Exploring the Equine Microbiome

Researchers are discovering how the vast and varied microbes in the horse’s gastrointestinal tract impact equine health.

Much like the intrepid explorers using the Curiosity rover to examine the surface of Mars, searching for evidence of microbes, veterinary researchers are investigating the depths of the equine gastrointestinal tract to learn about microscopic life forms residing there and what they are doing. Scientists are, quite literally, going where no one has gone before.

But unlike the surface of Mars, where scientists have only caught glimpses of proof of past microbial life, the horse’s large intestine hosts an extremely diverse range of microorganisms. Identifying all the species and their exact roles, however, has been slow going. Those microorganisms, together with their genomes and their interactions in a particular environment, are referred to as the microbiome.   

“Studying the equine intestinal microbiome is important because horses are hindgut fermenters,” says Scott Weese, DVM, MSc, Dipl. ACVIM, a professor in the Department of Pathobiology at the University of Guelph’s Ontario Veterinary College. “Changes in the microbiome can result in diseases such as colic, colitis, and laminitis, which are leading causes of morbidity and mortality in horses.”  

And much like Curiosity and its skilled drivers, Weese is a leader in microbiome exploration, which involves developing new techniques and tools. “Improving our current methods of identifying the trillions of microbes, their genomes, and learning how those microbes interact with their environment will help harness the power of the microbiome to both prevent and treat disease,” Weese says.

In this article we will describe scientists’ current knowledge of the equine intestinal microbiome, how it changes during times of stress and disease, and potential ways to maintain or restore a horse’s microbiome after it suffers an insult.  

The Microbiome in Health

“When we originally began studying the microbiome in the early 2000s, we were reliant on standard bacterial culture techniques,” says Weese. “Even though we knew we were going to miss a large number of organisms, it was a place to start assessing specific bacterial populations that we already knew existed. Those included lactobacilli, coliforms, and streptococci.”

Researchers developed techniques such as next generation sequencing—one of the most powerful DNA sequencing tools currently available—along the way, which allowed microbiome explorers to identify intestinal microbes based on their genes.  

For example, Weese and colleagues recently collected fecal samples from six healthy horses and characterized the bacterial composition using next generation sequencing. The most prominent phylum (the major division of living organisms below kingdom and above class) they identified was Firmicutes, followed by Bacteroidetes and Proteobacteria. Within those phyla, bacteria belonging to the genera (further down in the classification) Actinobacter, Lysinibacillus, and Carnobacterium were most common. Interestingly, Weese reports, bacterial populations were different among the six horses. This finding suggests that each horse’s microbiome is unique, adapted to each individual, and that a “standard” intestinal microbiome might not exist.

As they uncover the microbes in the ­intestine—which is as daunting a task as decoding the equine genome—­researchers are simultaneously attempting to determine what functions each serves. Currently, they believe on the whole the equine microbiome is responsible for:  

• Fermenting fiber in the hindgut to produce short-chain fatty acids (SCFAs), primarily propionate and butyrate, that the horse’s body absorbs into the bloodstream from the GI tract and uses for energy;
• Boosting the horse’s immune ­system;
• Producing antimicrobial products to control populations of pathogenic (disease-causing) microbes;
• Physically excluding pathogens; and
• Inhibiting the production and absorption of bacterial toxins (e.g., those produced by Clostridium difficile, a common cause of diarrhea in horses).

Development of the Microbiome

Another interesting aspect of the microbiome is learning where it came from, especially considering that a foal is conceived and develops in a sterile environment (the placenta). In other words, how does a foal’s gastrointestinal tract become colonized with such a rich array of life-saving microbes within hours of birth?

To answer this question, Weese and colleagues collected fecal samples from 11 Quarter Horse foals within 24 hours of birth and then routinely until the foals were weaned at about nine months of age. The team extracted and purified DNA from the microbes in those samples and identified the types of microorganisms based on their genome (specifically, the sequence of a region of a certain type of genetic material called 16S rRNA that is like a fingerprint for microorganisms). Microorganisms that have similar DNA are referred to as “operational taxonomic units” (OTUs).  

They found that:

• Newborn foals rapidly develop diverse and rich populations of fecal microbes;
• Those populations change markedly early in life. This is thought to be due to the acquisition of “transient” microbes that the foal is exposed to by his dam, her colostrum (first milk), and the environment that ultimately do not colonize the intestinal tract;
• The numbers of different OTUs decrease early during the foal’s life as the transient “pioneer” microbes clear and the true colonizers become established;
• By 60 days of age, foals have a relatively stable microbiome that is comparable to that of a mature horse;
• The Firmicutes and Verrucomicrobia phyla, together with other unclassified bacteria, comprise approximately 90% of the bacteria found in the feces of 60-day-old foals. The Firmicutes phylum includes a diverse group of bacteria, including beneficial species involved in fiber fermentation as well as the diarrhea-causing C. difficile and C. perfringens;

The change in the composition of the foal’s microbiome during the first month or so of his life is expected, considering the intestine goes from no microorganism exposure to an abundance following birth, when foals begin to nurse, graze, consume carbohydrates, and practice coprophagy (ingest feces).

The Microbiome During Disease, Stress

The intestinal microbiome is comprised of quadrillions of microbes, and keeping them all happy is imperative to horse health. In adult horses, colic and colitis, laminitis, foal heat diarrhea, and equine grass sickness are the most notable consequences of disrupting the equine intestinal microbiome.  

