INTRODUCTION
A huge number of various microorganisms live in the gastrointestinal tract of ruminants, about ten times their body’s own cells in number. Sheep is a ruminant animal where the gut is a fermentative chamber for a complex and dynamic microbial population. Symbiotic bacterial community is crucial for the host health in many aspects, such as in balancing the immune response, digesting the nutrients, and mediating the host physiology. In addition, symbioses between microbiota and host can facilitate the development of the latter’s gastrointestinal tract. Especially, bacteria in the ruminant gut play a major role in the biological degradation of dietary fibers. Ruminant digestion relies on the bulk of cellulose hydrolysis bacteria. However, the microbiota composition and diversity are affected by many factors, such as diet composition, host genetics, and environment, and so on. Therefore, it is now recognized that a better and sufficient understanding of the composition and diversity of the gastrointestinal microbial community is required to further enhance the growth and gut health of ruminants.
In the past, the conventional culture-based technique was used to isolate and characterize the gastrointestinal microbiota of ruminants. Indeed, even if the culture-based technique has successfully isolated key representative bacteria, it is not sufficient to characterize the entire microbial populations, because a large majority of gastrointestinal microbiota is not culturable. A recent article indicated that some unculturable microbiota were abundant in the rumen, which play an important role in the ruminal fermentation [
1]. Over the last 10 years the development of high-throughput sequencing techniques has allowed for a considerable increase in knowledge of the microbial diversity of the ruminant gut. Roche 454 pyrosequencing platform provides new approaches for researchers to investigate complex microbial communities. This platform does not required a clone library, and has high throughput efficiency and sensitivity. The application of this approach enabled us to successfully analyze several samples at a time, which significantly reduced experimental cost and improved efficiency.
Although the small tail Han sheep is an important sheep breed with many favorable features, including fast growth, strong reproduction, stable genetic performance, and good adaption, data on its gut microbiota are limited. Rumen contents and feces in ruminants are often used to assess gastrointestinal microbiota [
2,
3], however, these sections do not represent the composition and diversity of microbiota in the entire gastrointestinal tract. Especially, the forestomach of ruminants, namely, rumen, reticulum and omasum contribute to the digestion of the cellulose substances in the diet. The small intestine is responsible for absorption of water, nutrients, and electrolytes. However, knowledge on the microbiota in the gastrointestinal tract of ruminants is limited. Recent studies explored the composition of the gastrointestinal microbiota in dairy cattle and Brazilian Nelore breed of cattle [
4,
5]. However, no studies to date have evaluated the entire gastrointestinal bacterial community in adult sheep using pyrosequencing, especially small tail Han sheep, which is a main sheep breed in Tianjin. The objective of this study was to characterize the gastrointestinal bacterial community of small tail Han sheep using pyrosequencing of 16S rRNA gene amplicons. Understanding the composition and structure of the microbiota in the gastrointestinal tract of sheep may be useful for developing ruminant production and management.
DISCUSSION
In our study, higher bacterial richness and diversity were observed in the stomach and large intestine than in the small intestine, which was also previously observed in the dairy cattle and the Brazilian Nelore breed of cattle [
4,
5]. The distal gut environment of the ruminant is more complex than the proximal gut. Most of the dietary constituents are digested in the stomach. The small intestine is much longer than the other sections, and small intestine has high concentration of bile salt and digestive enzymes so that the bacteria are difficult to grow. The partial microbial digestion that also takes place in the large intestine of ruminants could also explains the detection of high richness and diversity in the large intestine. Different oxygen tension and physiological roles in different gastrointestinal tract sections may lead to this result. The PCoA plot in the present study is in agreement with on the Nelore cattle [
4] and shows that the samples from adjacent gastrointestinal tract segment (stomach, small intestine, and large intestine) harbor microbial communities more similar than from other segments.
The phylum level, the structure of bacterial community in the gastrointestinal tract was similar to that found in Chinese Mongolian sheep [
7] and Holstein dairy cattle [
5]. However, with regard to the abundance of these predominant phyla, there were some differences found among these studies. These differences might be due to variations in species, diets, living environment, and analysis methods. In general, the microbiota of the stomach, ileum, and large intestine exhibited greater abundances of
Firmicutes and
Bacteroidetes, while the duodenum and jejunum showed higher relative abundances of
Firmicutes and
Proteobacteria. A recent study on the dairy cattle Illumina-based method found that among the gastrointestinal tract, the high abundance of phylum
Proteobacteria was found in the small intestine [
5].
