Equine Gut Research Articles

Where are we with the equine gut microbiome?

Where are we with the equine gut microbiome?

Development of the equine gut microbiota

Shortly after birth the mammalian gut is colonized, by a transient microbiota, highly susceptible to environment and diet, that eventually stabilizes and becomes the resident gut microbiota. In a window of opportunity during the colonization, oral tolerance is established towards resident bacteria. In this study, the development of the equine gut microbiota was investigated in ten foals from parturition until post weaning. We found great differences in the core species of the gut microbiota composition between time-matched samples on Day 7 and 20 post-partum. Between day 20 and Day 50 post-partum, we saw the gut microbiota became increasingly dominated by fiber fermenting species. After Day 50, no significant changes in species abundance were observed. Gene expression analysis of pro- and anti-inflammatory cytokines in the blood revealed no significant changes before and after weaning. In summary, relative stability of the gut microbiota was reached within 50 days post-partum and, weaning did not have a major impact on the microbial composition.

A Metagenomic Analysis of the Equine Gut Microbiome with and Without Probiotic Supplementation (P09-006-19)

ObjectivesThe goal of this study is to compare the microbiome of domesticated horses with and without probiotic supplementation. MethodsOur University’s Institutional Animal Care and Use Committee has granted an exemption, as the horses were not housed on campus and there was no experimental manipulation to the horses’ feeding implemented. Fecal matter from six privately owned horses maintained on their standard grazing diet were collected. Three of the six horses received a probiotic supplement, SmartDigest, for several years prior to beginning the project. Supplementation ceased for one month, and samples were again obtained. The other three horses never received probiotics. Bacterial DNA was isolated from all fecal samples, the 16S rRNA gene amplified, tagged with index primers, and subsequently sequenced using the Illumina MiSeq. ResultsDominant groups from non-supplemented horses residing on the same property included the phyla bacteriodetes, firmicutes, proteobacteria, and verrucomicrobia. Interestingly, Sphingobacterium bambusae was identified in all three horses, even though this species has previously been isolated from the soil of bamboo plantations. Currently, samples from horses with probiotics are being analyzed. We are also employing qPCR analysis to validate the NextGen data, and to further investigate relative abundance of specific bacterial groups relevant to equine intestinal health. ConclusionsPreliminary NextGen sequence analysis of the relative abundance of bacterial phyla suggests that, as expected, the horses residing on the same property and thus receiving the same diet possess a similar intestinal microbiome composition. Similarities between horses persist down to the genus level, and are now being compared to samples from horses on a probiotic-supplemented diet. Funding SourcesFinancial support was provided by Towson University’s Fisher College of Science and Mathematics, and Jess and Mildred Fisher Endowed Chair funds to P. Tsuji.

The impact of DigestaWell Buffer on the equine gut microbiome of healthy horses

The impact of DigestaWell Buffer on the equine gut microbiome of healthy horses

Impact of endurance exercise on fecal indicators of equine gut health

Back pain is a significant factor for horses and is challenging for professionals to diagnose, with assessment frequently using subjective tools such as manual palpation. Reliable and valid objective measures are required and use of a pressure algometer (PA) has been investigated as an assessment tool; however, it has limitations, and other more realistic methods may be better suited for the task. The aim of the study was to establish inter- and intra-rater reliability for PA, FlexiForce Sensor (FFS), and manual palpation for equine epaxial soft tissue, measuring mechanical nociception threshold responses. In group 1, 10 horses underwent three repeated tests with PA and FFS, and once for manual palpation, with three Association of Chartered Physiotherapists in Animal Therapy (ACPAT) Chartered Physiotherapists in the right thoracic epaxial region. Group 2 followed the same protocol using one ACPAT Chartered Physiotherapist and 22 horses. The order of palpation was randomly applied for each test and each experimenter. Manual palpation showed excellent interrater reliability with no significant differences between scores (P = .64; intraclass correlation coefficient [ICC] 90.0%). PA (P = .002) and FFS (P = .025) scores significantly differed between experimenters. Intrarater testing showed significant differences (P = .014) with horses increasing sensitivity over repeated PA measures. The FFS showed no significant differences (P = .347; ICC 94.7%) in repeated measures with excellent reliability and consistency. The PA showed a lack of consistency in intrarater reliability conflicting with previous research findings, whereas the FFS showed greater reliability in comparison; however, it proved difficult to use in clinical practice. Manual palpation by physiotherapists was shown to have excellent interrater reliability when using a categorical scoring system.

