General characteristics of bovine fecal microbial communities
Deep sequencing of the fecal samples collected from the dairy cows fed with the two diets provided a detailed view of the cattle fecal microbiome in the samples tested. We detected members of 11 phyla of bacteria. The majority of the pyrotags belonged to the Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Tenericutes These phyla have been shown previously to constitute the majority of gut-associated phylotypes in a variety of different mammalian species [11–13], which suggests that these phyla, especially the Firmicutes, play a critical role in the microbial ecology of the mammalian gut, including the bovine gut. Other phyla represented were the Chloroflesi, Lentisphaerae, Planctomycetes, Spirochaetes, and Verrucomicrogia. This indicates that, even though all of the bacterial phyla contain a diverse range of taxa, the metabolic potential of some phyla most likely allows some to dominate in bovine feces while others remain less abundant.
A comparison of all the genera from this study identified only 41 and 31 genera that were present across the entire sample collection in the COD or the SAID group, respectively (Table 3). The majority of these genera were classified as Firmicutes and Proteobacteria. In addition, the characterization of the percentage abundance of all taxa in cattle given the two diets (Figure 4B) suggests that the population structure of the microorganisms, including those of the genera Solibacillus, Lysinibacillus, unclassified Lachnospiraceae, Stenotrophomonas, Blautia, and Prevotella, were dramatically different in the COD and the SAID group. Indeed, many Lachnospiraceae, Blautia, and Prevotella spp. are common inhabitants of the gastrointestinal tracts and feces of cattle and goats [14–16]. The shifts in the abundance of these microorganisms in the feces of the dairy cows in this study suggest that members of these taxa harbor the metabolic potential to thrive when the diet is transferred rapidly from a traditional COD to a high-concentrate diet.
Effects of SAID feeding on fecal microbiota
The SAID evaluated in this study seemed to have a complex effect on the fecal microbiota, and a common set of taxa seem to be responsive to the influence of SAID vs. traditional COD. Some of these taxa have been identified in other studies to be responsive to or seemingly influenced by a high-grain diet, regardless of the differences in experimental protocols and animals (beef vs. dairy cattle) [17, 18]. A more likely explanation for shifts in the microbial community structure in animals fed SAID may be the abundance and digestibility of starch in the SAID compared with that in the fibrous plant materials associated with the COD. The hindgut of cattle is ideally suited for the fermentation of sugars from fibrous plant materials . Bovine species themselves do not produce the required fiber-degrading enzymes; instead, they harbor fungi, protozoa, and bacteria in their guts that can ferment the fiber. Thus, bovine digestive physiology is dictated largely by the presence of fibrous materials in the rumen. If such animals are fed fiber-deficient diets, such as a high-grain diet, normal digestive processes can be disrupted, leading to the accumulation of fermentation acids, and thus lowering the ruminal pH [20–22]. In turn, these changes in the sources of sugar and starch, combined with shifts in pH, alter the digestive habitat, resulting in a fecal bacterial community that can have an impact on VFA production and pathogen shedding [8, 23]. In the present study, the concentration of starch in the feces from SAID-fed animals was 3 times higher than that in COD-fed cattle (4.43% vs 1.10%). Therefore, the large amount of starch in bovine feces during SAID feeding may be responsible for the changes in the composition of fecal microbial species. However, it should be noted that the SAID used in this study does not represent all types of SARA-inducing diet. A previous study showed that the rumen pH and fermentation variables obtained with wheat challenge were indicative of lactic acidosis, whereas butyric and propionic SARA was observed for corn and beet pulp challenges, respectively . In the present study, the SARA was induced by a corn-based diet. Thus, further work is required to determine whether particular grain sources can influence the ecology of bovine fecal bacterial communities. Nonetheless, this study indicated that there were fundamental differences in the fecal microbial communities of animals in the COD and SAID groups.
Feeding high grain to cattle has a significant effect on the animal health . Studies have indicated that varying the forage to grain ratio in cattle rations can have a marked effect on populations of E. coli. Some studies indicated that over feeding grain increased generic E. coli and/or O157:H7 populations [7, 26–28]. Although E. coli O157:H7 was not detected in any of the present samples and Streptococcus pluranimalium and Campylobacter spp. (Additional file 5: Table S1) were only detected in one fecal sample from the dairy cows fed with the SAID, the data presented herein demonstrate that changes in rations can affect the microbial ecology of the intestinal tract of cattle, which could potentially affect food safety.
