numbers of phyla and genera per sample identified were 12.3±1.2 (mean±sd) and 97.0±11.9, respectively. In contrast, a greater number of phyla were identified in the mature rumen of both dairy cows and beef steers (P <0.001; Table 1). Sequences assigned to the domain archaea were rare and detected in very low abundance only in 3 of 4 dairy cows and 2 of 8 beef steers analyzed. The primers used in this study to amplify the V3–V5 regions of the 16S rRNA genes were not designed specifically for archaea. This was confirmed by the fact that the very low abundance of Euryarchaeota was observed in our dataset.
The core rumen microbiome of both the developing and mature rumen consisted of 8 phyla, 11 classes, 15 fami-lies, and 17 genera. These 8 phyla were Bacteroidetes, Fir-micutes, Proteobacteria, Spirochaetes, Fibrobacteres, Verru-comicrobia, Synergistetes, and Actinobacteria, in descend-ing order of relative abundance. These 8 phyla, accountdescend-ing for 99.5% of input 16S sequences, are likely representing minimal components of the rumen bacterial community. The mean number of genera in the rumen microbiota under study was 79.9±14.0 (±sd). The 17 genera consisting of the core microbiome represented only 21.3% of all genera present in each sample. These genera, such as Butyrivibrio,
Fibrobac-ter, OscillibacFibrobac-ter, Paraprevotella, Prevotella, Ruminococ-cus, Succinivibrio, and Treponema, accounted for 67.6% of
16S sequence reads and likely contributed to the basic func-tion of the rumen microbial ecosystem.
Taxonomy-dependent algorithms such as RDP Classifier have been widely used to estimate bacterial community composition. However, their inherent limitation was obvious due to the fact that vast majorities of rumen microorganisms are not described and captured in the existing databases such as the Ribosomal Database Project and GreenGene databases. To overcome these limitations, we also surveyed the rumen microbial community composition using three taxonomy-independent clustering programs, CD-HIT-OTU, ESPRIT-tree, and CROP. The numbers of OTUs identified at the 0.03 and 0.05 distance levels (parameters loosely used to define bacterial species and genera, respectively) by the CD-HIT-OTU method in the developing rumen were 133.3±12.7 and 113.0±9.2, respectively. In the mature rumen of dairy and beef cattle, the numbers of OTUs identified using these two cutoffs were 512.3±39.2 and 343.0±21.7, respectively. The difference in the number of OTUs detected using both cutoff values between the developing and mature rumen was significant (P <1.0×10−9). However, no differences were detected in the number of OTUs at both the 0.03 and 0.05 levels between dairy and beef cattle (P >0.05). Of note, the number of genera (or OTUs at the 0.05 distance level) detected between RDP Classifier and taxonomy-independent methods in the rumen of either pre-ruminant calves or dairy and beef cattle was rather different. Taxonomy-independent methods tend to identify a significantly larger number of genera, especially in the mature rumen (343.0±21.7 vs 75.6±11.4 by RDP Classifier), suggesting that the rumen microbiota may harbor many novel microorganisms yet to be captured in the public database.
Figure 2: Clustering analysis based on the family-level Bray-Curtis similarity generated using the Primer 6 program. The relative abundance data were square-root transformed. The linkage function: complete linkage. A: pre-ruminant dairy calves (developing rumen); B: dairy cows (mature rumen); C: beef steers with abomasal casein infusion (mature rumen); and D: beef steers with abomasal starch infusion (mature rumen).
between clusters, as discussed [37]. Liberal applications of pre-defined nominal distance levels, such as 0.03 (or 97% similarity) and 0.05 (or 95% similarity) for taxonomic assignment at bacterial species and genus levels, respectively, often lead to inflated numbers of OTUs and subsequently overestimate microbial diversity in a given microbial community. In this study, the ESPRIT-tree program partitioned the 16S sequences into 28,495 and 14,186 OTUs using the 0.03 and 0.05 distance levels, respectively while CROP identified 5,434 and 1,215 OTUs from the same dataset using the same thresholds (at the 0.03 and 0.05 distance levels, respectively).
