The gut microbiome has become a popular topic of interest in recent years as scientists are beginning to understand the vast impact it can have on overall health and development in both humans and animals. A microbiome is defined as the collection of genomes of the microorganisms that reside in a specific environment . In regards to the gut microbiome, it is comprised of the genetic material of the microbes that inhabit an organism’s gastrointestinal system. In the horse, this microbiome includes bacteria, yeast, fungi and protozoa where the most functionally important microorganism is thought to be bacteria. Researchers have studied how this microbial community can affect not only the digestive tract, but also the immune response, endocrine system, behavior and even cognitive function.
The horse is a nonruminant herbivore, so its symbiotic relationship with the microbial population present in its stomach, small intestine and large intestine is imperative for survival. Horses do not have all of the digestive enzymes needed to process fibrous plants, so they use bacteria located in their gastrointestinal tract to do so. The homeostasis of the gut microbiome is also very important in order to prevent the overabundance of pathogenic bacteria and to protect the horse from gastrointestinal disease, which is a major issue in the horse industry.
Major Functions of the Horse’s Gut Microbiome
The horse has adapted to a fiber-rich diet with approximately 35-60% of their diet being composed of cell wall carbohydrates . The hindgut is the major compartment responsible for processing this type of carbohydrate because it is home to the bacteria that are able to break down and absorb the nutrients from fibrous plants using their own microbial enzymes.
Bacteria in the horse’s digestive system are able to hydrolyze plant fibers into soluble sugars and these sugars are then converted into short chain fatty acids by the process of fermentation. Short chain fatty acids, such as propionate, butyrate and acetate, can be readily utilized by the horse and provide them with approximately 60-70% of their energy [3, 4]. Therefore, it is apparent that they rely heavily on their gut microbiome to meet their dietary needs.
It is known that certain bacterial species have specific roles in the gut. Some are known for their function in digesting a certain type of feedstuff while others are proven to proliferate during particular states and their functions are not yet fully understood. For example, proteolytic bacteria are responsible for protein digestion and cellulolytic bacteria are the major fiber-digesters. It can be difficult to assign individual bacteria to specific functions manually, so bioinformatics programs like PICRUSt can be convenient for researchers to determine the function of their microbial community when transcriptomic data is not available . Although, there are still some limitations in predicting a bacterium’s function using extracted DNA rather than RNA. When determining function, it is usually better to sequence extracted RNA in order to achieve a better picture of what the sampled bacteria are actually doing in the digestive tract.
Effects of Management on the Horse and Their Microbiome
Management practices have been proven to greatly affect the horse’s gut microbiome. There have been studies addressing how factors such as weaning method in the foal, diet, exercise and general management influence the horse’s gut microbiota [6-10]. Management is a very important aspect of horse ownership and includes regulating the horse’s diet, exercise, social interaction and housing. It can be a major causal factor of many different diseases and behavioral abnormalities in the horse such as laminitis and stereotypic behaviors.
With the increased use of supplemental feeds in the domestic horse, overfeeding of carbohydrates, specifically starch and sugar, is becoming more common. Concentrate feeds can be helpful for horse owners when trying to achieve a balanced diet for their horse. However, when these feeds are administered inappropriately, there can be an increased risk for physiological issues such as laminitis and colic. Laminitis, a major persisting issue in the horse industry, can be triggered by overfeeding, high intake of soluble carbohydrates and severe concussion trauma to the laminae due to overworking.
Domesticated horses and ponies are thought to be more prone to major health issues than feral horses because of the way in which they are managed . Factors such as grazing access, exercise, social interaction and diet have been proven to be contributing factors to a horse’s health. Mainly due to the higher starch content commonly found in the domestic horse’s diet, there is a higher prevalence of diseases like starch-induced laminitis and gastric ulcers . Horses are adapted to be continuous grazers, which can be difficult to achieve in the domestic setting. Since diet and the microbiome are so interconnected, the gut microbial community and functionality may also be contributing factors to the higher prevalence of gastrointestinal-related disease in domesticated horses.