The correlation between composition of the bacterial community and concentration of volatile fatty acids
This study showed the presence of high concentrations of propionate and butyrate in the feces of dairy cattle fed with the SAID. The concentrations of volatile fatty acids in this study are similar to results obtained in other studies . Correlation analysis showed that a significant positive correlation was observed between the genus Stenotrophomonas and the levels of acetate, propionate, and TVFA (Figure 6). A previous study showed that Stenotrophomonas spp. were the predominant bacteria found in samples of feces from pigs and chickens and existed as opportunistic pathogens . Our present study showed that SAID feeding was significantly associated with the percentage of the genus Stenotrophomonas, and TVFA production, and this indicates that SAID feeding of livestock may increase the risk of human infection with these opportunistic pathogens.
In the present study, genera including Thalassospira, unclassified Cyanobacteria, unclassified Peptostreptococcaceae, Spirochaeta, Lysinibacillus, unclassified Bacillales, and Papillibacter were negatively correlated with the production of propionate, butyrate and TVFA, respectively, and we also observed that Thalassospira and unclassified Cyanobacteria were negatively correlated with acetate concentration, which may have been caused by the toxic effect of this VFA. This hypothesis is supported by a proposed mechanism for fatty acid toxicity, in which short-chain organic acids, including acetate, propionate and butyrate, can diffuse freely across the bacterial membrane into the cell. Inside the bacterial cell, the acid dissociates, thereby reducing the internal pH, which will cause internal cell damage [31–34].
Branched-chain VFA(BCVFA) are derived from branched-chain amino acids such as leucine, valine, and isoleucine, and some studies have shown that several species of rumen bacteria, including Prevotella ruminicola, Bacteroides ruminicola and Megasphaera elsdenii, have a specific requirement for one or more of the BCVFA [35, 36]. Allison and co-workers [37, 38] have indicated the importance of isovaleric and isobutyric acids in the synthesis of amino acids and lipids in two species of Ruminococcus found in the rumen. Interestingly, our studies have shown that a group of genera, including Holdemania, Subdoligranulum, Anaerovibrio, Desulfovibrio, Leucobacter, Moryella, Propionibacterium, Pseudoramibacter, Terribacillus, unclassified Actinobacteria, unclassified Anaerolineaceae, unclassified Proteobacteria, and Weissella, are positively correlated with isobutyrate and isovalerate. Other genera, such as Parabacteroides, unclassified Mollicutes, Solibacillus, Enterobacter, Paludibacter, unclassified Acetobacteraceae, Klebsiella, unclassified Lactobacillales, Phocaeicola, unclassified Lentisphaerae, Treponema, and unclassified Planctomycetaceae, were positively associated with valerate. However, to date, we know very little about the nutritional characteristics of these bacteria, and it is uncertain whether the growth of these bacteria is stimulated by BCVFA; therefore, the mechanism behind the association between the abundance of these bacteria and the concentrations of BCVFA is still not clear. Nonetheless, this study indicates that the concentrations of isobutyrate and isovalerate may be associated with a particular group of bacteria, and that valerate concentration is linked with a different bacteria group.
In the present study, although we have shown there are possible associations between some VFA and the microbial community, our data are limited and may be biased, because the VFA and the microbial community measured in this study were collected at a single time point. On the other hand, the decrease or increase in these particular genera may be dependent on many other variables (e.g. competition for substrates or production of antimicrobial substances). Similarly, the production of VFA is dependent on other variables (e.g. bacterial metabolism) [21, 39]. Therefore, it remains uncertain whether these significant correlations were influenced by other variables, and therefore if these significant correlation are causal. However, our results have revealed that the genera associated with propionate and butyrate producers may be similar, that the genus linked with the production of valerate is very distinctive, and that the species associated with isobutyate and isovalerate concentrations may belong to one group of ruminal bacteria.