Rumen Microbiome Composition Determined Using Two Nutritional Models of Subacute Ruminal Acidosis

Subacute ruminal acidosis (SARA) is a metabolic disease in dairy cattle that occurs during early and mid-lactation and has traditionally been characterized by low rumen pH, but lactic acid does not accumulate as in acute lactic acid acidosis. It is hypothesized that factors such as increased gut permeability, bacterial lipopolysaccharides, and inflammatory responses may have a role in the etiology of SARA. However, little is known about the nature of the rumen microbiome during SARA. In this study, we analyzed the microbiome of 64 rumen samples taken from eight lactating Holstein dairy cattle using terminal restriction fragment length polymorphisms (TRFLP) of 16S rRNA genes and real-time PCR. We used rumen samples from two published experiments in which SARA had been induced with either grain or alfalfa pellets. The results of TRFLP analysis indicated that the most predominant shift during SARA was a decline in gram-negative Bacteroidetes organisms. However, the proportion of Bacteroidetes organisms was greater in alfalfa pellet-induced SARA than in mild or severe grain-induced SARA (35.4% versus 26.0% and 16.6%, respectively). This shift was also evident from the real-time PCR data for Prevotella albensis, Prevotella brevis, and Prevotella ruminicola, which are members of the Bacteroidetes. The real-time PCR data also indicated that severe grain-induced SARA was dominated by Streptococcus bovis and Escherichia coli, whereas mild grain-induced SARA was dominated by Megasphaera elsdenii and alfalfa pellet-induced SARA was dominated by P. albensis. Using discriminant analysis, the severity of SARA and degree of inflammation were highly correlated with the abundance of E. coli and not with lipopolysaccharide in the rumen. We thus suspect that E. coli may be a contributing factor in disease onset.The bovine rumen is a classical host-microbe symbiotic system, and disturbances in this exquisitely balanced ecosystem may lead to disease in the host. An example is subacute ruminal acidosis (SARA), or non-lactic acid acidosis, which has a disease etiology distinct from that of acute lactic acid acidosis because there is no accumulation of lactic acid (35). Field studies in the United States estimated that 19% of early lactating cows and 26% of mid-lactation cows suffered from SARA (11). In Germany and The Netherlands, approximately 11% of early lactation and 18% of mid-lactation cows suffered from this disease (22). In the acute form, lactic acid accumulates in the rumen, causing metabolic acidosis, and it usually occurs when animals are abruptly transitioned to a high-grain diet from a predominantly forage diet (38). If, however, the adaptation is gradual, slower-growing lactic acid-consuming bacteria, like Megasphaera elsdenii, convert the lactic acid to propionic acid (29). In SARA, lactic acid does not accumulate during low-pH conditions and other factors, like microbial population shifts and immune responses, appear to be associated with the disease etiology (35).In both acute and subacute acidosis, there is an increase in lipopolysaccharide (LPS) concentrations in the rumen (8, 14, 16). LPS and/or the low-pH rumen conditions may increase the permeability of the gut to LPS, which could trigger systemic inflammation (4). We previously developed two animal models of SARA, one based on grain and one based on alfalfa pellets (20, 21). Even though both models resulted in substantial reductions in rumen pH and an accumulation of LPS, only the grain induction model resulted in inflammation and the appearance of LPS in the peripheral blood (20, 21).In contrast to the rumen microbiome during lactic acid acidosis, the rumen microbiome during SARA has not been evaluated (13, 28). Even in acute acidosis, studies are largely culture based, and the uncultured members of the community have not been extensively assessed (31, 46, 49). In this article, we describe the rumen microbiome when two SARA induction models were used. The shifts in microbial community structure were assessed using terminal restriction fragment length polymorphism (TRFLP) analysis and real-time PCR of key microbial populations.