Move Over Microbiota

PHOTOS BY ELIZABETH HAY PHOTOGRAPHY

While researchers’ ability to sequence the equine microbiome was an advancement, scientists have since learned that beyond the specific bacteria in a horse’s gut, it’s what the metabolome is producing that’s of greater importance.

By | Contributing Veterinarian:

Researchers Have Moved Beyond Simply Identifying the Microbiota Present in the Equine GI Tract and are Now Exploring Its True Function, Revealing It’s Not Necessarily What Bacteria are Present That Matters but Rather How Those Metabolites Produced Influence Not Just the Gut but Overall Health

There’s an intricate world, deep beneath the saddle, within the bowels of every horse. Researchers are seeking to unlock the mysteries of the massive gastrointestinal (GI) tract and gain a greater understanding that can lead to healthier horses. Over the past years, many studies have examined a range of factors affecting the gut microbiota — the microscopic communities that live along the digestive highway and play symbiotic functions from fermentation and digestion to various immune-modulatory roles.

Admittedly, it sounds wildly complex and begs the question of who wants to digest in-the-weeds science about this body of knowledge? Stay with me, don’t flip the page just yet. While scholars have just scratched the surface, this riveting exploration may profoundly change what we thought we knew regarding gut bacteria, its susceptibility to diet and lifestyle influences and its monumental impact on equine health.

Enter Professor Canaan Whitfield- Cargile, Crosswell Chair in Large Animal Medicine at the University of Georgia’s College of Veterinary Medicine in Athens. He received his DVM from the University of Georgia in 2006 and completed a large animal surgery residency at Texas A&M University four years later. Board-certified in equine surgery and sports medicine and rehabilitation, he earned his doctorate in biomedical sciences from Texas A&M in 2016. His research focuses on GI healing and intestinal inflammation. Other areas of interest include the impact of the GI microbiota on GI health with a secondary focus on treatment of infected, non-healing wounds.

Barely on the back side of 40 years old, Dr. Whitfield-Cargile has published numerous transformative equine research studies. Plainly, his work is reshaping what is known about the equine gut and its far-reaching impact on the overall health of the horse.

While he has been elbow-deep in work to more fully understand microbiota and the equine microbiome, the collection of all microbes (from bacteria and fungi to viruses and their genes), his focus now goes deeper. “Older studies have focused on describing the microbiota; simply telling us which bacteria were in the GI tract, but that information didn’t tell us much about why they matter, and how they’re impacting the health of the horse. As science has evolved, simply listing the microbiota, the types of bacteria in the GI tract, has become yesterday’s news.”

Defining the Players

Dr. Canaan Whitfield-Cargile outlines the components within the equine gut that perform a central role in horse (and human) health, impacting various body systems from the brain and lungs to the immune system and well beyond:

  • Microbiota: “We use the terms microbiota and microbiome interchangeably, but they are different things,” he says. “Microbiota refers to the individual bacteria, the microorganisms within a defined community.” In other words, all organisms within the equine gut environment. He continues: “Being at the University of Georgia, football is obviously a big deal. Let’s use the analogy of a football stadium. The microbiota would be all the people — the living things — within that stadium.”
  • Microbiome: “The microbiome, by contrast, represents the people in the stadium and everything associated with them and produced by them,” he continues. “It’s the entire environment, including each person, the breath they’re exhaling, the skin cells they’re exfoliating, the clothes they’re wearing, and the metabolites and proteins they produce. So, in reality, the microbiota consists of the organisms, and the microbiome includes the organisms and the products of their activity.” Past research on horses had some limitations. “The microbiota is not just bacteria; there are also fungal organisms, viruses, archaea (similar to but distinct from bacteria), phages and protozoa all found within the equine gut and making up the microbiota,” he says. “Almost all research conducted so far in horses is based on the bacterial component of the microbiota and has been further limited to simply describing the bacterial component and not examining function. Bacterial microbiota has historically been the easiest and most cost-effective thing to measure. It’s what we know and the research has been good and provided needed foundational knowledge.” But science and technology has evolved, now allowing researchers to look at how they impact the health and function of horses.
  • Metabolome: Keeping in mind the football stadium analogy, the metabolome is the small molecules produced by the people in the stands. “Small molecules in the GI tract are produced by the microorganisms, and the host (the horse),” Dr. Whitfield-Cargile explains. “The cells that line the GI tract are producing all sorts of small molecules that wind up within the gut, including metabolites that are a product of what the horse has eaten, the medication they’ve been given and things they’ve ingested or been exposed to.” Though these molecules are small, “it doesn’t mean they’re not important,” he adds. “These are responsible for cell signaling. Bacteria really don’t do much to a horse’s cell, but the chemicals they produce are what signals that cell to divide, produce, grow or die.”
  • Metatranscriptome: A central theme of biology states that all cells start with nuclear DNA, or deoxyribonucleic acid, the hereditary material that tells each cell how to grow, function and reproduce. Cellular transcription is the process of making an RNA copy of a gene’s DNA sequence, called messenger RNA (mRNA), which carries the gene’s protein information encoded in DNA. “The metatranscriptome is simply the list of all the genes that are being expressed by these bacteria or these microorganisms,” he adds. It’s an important distinction; if science only examined DNA, that would tell the singular story of the genetic capability of those bacteria. “The story DNA tells is essentially not a real, tangible thing,” Dr. Whitfield-Cargile explains. “It’s just telling us what could potentially happen according to the genes within the microbiota.” Although bacteria or other microorganisms have the ability to do something doesn’t mean it’s actually doing it. “That’s where the metatranscriptome comes into play. We’re looking at the RNA, and it’s telling us which genes specifically are being ‘turned on,’ in contrast to DNA, which only tells us a gene is present and has the potential to be turned on,” he says.
  • Exfoliome: “Examining the exfoliome is a newer technique that looks at the gene expression of the horse,” says Dr. Whitfield-Cargile of this novel science evaluating cells that are exfoliated from the lining of the GI tract. The GI tract of all animals, and people, is lined with a thin layer called the epithelium, and epithelial cells are constantly being replaced; in fact, the entire lining of the GI tract is replaced about every three days. The old cells are shed into the lumen (the interior of the GI tract) and ultimately discarded in the horse’s manure. “Through collaborators at Texas A&M, we have worked to figure out ways to get the mRNA out of the feces, so it can tell us what genes are being expressed by the cells that line the GI tract,” he says of his recent work. “We’re now seeing the function of the cells that line the GI tract throughout the whole length of it. It’s providing us with a global view of what’s happening inside the entire gastrointestinal system.”