Firmicutes in the ruminants is known to degrade the fibre and cellulose [
8].
Bacteroidetes is known to aid the digestion of complex carbohydrates, and also ferment organic matter [
9]. The cause of the high abundance of
Proteobacteria in the small intestine is not entirely clear, and future studies are needed to clarify this issue. To our knowledge, TM7 (0.0% to 4.6%) and SR1 (0.0% to 0.1%) phyla were found in the sheep gastrointestinal tract for the first time. TM7, which has not yet been cultivated in a laboratory, and is also reported in the feces of grass hay-fed horses [
10]. SR1 has been reported in the rumen of cow using pyrosequencing method [
11], however the role of this bacteria in the sheep gastrointestinal tract remains unknown.
Fifteen genera were considered as the core genera because these were existed in the all gastrointestinal tract sections. Of these 15 genera, several of unclassified genera, including S24-7, CF231, and RFP12, were detected. Therefore, this study provides detailed information regarding to both known bacteria and unclassified bacteria. A study on the rumen bacterial diversity of 80 to 110-day-old goats using 16S rRNA sequencing indicated that as the age of the goat increases, S24-7 showed an increasing trend [
12]. Therefore, that the 10-months sheep in the present study had a high abundance of S24-7 in the stomach is reasonable. CF231, family
Paraprevotellaceae, was the third top genus of the phylum
Bacteroidetes in the rumen of steers [
13]. However, in our present study, CF231 was high abundance in the ileum and large intestine, and the stomach was of low abundance. Different species could produce this discrepancy. The function of CF231 will need to be studied in the future. Family in the RFP12, order of
Verrucomicrobia was the second most abundant genus among all horse feces [
14]. However, in our present study, RFP12 was of high abundance in the jejunum, which was never reported before for ruminants. Therefore, the function of RFP12 will need to be characterized in the future. Of these 15 genera, a total of 11 genera, including
Prevotella, unclassified
Lachnospiraceae,
Ruminococcus, unclassified
Ruminococcaceae, CF231, unclassified RFP 12, unclassified
Clostridiaceae,
Clostridium,
Oscillospira, unclassified
Veillonellaceae, and
Coprococcus, have been reported to dominate the yaks rumen using Illumina MiSeq sequencing method (Majorbio Bio-Pharm Technology Co., Ltd, Shanghai, China) [
15].
Succinivibrio was previously detected in the rumen of cow using pyroseuquencing method [
11].
Anaerovibrio was seen throughout the gastrointestinal tract of 3-week-old preweaned calves using pyrosequencing method [
16]. Unclassified
Bifidobacteriaceae was previously found in the ileum of goats using pyrosequencing method [
17]. Therefore, some core genera could be shared in the most of the ruminants.
A primary finding of our study is that there are differences in microbiota composition between the segments of the gastrointestinal tract. This is in agreement with previous studies that have reported significant changes in the microbiota within the gastrointestinal tract as digesta passes from one segment to another [
4].
Prevotella in the stomach or the large intestine was identified the most abundant and important genus, which was in agreement with the sequencing of rumen or cecum samples of ruminants [
18,
19]. Prevotella, which has a unique mucin glycoprotein degradation capability, might exploit this, resulting in the host’s increased growth and survival, and also can degrade the hemicelluloses and xylans, which promotes the digestion of the feed [
20].
Butyrivibrio (average relative abundance is 9.5% of total sequences) dominated in the stomach of sheep in the present study. The abundance of
Butyrivibrio was in line with the previous report on the goat rumen microbiota, using cloned 16S rRNA gene analysis, where it accounted about 10.0% [
21] and dairy cattle four stomach microbiota, using Illumina MiSeq (Department of Computer Science, North Arizona University, Flagstaff, AZ, USA) platform analysis, where
Butyrivibrio accounted for about 5.1% [
5]. Therefore, even if the different methods and different ruminant breeds were used,
Butyrivibrio dominated the stomach of ruminants. Dunne et al. demonstrated that
Butyrivibrio could modulate the secretion of hemicelluloses-degrading enzymes [
22], which supported the notion that this organism made an important contribution to polysaccharide degradation in the rumen. In addition,
Butyrivibrio is an important butyrate producer, and promotes the stomach epithelium proliferation.
