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

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.”

Probiotics – The key to a well-balanced equine gut

Article by Kerrie Kavanagh

It is no surprise that the health maintenance of the racehorse is a top priority for trainers. And probiotics can be used as a treatment modality to manipulate the gut microbiome to improve or maintain health. Equine studies to date have shown that probiotic strains can offer an advantageous approach to minimising disturbances in the gut microbial populations, repair these deficiencies—should they occur—and re-establish the protective role of the healthy gut microbiome. Other probiotic-associated health benefits include reducing diet-related diseases such as colic and laminitis, preventing diarrhoea, conferring host resistance to helminth infection, improving stress-related behavioural traits (e.g., locomotion) and even promote the development of an effective gut-brain communication pathway. 

Probiotics have been used by humans for more than 5,000 years with their development closely linked to that of dairy products and fermented foods. Today, probiotics are seen as an excellent non-pharmaceutical way to improve the health of both humans and animals, and there are a plethora of products to choose from. But what exactly is a probiotic, and how do they work? Why would your horse need one? What types of probiotics are available for horses? These are all questions that horse trainers ask frequently, which we will attempt to answer here. 

The Equine Gut Microbiome

Probiotics and the equine microbiome can benefit from a valuable symbiotic relationship; probiotics are seen as a restorative treatment modality for the gut, to re-establish the bacterial populations there and also to re-establish the protective role that the health gut microbiome confers to the host. But when we discuss the equine microbiome, what are we really talking about? 

The gut microbiota/microbiome can be categorised by anatomical location such as the oral microbiota/microbiome in the mouth and the intestinal microbiota/microbiome in the intestines, etc. Therefore, the gut microbiome pertains to the microbiota in the gastrointestinal tract. This population of microorganisms (bacteria, fungi, viruses, protozoa) is referred to as the ‘microbiota’ of the gut, while the term ‘gut microbiome’ refers to the genetic material associated with these microorganisms. The microbiome can be defined as the sum of the microbes and their genomic elements in a particular environment. If we look at the definition of the microbiome having the propensity to an equation, then any equation must be balanced; to maintain that balance is key. If the microbial community exists in an environment in a balanced state, then any upset or disturbance to the microbial populations will cause the balance to shift (known as dysbiosis). To maintain the balance, we need to firstly understand the way the microorganisms exist within their community (i.e. their microorganism-to-microorganism interactions and also microorganism-to-environment interactions) and secondly, their functioning role. If we can understand their (microorganism) position and role, then we can maintain the balance or re-establish the balance if a shift occurs.  

The human intestinal microbiome is now recognised as an organ and likewise, the equine intestinal microbiome is deemed an ‘organ’ of the body and is vital for the breakdown of complex food and subsequent release of energy, protection against the pathogenic bacterial colonisation and in regulating the immune system and metabolic functions. There has been much debate regarding the content of the healthy equine microbiome, and even to deduce what ‘healthy’ or ‘normal’ is requires a level of understanding of the microbiota associated with healthy horses. This question has been posed by many researchers and frankly has yet to be answered with certainty. There are many reasons why the ‘normal’ microbiota keeps eluding us; and this can be attributed to the many reasons as to why the gut microbiota (of a healthy horse) can be affected (see Figure 1). It is thought that the diversity of the human gut microbiota and the general assembly of microbial communities within the gut (with the dominant phyla being classed as belonging to Firmicutes and Bacteroidetes) is a shared hypothesis across most species (i.e., humans and animals share a similar gut microbiome structure). Firmicutes and Bacteroidetes have been shown to constitute the main dominant phyla in equine, bovine, canine and feline gut microbiome studies indicating the cruciality of the role they play in the maintenance of a healthy microbial ecology in the gastrointestinal tract. Several studies do agree that dominant phyla of the equine gut microbiota are obligate anaerobes: the gram-positive Firmicutes and the gram-negative Bacteroidetes; other phyla are identified as Proteobacteria, Verrucomicrobia, Actinobacteria, Euryarchaeota, Fibrobacteres and Spirochaetes. Ninety-five percent of the  Firmicutes phyla contains the Clostridia genus in addition to genera related to gut health such as Lachnospiraceae, Faecalibacterium and Ruminococcaceae. The other main dominant phyla, Bacteroidetes, on the other hand contains a large variety of the genus. 

Role of the Equine Gut Microbiota

The role of the gut intestinal microbiota serves to protect and prevent disease. The gut microbiota has several purposes: prevention of pathogen colonisation by competing for nutrients, enrichment and maintenance of the intestinal barrier—their ability to renew gut epithelial cells and repair damage to the mucosal barrier, the breakdown of food and releasing energy and nutrients, such as synthesising vitamins D and K and also conserving and restoration of the immune system by the formation of antimicrobial metabolites and blocking access to the binding sites of the mucosal wall. The gut microbiota is also thought to play some role of influencing the neuro-active pathways that affect behaviour. It is not surprising to see that gut disorders and gastrointestinal diseases can arise when gut dysbiosis occurs. The role of the gut microbiota may have even more importance than is realised and may have a role to play with developing illness or disease later in life.