The Fickleness of the Microbiome

Over the past decade, there’s been considerable interest concerning the gut microbiome, with a number of research projects that thoroughly outlined the bacterial makeup of horses. “I remember that initial excitement. We so badly wanted to associate disease with changes in the microbiota,” Dr. Whitfield Cargile recalls. “We ended up with terabytes of data, but at the end of the day, it became hard to do much with it.” Nearly 10 years ago, Platinum Performance ® launched the Equine Microbiome Project, which sampled and sequenced the largest collection at the time of horses of diverse ages, breeds, disciplines, health statuses and geographic locations. The data was massive, and although researchers learned the bacterial makeup of nearly 1,000 horses, the same blunt question inevitably arose: So what? What did the data add up to and how could it be correlated to the health of the horse since no single standard exists? Researchers know that diversity in bacterial strains is important but the population of bacteria in each horse’s gut is constantly in flux and different from one horse to the next.

While researchers’ ability to sequence the equine microbiome was an advancement, scientists have since learned that beyond the specific bacteria in a horse’s gut, it’s what the metabolome is producing that’s of greater importance. Has science moved past studies that identify bacterial species? In many cases, yes. Yesterday’s advancement has yielded today’s breakthrough: specifics of metabolome, metatranscriptome and exfoliome. But why move on from the initial work surrounding the microbiome? “There are shortcomings there that limit the usefulness of the information,” Dr. Whitfield- Cargile explains. “We often use the 16s rDNA gene to study the microbiome. It’s a gene that all bacteria have, and it’s a cost-effective method by which we obtain a fecal sample from the horse, take a DNA sample, then do a PCR test (polymerase chain reaction) to give us a copy of that one gene.” That gene in turn can be sequenced and compared to a database to extrapolate the bacteria. “While fantastic, it has some limitations,” he offers. “We’re not able to tell for certain what type of bacteria came from that one gene; we don’t have great taxonomic resolution (clear visibility as to domain to genus to species).”

Why does that matter? Dr. Whitfield-Cargile cites the genetic ties between dolphins and humans to illustrate. “Dolphin DNA and human DNA are about 98% similar,” he says. Just using a DNA comparison, it may still be difficult to tell humans from other mammals despite profoundly obvious differences. So, DNA alone is insufficient. Another limitation is that not every bacterial strain is in a database, let alone have they all been discovered much less sequenced. “This means that we often can’t drill down far enough; some of these bacteria can only be classified to a certain level, to the phylum for instance,” he adds. “We’re usually missing the potential by only getting to that level.” While this approach is informative, it leaves out significant and crucial data to more fully understand the whole picture.

