Gut issue biomarkers and their use in signalling dysbiosis
/Article by Jackie Zions
Gastrointestinal issues (GI) are the number one cause of morbidity in horses other than old age. An unhealthy digestive system can cause poor performance, pain, discomfort, diarrhea, and a whole host of issues that can sideline your horse. It’s no wonder researchers are paying close attention to the ‘second brain’ and it’s billions of inhabitants. Ontario Veterinary College (OVC) researcher, Dr. Luis Arroyo has been studying the equine gastrointestinal systems for many years with several research projects receiving funding from Equine Guelph. Arroyo discusses what we know about equine gut health, causes of GI disorders and the extensive continuing research to understand what unstable and stable gut populations look like.
Starting with some basic anatomy Arroyo says, “The gastrointestinal tract of a horse is extremely large, and there are many things that can cause disturbances to the normal functioning or health of the gut.” A healthy gut microbiome is essential for the horse’s entire body to function optimally.
Signs of GI issues
Common signs of disorders could include abdominal pain, bloating, changes in fecal consistency (including diarrhea or constipation), excessive drooling, decrease in water consumption, lack of or poor appetite, weight loss and low body condition score.
“Some cases are more obvious to owners,” says Arroyo, “like poor performance, or acute or chronic diarrhea.”
Changes of behaviour such as becoming cranky or moody can be tell-tale signs there is unrest in the GI system. Biting at the flanks can signal abdominal pain as well as reactivity to being saddled. When the horse stops wanting to perform and athletic abilities suddenly decline, if there is no obvious lameness, GI issues are high among the considerations.
“Horses are herbivores, designed to consume a diet of forage, and to break down complex sugars within that forage.” says Arroyo. “The gut microbiota does this job and is very important for healthy digestion.” Recent research is connecting the changes in diversity of microbial communities to conditions like colic, colitis, and gastric ulcers.
Causes of GI Issues
Colic is the number one clinical condition occurring in horses. It is well-known that sudden dietary changes can be a major contributor as well as diets that are high in grain. This can create changes in the volatile fatty acids produced in the GI system, which in turn can lead to the development of gas colic. Arroyo provides the example of switching from dry hay fed in the winter, too rich, lush, spring grass as a big cause of rapid fermentation that can cause colic.
Any abrupt change, even if it’s a good quality feed to a different good quality feed, can be a source of colic. Then there is the more obvious consumption of moldy, poor, quality hay. So not only the quality but the transition/adaptation period needs to be considered when making feed changes and this goes for both changes to forage or concentrates.
A table of feed transition periods on the Equine Guelph website states an adaptation period of at least 10 – 14 days is recommended. Transition periods under seven days can increase colic risk over 22 times!
“Decrease in water consumption can be an issue, especially in countries with seasons,” says Arroyo. When water gets really cold, horses often drink less, and if it freezes, they don’t drink at all, which can lead to impaction colic. Parasite burden can also cause colic. If your horse lives in a sandy environment, like California, ingesting sand can cause impaction colic.
Non-steroidal anti-inflammatory drugs (NSAIDS) can cause colic or ulcers. NSAIDS can interfere with blood supply to the GI tract causing ulceration, for example in the mucosa of the stomach. Prolonged use can cause quite severe ulceration.
NSAIDS are not the only drugs that can contribute to GI issues. “Antibiotics - as the name says - kill many kinds of bacteria,” says Arroyo. “They are designed for that! Invariably they deplete some bacterial populations including in the intestine, and that is a problem because that may allow some other bacteria, potentially pathogenic or harmful, to overgrow, and that can cause dysbiosis.”
In a recent study, by fellow OVC researcher, Dr. Gomez and co-workers, it was determined that damage to the intestinal microbiota could occur after only 5 days of administering antibiotics to horses. Damage to the intestinal microbiota resembled dysbiosis that can potentially result in intestinal inflammation and colitis predisposing the horse to diarrhea. Judicious use of antibiotics and antimicrobials are advised.
There are infectious and non-infectious causes of colitis. Infectious examples include salmonella and then there is Neorickettsia risticii, which if ingested from contaminated sources, can cause Salmonellosis or Potomac horse fever, respectively.
“Any stress factors such as transportation, fasting or intense exercise like racing, can be a factor for developing stomach ulcers,” says Arroyo.
Current Diagnostics
Putting together a picture of the horse’s health status includes gathering clinical history from the horse owner and performing a physical examination for motility and hydration status. A biochemistry profile and complete set count can be gathered from blood testing.
Gastric ultrasound allows veterinarians to view the wall of the intestine, noting if it has thickened or distended, which could occur in cases when there is colic. They can assess appearance and find out if the intestine is displaced or if there is a twist. Gastroscopy is commonly used to find ulcers in the stomach and can reach as far as the first part of the duodenum.