Gut Dysbiosis in the Horse and Its Connections with Disease
There are many different gastrointestinal disorders common in the horse that have been associated with gut dysbiosis, including starch-induced laminitis, colitis, diarrhea and gastric ulcers [12-16]. These abnormalities have been proven to be correlated with differences in microbial diversity and abundances when compared to healthy horses.
Laminitis occurs when there is weakened adhesion between the distal phalanx and lamellae of the inner hoof wall. This inflammatory lesion can eventually cause complete detachment and rotation of the coffin bone as well as extreme pain for the horse. An excess of starch, which is commonly found in commercial concentrate feeds, is thought to be a contributing factor to dietary laminitis by way of the fermented components released by bacteria into the bloodstream during lactic acidosis. The specific processes involved are still unknown; however, gut bacteria and diet are known to have a large role in the onset of this disease [17, 18]. Bacteria in the hindgut are responsible for breaking down undigested sugar and starch. When there is a sudden increase in dietary starch, it can cause an excess of lactic acid bacteria in the hindgut. This can lead to lactate accumulation, gut acidity and the release of bacterial toxins into the bloodstream, which can trigger systemic inflammation.
Foal diarrhea is another gut microbiome-related disease that can cause worry and financial loss to horse owners. Gut dysbiosis is a common occurrence in the foal’s life and it has been found that diarrhea affects up to 60% of foals in their first 6 months . This type of diarrhea, also referred to as foal heat diarrhea, is a transient, non-infectious type. It is usually mild and does not require any veterinary treatment such as fluid administration or antibiotic treatment, however, in rare cases, the foal’s immune system can be compromised and their mild diarrhea can turn into a more life-threatening infection.
In a recent study using Standardbred and Shetland-type pony foals; it was found that there was not a significant difference between diarrheic and non-diarrheic foals when analyzing their hindgut communities as a whole . However, there were differences found in specific taxa and these small differences could help explain the events occurring during foal diarrhea. The findings pertaining to the depleted taxa in diarrheic foals could also provide more information on appropriate probiotic supplementation for immunologically compromised foals that may not have the ability to efficiently recover without intervention. Interestingly, two of the taxa found to be enriched in diarrheic foals were also discovered to have an increased abundance in human children with Irritable Bowel Syndrome when compared to healthy children [21-24].
There is still a lack of studies on the microbiome during cases of laminitis and foal diarrhea using next generation sequencing. The definitive cause of these diseases have still not yet been determined, so more studies characterizing the bacterial community present during these states may be helpful. Transcriptomic data can also allow for a deeper understanding of the events occurring in the gut when disease onset occurs.
Tools Used to Analyze the Microbiome
Many equine studies on the gut microbiome have used culture-based procedures to characterize the bacteria present. It is known, however, that a significant number of organisms present in the gut are unculturable using standard culture methods. Culture techniques can be helpful when trying to identify specific bacteria that cause disease or when trying to briefly analyze the microbiome as a whole. There are still many challenges in using cultures to analyze the microbiome because it can provide researchers with an inaccurate depiction of the microbial community. Therefore, the emergence of next generation sequencing techniques has been helpful in achieving a deeper understanding of the gut microbiome in horses as well as other animals.
16S rRNA gene sequencing is based on non-enriched PCR products and allows for a more reliable analysis of the microbiome. The 16S rRNA gene sequences are used to study bacteria because of its presence in virtually all bacteria, its function has been preserved over time and its size of 1,500 base pairs makes it large enough for informatics and analytics purposes . This type of next generation sequencing is very helpful in characterizing a microbial community in both its diversity and member abundance. In most cases, 16S rRNA gene sequencing is able to provide genus level identification and, in some cases, species level identification .
16S rRNA gene sequencing is slowly becoming more popular in horse microbiome studies. There are, however, still many researchers using culture-based methods for microbiome work. The use of next generation sequencing is imperative to provide horse owners with more definitive answers on the causes of equine gastrointestinal disease, which is a significant issue in the horse industry.
