B.E. Harlow

Diarrhea-associated pathogens, lactobacilli and cellulolytic bacteria in equine feces: Responses to antibiotic challenge

Antibiotics are important to equine medicine, but antibiotic-associated diarrhea (AAD) can lead to poor performance and even mortality. AAD is attributed to disruption of the hindgut microbiota, which permits proliferation of pathogenic microbes. The goal of this study was to evaluate the effects of common antibiotics on cellulolytic bacteria, lactobacilli, and AAD-associated pathogens in the feces of healthy horses. Fifteen horses were assigned to three treatment groups (blocked by age and sex): control (no antibiotics), trimethoprim-sulfadiazine (PO), or ceftiofur (IM). Fecal samples (n=8 per horse) were taken during dietary adaptation (3 weeks), antibiotic challenge (1 week), and withdrawal (1 week). Bacteria were enumerated by serial dilution and viable count. Cellulolytic bacteria decreased by >99% during administration of either antibiotic (P<0.0001) and were still less than controls at the end of the withdrawal period (P<0.0001). Fecal samples from horses challenged with ceftiofur had 75% fewer lactobacilli than those from control horses at the end of the antibiotic challenge period (P<0.05). Antibiotic challenged horses also shed more salmonella than control horses (P<0.05). Antibiotics had no effect on the number of Clostridium perfringens isolates. There was no detectable Clostridium difficile during adaptation or in any control horse. C. difficile increased (P<0.0001) to approximately 10(4)cfu/g when horses were challenged with antibiotics, and were still detectable 1 week after withdrawal. These results indicate that antibiotics can disrupt the normal gastrointestinal microbiota and allow proliferation of Salmonella spp. and C. difficile.

Inhibition of fructan‐fermenting equine faecal bacteria and Streptococcus bovis by hops (Humulus lupulus L.) β‐acid

The goals of this study were to determine if β‐acid from hops (Humulus lupulus L.) could be used to control fructan fermentation by equine hindgut micro‐organisms, and to verify the antimicrobial mode of action on Streptococcus bovis, which has been implicated in fructan fermentation, hindgut acidosis and pasture‐associated laminitis (PAL) in the horse.

Effect of Dietary Starch Source and Concentration on Equine Fecal Microbiota

Starch from corn is less susceptible to equine small intestinal digestion than starch from oats, and starch that reaches the hindgut can be utilized by the microbiota. The objective of the current study was to examine the effects of starch source on equine fecal microbiota. Thirty horses were assigned to treatments: control (hay only), HC (high corn), HO (high oats), LC (low corn), LO (low oats), and LW (low pelleted wheat middlings). Horses received an all-forage diet (2 wk; d -14 to d -1) before the treatment diets (2 wk; d 1 to 14). Starch was introduced gradually so that horses received 50% of the assigned starch amount (high = 2 g starch/kg BW; low = 1 g starch/kg BW) by d 4 and 100% by d 11. Fecal samples were obtained at the end of the forage-only period (S0; d -2), and on d 6 (S1) and d 13 (S2) of the treatment period. Cellulolytics, lactobacilli, Group D Gram-positive cocci (GPC), lactate-utilizers and amylolytics were enumerated. Enumeration data were log transformed and analyzed by repeated measures ANOVA. There were sample day × treatment interactions (P < 0.0001) for all bacteria enumerated. Enumerations from control horses did not change during the sampling period (P > 0.05). All treatments except LO resulted in increased amylolytics and decreased cellulolytics, but the changes were larger in horses fed corn and wheat middlings (P < 0.05). Feeding oats resulted in increased lactobacilli and decreased GPC (P < 0.05), while corn had the opposite effects. LW had increased lactobacilli and GPC (P < 0.05). The predominant amylolytic isolates from HC, LC and LW on S2 were identified by 16S RNA gene sequencing as Enterococcus faecalis, but other species were found in oat fed horses. These results demonstrate that starch source can have a differential effect on the equine fecal microbiota.

Effect of starch source (corn, oats or wheat) and concentration on fermentation by equine faecal microbiota in vitro.

The goal was to determine the effect of starch source (corn, oats and wheat) and concentration on: (i) total amylolytic bacteria, Group D Gram‐positive cocci (GPC), lactobacilli and lactate‐utilizing bacteria, and (ii) fermentation by equine microbiota.

