Year of Publication

2015

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department

Animal and Food Sciences

First Advisor

Dr. Laurie M. Lawrence

Second Advisor

Dr. Michael D. Flythe

Abstract

Grain inclusion in equine diets can allow starch to reach the hindgut where bacteria compete for the substrate. The hypothesis was that starch introduction would cause a source-dependent press disturbance in equine fecal microflora. Fecal cell suspensions were prepared by differential centrifugation and re-suspension in media with ground corn, oats or wheat. At 24 h, corn had more amylolytics and Group D Gram-positive cocci (GPC), and fewer lactate-utilizing (LU) bacteria and lactobacilli than oats, with wheat being intermediate. Predominant amylolytics were identified by their 16S RNA gene sequence as Enterococcus faecalis (corn, wheat) and Streptococcus bovis (oats). In an in vivo experiment, 30 horses were assigned to 1 of 6 treatments: CO (hay only), HC (high corn), HO (high oats), LC (low corn), LO (low oats), and LW (low wheat middlings). The study consisted of a 2 wk adaptation (forage only diet) followed by a 2 wk treatment period, during which horses were adapted to their final starch intake (high, 2 g kg BW-1; low, 1 g kg BW-1). Both HC and LC had fewer lactobacilli and LU and more GPC than CO. In contrast, LO and HO had more lactobacilli and LU, and fewer GPC. LW had higher lactobacilli and GPC than CO. The highest number of amylolytics was observed in HC, followed by LC and LW. The predominant amylolytic isolates from corn and wheat horses were E. faecalis. Both experiments identified a negative correlation between lactobacilli and amylolytics, indicating a potential competitive relationship (r = -0.89, in vitro; r = -0.95, in vivo). The next experiment was conducted to determine if a Lactobacillus addition would mitigate amylolytic proliferation, specifically GPC, with corn fermentation. This experiment was conducted as described above with ground corn ± live or dead (autoclaved) L. reuteri. The addition of L. reuteri, regardless of viability, decreased amylolytics and GPC. To identify the mechanism of action, an E. faecalis isolate was co-incubated with dead L. reuteri cells or supernatant. The supernatant depleted the intracellular K+ of E. faecalis. This result demonstrates that one aspect of competition between lactobacilli and enterococci could be a membrane active antimicrobial.

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