Aside from limitations in the ability to study the microbiome at a deeper level, the bacterial makeup of a microbiome varies wildly from one horse (or human) to the next. Moreover, that makeup is in a continual state of change, heavily influenced by the animal’s diet, medications, environment, seasonality, stress and so on. In other words, it’s studying a moving target with a glimpse at only one point in time. By the time sequencing results are available, the horse’s microbiome has already changed. “It becomes really difficult to interpret anything,” says Dr. Whitfield- Cargile plainly. “The information has very little meaning in terms of clinical implications. Despite all of our efforts, there isn’t much we can take home from it. We don’t know what normal is because normal for one horse can be so different from what’s normal for the next.”

“We have to understand not just what bacteria are present, but what they are doing and how that’s influencing gut function and overall health. That’s one reason we’re really excited about this study. It also exemplifies why we need to move on from describing the microbiota. None of us really care about the bacteria in the gut, what we care about is how that bacterium is affecting the horse. Microbiota data without in-depth horse data has limited usefulness.”
— Dr. Canaan Whitfield-Cargile, University of Georgia

Novel Research Points the Way Forward

While research using 16s rDNA has shortcomings, targeted studies using more concrete longitudinal data that is collected over time on the same equine subjects, offers better data. “We examined the potential for NSAIDs (non-steroidal anti-inflammatory drugs) — specifically phenylbutazone or ‘Bute’ — to cause GI injury in the horse,” he explains in reference to a 2019 study performed while still at Texas A&M, highlighting the effects of the anti-inflammatory alone or in combination with a nutritional supplement on gastric ulcers, leaky gut and the fecal microbiota in horses. “We all take NSAIDs all the time, and we certainly give them to our horses often,” he says. “Most times we don’t think anything of it, but they can definitely have a negative impact, and we learned that in this study.”

One goal of the study was to find a way to prevent NSAID-induced injuries using nutritional intervention with omega-3 fatty acids, antioxidants, L-glutamine and the trifecta of pre-, pro- and postbiotics. “We showed beneficial effects as far as reducing the severity of Bute-induced GI injuries,” he says summarizing the extensive findings. “While that study was exciting, we didn’t know the why.” As the science evolved, so did his methods and approach to the research. In a follow-up study, “Integrated Analysis of Gut Metabolome, Microbiome and Exfoliome Data in an Equine Model of Intestinal Injury,” published in April, he and his co-authors studied the microbiota combined with metabolomics and exfoliomics. “We can now see what metabolites were in the gut, and by examining the function of the horse’s GI tract through exfoliomics, we can see how one system changed the other,” he says. “We can see how the horses responded to changes in the metabolome.” Because of the longitudinal data, the team was able to chronicle how the bacteria behaved when exposed to phenylbutazone, as well as the subsequent effect on the health of the GI tract and the overall health of the horse. “We have to understand not just what bacteria are present but what they are doing and how that’s influencing gut function and overall health. That’s one reason we’re really excited about this study. It also exemplifies why we need to move on from describing the microbiota. None of us really care about the bacteria in the gut, what we care about is how that bacterium is affecting the horse. Microbiota data without in-depth horse data has limited usefulness.”

Be mindful of the horse’s gut by making dietary changes gradually; selecting high-quality hay; choosing feeds and supplements free of pro-inflammatory filler ingredients; hauling with care; and being selective with NSAID use.

Actionable Steps for Whole-Horse Health

A key question from veterinarians and riders is: How do we keep the gut healthy while understanding that healthy isn’t the same for every horse? “The answer, of course, is multifactorial and individual. I think one of the most important parts is to have some knowledge of the complexity and that nearly everything we do changes the makeup of the gut,” Dr. Whitfield-Cargile says. “Unless you appreciate that, you won’t make intentional decisions for the horse. It’s easy to forget that everything’s connected.” Be mindful of the horse’s gut, he cautions, by making dietary changes gradually; selecting high-quality hay; choosing feeds and supplements free of pro-inflammatory filler ingredients; hauling with care; and being selective with NSAID use. “A lot of it is common sense, but it’s also thinking ahead and taking preventive measures with trusted pre- and probiotics if you plan to give your horse Bute or even antibiotics under the care of your veterinarian,” he says.

One consideration about probiotics is it may be futile to use them if the environment within the GI tract isn’t favorable — unless the owner takes a whole-horse approach. “You can take a million polar bears and put them in the desert hoping to populate that landscape with them, but they’re still going to die because the environment isn’t right for them,” he offers as an example. “The same is true for the gut of the horse. I don’t care how many probiotics you put in there, if you don’t make the environment right, they’re all going die. That’s why the whole horse matters. You’ve got to feed the good bacteria in the gut, that’s what makes it live.” But “feeding” doesn’t simply mean nourishment. “Nutrition, of course, is the critical component,” he adds. “But it goes beyond that to include exercise, medications, stress, environment and so on. It all matters in terms of creating a healthy gut environment, and this is a primary reason why we really need to understand how our actions as veterinarians and horse owners can have a direct positive or negative influence on the horse’s gut, and in turn their happiness and welfare.”