3.2 Unique characteristics of the bacteria composition in the mature rumen
The bacterial composition between the developing and mature rumen displayed a striking difference. Bray-Curtis similarity matrix analysis and clustering using Primer v6 grouped all mature rumen samples together based on the relative abundances of bacterial composition at family level and the separation between the developing and mature rumen was distinct (Figure 2). PCA also confirmed the clustering results that the three groups of samples, from the developing rumen, dairy cows, and beef steers were clearly different, while the two treatment groups within the beef steers were indistinguishable (Figure3).
Among 15 phyla, 22 classes, and 436 OTUs consisting of a typical rumen bacterial community, 10 phyla (66%),
Figure 3: Differences in the microbial community compo-sition in the rumen of pre-ruminant dairy calves (group A, developing rumen), Dairy cows (group B), Beef steers with abomasal casein infusion (group C), and beef steers with abomasal starch infusion (group D). Principal component analysis was performed using the ade4 package in R based on relative abundance of 20 most abundant families. Of note, while the developing rumen and the mature rumen of dairy cows and beef steers were distinctly separate, the 2 groups of beef steers with abomasal infusion of casein and starch, respectively (C and D) were indistinguishable.
13 classes (61%), and 173 OTUs (41%) were different in their relative abundance (P <0.05) between the developing and mature rumen. At the family level, Bacteroidaceae was the most abundant family in the developing rumen, account-ing for 59.8% of the 16S sequences. However, its relative abundance in the mature rumen was 0.04% and 0.54% in the rumen of dairy cows and beef steers, respectively. The difference in the abundance of Bacteroidaceae between the developing and mature rumen was significant (P <0.0001). The family Comamonadaceae displayed a similar pattern; its abundance was higher in the developing rumen but barely detectable in the mature rumen (Table2). On the other hand, the relative abundance of the family Prevotellaceae was very great in the rumen of dairy cows (69.1%) and high in the rumen of beef steers (32.0%) but was small in the rumen of pre-ruminant calves (6.96%) (P <0.0001).
Table 2: The family-level percentage composition of the core microbiome of the bovine rumen. Group A: pre-ruminant dairy calves (N=3). Group B: dairy cows (N=4). Group C: beef steers with abomasal casein infusion (N=4). Group D: beef steers with abomasal starch infusion (N=4).
Rumen
Pvalues
Developing (D) Mature (M)
Dairy cows Beef steers
Family Group A Group B Group C Group D D vs M Dairy vs Beef
Bacteroidaceae 59.76±20.51 0.04±0.02 0.62±0.61 0.46±0.29 0.0000 0.0548 Comamonadaceae 5.65±7.88 0.01±0.01 0.03±0.01 0.01±0.00 0.0143 0.0900 Porphyromonadaceae 8.40±3.04 2.59±1.19 3.00±1.51 1.65±0.55 0.0001 0.7342 Lachnospiraceae 4.59±4.79 9.39±1.93 32.89±8.44 35.80±5.24 0.0198 0.0000 Prevotellaceae 6.96±2.40 69.10±3.34 35.93±11.81 28.05±3.76 0.0072 0.0000 Ruminococcaceae 3.69±0.80 5.82±1.47 14.94±2.23 19.63±3.79 0.0244 0.0002 Synergistaceae 1.20±0.13 0.09±0.02 0.18±0.08 0.18±0.06 0.0000 0.0386 Desulfovibrionaceae 0.29±0.38 0.01±0.00 0.02±0.02 0.02±0.01 0.0143 0.2964 Veillonellaceae 0.68±0.39 7.46±1.52 2.58±0.84 3.10±0.46 0.0260 0.0000 Erysipelotrichaceae 0.36±0.50 0.12±0.04 0.11±0.09 0.13±0.13 0.1055 0.9751 Clostridiales IS XIII 0.12±0.08 0.26±0.09 0.77±0.39 1.30±0.32 0.0542 0.0062 Succinivibrionaceae 0.01±0.00 0.61±0.32 1.28±0.83 0.56±0.60 0.0606 0.4735 Fibrobacteraceae 0.01±0.00 1.79±0.60 2.86±0.41 3.72±1.38 0.0014 0.0259 Spirochaetaceae 0.08±0.07 1.20±0.16 3.66±1.71 3.86±0.81 0.0110 0.0025 Coriobacteriaceae 0.06±0.09 0.04±0.01 0.11±0.06 0.10±0.05 0.5358 0.0282
in the mature rumen, including Fibrobacter (∼570-fold higher in the mature rumen), Prevotella (∼21-fold higher);
Succiniclasticum (∼36-fold higher) and Syntrophococcus (almost undetectable in the developing rumen).