To help characterize the exact changes in the equine intestinal microbiome under certain conditions, one research group, including Christopher Proudman, MA, Vet MB, PhD, Cert EO, FRCVS, of the equine division of the University of Liverpool’s Department of Veterinary Clinical Sciences, in the United Kingdom, collected and analyzed samples from the large intestines of horses:

1. Maintained on pasture;  
2. Consuming a concentrate diet; or  
3. Consuming a concentrate diet and diagnosed with simple colonic ­obstruction and distension (SCOD), a prevalent form of diet-induced intestinal disease.  

The researchers extracted and analyzed genetic material to identify the types of microbes present in the microbiomes and further analyzed the intestinal contents to identify the metabolites (e.g., short-chain fatty acids) present. They found:  

• A progressive and significant increase in Lachnospiraceae (in the Bacteroidetes phylum) and the lactic-acid producing bacteria Bacillus, Lactobacillus, and Streptococcus in response to a dietary switch from pasture to concentrates (Increased lactic acid results in a decreased pH in the large intestine [i.e., more acidic] and a shift in the microbe population in the microbiome.);
• A corresponding decrease in bacteria that can break down fiber with that switch; and,
• A higher concentration of lactic acid in samples from horses maintained on a concentrate diet and those with SCOD.  

In a separate study, a group of researchers from the Faculty of Veterinary Medicine at the University of Liège, in Belgium, analyzed intestinal microbiome samples collected from adult horses with and without diarrhea. They found that the intestinal microbial diversity was significantly less in horses with diarrhea than in horses without diarrhea. They also identified different species of bacteria in horses with diarrhea, including Fusobacteria, than in horses without diarrhea. Finally, they identified C. difficile in 3.7% of tested horses, none of which had diarrhea. Those horses did, however, have decreased “bacterial species richness” compared to horses that did not test positive for C. difficile.

Weese and colleagues also found an alteration in the microbiomes of horses with and without diarrhea. For example, they found predominantly Firmicutes phylum in healthy horses, whereas they most commonly found Bacteroidetes among horses with diarrhea. And they identified Clostridium spp even among healthy horses.

“Together these data suggest that diarrhea is not simply caused by the overgrowth of a single pathogen like C. difficile or Salmonella, but colitis may in fact be a disease of ‘gut dysbiosis’ (microbial imbalance) instead,” says Weese. “These results also highlight how important clostridia are. This broad group of often-maligned bacteria plays a critical role in horse health.”

Weese and his team have also noted alterations to the intestinal microbiome, resulting in reduced species richness, following systemic antimicrobial therapy. For example, in 2015 they reported that the intestinal microbiome changes following antimicrobial application, resulting in reduced species richness. Tested antimicrobials included intramuscular penicillin and ceftiofur and oral trimethoprim ­sulfadiazine.  

Finally, the same research team collected fecal samples from 26 mares both pre- and post-foaling, 13 of which developed colic postpartum. They found that the compositions of the mares’ microbiomes before foaling were notably different than they were after; however, foaling itself had a limited impact on the mares’ microbiomes. The team also identified notable differences between mares that did and did not develop colic following parturition. As such, they concluded, “Associations between Firmicutes (particularly Lachnospiraceae and Ruminococcaceae) and Proteobacteria and development of colic could lead to measures to predict and prevent colic.”

Keeping the Microbiome Happy

One potential way to maintain the equine intestinal microbiome’s health and integrity is through probiotic administration. Probiotics are “direct-fed microbials,” or live yeast and/or bacteria believed to help maintain or restore the health of the intestinal microbiome. Probiotics’ potential mechanisms of action include boosting the horse’s immune system, producing some antimicrobial products, excluding disease-causing microorganisms, and inhibiting bacterial toxins. Examples of probiotics include bacteria and yeast such as Bacillus subtilis, Bacillus licheniformis, Saccharomyces cerevisiae, Lactobacillus acidophilus, Enterococcus faecium, Lactococcus lactis, and Bifidobacterium longum, among others.

Weese explains, however, that despite probiotics’ popularity, the clinical evidence supporting their use is lacking.

“Whether probiotics are beneficial to horses or not remains debatable and is complicated by the fact that we simply do not know if we are administering the ‘right’ organisms to benefit the intestinal microbiome; if we are offering an adequate dose; if the products are of sufficient quality and actually contain the type and amount of live organisms described on the label; or if it is even possible to orally administer microorganisms to alter the equine intestinal microbiome,” he says.   

In lieu of a fecal microbiota transplant (yes, this refers to administering “healthy” feces by enema or nasogastric tube), there are ways to help protect a horse’s microbiome, says Kathleen Crandell, PhD, equine nutritionist and consultant to Kentucky Equine Research, in Versailles.

“Maintaining horses on forage-based diets with minimal amounts of concentrates and avoiding abrupt changes in diet are key factors involved in gastrointestinal health,” she says.

When horses consume high levels of concentrates, some of the starch and sugar therein is fermented in the large intestine rather than digested in the stomach and small intestine. Unlike fiber that is fermented to produce SCFAs that the horse uses for energy, fermented starch and sugar from concentrates produce lactic acid. Remember that abnormal lactic acid production causes a shift in the population of microbes that make up the microbiome.

“Hindgut buffers are, therefore, another potential means of maintaining a normal pH in the large intestine, which helps stabilize the microbiome in horses fed high amounts of concentrate feeds to supply adequate energy,” says Crandell.

Take-Home Message

While Weese and other researchers throughout the world are working to better understand the intestinal microbiome, the number of questions each discovery generates is as vast as the intestinal microbiome itself.

“Although we have made a progress in identifying parts of the equine intestinal microbiota, we still only have a superficial understanding of this complex microbial population,” Weese says, adding, “Continued research in this field will revolutionize our understanding of the role of the microbiome in health and disease.”