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.”

The use of probiotics as an alternative to antibiotics to reduce resistance in the gut

Article by Kerrie Kavanagh

The leading causes of horse mortality can be attributed to gastrointestinal diseases. Therefore, maintaining the balance of the gut microbiota and avoiding a shift in microbial populations can contribute to improved health status. The gut microbiota, however, can be influenced by countless dynamic events: diet, exercise, stress, illness, helminth infections, aging, environment and notably, antimicrobial therapy (antibiotics). These events can lead to gut dysbiosis—a fluctuation or disturbance in the population of microorganisms of the gut, which can contribute to a wide range of disease. The use of antibiotics in horses is thought to have one of the most notable effects on the gut microbiota (gut dysbiosis), which can lead to diseases such as colitis, colic and laminitis.

Antibiotics, which are antimicrobial agents active against bacteria, are important to equine medicine; and bacterial infections can be resolved quite successfully using antibiotics for antimicrobial therapy, but there are consequences to their use. An antimicrobial agent can be defined as a natural or synthetic substance that kills or inhibits the growth of microorganisms such as bacteria, fungi and algae. One of the consequences of antibiotic use is that of antibiotic-associated diarrhea, which can contribute to poor performance in the horse and even mortality. In antimicrobial therapy, the target organism is not the only organism affected by the antimicrobial agent but also the commensal microbiota too (the normal flora of the equine gut). Antibiotics can promote fungal infections and resistant organisms and impede or even eliminate the more sensitive organisms; and they can have both short- and long-term consequences on the gut microbiota composition and function. 

Research has indicated that antibiotic treatment may adversely affect metabolic function in the gut by decreasing protein expression responsible for biochemical pathways such as glycolysis, iron uptake, glutamate hydrolysis and possibly even more metabolic functions. The use of antimicrobial drugs directly impacts and possibly contributes to the most notable effect on the gut microbiota of the host, leading to gut dysbiosis; and certain antibiotics can have further-reaching consequences on the microbiota than others. The type of antibiotic and mode of action (bacteriostatic versus bactericidal) will differ in their influences on the gut microbiota composition, e.g., clindamycin operates a bacteriostatic mode of action by inhibiting protein synthesis and exerts a larger impact on the gut microbiota compared to other antimicrobials. These influential consequences that are imparted by the antimicrobial agent are relatively yet to be elucidated and may result in the manifestation of illness or conditions later in life. For example, the development of asthma in humans has been linked to antibiotic treatment in early childhood as a result of bacterial infections. It may yield interesting results if researchers were to examine the gut microbiome of horses suffering from chronic obstructive pulmonary disease (COPD) and other chronic respiratory illnesses and to establish if there is indeed a link with antibiotic therapy used in horses from an early age. 

In comparison to the vast wealth of human studies conducted so far, the volume of equine studies falls disappointingly far behind, but that is changing as researchers focus their interest on developing and filling this gap of knowledge. One such study, which examined the effect of antibiotic use on the equine gastrointestinal tract, demonstrated a significant reduction in culturable cellulolytic bacteria (>99%) from equine feces during the administration period of trimethoprim sulfadiazine and ceftiofur in a study comparing responses to antibiotic challenge. That reduction was still evident at the end of the withdrawal period when compared to the control group. In other words, there was a significant reduction in the ‘normal’ bacteria of the gut. The ability of antibiotics to modulate the gut microbiota was evidenced by the proliferation of pathogenic Salmonella and Clostridia difficile (commonly associated with diarrhea in horses) in the antibiotic challenged horses. This trend of reduction in cellulolytic bacteria associated with antibiotic use was also mirrored in a relatively recent study conducted in 2019, where a short-term reduction in culturable cellulolytic bacteria was combined with a progressive increase in amylolytic bacteria. The heavy reliance on cellulolytic bacteria in the role of equine digestion (without these types of bacteria the horse cannot break down their food) may, therefore, adversely affect the dietary energy available from forage during antimicrobial therapy and may therefore impact performance.