It is apparent that the horse’s gut microbiome plays a very important role in the development and health of the horse. In the future, there will hopefully be a better understanding of the gut microbiome’s role in disease and in any other abnormalities that can negatively affect the horse. Future research will provide horse owners with a better understanding of the gut microbiome’s impact on their horse and with better ways to manage them.
Meredith is a graduate of the University of Delaware with a B.S. in Pre-Veterinary Medicine and Animal Biosciences as well as a M.S. in Animal Science. Her Master's thesis research was focused on the effects of domesticity on the development of the equine gut microbiome. She joined the SBS-MD team in March of 2017 and her main responsibilities include assisting with semen collection, processing and distribution.
 “Microbiome.” Merriam-Webster, Merriam-Webster, Aug. 2018, www.merriam-webster.com/dictionary/microbiome.
 Julliand, V., & Grimm, P. (2017). The Impact of Diet on the HindgutMicrobiome. Journal of Equine Veterinary Science, 52, 23-28. doi:10.1016/j.jevs.2017.03.002
 Argenzio, R.A, Southworth, M, Stevens, C.E (1974) Sites of organic acid production and absorption in the equine gastrointestinal tract. Am. J. Physiol. 226, 1043–1050.
 Argenzio, R.A (1975) Functions of the equine large intestine and their interrelationship in disease. Cornell Vet. 65, 303–327.
 Langille, M. G. I., Zaneveld, J., Caporaso, J. G., McDonald, D., Knights, D., Reyes, J. A., ... Huttenhower, C. (2013). Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences. Nature Biotechnology, 31(9), 814-821. DOI: 10.1038/nbt.2676
 Grimm, P., Philippeau, C., & Julliand, V. (2017). Faecal parameters as biomarkers of the equine hindgut microbial ecosystem under dietary change. Animal, 11(07), 1136-1145. doi:10.1017/s1751731116002779
 Janabi, A., Biddle, A., Klein, D., & Mckeever, K. (2016). Exercise traininginduced changes in the gut microbiota of Standardbred racehorses. Comparative Exercise Physiology, 12(3), 119-130. doi:10.3920/cep160015
 Almeida, M. L., Feringer, W. H., Carvalho, J. R., Rodrigues, I. M., Jordão, L. R., Fonseca, M. G., . . . Ferraz, G. D. (2016). Intense Exercise and Aerobic Conditioning Associated with Chromium or L-Carnitine Supplementation Modified the Fecal Microbiota of Fillies. Plos One, 11(12). doi:10.1371/journal.pone.0167108
 Jacquay, E. (2017). Colonization and maturation of the foal fecal microbiota from birth through weaning and the effect of weaning method (Unpublished master's thesis).
 Metcalf, J. L., Song, S. J., Morton, J. T., Weiss, S., Seguin-Orlando, A., Joly, F., . . . Orlando, L. (2017). Evaluating the impact of domestication and captivity on the horse gut microbiome. Scientific Reports, 7(1). doi:10.1038/s41598-017-15375-9
 Ward, S., Sykes, B. W., Brown, H., Bishop, A. and Penaluna, L. A. (2015), A comparison of the prevalence of gastric ulceration in feral and domesticated horses in the UK. Equine Vet Educ, 27: 655–657. doi:10.1111/eve.12491
 Steelman, S. M., Chowdhary, B. P., Dowd, S., Suchodolski, J., & Janečka, J. E. (2012). Pyrosequencing of 16S rRNA genes in fecal samples reveals high diversity of hindgut microflora in horses and potential links to chronic laminitis. BMC Veterinary Research, 8(1), 231. doi:10.1186/1746-6148-8-231 74
