By Meredith Bonnell MS

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 [1].

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 [2]. 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 [5]. 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.

Management effects on the 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 [11]. 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 [11]. 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 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 six months [19]. 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 [20]. 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.
The 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 [25]. 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 [25].
The 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.

Establishing a foal’s healthy gut microbiome

The mare-foal bond is a special connection that’s normally associated with the behavioral interactions between a mare and her foal. What’s not usually thought of is the bond formed between their immune systems and microbiomes. A healthy gut microbiome goes hand in hand with a strong immune system. Foals have innate immunity at birth, but several adaptive immune responses can take up to a year to develop to those of an adult horse. The correct development of a foal’s immune system is very important in protecting them from microbial pathogens and, in turn, gastrointestinal disease.
Horses have epitheliochorial placentation, which prevents the transfer of immunoglobulins from the mare to the fetus in utero. Therefore, a foal’s ingestion of colostrum is imperative before the foal is no longer able to absorb the immunoglobulins, maternal immune cells, and cytokines from their dam and before the colostrum transitions to milk [1*]. Colostrum is also very nutrient rich and decreases in nutrients throughout the first 24 hours following birth, which is another reason why timely ingestion of it is so important.
Mares not only share their immunity with their foal, but are also thought to affect their foal’s gut microbiome. A foal’s development of this microbial community in their gastrointestinal tract has been shown to be important in keeping them healthy. The proliferation of the wrong kinds of bacteria can cause serious illness and even death in foals.
A few recent studies have looked into how the foal’s gut is colonized by bacteria before and after birth. A group of researchers sampled amniotic fluid, mare feces and colostrum in order to compare it to foal meconium [2*]. They found that foal meconium shared bacterial species with amniotic fluid, mare feces and colostrum. The group suggested that this may be due to two different processes that have been shown in humans. Their theory was that the mare’s dendritic cells took bacteria from their gut and transferred it to their amniotic fluid or to their mammary glands during late pregnancy via their bloodstream [3*, 4*]. In humans, this process has been hypothesized to help the neonate adapt outside of their mother by starting their gut bacterial colonization in the womb. This colonization is also known to jumpstart the development of important immune system tissue in the gut.
The transfer of bacteria from the mare’s gut to her mammary glands would explain the similarities in bacteria between colostrum, foal meconium and mare feces while the similarities in bacteria between amniotic fluid, mare feces and foal meconium could be due to bacteria being transported to the amnion. However, more research needs to be conducted in order to determine the actual processes taking place since humans and horses have huge differences in their types of placentation and transfer of immunity from mom to baby. If these theories are proven to be true, then this may indicate that the health of a mare’s gut microbiome affects her foal a lot more than previously thought.
Even before birth, the colonization of a foal’s gut is an important step in their development. New knowledge about the effect of a mare’s microbiome on their foal in utero may give rise to new ways in which we can support a healthy foal. Since mares aren’t able to transfer antibodies to their foal in utero, it’s very interesting that it might be possible for their immune system to transfer bacteria to them. This could be a process that has adapted in the horse to stimulate the development of important immune system tissues in the foal before birth and to better prepare them for life outside of the womb.

Meredith Bonnell 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.

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