Another study that compared the effect of penicillin, ceftiofur and trimethoprim sulfadiazine (TMS) on the gut microbiota in horses using next-generation sequencing showed that TMS had the most profound impact on the microbiota, in particular the phylum verrucomicrobia. This same study also reported a significant decrease in bacterial richness and diversity of the fecal microbiota. A reduction in bacterial diversity is certainly a trend that is commonly seen in gastrointestinal disease in horses. The restoration of the normal gut microbiota after completion of antibiotic treatment can take up to 40 days, but the organizational structure of the bacterial populations can take many years to re-establish the original structure map that was laid out in treating the pre-antibiotic gut. 

Equine studies certainly show similarities to human studies, indicating the consequences of antibiotics that can be seen across more than one species. Human studies have reported long-term consequences of antibiotic treatment on the human microbiota. One such human study investigated a seven-day clindamycin treatment and monitored the patients for two years. The impact on the human microbiota remained evident two years post-treatment, where a reduction in bacterial diversity and detection of high-resistance to clindamycin were detected. 

Interestingly, no resistant clones were detected in the control group over the two-year sampling period. Another study focusing on the effects of antibiotic treatment for Helicobacter pylori showed findings mirrored in similar studies of that field. The findings demonstrated the rapidly reducing bacterial diversity (one week) after antibiotic treatment and found that disturbances in the microbiota and high levels of macrolide resistance were evident four years post-treatment. Human studies may predict that equine studies will find similar trends with equine antimicrobial therapy. These studies highlight the impact of antibiotic use and the long-term persistence of antibiotic resistance remaining in the intestinal microbiome, which is a concern for both humans and animals. 

Antibiotics can lead to the selectivity and proliferation of resistant bacteria, which is evidenced by the long-term effects observed on the gut microbiota harboring drug-resistant encoded genes. Horizontal gene transfer (HGT) commonly occurs in the gut (can be up to 25 times more likely to occur in the gut than in other environments). HGT can be attributed to the close proximity of the microbiota in the gut, allowing the transfer of genetic material via routes such as plasmids and conjugation; in other words, the bacteria in the gut have developed a pathway to transfer antibiotic-resistant genes from one generation to another. Resistance to antibiotics is now a global issue for the treatment of many diseases. 

With the unfavorable association tied to Clostridium difficile infections (CDI) and the onset of colitis, particularly in mature horses treated with β-lactam antibiotics (commonly used for equine infections), the incidences in which antimicrobial therapy is considered should be minimized and only used if entirely necessary. The use of broad-spectrum antibiotics in recurrent presentations of symptoms of disease such as urinary tract infections in humans or diarrhea as a result of CDI in both humans and horses is promoting drug resistance. The antibiotics, by disrupting the gut microbiota (which act as a defense against the establishment and proliferation of such pathogenic bacteria) are allowing the opportunity of growth for these multi-resistant microorganisms such as C. difficile, vancomycin-resistant enterococci (VRE), and multi-resistant Staphylococcus aureus (MRSA). The organism C. difficile and its antibiotic resistance has been demonstrated in the treatment of CDI for both humans and animals. The introduction of vancomycin (a glycopeptide antibiotic) in 1959 for the control of CDI remained effective until the 1990s when a more virulent form of C. difficile emerged. This new form of C. difficile with reported broad-spectrum antibiotic resistance resulted in chronic conditions and increased human mortality. C. difficile is most noted with human hospital-acquired infections. C. difficile BI/NAP1/027 has been shown to have resistance to fluoroquinolone antibiotics, moxifloxacin and gatifloxacin, which was not seen in historical genotypes. As C. difficile infections are found to cause gastrointestinal disease in horses as well as humans, this is certainly of concern.