The 10 most abundant OTUs at the 0.03 distance level in the developing rumen accounted for 82.6% of 16S sequences from the community. All 10 OTUs displayed a significant difference in their abundance between the developing and mature rumen (P <0.01). Annotation of the consensus sequences representing these OTUs indicated that the 2 most abundant OTUs were dominant in the community and represented by ∼55.9% sequences. Not surprisingly, both OTUs were annotated to the genus
Bacteroides, the most abundant genus in the developing
rumen based on the RDP Classifier results. The third most abundant OTU belonged to the genus Prevotella, which comprised 5.35% of the 16S sequences. This number was somewhat higher than that derived from the taxonomy-dependent RDP Classifier (Table 3). Interestingly, the consensus sequences from OTU clusters detected by CD-HIT-OTU allowed for species identification. Two species,
Comamonas kerstersii and Porphyromonas levii, comprising
5.19 and 5.14% in the community in the developing rumen, respectively, were barely detectable in the mature rumen. Intriguingly, only 4 OTUs were shared by all samples tested (of pre-ruminant calves, dairy cows, and beef steers). Two of the OTUs can be annotated to the family Ruminococcaceae while the remaining two were related to the order Bacteroidales and the genus Paludibacter (in the family Porphyromonadaceae). Together, our results demonstrate that the microbial communities in
the rumen from different developmental stages have unique compositional characteristics.
3.3 Microbiological distinctions in the rumen between dairy and beef cattle
Table 3: Genera with a significant difference in their relative abundance between the developing and mature rumen (P <0.01). The numbers denote mean ± sd (percentage composition).
Genus Developing Mature Pvalue
Acinetobacter 0.034±0.031 0.003±0.005 0.0028 Alistipes 0.206±0.210 0.002±0.006 0.0021 Bacteroides 70.420±21.360 0.687±0.864 0.0000 Butyricimonas 2.029±0.982 0.016±0.035 0.0000 Campylobacter 0.531±0.203 0.020±0.025 0.0000 Cloacibacillus 1.031±0.725 0.004±0.009 0.0001 Clostridium XIX 0.006±0.007 0.000±0.000 0.0047 Eggerthella 0.005±0.006 0.000±0.000 0.0051 Fibrobacter 0.010±0.004 5.703±3.010 0.0072 Flavonifractor 0.193±0.125 0.007±0.011 0.0001 Gallibacterium 0.321±0.276 0.001±0.002 0.0005 Guggenheimella 0.654±0.704 0.012±0.028 0.0033 Lachnospiracea i.s. 0.206±0.161 1.683±0.769 0.0066 Mannheimia 0.018±0.016 0.000±0.000 0.0005 Megamonas 0.003±0.002 0.000±0.000 0.0009 Morganella 0.003±0.003 0.000±0.000 0.0010 Odoribacter 0.110±0.030 0.001±0.002 0.0000 Parabacteroides 1.080±0.615 0.007±0.010 0.0000 Parasutterella 0.006±0.005 0.000±0.000 0.0005 Pelistega 0.052±0.027 0.002±0.005 0.0000 Phascolarctobacterium 0.116±0.047 0.004±0.008 0.0000 Porphyromonas 5.745±3.951 0.041±0.063 0.0001 Prevotella 2.124±0.483 42.945±19.788 0.0041 Pseudoflavonifractor 0.005±0.006 0.000±0.000 0.0065 Sphingobacterium 0.005±0.002 0.000±0.000 0.0000 Sphingomonas 0.014±0.016 0.001±0.002 0.0084 Streptococcus 0.122±0.072 0.014±0.043 0.0042 Succiniclasticum 0.170±0.150 6.052±2.195 0.0006 Syntrophococcus 0.000±0.000 0.103±0.051 0.0047 Turicibacter 0.003±0.003 0.000±0.000 0.0006
