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

turn out and exercise are extremely important to gut function
  1. Horses are hind gut fermenters who rely on adequate amounts of fiber in the diet to maintain healthy gut function.

  2. Make dietary changes slowly as abrupt changes disrupt the microbiota.

  3. 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.

  4. 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.

  5. Have a parasite prevention program.

  6. Provide fresh water 24/7 to maintain good hydration and keep contents moving smoothly through the GI tract.

  7. Keep up to date on dental appointments. 

  8. 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.”

A New Look at Lameness

Words - Jackie Zions (interviewing Dr. Koenig)

Prevention is the ideal when it comes to lameness, but practically everyone who has owned horses has dealt with a lay-up due to an unforeseen injury at some point. The following article will provide tools to sharpen your eye for detecting lameness, review prevention tips and discuss the importance of early intervention. It will also begin with a glimpse into current research endeavouring to heal tendon injuries faster, which has obvious horse welfare benefits and supports horse owners eager to return to their training programs. Dr. Judith Koenig of Ontario Veterinary College (OVC) spends half of her time as a surgeon and teacher with a strong interest in equine sports medicine and rehabilitation, and the other half as a researcher at the OVC.

Lameness is a huge focus for Koenig, whose main interest is in tissue healing. “I think over the past 20 or 30 years we have become very, very good in diagnosing the cause of lameness,” says Koenig. “In the past, we had only radiographs and ultrasound as a diagnostic tool, but by now most referral centers also have MRI available; and that allows us to diagnose joint disease or tendon disease even more. We are much better now [at] finding causes that previously may have been missed with ultrasound.” 


Improvements in diagnostics have resulted in increased ability to target treatment plans. With all the different biologics on the market today, Koenig sees a shift in the management of joint disease with more people getting away from steroids as a treatment.

The following list is excerpted from Equine Guelph’s short course on lameness offered on TheHorsePortal.ca. It outlines the different diagnostics available:

Stem Cell Therapy
When asked for the latest news on research she has been involved in, Koenig proclaims, “I'm most excited about the fact that horses are responding well to stem cell treatment—better than I have seen any response to any other drug we have tried so far!”

Koenig has investigated the use of many different modalities to see if they accelerate tissue healing and has studied which cellular pathways are affected. Two recent collaborative studies have produced very exciting findings, revealing future promise for treating equine osteoarthritis with stem cell therapy.  

In a safety study, Koenig and her team at the Ontario Veterinary College have shown equine pooled cryopreserved umbilical cord blood, (eCB) MSC, to be safe and effective in treatment of osteoarthritis.  

“These cells are the ones harvested from umbilical cord blood at the time of foaling and then that blood is taken to the lab and the stem cells are isolated out of it,” explains Koenig. The stem cells are then put through a variety of tests to make sure they are free of infectious diseases. Once given a clean bill of health, they are expanded and frozen. 
The stem cells harvested from multiple donors of equine umbilical cord blood [eCB, (kindly provided by eQcell), MSC] were compared to saline injections in research horses. “This type of cells is much more practical if you have a cell bank,” says Koenig. “You can treat more horses with it, and it’s off the shelf.” There were no systemic reactions in the safety study. Research has also shown no different reactions from sourcing from one donor or multiple donors.  

In the second study, 10 million stem cells per vial were frozen for use in healing OA from fetlock chips in horses that were previously conditioned to be fit. After the fetlock chip was created, exercise commenced for six more weeks, and then osteoarthritis was evaluated by MRI for a baseline. Half the horses were treated with the pooled MSC stem cells, and the control group received saline before another month of exercise. Then MRI and lameness exams were repeated, and arthroscopy was repeated to score the cartilage and remove the chip.

Lameness was decreased and cartilage scores were improved in the group that received stem cell therapy at the time of the second look with arthroscopy.

Many diagnostics were utilized during this study. MRIs, X-rays, ultrasounds and weekly lameness evaluations all revealed signs of osteoarthritis in fetlock joints improved in the group treated with (eCB) MSCs. After six weeks of treatment, the arthroscopic score was significantly lower (better cartilage) in the MSC group compared to the control group. 

“Using the MRI, we can also see a difference that the horses treated with stem cells had less progression of osteoarthritis, which I think is awesome,” says Koenig. “They were less lame when exercised after the stem cell therapy than the horses that received saline.”
This research group also just completed a clinical trial in client-owned horses diagnosed with fetlock injuries with mild to moderate osteoarthritis changes. The horses were given either 10 million or 20 million stem cells and rechecked three weeks and six weeks after the treatment. Upon re-evaluation, the grade of lameness improved in all the horses by at least one. Only two horses presented a mild transient reaction, which dissipated after 48 hours without any need for antibiotics. The horse’s joints looked normal, with any filling in the joint reduced.
There was no difference in the 18 horses, with nine given 10 million stem cells and the other nine 20 million stem cells; so in the next clinical trial, 10 million stem cells will be used.

The research team is very happy with the results of this first-of-its-kind trial, proving that umbilical cord blood stem cells stopped the progression of osteoarthritis and that the cartilage looked better in the horses that received treatment. The future of stem cell therapy is quite promising!

