INTRODUCTION
Recent years have witnessed growing interest in the diversity and function of ruminal epithelial bacteria. It has been demonstrated that ruminal epithelial bacteria are involved in oxygen scavenging, tissue recycling, and urea digestion (
1). Furthermore, in steers, the ruminal epithelial bacterial communities of acidosis-resistant and acidosis-susceptible groups were different during subacute ruminal acidosis development, and this difference could be recognized by the host TLRs (Toll-like receptors), which are associated with changes in the function of the rumen epithelial tissue barrier (
2). Similarly, in the mouse colon, epithelial bacterial diversity correlated with TLR2 and TLR4 gene expression (
3). These findings reveal that since epithelial bacteria are directly attached to the epithelial surface, their end products may play a direct or indirect role in host immune responses and tissue barrier function.
Ruminal epithelial bacteria are distinctly different at the taxonomic level from bacteria associated with rumen contents (
4,
5). By use of culture-based techniques, the ruminal epithelial communities have been found to comprise predominantly Gram-positive, facultatively anaerobic flora, among which
Butyrivibrio spp. (31.1%),
Bacteroides spp. (22.4%),
Selenomonas ruminantium (9.9%),
Succinivibrio dextrinosolvens (8.7%) and
Streptococcus bovis (8.1%) predominate (
6). At the class level, ruminal epithelial bacteria were found by pyrosequencing to be composed mainly of
Clostridia (67%),
Bacteroidia (9%),
Deltaproteobacteria (4%), and
Erysipelotrichia (3%) (
7).
Diet is an indispensable determinant of the structure and function of the diverse microbial populations in rumen contents and attached to rumen tissue. In the rumen contents of dairy cows, deep amplicon sequencing of the 16S rRNA gene revealed a higher abundance of members of the
Fibrobacteraceae family in total mixed-ration samples and a lower abundance of members of the propionate-producing
Veillonellaceae than in pasture samples (
8). Moreover, based on sequence information from the reference marker of PCR-denaturing gradient gel electrophoresis (DGGE), abundances of the phyla
Firmicutes and
Proteobacteria in the heifer ruminal epithelial bacterial community decreased in response to a rapid transition from a diet containing 97% hay to a diet containing 8% hay (
9).
The process of bacterial colonization in the developing rumen is important for the achievement of rumen functions. Bacterial colonization can influence the early development and productive efficiency of the mature animal (
10). In cow rumen contents, the bacterial composition underwent dynamic changes during the first 2 years; levels of members of the phylum
Proteobacteria decreased with age, while those of
Bacteroidetes and
Firmicutes increased with age (
11). Our earlier study showed that during the three typical phases of the rumen development process (nonrumination, 0 to 3 weeks; transition, 3 to 8 weeks; rumination, from 8 weeks on) in goats, microbial colonization of the rumen contents occurs at 1 month, functional achievement at 2 months, and anatomic development after 2 months (
12). Nonetheless, research into age-related dynamic colonization by ruminal epithelial bacterial populations has been very limited. Therefore, we aimed to characterize the sequential dynamics of ruminal epithelial bacterial colonization and to explore its relationship with previously published rumen anatomic and functional parameters.
DISCUSSION
Relationships between gastrointestinal microbial communities and their hosts have been shown in recent years to play an important role in the host's well-being and proper function. The objective of this study was to evaluate how bacteria colonized the rumen epithelium during normal development. Using MiSeq sequencing, we obtained 62,641 reads on average for each sample (with a minimum of 27,120 reads) with good coverage (>99.6%). Furthermore, our results suggest that each age group has its own distinct epithelial microbiota, as reflected by the clustering of samples by age group using PCoA and tb-PCA. Meanwhile, most of the alpha diversity indices (except ACE and the Simpson index) increased with age, suggesting that the microbiota in older age groups is more diverse than that in earlier age groups. This is similar to the process of microbial colonization of the rumen contents (
11,
26).
Individual variation is an indispensable determinant of microbiota taxa. Although the experimental conditions, diet, and sampling procedures were similar for bovine rumen samples, a large fraction of the OTUs (50%) occurred in only a small number of samples (0 to 30%), implying that bacterial taxa differed considerably between individual bovine rumen contents (
27). Similarly, within-group similarity in the rumen epithelial bacterial community in our study during the first week was quite low (around 0.2). Despite this, we observed a drastic increase in within-group similarity with age. This suggests that the rumen epithelium in older groups is a more restricted environment, housing a bacterial community more homogeneous than the earlier, heterogeneous community.
Host genetics is another factor affecting gastrointestinal microbial adaptation and evolution (
28). In newborn calves, the bacterial communities of fecal content samples revealed higher similarity in PCR–single-strand conformation polymorphism (PCR-SSCP) profiles for twins than for their coresidents of the same age, indicating that individual microbial diversity might be genetically influenced (
29). In our study, the effects of age on rumen epithelial bacterial diversity were compared using different groups of kids, which also accounted for the variation. Moreover, total epithelial bacterial populations for alfalfa hay- and oat-fed dairy calves (
30), hay-fed sheep (
31), and concentrate- and fresh-grass-fed goat kids (this study) ranged from 4.4 × 10
9 to 4.2 × 10
10, 4.4 × 10
7 to 2.2 × 10
8, and 1.4 × 10
8 to 4.4 × 10
7 copy numbers per g of wet tissue, respectively. Besides the differences in model animals, diet, the DNA extraction method, and PCR primers and conditions, the host might also be an important determinant in regulating bacterial density.