Removal of adult cyathostomins alters faecal microbiota and promotes an inflammatory phenotype in horses

The interactions between parasitic helminths and gut microbiota are considered to be an important, although as yet incompletely understood, factor in the regulation of immunity, inflammation and a range of diseases. Infection with intestinal helminths is ubiquitous in grazing horses, with cyathostomins (about 50 species of which are recorded) predominating. Consequences of infection include both chronic effects, and an acute inflammatory syndrome, acute larval cyathostominosis, which sometimes follows removal of adult helminths by administration of anthelmintic drugs. The presence of cyathostomins as a resident helminth population of the equine gut (the “helminthome”) provides an opportunity to investigate the effect helminth infection, and its perturbation, has on both the immune system and bacterial microbiome of the gut, as well as to determine the specific mechanisms of pathophysiology involved in equine acute larval cyathostominosis. We studied changes in the faecal microbiota of two groups of horses following treatment with anthelmintics (fenbendazole or moxidectin). We found decreases in both alpha diversity and beta diversity of the faecal microbiota at Day 7 post-treatment, which were reversed by Day 14. These changes were accompanied by increases in inflammatory biomarkers. The general pattern of faecal microbiota detected was similar to that seen in the relatively few equine gut microbiome studies reported to date. We conclude that interplay between resident cyathostomin populations and the bacterial microbiota of the equine large intestine is important in maintaining homeostasis and that disturbance of this ecology can lead to gut dysbiosis and play a role in the aetiology of inflammatory conditions in the horse, including acute larval cyathostominosis.

The impact of seasonal changes on the equine gut

Briony Witherow MSc RNutr. Practical Equine Nutrition, outlines how to minimise nutritional challenges to the equine gut from spring grass and changes to routine

Modulation of the equine microbiome by pasture and feed supplements: A metabolomics approach

Faecal VOCs (volatile organic compounds) were compared in horses and ponies before and after 6 weeks on Spring pasture and with and without a dietary supplement (ERME). The supplement was derived from malted barley and contained enzymes which degrade complex carbohydrates, including amylase, maltase, glucanases and fructanases. VOCs of faecal samples were analysed by selected ion flow tube mass spectrometry (SIFT-MS) and the results showed that the faecal microbiome was altered in different ways both by feeding on Spring pasture and by the dietary supplement. The composition of the equine gut microbiome (EGM) is therefore potentially a reflection of dietary constituents, both pasture and supplements; any metabolomic approach should take this into account since the EGM may have modulating effects on physiological parameters such as diabetes and obesity.