GI Research
“DNA sequencing has been a breakthrough in science in terms of understanding the communities of different microorganisms living in many different niches from the skin to the lungs to the upper airways to the intestine,” says Arroyo.
It has allowed in-depth study of the population of microorganisms, providing a big picture of the different inhabitants in various areas of the GI tract, such as the lumen of the small intestine and the small and large colon. “The microorganisms vary, and they have different functions in each compartment,” says Arroyo.
DNA sequencing has allowed researchers to study microbial populations and gather information on what happens to bacterial communities when impacted by diseases like colitis. “We can see who is down, and who is up,” explains Arroyo, “and determine what populations have been depleted.” It has led to a better knowledge of which of the billions of factors are harmful to the system and which can compromise the health of the horse.
Robo-gut is one example of a fantastic system where bacterial communities are being replicated in the lab to mimic what would be found in a natural environment.
Researchers at the University of Guelph have measured metabolic profiles of the bacterial population after the addition of supplements like probiotics and prebiotics. They found they can dramatically change the metabolites that are being produced, according to what is being added to the system.
Exciting new research that could impact the future of diagnostics includes screening for biomarkers as indicators of intestinal health among equine microbiota. Dr. Arroyo is currently working with research partner, Dr. Marcio Costa, from the University of Montreal, looking for biomarkers that indicate changes in the inhabitants of the equine gut that take place during the early onset of illness.
“A biomarker is a biological molecule that you can find in different places,” explains Arroyo. “For example, you might find them in tissue, blood, urine, or different body fluids. They can signal normal or abnormal processes or could reveal a marker of a disease. For example, a biomarker can be used to see how well the body might respond to a treatment or to a disease condition.”
“The objective of a dysbiosis index is quantifying ‘X’ number of certain bacteria that are important to us,” says Arroyo. In this case, the dysbiosis derives from sequencing of the bacterial population in fecal samples.
Changes in the intestinal microbiota (dysbiosis) are present before and during the outset of diseases and after treatment with antibiotics. Arroyo cites the example of decreased Lachnospiraceae commonly observed when there is intestinal inflammation.
Bacterial biomarkers are currently being used in other species to accurately predict intestinal dysbiosis, for example in cats and dogs. One canine study quantified the number of seven different taxa of importance of the total bacterial populations. This information is entered into a mathematical algorithm that comes up with results explaining which bacteria have increased or decreased. Based on those numbers, one can use a more specific taxa to identify dysbiosis. In a feline study, it was discovered that six bacterial taxa could be accurately used to predict diarrhea in 83% of cases.
It is hoped the same results could be accomplished for horses. Developing PCR testing to screen for biomarkers could be a game changer that could potentially provide speedy, economical early diagnostics and early treatment.
So far, the most remarkable finding in the preliminary data reveals that in horses with colitis, the whole bacterial population is very depleted.
“At this stage we are in the process of increasing our numbers to find significant differences in which bacterial taxa are more important,” says Arroyo. “Soon we hope to share which bacteria taxa are more promising for predicting dysbiosis in horses with gastrointestinal disease.”
The researchers are delving into a huge biobank of samples to identify potential markers of intestinal dysbiosis in horses, utilizing PCR testing as a faster and more economical alternative to the complex DNA sequencing technologies that have been used to characterize changes in microbiota thus far. The goal is to develop simple and reliable testing that veterinarians can take right to the barn that will result in early treatment and allow closer monitoring of horses at the first onset of GI disease.
Top Tips to Protect Digestive Health
Horses are hind gut fermenters who rely on adequate amounts of fiber in the diet to maintain healthy gut function.
Make dietary changes slowly as abrupt changes disrupt the microbiota.
Avoid large grain meals as huge portions of highly fermentable diets can be quite harmful to the microbiota and can also be a source of risk for developing gastric ulcers. Opt to spread out concentrates into several smaller rations.
Prevent long periods of fasting which can also lead to ulcers. Horses are continuous-grazers, and they need to have small amounts of feed working through their digestive system to keep it functioning optimally.
Have a parasite prevention program.
Provide fresh water 24/7 to maintain good hydration and keep contents moving smoothly through the GI tract.
Keep up to date on dental appointments.
Motion is lotion – turn out and exercise are extremely important to gut function.
In closing, Arroyo states, “These top tips will help keep the horse happy and the gastrointestinal tract functioning properly.”