Ruminococcus dominated in the small intestine and large intestine, and it account for 6.3%±1.9% and 13.0%±2.9% of total sequences, respectively.
Ruminocuccus is also found in the 3-week old preweaned calves, and it dominated in the jejunum, however it is less abundant genus in the large intestine [
16]. This disagreement may be due to the variance of the host age, diet and breed.
Ruminococcus was found to produce carbohydrate active enzymes, and degraded the carbohydrate from the diet [
23]. It is noteworthy that
Escherichia (12.6%±3.0%) accounted a large amount in the small intestine, which is unexpected before. A review study [
24] indicated
Proteobacteria represented between 5% and 40% of the bacteria detected in the ileum of pigs, and it is well known that
Escherichia was the largest genus in the
Proteobacteria. Therefore, whether pigs or ruminants, Escherichia may have the same physiological roles in the small intestine. A high abundance of unclassified
Lachnospiraceae was observed in the stomach and small intestine in the present study, which agrees with a review on the status of the phylogenetic diversity census of ruminal microbiomes that summarized
Lachnospiraceae was one of the largest taxons [
1]. A study on the ileum of goats using pyrosequencing also indicated that unclassified
Lachnospiraceae dominated in the ileum [
17].
Lachnospiraceae is known to be a beneficial bacterium in the human intestine, because of the role it plays in the fermentation of carbohydrates to short chain fatty acids [
25]. Maybe
Lachnospiraceae has the similar role in the gastrointestinal tracts of sheep. A high abundance of unclassified
Ruminococcaceae (12.3%±2.6%) was observed in the large intestine from our study, which is in accordance with the report on adult goat cecal luminal content microbiota [
26]. Most of the
Ruminococcaceae also act as major degraders of resistant polysaccharides, such as starch and cellulose, and contributes a range of degradative enzyme systems that allow the host to break up plant cell walls [
27]. In our study, the abundant genera in the large intestine does not in agree with the cattle fecal microbiota identified using pyrosequencing [
28]. They suggested that
Clostridium (19.7%) and
Bacteroides (10.4%) predominated in the feces of cattle. However, in the present study,
Clostridium and
Bacteroides only accounted 0.6% and 1.3% of total sequences in the rectum, respectively. The large variation in the abundant genera could be attributed to the differences in the breed, and differences between feces and rectal samples. Therefore, the feces should not be used instead of rectal samples.
Perhaps, the most salient finding in the present study was the characterization of the microbial composition in the sheep gastrointestinal tract at the species level. As Illumina sequencing, the most popular method nowadays, would not be able to identify to the species owing to short sequencing length. However, pyrosequening has a long sequencing length, so it overcomes this problem.
R. flavefaciens,
R. bromii, and
F. succinogenes are the most dominant cellulolytic bacteria in the rumen and feces of ruminant [
1,
7]. Our results demonstrated that
R. flavefaciens was the most abundant species in the gastrointestinal tract. This result supports the previous finding that
R. flavefaciens dominated in the rumen of goats [
21]. However, the abundances of
R. bromii and
F. succinogenes were different in different ruminant species. The three dominant cellulolytic bacteria are consistently higher in the stomach than in the large intestine and small intestine. The stomach provides low pH and cellulosic materials, therefore is convenient for the cellulolytic bacteria growth. Another species,
B. fibrisolvens, dominated in the stomach and small intestine of sheep, which is a hemicellulolytic bacterium commonly isolated from the rumen of cattle, sheep, and deer [
29].
B. pseudolongum dominated in the small intestine and large intestine, which has the activity of degrading the pectin and glucose in the rabbit cecum [
30].
P. ruminicola is also reported in the rumen of cattle, and it is associated with ruminal carbohydrate and protein fermentation [
2].