Rehabilitation


Research has shown adhering to a veterinary-prescribed rehabilitation protocol results in a far better outcome than paddock turn out alone. It is beneficial for tendon healing to have a certain amount of controlled stimulation. “These horses have a much better outcome than the horses that are treated with just being turned out in a paddock for half a year,” emphasizes Koenig. “They do much better if they follow an exercise program. Of course, it is important not to overdo it.”

For example, Koenig cautions against skipping hand-walking if it has been advised.  It can be so integral to stimulating healing, as proven in recent clinical trials. “The people that followed the rehab instructions together with the stem cell treatment in our last study—those horses all returned to racing,” said Koenig.  

“It is super important to follow the rehab instructions when it comes to how long to rest and not to start back too early.”

Another concern when rehabilitating an injured horse would be administering any home remedies that you haven't discussed with your veterinarian. Examples included blistering an area that is actively healing or applying  shockwave to mask pain and then commence exercise.

Prevention and Training Tips


While stating there are many methods and opinions when it comes to training horses, Koenig offered a few common subjects backed by research. The first being the importance of daily turnout for young developing horses.  

Turnout and exercise
Many studies have looked at the quality of cartilage in young horses with ample access to turn out versus those without. It has been determined that young horses that lack exercise and are kept in a stall have very poor quality cartilage.
Horses that are started early with light exercise (like trotting short distances and a bit of hill work) and that have access to daily paddock turnout, had much better quality of cartilage. Koenig cited research from Dr. Pieter Brama and similar research groups.

Another study shows that muscle and tendon development depend greatly on low grade exercise in young horses.  Evaluations at 18 months of age found that the group that had paddock turnout and a little bit of exercise such as running up and down hills had better quality cartilage, tendon and muscle.  

Koenig provides a human comparison, with the example of people that recover quicker from injury when they have been active as teenagers and undergone some beneficial conditioning. The inference can be made that horses developing cardiovascular fitness at a young age stand to benefit their whole lives from the early muscle development.

Koenig says it takes six weeks to regain muscle strength after injury, but anywhere from four to six months for bone to develop strength. It needs to be repeatedly loaded, but one should not do anything too crazy! Gradual introduction of exercise is the rule of thumb.

Rest and Recovery


“Ideally they have two rest days a week, but one rest day a week as a minimum,” says Koenig. “I cannot stress enough the importance of periods of rest after strenuous work, and if you notice any type of filling in the joints after workout, you should definitely rest the horse for a couple of days and apply ice to any structures that are filled or tendons or muscles that are hard.” 

Not purporting to be a trainer, Koenig does state that two speed workouts a week would be a maximum to allow for proper recovery. You will also want to make sure they have enough access to salt/electrolytes and water after training.

During a post-Covid interview, Koenig imparted important advice for bringing horses back into work methodically when they have experienced significant time off.


“You need to allow at least a six-week training period for the athletes to be slowly brought back and build up muscle mass and cardiovascular fitness,” says Koenig.  “Both stamina and muscle mass need to be retrained.”

Watch video: “Lameness research - What precautions do you take to start training after time off?”

The importance was stressed to check the horse’s legs for heat and swelling before and after every ride and to always pick out the feet. A good period of walking is required in the warmup and cool down; and riders need to pay attention to soundness in the walk before commencing their work out.

Footing and Cross Training


With a European background, Koenig is no stranger to the varying track surfaces used in their training programs. Statistics suggest fewer injuries with horses that are running on turf, like they practice in the UK.  

Working on hard track surfaces has been known to increase the chance of injury, but delving into footing is beyond the scope of this article.

“Cross training is very important,” says Koenig. “It is critical for the mental and proper musculoskeletal development of the athlete to have for every three training days a day off, or even better provide cross-training like trail riding on these days." 

Cross-training can mitigate overtraining, giving the body and mind a mental break from intense training. It can increase motivation and also musculoskeletal strength. Varied loading from training on different terrain at different gaits means bone and muscle will be loaded differently, therefore reducing repetitive strain that can cause lameness.

Hoof care


Whether it is a horse coming back from injury, or a young horse beginning training, a proficient farrier is indispensable to ensure proper balance when trimming the feet. In fact, balancing the hoof right from the start is paramount because if they have some conformational abnormalities, like abnormal angles, they tend to load one side of their joint or bone more than the other. This predisposes them to potentially losing bone elasticity on the side they load more because the bone will lay down more calcium on that side, trying to make it stronger; but it actually makes the bone plate under the cartilage brittle.  

Koenig could not overstate the importance of excellent hoof care when it comes to joint health and advises strongly to invest in a good blacksmith. Many conformational issues can be averted by having a skilled farrier right from the time they are foals. Of course, it would be remiss not to mention that prevention truly begins with nutrition. “It starts with how the broodmare is fed to prevent development of orthopedic disease,” says Koenig. Consulting with an equine nutritionist certainly plays a role in healthy bone development and keeping horses sound.