498 Saccharomyces Cerevisiae Supplementations Effect on Fiber Digestibility in Equine.

Probiotics have become increasingly common in equine diets to aid in fermentation in the hindgut. Yeast culture additive, such as Saccharomyces cerevisiae, is a common probiotic that has recently been used to optimize equine diets by enhancing nutrient digestibility. Yeast culture has been shown to increase the activity of cellulolytic bacteria in the hindgut by stabilizing cecal pH, which maximizes fiber fermentation. Dietary energy, dry matter, crude protein, NDF, ADF, hemicellulose, and cellulose apparent digestibility also increased when yeast culture was supplemented. However, studies examining the effect of yeast culture additive have been conflicting and inconsistent. The objective of the current study was to investigate the effect of Saccharomyces cerevisiae supplementation on weight gain and nutrient digestibility, focusing on NDF and ADF levels in healthy horses. A live strain of Saccharomyces cerevisiae product was provided to twelve horses of varying age, breed, and weight. The current study utilized an 88 d switchback design sectioned into two 44 d trial periods, including a 14 d adjustment period. Horses were randomly sectioned into two groups; supplemented then control, or control then supplemented. Horses were supplemented at a rate of 1 ounce twice daily with feed, at 0730 and 1700, as suggested by the product. Body weights were recorded weekly and a biweekly fecal grab sample was collected from each horse. Fecal samples were frozen at -80⁰C and then dried for 36 hours in a 70⁰C oven. Samples were sent to Cumberland Valley Labs for analysis of ADF, NDF, and ADL, using ADL as an internal marker. Weight and digestibility analysis was done using LSM in Proc GLM through SAS 9.0. There was no significant change in weight (P>.05) due to yeast supplementation. Apparent NDF digestibility was not effected by treatment (P=.296), while apparent ADF digestibility was increased due to supplementation (P<.0001). Overall, Saccharomyces cerevisiae, as supplemented in the current study, increased ADF, but did not increase NDF or cause a change in weight. An increase in ADF correlates to an increase in forage digestibility, enhancing overall equine gut health.

The association between gut microbiome, sex, age and body condition scores of horses in Maiduguri and its environs

The equine gut harbours complex microbial populations which influence physiology, metabolism, nutrition and immune functions, while disruption to the gut microbiota has been linked with conditions such as lameness, diabetes and obesity. Therefore, the present study was conducted to determine the association between microbial dysbiosis, sex, age and body condition scores (BCS) of horses (Equus equus caballus) in Maiduguri and its environs. Forty horses were assessed by convenient sampling, while faecal samples were collected and analyzed to determine the microbiomes in the various age groups with variable BSC in stallions and mares. The present study revealed that there was strong and not significant association between the microbial populations in the gut of horses and the various age groups (Chi-Squared test = 11.809; df = 18; P = 0.8570). There were significant (p < 0.0001) differences in the microbiome population in the guts of the horses. There were significant (p < 0.05) differences in the microbiomes in the various body scores. There were significant (P < 0.047) differences in the microbiomes of the mares and stallions. E. coli had the highest burden of microbial populations with 30.38*103 ± 1.04*103 in the guts of horses in the study area, while statistically there were no differences in the populations of Corynebacterium, Klebsiella, Salmonella, Staphylococcus and Streptococcus in the gut of horses in the study area relative to E. coli. This study provides information on the nature of bacterial population present in horses in Maiduguri and its Environs. Therefore, further high-throughput characterization of the faecal bacteria microbiome of horses is essential to enable comparison to disease state and thus develop diagnostic tests, prophylactic measures and appropriate treatments.

The relationships between faecal egg counts and gut microbial composition in UK Thoroughbreds infected by cyathostomins

A growing body of evidence, particularly in humans and rodents, supports the existence of a complex network of interactions occurring between gastrointestinal (GI) helminth parasites and the gut commensal bacteria, with substantial effects on both host immunity and metabolic potential. However, little is known of the fundamental biology of such interactions in other animal species; nonetheless, given the considerable economic losses associated with GI parasites, particularly in livestock and equines, as well as the global threat of emerging anthelmintic resistance, further explorations of the complexities of host-helminth-microbiota interactions in these species are needed. This study characterises the composition of the equine gut commensal flora associated with the presence, in faecal samples, of low (Clow) and high (Chigh) numbers of eggs of an important group of GI parasites (i.e. the cyathostomins), prior to and following anthelmintic treatment. High-throughput sequencing of bacterial 16S rRNA amplicons and associated bioinformatics and statistical analyses of sequence data revealed strong clustering according to faecal egg counts (P = 0.003). A trend towards increased populations of Methanomicrobia (class) and Dehalobacterium (genus) was observed in Clow in comparison with Chigh. Anthelmintic treatment in Chigh was associated with a significant reduction of the bacterial Phylum TM7 14 days post-ivermectin administration, as well as a transient expansion of Adlercreutzia spp. at 2 days post-treatment. This study provides a first known insight into the discovery of the intimate mechanisms governing host-parasite-microbiota interactions in equines, and sets a basis for the development of novel, biology-based intervention strategies against equine GI helminths based on the manipulation of the commensal gut flora.