Degradation of spent craft brewer's yeast by caprine rumen hyper ammonia‐producing bacteria

Spent yeast from craft beers often includes more hops (Humulus lupulus L.) secondary metabolites than traditional recipes. These compounds include α‐ and β‐ acids, which are antimicrobial to the rumen hyper ammonia‐producing bacteria (HAB) that are major contributors to amino acid degradation. The objective was to determine if the hops acids in spent craft brewers yeast (CY; ~ 3·5 mg g−1 hops acids) would protect it from degradation by caprine rumen bacteria and HAB when compared to a bakers yeast (BY; no hops acids). Cell suspensions were prepared by harvesting rumen fluid from fistulated goats, straining and differential centrifugation. The cells were re‐suspended in media with BY or CY. After 24 h (39°C), HAB were enumerated and ammonia was measured. Fewer HAB and less ammonia was produced from CY than from BY. Pure culture experiments were conducted with Peptostreptococcus anaerobiusBG1 (caprine HAB). Ammonia production by BG1 from BY was greater than from CY. Ammonia production was greater when exogenous amino acids were included, but similar inhibition was observed in CY treatments. These results indicate that rumen micro‐organisms deaminated the amino acids in CY to a lesser degree than BY.

Phosphorus digestibility and phytate degradation by yearlings and mature horses.

Inorganic P is often added to growing horse diets because organic P, or phytate-P, is believed to have lower digestibility. If horses can efficiently digest organic P, then the need for inorganic P may be reduced. Much of the P in grain-based concentrates fed to growing horses is in the form of phytate-P. Little is known about the ability of growing horses to degrade phytate-P or whether horse age affects mineral digestion in horses. The objective of this study was to examine the effect of age on P, Ca, and Mg digestibility as well as phytate-P degradation. Four yearling geldings and 4 mature geldings were fed a diet of alfalfa cubes, timothy cubes, and a pelleted concentrate. The diet contained 0.28% total P and 17.4% of that P was in the phytate form. There was a 14-d diet adaptation period followed by a 4-d fecal collection period. Apparent total tract P digestibility was higher for yearlings than mature geldings ( = 0.036; 7.7 and -6.6% for yearlings and mature geldings, respectively). Phytate-P disappearance was 94.8% and did not differ between ages ( = 0.190). Apparent Ca digestibility was lower in mature geldings ( = 0.043), but apparent Mg digestibility did not differ between ages ( = 0.414). Phytate is broken down in the gastrointestinal tract, but the low P digestibilities suggest that either degradation occurs after the site of P absorption or liberated P is recycled back into the gastrointestinal tract. Yearlings can utilize organic P as well as mature horses; therefore, diets without inorganic P are acceptable for growing horses.

Exogenous Lactobacilli Mitigate Microbial Changes Associated with Grain Fermentation (Corn, Oats, and Wheat) by Equine Fecal Microflora Ex Vivo

Cereal grains are often included in equine diets. When starch intake exceeds foregut digestion starch will reach the hindgut, impacting microbial ecology. Probiotics (e.g., lactobacilli) are reported to mitigate GI dysbioses in other species. This study was conducted to determine the effect of exogenous lactobacilli on pH and the growth of amylolytic and lactate-utilizing bacteria. Feces were collected from 3 mature geldings fed grass hay with access to pasture. Fecal microbes were harvested by differential centrifugation, washed, and re-suspended in anaerobic media containing ground corn, wheat, or oats at 1.6% (w/v) starch and one of five treatments: Control (substrate only), L. acidophilus, L. buchneri, L. reuteri, or an equal mixture of all three (107 cells/mL, final concentration). After 24 h of incubation (37°C, 160 rpm), samples were collected for pH and enumerations of total amylolytics, Group D Gram-positive cocci (GPC; Enterococci, Streptococci), lactobacilli, and lactate-utilizing bacteria. Enumeration data were log transformed prior to ANOVA (SAS, v. 9.3). Lactobacilli inhibited pH decline in corn and wheat fermentations (P < 0.0001). Specifically, addition of either L. reuteri or L. acidophilus was most effective at mitigating pH decline with both corn and wheat fermentation, in which the greatest acidification occurred (P < 0.05). Exogenous lactobacilli decreased amylolytics, while increasing lactate-utilizers in corn and wheat fermentations (P < 0.0001). In oat fermentations, L. acidophilus and L. reuteri inhibited pH decline and increased lactate-utilizers while decreasing amylolytics (P < 0.0001). For all substrates, L. reuteri additions (regardless of viability) had the lowest number of GPC and the highest number of lactobacilli and lactate-utilizers (P < 0.05). There were no additive effects when lactobacilli were mixed. Exogenous lactobacilli decreased the initial (first 8 h) rate of starch catalysis when wheat was the substrate, but did not decrease total (24 h) starch utilization in any case. These results indicate that exogenous lactobacilli can impact the microbial community and pH of cereal grain fermentations by equine fecal microflora ex vivo. Additionally, dead (autoclaved) exogenous lactobacilli had similar effects as live lactobacilli on fermentation. This latter result indicates that the mechanism by which lactobacilli impact other amylolytic bacteria is not simple resource competition.