![Promising developments in quest to prevent catastrophic racehorse injuriesUniversity of Kentucky study shows association between mRNA biomarkers and catastrophic injuries in Thoroughbred racehorses—a positive step forward in the development of a pre-race screening toolCatastrophic injuries in Thoroughbred racehorses is a top-of-mind concern for the global racing industry and its fans. That sentiment is shared by researchers at the University of Kentucky and their collaborators, who are working to learn more about changes happening at a cellular level that might indicate an injury is lurking before it becomes career or life ending. Could it be possible to identify an early marker or signal in horses at risk of catastrophic injury, allowing for intervention before those injuries happen? And, if so, might this type of detection system be one that could be implemented cost effectively on a large scale?According to Allen Page, DVM, PhD, staff scientist and veterinarian at UK’s Gluck Equine Research Center, the short answer to both questions is that it looks promising. To date, attempts to identify useful biomarkers for early injury detection have been largely unsuccessful. However, the use of a different biomarker technology, which quantifies messenger RNA (mRNA), was able to identify 76% of those horses at risk for a catastrophic injury. An abstract of this research was recently presented at the American Association of Equine Practitioners’ annual meeting in December 2020 and the full study published January 12 in the Equine Veterinary Journal (https://beva.onlinelibrary.wiley.com/journal/20423306). In this initial research—which looked at 21 different mRNA markers selected for their roles in encoding proteins associated with inflammation, bone repair and remodeling, tissue repair and general response to injury—three markers showed a large difference in mRNA levels between injured and non-injured horses. For almost four years, Page and his University of Kentucky colleagues have been analyzing blood samples from almost 700 Thoroughbred racehorses. The samples, collected by participating racing jurisdictions from across the United States, have come from both catastrophically injured and non-injured horses in a quest to better understand changes that might be happening at the mRNA level and if there are any red flags which consistently differentiate horses that suffer a catastrophic injury.According to Page, the ultimate hope is to develop a screening tool that can be used pre-race to identify horses at increased risk for injury. The results of this study, which was entirely funded by the Kentucky Horse Racing Commission’s Equine Drug Research Council, suggest that analysis of messenger RNA expression could be an economical, effective and non-invasive way to identify individual racehorses at risk for catastrophic injury.Joining Page in the research from UK’s Gluck Center are Emma Adam, BVetMed, PhD, DACVIM, DACVS, assistant professor, research and industry liaison, and David Horohov, PhD, chair of the Department of Veterinary Science, director of the Gluck Center and Jes E. and Clementine M. Schlaikjer Endowed Chair.Previous research has shown that many catastrophic injuries occur in limbs with underlying and pre-existing damage, leading to the theory that these injuries occur when damage accumulation exceeds the healing capacity of the affected bones over time. Since many of these injuries have underlying damage, it is likely that there are molecular markers of this that can be detected prior to an injury.The identification of protein biomarkers for these types of injuries has been explored in previous research, albeit with limited success. The focus of this project, measuring messenger RNA, had not yet been explored, however. The overall objective was to determine if horses that had experienced a catastrophic injury during racing would show increased inflammatory mRNA expression at the time of their injury when compared to similar horses who were not injured.The genetic acronyms: A primer on DNA, RNA, mRNA and PCRThis research leverages advances made in genetics during the last several decades, both in a greater understanding of the field as well as in applying that knowledge to specific issues facing the equine industry, including catastrophic breakdown in racehorses.The genetic code of life is made up of genes and regulatory elements encoded by DNA, or deoxyribonucleic acid, which is found in the nucleus of cells in all living organisms. It is arranged in a double helix structure, similar to a twisted ladder. The rungs of that ladder are nucleotide base pairs, and the ordering of those base pairs results in the specific genetic code called a gene. The genetic code in the genes and the DNA tell the body how to make proteins. RNA (ribonucleic acid) is created by RNA polymerases, which read a section of DNA and convert it into a single strand of RNA in a process called transcription. While all types of RNA are involved in building proteins, mRNA is the one that actually acts as the messenger because it is the one with the instructions for the protein, which is created via a process called translation. In translation, mRNA bonds with a ribosome, which will read the mRNA’s sequence. The ribosome then uses the mRNA sequence as a blueprint in determining which amino acids are needed and in what order. Amino acids function as the building blocks of protein (initially referred to as a polypeptide). Messenger RNA sequences are read as a triplet code where three nucleotides dictate a specific amino acid. After the entire polypeptide chain has been created and released by the ribosome, it will undergo folding based on interactions between the amino acids and become a fully functioning protein. While looking at inflammation often involves measuring proteins, Page and his collaborators opted to focus on mRNA due to the limited availability of reagents available to measure horse proteins and concerns about how limited the scope of that research focus would be. Focusing on mRNA expression, however, is not without issues. According to Page, mRNA can be extremely difficult to work with. “A normal blood sample from a horse requires a collection tube that every veterinarian has with them. Unfortunately, we cannot use those tubes because mRNA is rapidly broken down once cells in tubes begin to die. Luckily, there are commercially-available blood tubes that are designed solely for the collection of mRNA,” he said.