Morphometric comparisons of draft, pony, and horse breeds

The equine gut harbours complex microbial populations which influence physiology, metabolism, nutrition and immune functions, while disruption to the gut microbiota has been linked with conditions such as lameness, diabetes and obesity. Therefore, the present study was conducted to determine the association between microbial dysbiosis, sex, age and body condition scores (BCS) of horses (Equus equus caballus) in Maiduguri and its environs. Forty horses were assessed by convenient sampling, while faecal samples were collected and analyzed to determine the microbiomes in the various age groups with variable BSC in stallions and mares. The present study revealed that there was strong and not significant association between the microbial populations in the gut of horses and the various age groups (Chi-Squared test = 11.809; df = 18; P = 0.8570). There were significant (p < 0.0001) differences in the microbiome population in the guts of the horses. There were significant (p < 0.05) differences in the microbiomes in the various body scores. There were significant (P < 0.047) differences in the microbiomes of the mares and stallions. E. coli had the highest burden of microbial populations with 30.38*103 ± 1.04*103 in the guts of horses in the study area, while statistically there were no differences in the populations of Corynebacterium, Klebsiella, Salmonella, Staphylococcus and Streptococcus in the gut of horses in the study area relative to E. coli. This study provides information on the nature of bacterial population present in horses in Maiduguri and its Environs. Therefore, further high-throughput characterization of the faecal bacteria microbiome of horses is essential to enable comparison to disease state and thus develop diagnostic tests, prophylactic measures and appropriate treatments.

A Microbiological Map of the Healthy Equine Gastrointestinal Tract.

Horses are exquisitely sensitive to non-specific gastrointestinal disturbances as well as systemic and extraintestinal conditions related to gut health, yet minimal data are available regarding the composition of the microbiota present in the equine stomach, small intestine, and cecum and their relation to fecal microbiota. Moreover, there is minimal information regarding the concordance of the luminal and mucosal microbial communities throughout the equine gut. Illumina-based 16S rRNA gene amplicon sequencing of the luminal and mucosal microbiota present in seven regions of the gastrointestinal tract of nine healthy adult horses revealed a distinct compositional divide between the small and large intestines. This disparity in composition was more pronounced within the luminal contents, but was also detected within mucosal populations. Moreover, the uniformity of the gut microbiota was much higher in the cecum and colon relative to that in the stomach, jejunum and ileum, despite a significantly higher number of unique sequences detected in the colon. Collectively, the current data suggest that while colonic samples (a proxy for feces) may provide a reasonable profile of the luminal contents of the healthy equine large intestine, they are not informative with regard to the contents of the stomach or small intestine. In contrast to the distinct difference between the highly variable upper gastrointestinal tract microbiota and relatively uniform large bowel microbiota present within the lumen, these data also demonstrate a regional continuity present in mucosal microbial communities throughout the length of the equine gut.

Molecular Phylogeny of Spirodinium equi, Triadinium caudatum and Blepharocorys sp. from the Equine Hindgut

Single cell morphotypes of the species Triadinium caudatum and Spirodinium equi, together with a representative of the genus Blepharocorys (Blepharocorys sp.) were used for phylogenetic analysis based on their 18S rRNA genes. Spirodinium equi clustered with sequences already described for the entodiniomorphs isolated from horses and the Blepharocorys sp. also grouped within the Entodiniomorphida clade, although both sequences were distinct from those described from rumen ciliates. Triadinium caudatum clustered within the Vestibuliferida, and most closely to that of Paraisotricha, only other member of this order which has been described in the horse. It was concluded that although members of the orders Entodiniomorphida and Vestibuliferida are present in the equine gut, and that they share an ancient linage with their rumen counterparts, they are ancestrally different groups.

