Microbial community composition along the digestive tract in forage- and grain-fed bison

Ruminants, like all vertebrate herbivores, rely on vegetation for nourishment, but are unable to digest plant fibers without the aid of symbiotic microbes in their digestive tract [1]. Most of the ruminant digestive system is a favorable environment for microbes, as body temperature is warm and stable at around 39 °C [2]. However, the digestive tract is also a challenging environment for microbes given that they must compete for space and nutrients [3] Moreover, although ruminants provide their symbionts with abundant resources, most are in the form of cellulose, which is difficult to break down [4]. The reticulorumen and omasum are weakly acidic with a pH of 5.5–6.5, which is favorable for many microbes [5]. These sections house most of the ruminant’s symbiotic microbiota. Bacteria and protozoa are predominant, accounting for 40–60% of microbial biomass [6]. Bacteria fermenting this material release the volatile fatty acids (VFAs) acetic, propionic, and butyric acid, which the host absorbs and metabolizes [1].

As in other mammals, the ruminant gut microbiota is dominated by the bacterial phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria (especially in the class Gammaproteobacteria) and Verrucomicrobia [7], while most archaea are methanogens in the phylum Euryarchaeota [6, 8–10]. Because the different sections of the ruminant gut present different environmental conditions, the composition of the gut microbiota changes from one section to another. The first region (a functional grouping of sections) is the foregut, which houses several common fibrolytic species [11–13], as well as amolytic, saccharolytic, lactolytic, and proteolytic species [14–17]. Second, the abomasum has a pH of 2–4, which kills and digests many of the microbes entering from the omasum, supplying the host with 60–90% of its amino acids, which are in turn absorbed in the small intestine [18–20]; [21]. Third, the small intestine is responsible for neutralizing acid from the stomach, breaking down macromolecules with enzymes, and absorbing nutrients. Microbial biomass drops sharply between the foregut and small intestine because of the acidity of the abomasum, but then increases caudally as pH rises again from 2 to 4 in the duodenum, to 4–7 in the jejunum, and finally to 7–8 in the ileum [5]. Finally, the hindgut is the last site for salt and water balance. In ruminants, this region is second to the foregut in microbial biomass. Microbiota in the cecum, colon, and rectum ferment remaining fiber and produce a variety of vitamins for their host [1, 22].

Community composition was compared along the entire digestive tract of two captive populations of plains bison (Bison bison bison). In domestic cattle (Bos taurus taurus), gut microbiota have been explored in the reticulorumen and feces [23–26], as well as across more sites along the digestive tract [5, 27]. Microbial diversity along the digestive tract has been researched in humans, rodents, and horses as well [28–30]. However, studies of the gut microbiota of bison have been limited to the reticulorumen [31–33] and feces [34]. The earlier research on ruminal microbiota predates high-throughput sequencing, so it relied on culture-dependent methods that could not detect much of the microbial diversity found in the gut. The study by [34] used culture-independent methods to characterize fecal microbiota in semi-free-ranging wood bison (B. bison athabascae). In the present study, two related questions were addressed. One, to what degree does microbial community composition vary along the length of the digestive tract in plains bison? Two, does the relative abundance of a given microbial taxon in a given gut section differ between two bison populations? It was hypothesized that different sections of the digestive tract would exhibit differences in microbial diversity and community composition. Likewise, for a given digestive tract section, these variables were expected to differ between the two bison populations.

In each population, microbial community composition was significantly different among gut sections from different regions, but similar among those within the same region, namely the multi-chambered stomach and large intestine. This is probably because, although anatomically partitioned, each of these two regions probably functions as a unit [44] The present study on bison, like previous work on cattle [44, 45], sheep [46], and moose [27, 47] also shows that the microbial communities of the rumen and colon are distinct. Thus, while fecal samples can be used as noninvasive colonic samples, they do not represent ruminal communities. Further work is needed to determine if microbial taxa or their ratios in fecal samples may be used as indicators of these or other taxa in the rumen.

For both bison populations studied, diversity as indicated by the Shannon index appears to be lowest in the small intestine, and for bison from Population A (forage-finished), diversity was lowest in the ileum specifically, although the other sections showed no clear trend. The two dominant bacterial phyla in the digestive tract were the Bacteroidetes and Firmicutes, as is the case in all known mammals [48–51]). Bacteroidetes exhibited their lowest relative abundance in the small intestine, while Firmicutes reached their highest relative abundance there.