Alternative therapies to antibiotic therapy to restore or modulate the gut microbiome after a gut dysbiosis event could be considered in certain circumstances where antibiotics are no longer effective (e.g., CDI), if they’re not the best course (presence of extended-spectrum -β-lactamase (ESBL) producing  organisms) or if they’re not essential for example, when the diagnosis of the bacterial cause is uncertain. The rationale to using probiotic treatment along with antimicrobial treatment is that the antibiotic will target the pathogenic bacteria (e.g., C. difficile) and also the commensal microbiota of the gut, but the probiotic bacteria will help to re-establish the intestinal microbiota, and in turn, prevent the re-growth of the pathogenic bacteria in the case or residual spores of C. difficile surviving the antibiotic treatment. Alternative therapies such as fecal microbiota transplant (FMT) or probiotic solutions can reduce the risk of proliferation of antibiotic-resistant bacteria and also have fewer implications on the gut microbiome as evidenced by antibiotic use. 

Probiotics have been defined by the Food and Agricultural Organization (FAO) and the World Health Organization (WHO) as “live non-pathogenic microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” The word probiotic is Greek in origin, meaning, “for life”; and the term was coined by Ferdinand Vergin in 1954. While the mechanisms of action of probiotics are complex and require a deeper knowledge of the modulations of the gastrointestinal microbiota, and the health benefits due to their use are the subject of some debate, there is no doubt that probiotics are considered by many as a vital resource to human and animal health.   

The use of probiotics in animal production, particularly in intensive swine and poultry production, has increased in recent years, primarily as an alternative to the use of antimicrobials in the prevention of disease. The problem of antibiotic resistance and antimicrobial residues in food-producing animals (the horse is considered a food-producing animal), as a result of historical antibiotic use with the corresponding reduction in antibiotic efficacy in humans, leads to having to look at more sustainable options, such as probiotic use, to combat disease. Probiotics in horses are predominantly used as a treatment modality in the gastrointestinal microbial populations to combat illnesses such as diarrhea—to prevent diarrhea (particularly in foals) or help improve digestibility.  Shifts or fluctuations in the microbial populations of the equine gastrointestinal tract have been associated with diseases such as laminitis and colic.  

Gut dysbiosis, as mentioned previously, is a fluctuation or disturbance in the population of microorganisms of the gut. It is now being recognized as a cause of a wide range of gastrointestinal diseases; and in horses, it is one of the leading causes of mortality. The ability of probiotics in conferring health benefits to the host can occur via several different mechanisms: 1) inhibiting pathogen colonization in the gut by producing antimicrobial metabolites or by competitive exclusion by adhering to the intestinal mucosa, preventing pathogenic bacteria attachment by improving the function and structure; 2) protecting or restabilizing the commensal gut microbiota; 3) protecting the intestinal epithelial barrier; 4) inducing an immune response.

It is known that there are a wealth of factors that will adversely affect the gut microbiome: antibiotics, disease, diet, stress, age and environment are some of these compounding contributors. To mirror one researcher’s words, echoing from an era where antibiotics were used as growth promoters in the animal industry, “The use of probiotic supplements seeks to repair these deficiencies. It is, therefore, not creating anything that would not be present under natural conditions, but it is merely restoring the flora to its full protective capacity.” In the case of using concurrent antibiotic and probiotic treatment, this strategic tweaking of the microbiota could be used as a tool to prevent further disease consequences and perhaps help improve performance in the horse.

The benefits of probiotic use in horses have not been investigated extensively, but as mentioned previously, they are now being focused upon by researchers in the equine field. The most common bacterial strains used in equine probiotic products are Lactobacillus, Bifidobacterium, Streptococcus, Enterococcus, Bacillus and yeast strains of Saccharomyces. Lactobacillus, Bifidobacterium and Enterococcus strains typically account for less than 1% of the microbiota’s large gastrointestinal populations. Regulation is lacking regarding labeling of probiotic products, often not displaying content with clarification and quality control (such as confirmed viability of strain[s]) not excised with over-the-counter probiotic products. There is evidence to suggest that host-adapted strains of bacteria and fungi enjoy a fitness advantage in the gut of humans and animals.  Therefore, there may be an advantage in using the individual animal’s own bacteria as potential probiotics. Probiotics and antibiotics used concurrently could be the way to minimize the introduction of antibiotic-resistant bacterial strains in the gut, and in turn, protect future antibiotic efficacy.