Expression of sweet receptor components in equine small intestine: relevance to intestinal glucose transport

The heteromeric sweet taste receptor T1R2-T1R3 is expressed on the luminal membrane of certain populations of enteroendocrine cells. Sensing of sugars and other sweet compounds by this receptor activates a pathway in enteroendocrine cells, resulting in secretion of a number of gut hormones, including glucagon-like peptide 2 (GLP-2). This subsequently leads to upregulation in the expression of intestinal Na(+)/glucose cotransporter, SGLT1, and increased intestinal glucose absorption. On the basis of the current information available on the horse genome sequence, it has been proposed that the gene for T1R2 (Tas1R2) is absent in the horse. We show here, however, that horses express both the mRNA and protein for T1R2. Equine T1R2 is most closely homologous to that in the pig and the cow. T1R2 protein, along with T1R3, α-gustducin, and GLP-2 proteins are coexpressed in equine intestinal endocrine cells. Intravenous administration of GLP-2, in rats and pigs, leads to an increase in the expression of SGLT1 in absorptive enterocytes and enhancement in blood glucose concentrations. GLP-2 receptor is expressed in enteric neurons, excluding the direct effect of GLP-2 on enterocytes. However, electric stimulation of enteric neurons generates a neural response leading to SGLT1 upregulation, suggesting that sugar in the intestine activates a reflex increase in the functional expression of SGLT1. Horses possess the ability to upregulate SGLT1 expression in response to increased dietary carbohydrates, and to enhance the capacity of the gut to absorb glucose. The gut sweet receptor provides an accessible target for manipulating the equine gut to absorb glucose (and water), allowing greater energy uptake and hydration for hard-working horses.

Bacterial Colonization of the Equine Gut; Comparison of Mare and Foal Pairs by PCR-DGGE

Horses, like all animals, are born without the symbiotic microbes that occupy the gastrointestinal tracts of mature animals. As grazing animals, horses rely on these microbes to fully utilize the grasses and other cellulosic feeds that they consume. Thus, colonization of the foal's gastrointestinal tract must occur between birth and weaning. The feces of nine mare and foal pairs were sampled from the day of parturition until 12 weeks of age, and the samples were analyzed by polymerase chain reaction amplification of the bacterial 16S rRNA gene and denaturing gradient gel electrophoresis (PCR-DGGE). The gels from feces of day (d) 0 foals had no or very few ( x = 3, n = 6) bands, which indicates that species richness was low. The number of bands increased during the first 4 days of life, and by d 14 the foals and mares had similar numbers of bands ( x = 28, n = 23). Some bands were present in young foals, but not in mares or in foals on d 42 or d 84, which indicated succession of bacterial species. When the PCR-DGGE profiles were compared with Dice's algorithm, all mare-foal pairwise similarities on d 14 and later were as great as the pairwise similarities between mares. These results are consistent with the idea that foals are born with a sterile gut, colonization proceeds rapidly, and a mature microbial community is present in the first few weeks of life.

Alterations in microbiota and fermentation products in equine large intestine in response to dietary variation and intestinal disease

We aimed to determine the effects of variations in dietary composition on equine gut microbiota and their fermentation products, and proposed that dietary modifications profoundly affect microbial ecosystems and their metabolites. Bacterial communities within the large intestine of three groups of horses were compared using oligonucleotide-RNA hybridisation methodology. Each group consisting of six horses was maintained on (1) a grass-only diet, (2) a concentrate diet (i.e. supplemented with hydrolysable carbohydrates) and (3) a concentrate diet but horses were affected by simple colonic obstruction and distension (SCOD), a prevalent form of dietary-induced intestinal disease. We show that in response to dietary change and intestinal disease, there is a progressive and significant increase in Lachnospiraceae, the Bacteroidetes assemblage and the lactic acid-producing, Bacillus-Lactobacillus-Streptococcus (BLS) group. In contrast, there is a corresponding decrease in the proportion of obligate fibrolytic, acid-intolerant bacteria, Fibrobacter and Ruminococcaceae. Assessment of monocarboxylic acids indicated that there are significantly higher concentrations of lactic acid in the colonic contents of horses maintained on a concentrate diet and those suffering from SCOD, correlating with the observed increase in the population abundance of the BLS group. However, the population size of the Veillonellaceae (lactate utilisers) remained constant in each study group. The inability of this group to respond to increased lactic acid may be a contributory factor to the build-up of lactic acid observed in horses fed a concentrate diet and those suffering from SCOD.