Earlier research has shown that, despite high resource availability, the small intestine tends to harbor the lowest levels of microbial biomass and diversity of any gastrointestinal section, for two main reasons [52]. The first is that digesta have a relatively short retention time in the small intestine due to peristaltic movement, which gives microbes less time to proliferate there [53]. The second is the influence of the section just anterior to the small intestine, the abomasum (true stomach). While the pH of most gut sections ranges from 5 to 7, the abomasum has pH levels of 2–4, the lowest of the ruminant digestive tract [6, 54, 55]. Although the small intestine receives this gastric acid and begins buffering it, that takes time. Digesta pass through the long ruminant small intestine, which is about 20 times the length of the animal, or some 40 m long in bison and cattle. During this time, pH remains relatively low in the duodenum and jejunum, and does not return to near neutral levels until the ileum. In addition to acid, the small intestine also receives bile from the gall bladder and enzymatic secretions from the pancreas. Together, these three inputs create a relatively harsh environment for most microbes [53]. In this environment, the Gram- Bacteroidetes could be at a disadvantage compared to the Firmicutes, which have a thick peptidoglycan Gram + cell wall [56].

The effect of gut region on community composition was stronger than that of population, as microbial communities were more similar in the same region between populations, than to another region within the same population (Fig. 3). Nevertheless, the bison from Population A (forage-finished) appeared to support greater microbial diversity in most sections of the digestive tract, except the ileum. Previous work with captive domestic cattle has shown that age, sex, and location have less of an effect on microbial community composition than diet [25, 57], although the ruminal microbiota of cattle have been observed to vary with both age and diet [58], and those of moose (Alces alces) with age, weight, and location, but not sex [27, 47]. Like other sexually dimorphic ungulates, bison exhibit sexual segregation for most of the year [59]. Accordingly, isotopic and chemical analyses have shown that bison diets differ with age and sex [60, 61] although chloroplast gene sequence-based research has shown that the bison diet does not vary with age or sex [62]. Differences in age, sex, and location between bison populations in the present study make interpretation of the results more difficult, but dietary differences could have had a greater effect on gut community composition than these other factors. Thus, differences in community composition between these populations for a given gut section may be due primarily to forage-finishing in Population A and grain-finishing in Population B. Specifically, the present study suggests that a forage-based diet may be associated with overall greater gut microbial diversity in bison (Fig. 1). This finding is consistent with those of other studies, which have shown that fiber-based diets promote higher levels of microbial diversity than finishing diets, because the fermentation of fiber generates more byproducts than that of starch [63, 64].In many gut sections, Bacteroidetes were more abundant with a forage diet, while Firmicutes were more abundant with a grain diet. This mirrors what has been found in studies on obesity in humans and mice, where a more natural, less calorific diet that is rich in protein and fiber seems to favor Bacteroidetes, while an artificial, energy-rich diet of starch and fats seems to favor Firmicutes [22, 65, 66]; [67, 68]. Thus, the gut microbiota of grain-fed bison appears to resemble those of other animals fed a diet high in starchy, processed foods. At the phylum level, Proteobacteria were more abundant with a grain diet only in the omasum. Moreover, at the class level, a higher relative abundance of Gammaproteobacteria was associated with the grain diet throughout much of the digestive tract.

Within a given population, microbial community composition differed among sections of the digestive tract, but was similar for sections within the same region, especially the foregut and hindgut. The sections of the small intestine or midgut were overall similar to one another, but exhibited some similarities to the other regions as well. Shannon diversity was lowest in the small intestine, likely because of a generally short retention time in the small intestine, and the influence of low pH from the stomach, bile from the gall bladder, and digestive enzymes from the pancreas. This may be why Firmicutes, with their thick cell wall, dominate in the small intestine. The present study also found microbial community compositional differences for a given gut section from two bison populations, possibly due to differences in diet (forage- vs. grain-fed). However, microbial community composition was more divergent among regions of the gut than between dietary groups, indicating that physiological conditions along the digestive tract play a larger role in structing microbial communities than does diet.

It is noteworthy that certain bacterial groups exhibited differences in relative abundance between the rectum and elsewhere in the GI tract. Thus, although fecal sampling is convenient and noninvasive, one must be cautious when extrapolating abundances of bacteria in fecal samples to elsewhere in the alimentary canal [44]. Given previous research on grain-fed cattle and bison, as well as on obese mice and humans, it is likely that the higher levels of Firmicutes and Gammaproteobacteria found in the grain-finished bison represented dysbiosis [69, 70]; [71]. However, the bison in this study were not assessed for ruminal acidosis, anorexia, or the shedding of enterohemorrhagic bacteria to their environment. Additional research is needed to evaluate the clinical relevance of symbiotic communities brought about by artificial feeding methods in bison.