Host immunity and the colon microbiota of mice infected with Citrobacter rodentium are beneficially modulated by lipid-soluble extract from late-cutting alfalfa in the early stages of infection

Alfalfa is a forage legume commonly associated with ruminant livestock production that may be a potential source of health-promoting phytochemicals. Anecdotal evidence from producers suggests that later cuttings of alfalfa may be more beneficial to non-ruminants; however, published literature varies greatly in measured outcomes, supplement form, and cutting. The objective of this study was to measure body weight, average daily feed intake, host immunity, and the colon microbiota composition in mice fed hay, aqueous, and chloroform extracts of early (1(st)) and late (5(th)) cutting alfalfa before and after challenge withCitrobacter rodentium. Prior to inoculation, alfalfa supplementation did not have a significant impact on body weight or feed intake, but 5(th)cutting alfalfa was shown to improve body weight at 5- and 6-days post-infection compared to 1(st)cutting alfalfa (P= 0.02 and 0.01). Combined with the observation that both chloroform extracts improved mouse body weight compared to control diets in later stages ofC.rodentiuminfection led to detailed analyses of the immune system and colon microbiota in mice fed 1(st)and 5(th)cutting chloroform extracts. Immediately following inoculation, 5(th)cutting chloroform extracts significantly reduced the relative abundance ofC.rodentium(P= 0.02) and did not display the early lymphocyte recruitment observed in 1(st)cutting extract. In later timepoints, both chloroform extracts maintained lower splenic B-cell and macrophage populations while increasing the relative abundance of potentially beneficially genera such asTuricibacter(P= 0.02). At 21dpi, only 5(th)cutting chloroform extracts increased the relative abundance of beneficialAkkermansiacompared to the control diet (P= 0.02). These results suggest that lipid soluble compounds enriched in late-cutting alfalfa modulate pathogen colonization and early immune responses toCitrobacter rodentium, contributing to protective effects on body weight.