The 42 genera with different relative abundance between dairy and beef cattle were represented by 92.2% and 87.4% of 16S sequences analyzed, respectively. Of them, 12 genera had a relative abundance greater than 1.0% in the rumen of beef steers, such as Prevotella (30.17%),
Butyrivibrio (12.89%), Treponema (7.73%), Fibrobacter
(7.11%), Saccharofermentans (5.43%), Succiniclasticum (5.11%), Sporobacter (4.87%), Moryella (3.61%),
Pseu-dobutyrivibrio (2.94%), Anaerovorax (2.09%), a potentially
novel genera in the family Lachnospiraceae (2.02%), and
Bacteroides (1.03%). Sporobacter was a genus with a single
anaerobic species isolated from the paunch of a wood-feeding termite and may contribute to the degradation of lignocellulosic matter in the digestive tract of the termite [12]. The presence of this genus in the rumen or gastroin-testinal tract of ruminants has not been reported previously. Similarly, little is known about the genus Moryella, a recently described genus containing a single anaerobic species of putatively intestinal origin [5]. Our results
Table 4: Families with a significant difference in their percentage composition in the rumen microbiota between mature dairy and beef cattle. The numbers denote mean± sd (N=4 for dairy;N=8 for beef).
Family Dairy Beef Pvalue
Acetobacteraceae 0.118±0.057 0.001±0.001 0.0001 Anaeroplasmataceae 0.653±0.068 0.397±0.215 0.0470 Bifidobacteriaceae 0.024±0.016 0.000±0.000 0.0012 Chloroplast 0.010±0.006 0.111±0.053 0.0040 Clostridiales is XIII 0.257±0.088 1.035±0.436 0.0062 Coriobacteriaceae 0.038±0.010 0.106±0.051 0.0282 Desulfobulbaceae 0.034±0.019 0.008±0.010 0.0135 Fibrobacteraceae 1.794±0.597 3.294±1.049 0.0259 Lachnospiraceae 9.389±1.929 34.344±6.691 0.0000 Methanobacteriaceae 0.004±0.003 0.001±0.002 0.0290 Methylobacteriaceae 0.078±0.016 0.000±0.000 0.0000 Planctomycetaceae 0.024±0.021 0.005±0.004 0.0318 Prevotellaceae 69.097±3.338 31.992±9.139 0.0000 Rikenellaceae 0.003±0.002 0.048±0.036 0.0367 Ruminococcaceae 5.818±1.471 17.287±3.815 0.0002 Sphingomonadaceae 0.003±0.002 0.000±0.000 0.0016 Spirochaetaceae 1.197±0.158 3.762±1.245 0.0025 Synergistaceae 0.092±0.022 0.178±0.069 0.0386 Veillonellaceae 7.464±1.518 2.839±0.685 0.0000 Xanthomonadaceae 0.005±0.006 0.000±0.000 0.0401
Figure 4: The relative composition of 2 classes in the rumen microbiome. Boxes denote the inter-quartile range between the 1st and 3rd quartiles (25 and 75%, respectively). Red: pre-ruminant dairy calves; blue: dairy cows; and green: beef steers.
suggest that both genera were abundant in the rumen of beef steers. The ecological role of these bacteria in the rumen microbial ecosystem is worthy of further investigation.
In the rumen of dairy cows, 3 of the 5 most abundant OTUs were related to Prevotella. Together, these 3 OTUs comprised 22.3% of the rumen microbial community in dairy cows. In the rumen of beef cattle, a single species,
Butyrivibrio hungatei, accounted for 2.8% of the 16S
and dairy cows. Eight of the 10 most abundant OTUs in the rumen of dairy cows exhibited significant differences in abundance between dairy and beef cattle.