His starting point is a diet based on high-quality forage with minimal processed grains and concentrates, supplements with trusted pre-, pro- and even postbiotics from reputable companies. Stress management, being mindful of sleep and hauling practices and becoming an attuned observer of your horse’s behavior combine in building a healthy gut environment. “The critical part is to understand that you have to feed these bacteria via the lifestyle and nutrition choices you make for these horses,” he counsels.

“Choose a (supplement) company based on their reputation and the science they can produce to support their formulas. I also look for companies — for these types of products and supplements in general — that have been around for a long time and have both hard science but also time-tested anecdotal evidence. We’re never going to be able to thoroughly study every ingredient and every product, so that volume of anecdotal data, the reputation of the company, the support of veterinarians and time-tested formulas are all key factors to weigh when making a decision. Just because something’s out there and available for sale doesn’t mean it’s a good choice or even a safe choice — not all are created equal.”
— Dr. Canaan Whitfield-Cargile, University of Georgia

Understanding Probiotics, Prebiotics and Postbiotics

From research by Dr. Whitfield-Cargile and others, certain pre- and probiotics can have a positive effect on the equine gut in healthy horses and those with GI disruption or while being given antibiotics or NSAIDs. The real key to understanding their impact on the microbiota and metabolome, however, is first comprehending their function and individual roles:

  • Probiotics are live microorganisms that, ideally, when ingested, travel to the horse’s GI tract, manage to stay alive and exert a beneficial effect on the gut environment. Supplemental probiotics, when chosen correctly, help maintain a healthy balance of bacteria within the equine gut.
  • Prebiotics, in contrast, are non-living, often fiber-based products, that feed the beneficial bacteria living in the hind gut. They aid the bacteria that break down prebiotic fiber. Prebiotics essentially nourish and promote growth in beneficial bacteria already present in the gut.
  • Postbiotics are a newer category that refers to a preparation of non-living microorganisms that ultimately confers a health benefit to the horse.

When choosing pre-, pro- and postbiotics, be aware that while numerous options are available, there’s “very little research to support them,” cautions Dr. Whitfield-Cargile. “Choose a company based on their reputation and the science they can produce to support their formulas. I also look for companies — for these types of products and supplements in general — that have been around for a long time and have both hard science but also time-tested anecdotal evidence. We’re never going to be able to thoroughly study every ingredient and every product, so that volume of anecdotal data, the reputation of the company, the support of veterinarians and time-tested formulas are all key factors to weigh when making a decision. Just because something’s out there and available for sale doesn’t mean it’s a good choice or even a safe choice — not all are created equal.”

The Next Frontier

While science has evolved beyond simply identifying which bacteria are present within a horse’s unique GI tract. Ultimately, researchers and veterinarians are aligned to more fully understand these metabolites and correlate them as clearly measurable health markers in the horse. “We’re working on the ability to combine something like metabolomics data with biomarkers and other diagnostics that speak to the health of the GI tract directly,” says Dr. Whitfield-Cargile of the forward trajectory of research. “Those markers could be specific to leaky gut, for instance, and in combination with this metabolite or that metabolite, it could give us an understanding of where we need to go to identify horses that can benefit from appropriate intentional alteration of the microbiome. We can then intervene and exert a positive effect in horses by altering gut bacteria in a beneficial way.”

Research has clearly shown that the gut is the epicenter of health in both horses and humans — for diet utilization and as the principal immunity organ in the body. Greater understanding of the bacteria and resulting metabolites within the gut can help veterinarians and horse owners maintain their animals’ health, optimize performance and support more successful outcomes. For researchers like Dr. Whitfield-Cargile, it all matters. Every choice made for the horse plays a role in its gut environment, which then plays an even greater role in overall health, capacity for performance and potential for longevity. As veterinarians and riders, the more thoughtful the decision, the bigger the benefit to the horse. From forage and feed to supplementation and on to management, training, stress, hauling practices, sleep and medications, “it all matters” to not just the makeup of the equine gut but how it will in turn impact other body systems.

As transformative as his work has been, Dr. Whitfield-Cargile is the first to keep things in perspective: “I’m fascinated by this work and fascinated by the equine gut, but, really, it’s not the gut bacteria that we care about. We care about the horse.”

Platinum Podcast

Platinum Podcast

Microbiota

Research has moved on from the microbiota to the metabolome, metatranscriptome and exfoliome, revealing it’s not necessarily what bacteria are present but how they’re contributing to each horse’s GI function and overall health that really matters. Featuring renowned equine veterinarian and researcher, Canaan Whitfield-Cargile.

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