Chris Proudman

Introduction. The composition of the equine gut microbiome is associated with many aspects of gastrointestinal, respiratory and musculoskeletal health that have been reported in the horse. Scientific studies exploring the microbiome non-intestinal ecological niches in or on horses are lacking. The clinical use of bacterial community profiling in horses is currently limited by cost and by slow analytical workflows. Hypothesis/Gap Statement. Most equine microbiome studies have relied on 16S rRNA amplicon sequencing of bacterial DNA, using high-throughput short-read sequencing technologies. This is often provided by an external service due to the cost of Illumina and other sequencers. Analysis of such sequencing files relies upon the researcher to have prior experience of coding-based programs. Aim. To explore the utility of Oxford Nanopore long-read sequencing in the analysis of microbiomes from several anatomical sites of the horse as a quicker and cheaper alternative to short-read sequencing. Methodology. Bacterial DNA was extracted from horse (udder) skin swabs, saliva swabs, faecal samples and milk samples. Samples were prepared for Oxford Nanopore long-read sequencing and sequenced using a flow cell on the MinION Mk1D. Sequencing data were analysed using EPI2ME, along with extra analyses on exported taxa abundance data in R. Results. Diversity measures and taxonomic relative abundance from phylum to family level were comparable to previously published equine studies that used Illumina sequencing. Sequencing data were acquired within 3 days costing around £30 per sample. Long-read sequencing gave accurate taxa assignment for two positive controls included at phylum, class, order and family levels of taxonomic classification. Conclusion. This work demonstrates that long-read technologies such as Oxford Nanopore MinION sequencing can provide a reliable, quick and cost-effective alternative to short-read Illumina sequencing when characterizing microbial communities from a range of anatomical locations on/in the horse.

Gut bacterial communities have a profound influence on the health of humans and animals. Early-life gut microbial community structure influences the development of immunological competence and susceptibility to disease. For the Thoroughbred racehorse, the significance of early-life microbial colonisation events on subsequent health and athletic performance is unknown. Here we present data from a three-year cohort study of horses bred for racing designed to explore interactions between early-life gut bacterial community structure, health events in later life and athletic performance on the racetrack. Our data show that gut bacterial community structure in the first months of life predicts the risk of specific diseases and athletic performance up to three years old. Foals with lower faecal bacterial diversity at one month old had a significantly increased risk of respiratory disease in later life which was also associated with higher relative abundance of faecal Pseudomonadaceae. Surprisingly, athletic performance up to three years old, measured by three different metrics, was positively associated with higher faecal bacterial diversity at one month old and with the relative abundance of specific bacterial families. We also present data on the impact of antibiotic exposure of foals during the first month of life. This resulted in significantly lower faecal bacterial diversity at 28 days old, a significantly increased risk of respiratory disease in later life and a significant reduction in average prize money earnings, a proxy for athletic performance. Our study reveals associations between early-life bacterial community profiles and health events in later life and it provides evidence of the detrimental impact of antimicrobial treatment in the first month of life on health and performance outcomes in later life. For the first time, this study demonstrates a relationship between early-life gut bacterial communities and subsequent athletic performance that has implications for athletes of all species including humans.

Background: Gut microbial communities are increasingly being linked to diseases in animals and humans. Obesity and its associated diseases are a concern for horse owners and veterinarians, and there is a growing interest in the link among diet, the intestinal microbiota and metabolic disease. Objectives: Assess the influence of long‐term hay or haylage feeding on the microbiota and metabolomes of 20 Welsh mountain ponies. Study design: Longitudinal study. Methods: Urine, faeces and blood were collected from 20 ponies on a monthly basis over a 13‐month period. Urine and faeces were analysed using proton magnetic resonance ( ¹ H NMR) spectroscopy and faecal bacterial DNA underwent 16S rRNA gene sequencing. Results: Faecal bacterial community profiles were observed to be different for the two groups, with discriminant analysis identifying 102 bacterial groups (or operational taxonomic units, OTUs) that differed in relative abundance in accordance with forage type. Urinary metabolic profiles of the hay‐ and haylage‐fed ponies were significantly different during 12 of the 13 mo of the study. Notably, the urinary excretion of hippurate was greater in the hay‐fed ponies for the duration of the study, while ethyl‐glucoside excretion was higher in the haylage‐fed ponies. Main limitations: The study was undertaken over a 13‐month period and both groups of ponies had access to pasture during the summer months. Conclusions: The data generated from this study suggest that the choice of forage may have implications for the intestinal microbiota and metabolism of ponies and, therefore, potentially their health status. Understanding the potential implication of feeding a particular type of forage will enable horse owners to make more informed choices with regard to feed, especially if their horse or pony is prone to weight gain.