In all age groups,
Firmicutes,
Bacteroidetes, and
Proteobacteria were the dominant phyla in the epithelial microbiota, and their abundances and genus compositions differed remarkably, in line with previous studies (
7,
32). Similarly, at the genus level, although the abundances of some genera changed with age, they were present in all groups, representing a core microbiome in the ruminal epithelial microbiota of goats. The current study showed that after 20 days, the epithelial microbiota harbored more members of the genera
Butyrivibrio and
Campylobacter than the bacterial population in the rumen contents (
11), indicating that these two genera might be uniquely abundant epithelial bacteria.
The ruminal epithelial bacterial composition of goat kids undergoes sequential changes during the rumen development process. On the day of birth, the phylum
Proteobacteria (90.13%) far exceeded other phyla, with the majority belonging to the genus
Escherichia. This microbiota might derive from the microbiota of the mother's vagina (
33), skin, colostrum (
34), and environment. The genus
Escherichia comprises facultatively anaerobic bacteria. Some commensal strains of
Escherichia coli can reduce intestinal epithelial inflammatory signaling
in vitro (
35), while some pathogenic
E. coli strains can invade host cells (
36). As reflected by tb-PCA, two dominant OTUs that separated samples at day zero from others also displayed high identity to this genus. These facultative anaerobes could create the reduced environment that is required for anaerobic microbes (
37). Further work would be needed to ascertain the functions of this genus in the rumen. Moreover, this genus declined considerably in abundance at 7 days, and a great many genera belonged to unclassified
Proteobacteria. The remarkable changes observed in ruminal epithelial bacterial communities during the first week after birth indicated a rapid change in the rumen environment.
A noticeable change occurring at 28 days after solid food was offered (at 20 days) was the increase in ruminal epithelial bacterial density, which was reflected by qPCR (almost three times the copy numbers). Similar observations have been reported for rumen and gut contents (
37). Moreover, the changes in rumen tissues were similar to the compositional changes in rumen contents (
11): the genus
Bacteroides declined in abundance in rumen tissues, while the proportions of the genera
Prevotella,
Butyrivibrio,
Campylobacter, and
Succinivibrio surged. Taking the findings together, solid food caused disruption in the ruminal epithelial microbiota by selecting bacterial taxa that were more specific and were adapted to new substrates. Members of the genus
Prevotella, numerically predominant in ruminants, are capable of utilizing starches, other noncellulosic polysaccharides, and simple sugars as energy sources (
24). Members of the
Butyrivibrio group (including the genera
Butyrivibrio and
Pseudobutyrivibrio) represented 12.98% of total bacteria in the rumina of goats (
38) and were involved in the biohydrogenation of unsaturated C
18 fatty acids (
39). Although some species of
Campylobacter, such as
Campylobacter concisus and other non-
Campylobacter jejuni Campylobacter species, have been implicated in the initiation of gastrointestinal diseases (
40), they represent persistent residents of rumen microbial communities (
10). Some species of the genus
Succinivibrio could ferment maltose and glucose, thus participating in the last stage of starch digestion (
41). From 28 to 70 days, the abundances of the genera
Prevotella,
Butyrivibrio, and
Campylobacter still increased with age, implying that they were dominant genera in ruminal epithelial microbiota after the provision of solid feed. However, what role these genera played and how they interacted with the host have not been explored and remain to be elucidated.
Based on the data already published on rumen functional variables in the same goat kids (
12), we explored the relationship between ruminal epithelial microbiota and rumen functions. The abundances of the genera
Butyrivibrio,
Campylobacter,
Desulfobulbus, and
Ruminococcus were positively correlated with rumen weight, rumen papilla length, ammonia concentration, and total volatile fatty acid concentration, suggesting that they might be involved in ammonia and volatile fatty acid metabolism as well as in the anatomic development of the rumen. Similarly, the genera
Butyrivibrio,
Campylobacter, and
Desulfobulbus might also participate in fibrolytic enzyme secretion, and the genus
Fusobacterium is related to starch degradation. Furthermore, specific commensal microbes have been reported to modulate host immune responses via proinflammatory and anti-inflammatory pathways, as well as epithelial-cell-mediated signals (
42,
43). Therefore, further investigation to better explain the symbiotic relationship between the ruminal epithelial microbiota and the host is warranted.
Conclusion.
In conclusion, ruminal epithelial bacterial communities were more diverse in older groups than in earlier groups, and each age group had its distinct microbiota. Of the three dominant phyla, Proteobacteria declined in abundance with age, while Bacteroidetes and Firmicutes increased in abundance with age. The genus Escherichia dominated at the day of birth, while Prevotella, Butyrivibrio, and Campylobacter surged in abundance at around 2 months. Furthermore, the correlations between particular genera of the ruminal epithelial bacteria and anatomic and functional variables might indicate different types of relationships with the host. This study provides some preliminary information on the potential role of the ruminal epithelial bacteria, and further investigations are required to determine their actual roles and interactions with the host.