3.4 Abomasal substrate availability had a minimal impact on the ruminal bacterial composition
Two treatments were administrated per abomasum in beef steers to assess the impact of post-ruminal delivery of selected nutrients, starch and casein (N=4, respectively). The objective of this experiment was to examine any possible systematic effect of substrate availability in the lower gastrointestinal tract on the rumen microbiome. The ruminal bacterial composition between the two groups was compared. As expected, PCA analysis failed to separate these 2 groups, suggesting that the overall bacterial community structure and composition between the 2 groups were indistinguishable (Figure3). However, further analysis showed that the relative abundance of 7 genera displayed marginally differences between the 2 groups (P < 0.05) (Table 5). Of note, the presence of Campylobacter in the rumen was rare but reliably detectable, in agreement with the previous findings [26]. The abundance of this genus was greater in the rumen of the beef steers with abomasal infusion of casein than with abomasal infusion of starch (Table 5). The ecological and potential pathological significance of this observation remained unknown. On the other hand, the greater abundance of Sporobacter was seemingly associated with abomasal infusion of starch (P <0.05). Our results suggest that down-stream infusion of substrates along the gastrointestinal tract may have a minimal effect on the bacterial composition in the rumen microbial ecosystem although nutrient recycling of nitrogen (N) could have the potential to alter the ruminal environment and cause adaptation of certain microorganisms.
4 Discussion
Microorganisms in the rumen perform many important bio-chemical processes that are beneficial to the host, including fibrolytic reactions that convert plant fiber to short-chain fatty acids and vitamin and detoxification of secondary com-pounds in plants [20]. The dazzling microbial diversity and complexity of microbial interactions, both synergistic and antagonistic, in the rumen have been long appreciated [23]. As a result of co-evolution between microbial communities and their host, the rumen microbial ecosystem represents a temporal equilibrium between functional redundancy of a stable community and niche specialization. A comprehen-sive survey of microorganisms in the rumen is necessary to facilitate a better understanding of the biological function at the whole rumen ecosystem level. Towards this end, we investigated the bacterial composition in the rumen of both pre-ruminant dairy calves and dairy cows and beef steers using bar-coded pyrosequencing of the 16S rRNA gene.
Table 5: Substrate availability in the post-ruminal abo-masum may have a minimal impact on the microbial composition in the rumen in beef steers. The numbers represent the percentage composition (mean±sd; N =4 in each group).
Genus Casein Starch Pvalue
Campylobacter 0.046±0.03 0.007±0.01 0.0392 Catenibacterium 0.006±0.00 0.000±0.00 0.0251 Dehalogenimonas 0.006±0.00 0.000±0.00 0.0259 Pelospora 0.000±0.00 0.010±0.01 0.0255 Roseburia 0.305±0.14 0.092±0.05 0.0303 Schwartzia 0.020±0.01 0.000±0.00 0.0212 Sporobacter 3.603±0.76 6.144±1.92 0.0487
However, the relative abundance of Desulfovibrio in hay-free, sulfate-low habitats such as the developing rumen was much greater than in the mature rumen, suggesting that this group of rumen bacteria may have other biological functions in the rumen microbial ecosystem. Furthermore, plant fiber was absent in the diet of pre-ruminant calves and major substrates for fibrolytic bacteria such as Fibrobacter and Ruminococcus are lacking in the developing rumen. These bacteria were nevertheless reliably detected in the rumen of pre-ruminant calves. This observation provided a strong piece of evidence that the developing rumen may possess sufficient microbial diversity. When substrates become available or in response to changes in the environment or diet, these bacteria can rapidly expand their populations to exploit novel niche space available. Indeed, a significant population expansion of Fibrobacter (722 fold) and Ruminococcus (415 fold) was observed in the rumen of beef steers fed a hay-based diet, compared to in the developing rumen of pre-ruminant calves.