“One of the early concerns people had about this project when we talked with them was whether we were going to try to link catastrophic injuries to the presence or absence of certain genes and familial lines. Not only was that not a goal of the study, [but] the samples we obtain make that impossible,” Page said. “Likewise for testing study samples for performance enhancing drugs. The tubes do an excellent job of stabilizing mRNA at the expense of everything else in the blood sample.”In order to examine mRNA levels, the project relied heavily on the ability to amplify protein-encoding genes using a technique called the Polymerase Chain Reaction (PCR). By using a variety of techniques, samples from the project were first converted back to DNA, which is significantly more stable than mRNA, and then quantified using a specialized machine that is able to determine the relative amount of mRNA initially present in the individual samples. While it is easy to take for granted the abilities of PCR, this Nobel Prize winning discovery has forever changed the face of science and has enabled countless advances in diagnostic testing, including those used in this study.The research into mRNA biomarkers Catastrophic racing and training injuries have long been a target for researchers due to the high societal and welfare impacts on the racing industry. With the nearly universal requirement for necropsies on horses that succumb to these injuries, work by researchers has demonstrated that most horses with catastrophic injuries have pre-existing damage in their legs. This pre-existing damage presents an opportunity to detect injuries before they occur, whether that be with advanced imaging or less invasive techniques, such as screening of blood for injury biomarkers. Horses eligible for inclusion in the study were Thoroughbreds entered into any race in one of five participating jurisdictions from September 2017 to June 2020. To look at the mRNA, these jurisdictions collected specific blood samples either pre-race or post-race from a selection of non-injured horses or immediately from a horse after a catastrophic injury. Once collected, samples were sent to the Gluck Center where they were analyzed using PCR. The names of horses and sample types (injured, pre-race or post-race) were kept from the researchers until the samples had been fully analyzed.Once the names and dates of samples were revealed, public records were then used to learn more about each horse. Information examined included the horse’s sex, age, race type and whether non-injured horses raced again within three months of the sampled race. For horses who had been catastrophically injured, necropsy results were used to categorize the type of musculoskeletal injury that occurred. “Out of the 21 markers (genes) that were measured, three of them immediately stood out as being able to predict injury. The three individual markers of interest were Insulin-like Growth Factor 1 (IGF-1), Matrix Metalloproteinase-2 (MMP2) and IL-1 Receptor Antagonist (IL1RN). Taken together, the changes seen in all three of these markers suggest that there is increased inflammation in the injured horses and that the inflammation arises from bone, just as was suspected,” Page said.“Based only on these three markers, we were able to correctly identify horses at risk for injury 76% of the time and exclude horses for being at risk 88% of the time,” Page said. “Obviously, we want to maximize those numbers as much as possible, so while there’s room for improvement, this is significantly better than any other option currently available.”One of the limitations of the study was that horses were only sampled once, so there was no ability to identify changes in individual horses over a period of time. Once horses start being sampled repeatedly on a regular basis with this testing, Page said he believes the ability to identify at-risk horses will improve dramatically.What does the future hold?“Since the ultimate hope is to develop a commercially-viable screening tool that can be used pre-race to identify horses at increased risk for injury, we anticipate adding multiple other markers with a new study that is just getting started,” Page said.As part of the new study, also funded by the Kentucky Horse Racing Commission, Page and two Gluck Center colleagues, James MacLeod, VMD, PhD, John S. and Elizabeth A. Knight chair and director of UK Ag Equine Programs, and Ted Kalbfleisch, PhD, associate professor, plan to utilize RNA-sequencing, a relatively new technology, to expand their search to all of the approximately 22,000 protein-coding genes horses have. This will dramatically increase the likelihood that they will be able to identify additional markers for horses at risk of injury. They plan to do this by using the large number of samples that have already been collected, further leveraging their initial research and decreasing the amount of time it will take to complete their new study.“We are really excited about this new project and the promise that it holds,” Page said. “In our first study, we drove the data because we had to select which mRNA markers we wanted to examine. In our new study, the RNA-sequencing data is really what will be driving us.”While that project is ongoing, Page and his colleagues continue to refine and improve upon the various laboratory steps required to isolate and analyze mRNA. Guided by the hope of providing the racing industry with a high-throughput screening tool, the group has employed multiple robotic platforms that can already handle 100 samples per day and be easily scaled up to handle more.“As a researcher, I see it as being my job to provide practical and reliable solutions to the horse racing industry,” Page said. “I know that change can be scary, but we can all agree that something needs to change to help better protect racehorses and the jockeys who ride them. Ultimately, the racing industry will decide when it wants to give this screening tool a chance. I’m confident that, when the industry is ready, we will be too.” The full study published in the Equine Veterinary Journal can be found here: https://doi.org/10.1111/evj.13423](https://images.squarespace-cdn.com/content/v1/517636f8e4b0cb4f8c8697ba/1619181103562-L9SRTHKK0L6C0FFYFXA2/Screenshot+2021-04-23+at+13.31.27.png)