 Oliver, O. E., & Stämpfli, H. (2006). Acute Diarrhea in the Adult Horse: Case Example and Review. Veterinary Clinics of North America: Equine Practice, 22(1), 73-84. doi:10.1016/j.cveq.2005.12.008
 Costa MC, Arroyo LG, Allen-Vercoe E, Stämpfli HR, Kim PT, Sturgeon A, et al. (2012) Comparison of the Fecal Microbiota of Healthy Horses and Horses with Colitis by High Throughput Sequencing of the V3-V5 Region of the 16S rRNA Gene. PLoS ONE 7(7): e41484. https://doi.org/10.1371/journal.pone.0041484
 Milinovich, G. J., Trott, D. J., Burrell, P. C., Van Eps, A. W., Thoefner, M. B., Blackall, L. L., Al Jassim, R. A. M., Morton, J. M. and Pollitt, C. C. (2006), Changes in equine hindgut bacterial populations during oligofructose-induced laminitis. Environmental Microbiology, 8: 885–898. doi:10.1111/j.1462-2920.2005.00975.x
 Jassim, R. A., & Andrews, F. M. (2009). The Bacterial Community of the Horse Gastrointestinal Tract and Its Relation to Fermentative Acidosis, Laminitis, Colic, and Stomach Ulcers. Veterinary Clinics of North America: Equine Practice, 25(2), 199- 215. doi:10.1016/j.cveq.2009.04.005
 Bailey SR, Baillon M-L, Rycroft AN, Harris PA, Elliott J (2003) Identification of Equine Cecal Bacteria Producing Amines in an In Vitro Model of Carbohydrate Overload. Appl Environ Microbiol 69: 2087–2093. doi:10.1128/AEM.69.4.2087- 2093.2003
 Bailey SR, Adair HS, Reinemeyer CR, Morgan SJ, Brooks AC et al. (2009) Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis. Vet Immunol Immunopathol 129: 167–173. doi:10.1016/j.vetimm.2008.11.009
 Schoster, A., Guardabassi, L., Staempfli, H. R., Abrahams, M., Jalali, M., & Weese, J. S. (2016), The longitudinal effect of a multi-strain probiotic on the intestinal bacterial microbiota of neonatal foals. Equine Vet J, 48: 689–696. doi:10.1111/evj.12524
 Bonnell, Meredith. “Early Functional and Community Development of The Equine Hindgut Microbiome in Semi-Feral- and Domestic Conventionally-Managed Foals Including Cases of Foal Diarrhea.” University of Delaware, Udspace.udel.edu, 2018, pp. 1–120.
 Rigsbee, L., Agans, R., Shankar, V., Kenche, H., Khamis, H. J., Michail, S., & Paliy, O. (2012). Quantitative Profiling of Gut Microbiota of Children With Diarrhea Predominant Irritable Bowel Syndrome. The American Journal of Gastroenterology, 107(11), 1740-1751. doi:10.1038/ajg.2012.287
 Tana, C., Umesaki, Y., Imaoka, A., Handa, T., Kanazawa, M. and Fukudo, S. (2010), Altered profiles of intestinal microbiota and organic acids may be the origin of symptoms in irritable bowel syndrome. Neurogastroenterology & Motility, 22: 512– e115. doi:10.1111/j.1365-2982.2009.01427.x
 Saulnier, D. M., Riehle, K., Mistretta, T., Diaz, M., Mandal, D., Raza, S., . . . Versalovic, J. (2011). Gastrointestinal microbiome signatures of pediatric patients with irritable bowel syndrome. Gastroenterology, 141(5), 1782-1791. doi:10.1053/j.gastro.2011.06.072
 Malinen, E., Rinttila, T., Kajander, K., Matto, J., Kassinen, A., Krogius, L., . . . Palva, A. (2005). Analysis of the Fecal Microbiota of Irritable Bowel Syndrome Patients and Healthy Controls with Real-Time PCR. The American Journal of Gastroenterology, 100(2), 373-382. doi:10.1111/j.1572-0241.2005.40312.x
 Janda, J. M., & Abbott, S. L. (2007). 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls . Journal of Clinical Microbiology, 45(9), 2761–2764. http://doi.org/10.1128/JCM.01228-07