Background: Anthelmintic treatment is a risk factor for intestinal disease in the horse, known as colic. However the mechanisms involved in the onset of disease post anthelmintic treatment are unknown. The interaction between anthelmintic drugs and the gut microbiota may be associated with this observed increase in risk of colic. Little is known about the interaction between gut microbiota and anthelmintics and how treatment may alter microbiome function. The objectives of this study were: To characterise (1) faecal microbiota, (2) feed fermentation kinetics in vitro and (3) metabolic profiles following moxidectin administration to horses with very low (0 epg) adult strongyle burdens. Hypothesis: Moxidectin will not alter (1) faecal microbiota, (2) feed fermentation in vitro, or, (3) host metabolome.Results: Moxidectin increased the relative abundance ofDeferribacterspp. andSpirochaetesspp.observed after 160 h in moxidectin treated horses. Reduced in vitro fibre fermentation was observed 16 h following moxidectin administration in vivo (P = 0.001), along with lower pH in the in vitro fermentations from the moxidectin treated group. Metabolic profiles from urine samples did not differ between the treatment groups. However metabolic profiles from in vitro fermentations differed between moxidectin and control groups 16 h after treatment (R² = 0.69, Q²Y = 0.48), and within the moxidectin group between 16 h and 160 h post moxidectin treatment (R² = 0.79, Q²Y = 0.77). Metabolic profiles from in vitro fermentations and fermentation kinetics both indicated altered carbohydrate metabolism following in vivo treatment with moxidectin.Conclusions: These data suggest that in horses with low parasite burdens moxidectin had a small but measurable effect on both the community structure and the function of the gut microbiome.

The equine gut model that we have developed and describe has the potential to facilitate the exploration of how the equine gut microbiota is affected by diet, disease, and medication. It is a convenient, cost-effective, and welfare-friendly alternative to in vivo research models.

Perioperative lidocaine treatment is commonly used in horses that undergo surgical treatment of colic, to prevent or treat postoperative ileus and reduce the effects of intestinal ischaemia-reperfusion injury. However, its clinical efficacy has not been evaluated in a large population of horses undergoing small intestinal surgery. The aim of the current study was to evaluate whether systemic lidocaine administration reduced the prevalence, volume and duration of postoperative reflux and improved rates of survival following surgical treatment of small intestinal lesions. Data were collected as a part of two prospective studies investigating postoperative survival of surgical colic patients admitted to a UK equine referral hospital during the periods 2004–2006 and 2012–2014. Kaplan-Meier plots of cumulative probability of survival and the log-rank test were used to compare survival between horses that did or did not receive lidocaine. The Wilcoxon rank-sum test was used to compare the total reflux volume and duration of reflux between the groups. A multivariable Cox proportional hazards model was used to identify pre- and intraoperative risk factors for non-survival. Data from 318 horses were included in the final analysis. The overall prevalence of postoperative reflux was 24.5 %. This was significantly higher (34.8 %) in horses admitted in 2012–2014 compared to the 2004–2006 cohort (16.7). Perioperative lidocaine treatment had no effect on total reflux volume, duration of reflux or rates of postoperative survival nor was it a risk factor associated with altered postoperative survival. Variables identified to be associated with increased risk of postoperative mortality included packed cell volume on admission (hazard ratio [HR] 1.03 95 %, 95 % confidence interval [CI] 1.004–1.06, p = 0.024), heart rate on admission (HR 1.014, 95 % CI 1.004–1.024, p =0.008) and duration of surgery (HR 1.007, 95 % CI 1.002–1.01, p = 0.008). Lidocaine therapy had no effect on the prevalence of postoperative reflux, total reflux volume and duration of reflux nor did it have any effect on postoperative survival in horses undergoing surgical management of small intestinal disease for treatment of colic. There is a need for a well-designed multicentre, prospective randomised controlled trial to fully investigate the efficacy of lidocaine across different hospital populations.