The microbial colonisation of the intestinal tract begins at birth. The foal begins its colonisation through contact with the microbiota of the mare’s vaginal and skin surfaces plus the surrounding environments to which the foal is exposed and reaches a relatively stable population by approximately 60 days in age. It is perhaps a fight for dominance to achieve establishment in the gut among the bacterial populations that sees the foal’s microbiota as being more diverse and quick to change when compared to that of the older horse. The subsequent colonisation of the intestinal tract will reflect the foal’s diet, changing environment, introduction to other animals, ageing and health.

Figure 1: Factors that can lead to gut dysbiosis

What exactly is a probiotic?

The word ‘probiotic’ is of Greek origin meaning ‘for life’ and the WHO/FAO have defined probiotics as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’.  People have long believed that exposure to non-pathogenic microorganisms can benefit the health of humans and animals. The thinking behind this is that daily consumption of sufficient numbers of ‘good’ microorganisms (either bacteria or fungi) can maintain a healthy population of microorganisms in the gut and benefit overall health.  

Probiotics are used to manipulate the bacterial populations of the gut in order to re-establish the delicate microbial balance there which, in turn, confers health benefits on the host. As the benefits associated with some of the ‘good’ bacteria within the gut became known, these were referred to as probiotic bacteria. 

How do probiotics work?

There are 4 main mechanisms by which probiotics are thought to exert their effects.

1. By inhibiting pathogen colonisation in the gut through the production of antimicrobial metabolites or by competitive exclusion; in other words, they prevent the ‘bad’ bacteria from growing in the gut.

2. By protecting or re-stabilising the commensal gut microbiota, probiotics can be a means to re-establish the balance of the gut microbial populations.

3. By protecting the intestinal epithelial barrier, they maintain the health of the intestinal wall.

4. By inducing an immune response, probiotics can boost the immune response and help prevent disease.

If we consider the definition of a probiotic as ‘live non-pathogenic microorganisms that, when administered in adequate amounts, confer a health benefit on the host’, then this reference to ‘adequate amounts’ must be emphasised, and the dose administered is critical to ensure that the probiotic has the desired effect. For horses, we must consider the route through the digestive tract that the probiotic strains must travel to arrive at their destination is a distance over 15 metres long. It is a race for survival! The gastrointestinal system has many obstacles along the passage such as the acidic stomach environment and the dangers of exposure to bile and digestive enzymes, in which they must survive. The initial dose of ‘live’ probiotic strains is therefore crucial to ensure survival in the gut. Prebiotics are ingredients such as carbohydrates and fibre, which promote the growth of these probiotic bacterial/yeast strains in the gut. Prebiotics are essentially the food for the probiotic strains and can help form a symbiotic relationship with the probiotic to improve the overall health status of the horse. 

Why would you need to give your horse a probiotic?

Gut dysbiosis is a fluctuation or disturbance in the population of microorganisms of the gut, which may be linked to a wide range of diseases in horses. Gut dysbiosis can be caused by many factors ranging from dietary changes, antibiotics, disease, intense exercise and training, age, worms, environment, travel, or even minor stress events—resulting in major consequences such as colic. Dysbiosis is generally associated with a reduction in microbial species diversity. 

Diet is one of the major factors contributing to gut dysbiosis. Unlike the ruminant cattle and sheep that use foregut fermentation, horses are hindgut fermenters. The large intestine is the main area where fermentation occurs. The horse utilises the microbial enzymes of the hindgut microbial population in the colon and caecum to break down the plant fibres (cellulose fermentation) sourced mainly from grasses and hay. The horse itself does not possess the hydrolytic enzymes that are required to break the bonds of the complex structures of the plant carbohydrates (in the form of celluloses, hemicelluloses, pectins) and starch; so therefore, it strongly relies on the microbiota present to provide those critical enzymes required for digestion. The main phyla Firmicutes and Bacteroidetes possess enzymes capable of breaking down the complex carbohydrates (such as starch and cellulose).

Research has shown that forage-based diets (grasses and hay) promote the most stable gut microbiomes, but ultimately the equine athlete requires far more energy than a forage-based diet can supply. Supplementing the diet with concentrates containing starch such as grain, corn, barley and oats can affect the number and type of bacteria in the gut. Optimising diet composition is so important as carbohydrate overload—as seen with high-starch diets (>1g/kg body weight per meal)—can change the populations of bacteria in the gut, alter pH, upset digestion and the gut environment, and ultimately result in diseases such as colitis, colic and laminitis. The correct diet is essential for maintaining the delicate balance of bacterial populations. Probiotics can be used to either replace the bacteria missing in the gut and/or can help maintain the delicate microbial balance even in the face of adversity such as abrupt dietary changes, antibiotic treatment and stress.

What types of probiotics are available for horses?

There are several probiotic products on the market, and most are in powder or liquid form. There are two main categories of probiotics: generic and autogenous. Generic probiotics are off-the-shelf products that contain specific strains of bacterial or yeast, singularly or in combination. The Lactobacillus and Bifidobacterium families, Enterococci and yeasts such as Saccharomyces cerevisae and boulardii are the most common equine probiotic strains. Advantages of generic probiotics are that they are widely available, easy to administer, and they may be beneficial to horse health (if the strains are alive in sufficient numbers). Autogenous probiotics are specifically formulated using bacteria obtained from the horse’s own faecal sample and, as such, are uniquely adapted to that individual animal. These host-adapted bacteria are more likely to survive in the gut than non-adapted generic strains and can quickly replenish absent or low levels of bacteria unique to the individual horse, thus maintaining health.