The microbial composition in the rumen of dairy cows has been extensively surveyed using next-generation sequencing technologies [19,24]. However, the rumen microbiota of beef cattle has not received similar attention. In this study, we conducted a detailed comparison in the rumen bacterial community composition between dairy and beef cattle. In a typical rumen microbial community of mature cattle, the mean number of taxa included approximately 16.2 phyla (±1.4), 22.4 classes (±2.1), 39.9 families (±4.7), 75.6 genera (±11.4), and 512.3 OTUs (±39.2). The percentage of taxa displaying a significant difference in the relative abundance between dairy and beef cattle ranged from 44.6% at a class level to 67.3% in an OTU level. Twenty families,∼50% of all families present in a typical rumen microbial community in mature cattle, varied significantly in abundance between dairy and beef cattle (Table 4). For example, the most abundant family in the rumen of dairy cows, Prevotellaceae, which accounted for ∼69% of 16S sequences, was much less in the rumen of beef cattle (∼32%), where Lachnospiraceae became the most dominant (and accounted for 34.3% of all 16S sequences). The significant change in the abundance was also observed for several other predominant families, such as Veillonellaceae, Ruminococcaceae, Spirochaetaceae, and Fibrobacteraceae (Table3). The 15 most abundant genera, such as Prevotella, Butyrivibrio, Treponema, Fibrobacter, and Saccharofermentans, in the descending order of the abundance, accounted for 92.9% of 16S sequences. Of them, 12 genera had significant alterations in their relative abundance when the rumen microbial populations of dairy and beef cattle were compared. For example, like in many other gut microbial ecosystems, such as in the porcine proximal colon [26], Prevotella was the most abundant genus in the rumen of beef steers. However, its relative
abundance was much less than in the rumen of dairy cows, where it accounted for 68.5% of 16S sequences. Our findings were quantitatively similar to a previous report in which Prevotella was reported to comprise 42% to 60% of the bacterial rRNA gene copies in lactating cows detected using quantitative PCR [36]. The significant difference in the abundance of Prevotella between dairy and beef cattle (P=1.60×10−6) is likely a result of their respective diets (concentrate vs hay). In agreement with several previous reports [3], the bovine rumen may harbor a much higher level of Prevotella genetic diversity because the cultured or known Prevotella species accounted for only a small portion of the observed predominance in the rumen. The genus Prevotella includes a group of bacteria with great genetic divergence and functional versatility [32]. Several species, such as P. ruminicola, are commonly thought to play a major role in initial dietary protein breakdown in the rumen [40] because they possess a rate-limiting dipeptidyl peptidase type IV. Prevotella species may contribute to ruminal fibrolytic capacity by acting synergistically with the cellulolytic species Fibrobacter succinogenes for efficient utilization of hemicellulose [30]. Recently, it has been found that certain Prevotella species may act as opportunistic human pathogens and are associated with dysbiosis-associated diseases [1]. Because of the fact that
Prevotella may serve as a reservoir for resistance genes
in the gut microbiota and its high predominance in the rumen and hindgut, this group of bacteria may deserve a special attention from the microbiological community, especially their eco-physiological roles in the rumen microbial ecosystem, relationships between the abundance and metabolic activities, and their potential pathological implications in human health.
unknown. Intriguingly, Acetobacter and Methylobacterium were moderate in their relative abundance but persistent the rumen of dairy cows. However, these genera were not detected in beef steers. Methylobacterium species are able to utilize oxalate, widespread two-carbon compounds produced in large quantity by certain plants [34]. Oxalate is toxic to mammals; and some rumen microorganisms in the rumen and colon in mammals possess the ability to degrade oxalate. Additionally, Methylobacterium has been shown to degrade nitroaromatic explosives, such as RDX [38]. Acetobacter species belong to a group of acetic acid bacteria and are aerobic. Acetobacter can be readily found in nature, especially in the alcohol-enriched environment [33]. At least one Acetobacter species is known to synthesize crystalline cellulose [42]. However, little is known about these 2 genera of rumen origin.
In conclusion, we investigated the rumen microbial community composition and diversity in the bovine rumen and compared the similarities and differences of the rumen microbiota between the developing and mature rumen, as well as between dairy and beef cattle. The rumen microbiota consists of a complex community of microorganisms with a myriad of microbial interactions. Our findings provide a glimpse of the dazzling microbial diversity in the rumen and will undoubtedly contribute to the understanding of ruminal function and ruminant nutrition.
Acknowledgments The authors thank Ashley Sperling and Alicia
Beavers for their excellent technical assistance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer. Sitao Wu and Weizhong Li were partly supported by Award R01HG005978 to Weizhong Li from the National Human Genome Research Institute (NHGRI). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NHGRI or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.
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