Summary: Reasons for performing study: The intestinal bacterial community of the horse is a key determinant of intestinal and whole body health. Understanding the bacterial community structure and function is an important foundation for studies of intestinal health and disease. Objectives: To describe the faecal bacterial community and volatile organic compounds ( VOCs ) of the faecal metabolome of healthy Thoroughbred racehorses and to characterise responses to dietary supplementation with amylase‐rich malt extract. Study design: Intervention study. Methods: Faecal samples were collected noninvasively before and 6 weeks after supplementation in 8 privately owned Thoroughbred racehorses in active race training. Faecal metabolome was characterised using thermal desorption‐gas chromatography‐mass spectrometry ( TD‐GC ‐ MS ), with spectral analysis performed using AMDIS and compared against the NIST database. Taxonomic description of the faecal microbiota was achieved using error‐corrected 454 pyrosequencing data from 16S rRNA gene amplicons. Results: The faecal metabolome of our study population was dominated by organic acids, alcohols and ketones. We identified 81 different VOCs only 28 of which were present in >50% of samples indicating functional diversity. Faecal VOC profiles differed between first and second sampling point, some VOCs being significantly reduced post supplementation, consistent with a marked response to dietary amylase‐rich malt extract. Faecal microbiota was characterised as highly diverse; samples demonstrated verifiable diversity in the range 1200–3000 operational taxonomic units ( OTUs ) per individual. The methods used also describe high levels of infrequent, low abundance OTUs . Faecal microbial community structure was found to be different following dietary supplementation. Differences in several low abundance bacterial taxa were detected and also some evidence of interhorse variation in response. Conclusions: The volatile faecal metabolome of Thoroughbred racehorses is dominated by organic acids, alcohols and ketones; this study demonstrates that dietary supplementation with amylase‐rich malt extract may significantly alter the profile of VOCs . The faecal microbiome is highly diverse, dominated by Firmicutes and Bacteroidetes. Small but significant changes in microbial community structure were detected following dietary supplementation. This study describes the faecal metabolome and microbiome of healthy Thoroughbred racehorses against which future studies of disease and dietary intervention can be benchmarked.

Introduction: The horse has evolved to survive on a low‐energy, high‐fibre diet. The addition of starch, in the form of cereals, to the diet of racehorses to satisfy the high energy demands of extreme exercise has been identified as a risk factor for intestinal dysfunction. In this study we use 454 pyrosequencing of 16S rRNA genes to characterize the gut microbiome of 8 T horoughbred ( T b) racehorses and study the response to dietary supplementation. Methods: Faecal samples were collected from 8 T b racehorses in training. A second sample was collected from all horses six weeks after commencement of twice daily dietary supplementation with an amylase‐rich malt extract, designed to enhance pre‐caecal digestion. Amplicons for the V1‐V3 region of the 16S rDNA gene were multiplexed and sequenced on the 454 resulting in ∼25,000 reads per sample. Differences in bacterial community structure were detected by linear discriminant analysis. Results: Communities were composed of an equal balance between B acteroidetes and F irmicutes with a ∼5% contribution from other bacterial phyla. The number of observed operational taxonomic units sharing ≥97% nucleotide sequence in our racehorse population was 6000–8000. Horses showed no significant change in the number of OTUs observed after diet supplementation, but significant changes in the relative abundance of both B acteroidetes and F irmicutes families were observed. Conclusions: We demonstrated a highly diverse and complex faecal microbial community in T b racehorses in the UK . Inter‐horse variability is low amongst horses in the same training yard. We identified significant shifts in community profile following dietary supplementation and speculate that these may enhance intestinal health. Ethical Animal Research: This study was conducted under the U niversity of L iverpool research ethics approval: RETH 000363. The informed consent of the trainer was obtained and all samples were obtained non‐invasively. Sources of funding: private donation. Competing interests: none.