Black walnut extract inhibits secretory pathways in equine cecum and colon via mechanisms that overlap with cyclooxygenase signaling

Disturbances in the hind‐gut have been linked to the induction of equine laminitis, presumably by initiating enteric signaling pathways that result in release of mediators that affect distant fragile vascular beds such as that in the hoof. Laminitis has been experimentally induced by gavaging with black walnut extract (BWE) toxin, leading to changes in both the gut and laminae. Cecal and colonic tissue were mounted on Ussing chambers and treated with escalating doses of BWE (0.6, 1.25, 2.5 and 5%). Changes in short circuit current (Isc), transepithelial electrical resistance, and 3H‐mannitol fluxes were measured. BWE treatment did not affect gut barrier function as there were no significant changes in resistance or mannitol flux. However, BWE caused significant decreases in Isc when compared with controls, most likely due to inhibition of basal Cl− secretion. Pre‐treatment with the cyclooxygenase inhibitor flunixin (2.7μM) resulted in a similar reduction in Isc, which has previously been shown to inhibit Cl− secretion. Treatment with both BWE and flunixin had no additive effect, further suggesting that BWE and flunixin have overlapping signaling pathways. The precise mechanism by which BWE reduces Isc in the equine gut will require further study, but early evidence is suggestive of an inhibitory effect on Cl− secretion. This in turn may affect colonic luminal contents, with systemic effects such as laminitis.

Molecular insights into dietary induced colic in the horse

Equine colic, a disorder manifested in abdominal pain, is the most frequent cause of emergency treatment and death in horses. Colic often requires intestinal surgery, subsequent hospitalisation and post operative care, with a strong risk of complications arising from surgery. Therefore strategies that explore approaches for preventing the condition are essential. To this end, a better understanding of the factors and mechanisms that lead to the development of colic and related intestinal diseases in the horse allows the design of preventive procedures. Colic is a multifactorial disorder that appears to be induced by environmental factors and possibly a genetic predisposition. One factor that seems to influence the risk of developing colic is the excessive consumption of diets containing high levels of carbohydrates. Therefore, major efforts have been made by various laboratories and institutions across the world to study the type and digestibility of various feed in order to formulate accurate and safe feed components and proportions. However, relatively little work has been carried out to characterise, in detail, the carbohydrate digestive and absorptive capacity and mechanisms underlying the potential adaptive response of equine gut epithelium to a changing diet. This review focuses on advances made towards understanding the molecular and cellular mechanisms involved in digestion and absorption of dietary carbohydrates in the equine gastrointestinal tract and the implication of these processes for the whole body physiology. It addresses the underlying mechanisms that may govern the adaptive response of equine small intestine to increased dietary hydrolysable carbohydrates. Furthermore, it describes changes that occur in the equine large intestinal microbiology and host tissue biology brought about by alterations in diet and in colic. It is hoped that a better understanding of the molecular and cellular processes that play important roles in the physiology and pathology of the equine gastrointestinal tract will assist the development of effective strategies to prevent equine colic.

Fermentative activity of horse gut contents assessed in vitro

There is little published information concerning microbial diversity in the horse gut. However, microbial instability can influence gut lumen pH and lactate accumulation, both of which have been implicated in the occurrence of laminitis and colic. In this study three substrates, hay (H), oats (O) and a 50:50 hay:oats (DM basis) mixture (HO) were used to evaluate in vitro microbial fermentative activity in four sections of the equine gut.