Effects of Inulin Chain Length on Fermentation by Equine Fecal Bacteria and Streptococcus bovis

&NA; Grass fructans can be fermented by Gram‐positive bacteria (e.g., Streptococcus bovis) in the equine hindgut, increasing production of lactic acid and decreasing pH. The degree of polymerization (DP) of fructans has been suggested to influence fermentation rates. The objective of the present study was to determine how DP impacts fermentation by equine fecal bacteria and a model S. bovis. Fecal microbes from three mares were harvested by differential centrifugation, washed, and resuspended in anaerobic media containing short‐chain (SC; DP ≤ 10) or long‐chain (LC) inulin (DP ≥ 23) from 0% to 2% wt/vol. After 24 hours of incubation (37°C), samples were collected for pH determination. Data were analyzed using the general linear models (GLM) procedure testing for the effect of treatment, concentration, and treatment × concentration (SAS v. 9.3). At all concentrations, the pH was lower in SC fermentations than in LC (P < .0001, in all cases). To determine the effect of DP on S. bovis, cultures were grown (39°C, 9 hours) with 0.1%, 0.5%, or 1.3% SC or LC inulin. Optical density (600 nm) was determined by spectrophotometry. Maximum specific growth rates (&mgr;) were determined by linear regression (2–5 hours). Data were analyzed using the one‐way analysis of variance procedure (SAS v. 9.3). The final optical density (600 nm), &mgr;, and yield were higher with SC than with LC fermentation (P < .05). These results indicate that SC inulin may be more available for fermentation than LC inulin by equine fecal bacteria and S. bovis, specifically. HighlightsInulin degree of polymerization (DP) influences equine fecal bacteria and Streptococcus bovis fermentations in vitro.Short‐chain inulin is more available for fermentation than long‐chain inulin.Streptococcus bovis JB1 grows faster on low DP inulin than on high DP inulin.

Effect of selenium status on the response of unfit horses to exercise

Exercise is known to increase reactive oxygen species and alter glutathione peroxidase activity (GPx), a selenoenzyme responsible for neutralising hydrogen peroxide. This study evaluated the effect of selenium (Se) status on the response of unfit horses to mild exercise. 25 mature horses received one of four dietary treatments for 29 weeks: low Se (LS, n=6), adequate Se (AS, sodium selenite, n=6), high organic Se (SP; Sel-Plex®, n=7) or high inorganic Se (SS, sodium selenite, n=6). Total dietary Se concentration for LS, AS, SP and SS was 0.06, 0.12, 0.3 and 0.3 mg/kg respectively. Blood samples were collected before and at 0, 4 and 24 h after a 36 min exercise test covering 4.41 km. Blood samples were evaluated for blood GPx, pro-inflammatory cytokine expression, serum malondialdehyde (MDA), creatine kinase (CK), aspartate aminotransferase (AST) and blood pro-inflammatory cytokine expression. Data were analysed as ANOVA with repeated measures. Prior to the exercise test blood Se was higher (P<0.01) for SP...

Effect of starch source in pelleted concentrates on fecal bacteria in pre- and postpartum mares

&NA; Dietary starch source has been shown to affect fecal bacterial communities of horses fed minimally processed cereal grains. However, processing may increase foregut starch digestibility, reducing effects of starch source on fecal bacterial communities. This study aimed to determine the effect of starch source in pelleted concentrates on fecal Lactobacillus spp., amylolytic bacteria, and cellulolytic bacteria in broodmares mares, during the prepartum and postpartum period. Thoroughbred mares (n = 18) were paired by last breeding date then randomly assigned to either an oat‐based or a corn and wheat middlings‐based pelleted concentrate fed with forage. Mares were fed their assigned concentrates beginning on 310 days of gestation, and fecal samples were collected at 324 days of gestation, before parturition, 1 day, 14 days, and 28 days postpartum. Fecal samples were enumerated by serial dilution and inoculation into selective, enriched media for Lactobacillus spp., amylolytic bacteria, and cellulolytic bacteria. Data were log transformed then analyzed using a mixed model ANOVA with repeated measures (SAS 9.3) to test the main effects of treatment, time of sample, and treatment by time interaction. Starch source did not affect enumerated bacterial communities (P > .05); thus, pelleting concentrates may alter some of the effects of starch sources on the hindgut microbiota. Sample date did not affect amylolytic bacteria (P > .05); however, lactobacilli and cellulolytic bacteria decreased 1 day postpartum (P < .05). Although we did not observe an effect of starch source on fecal bacteria in mares, parturition did appear to alter the hindgut microbiota.