Nutrition and the new science of the "Gut-Brain connection"

Article by Scott Anderson

Trainers are always looking to gain an edge in performance. But what about their mental state? Are they jittery, distracted or disinterested? No matter how strong the horses, their heads must be in the game to succeed.

Surprisingly, much of that mental attitude is driven by gut health, which in turn depends on the collection of microbes that live there: the microbiota. In a horse, the microbiota is a tightly packed community of about 100 trillion microbes, composed of bacteria, archaea, fungi and protozoa. It colonises the entire GI tract but is largely concentrated in the hindgut, where it works to ferment the prebiotic fibre in forage. The microbial fermentation of fibre into fatty acids produces 70% of the animal’s energy requirements and without it, the horse couldn’t get sufficient energy from simple forage. Intriguingly, byproducts of that fermentation can affect the brain. 

It is easy to be sceptical about this gut-brain connection, but over the last decade, research has made it clear that gut microbes have an outsized influence on mood and behaviour. Microbes that improve mental state are called psychobiotics, and they may completely change the way you train and manage your horses. A horse’s health – and consequently its performance – starts in the gut.

Inflammation

When the microbiota is unbalanced by stress, diet or sickness, it is said to be dysbiotic. It loses diversity, and a handful of bacterial species compete for domination. Without the pushback of a diverse population, even beneficial bacteria can become pathogenic. Surprisingly, that can affect the brain. Multiple studies in various animal models have shown that transmitting faecal matter from one animal to another also transmits their mood. This demonstrates that a dysbiotic microbiota can reliably cause mental issues including anxiety and depression, thereby affecting performance. 

An important function of the microbiota is to fight off pathogens by outcompeting, starving or killing them. However, a dysbiotic microbiota is less diligent and may permit pathogens to damage the gut lining. A degraded gut lining can leak, allowing bacteria and toxins into the bloodstream. The heart then unwittingly pumps them to every organ in the body, including the brain. This makes the gut the primary source of infection in the body, which explains why 80% of the immune system is located around the intestines. Over time, a leaky gut can lead to chronic systemic inflammation, which weakens the blood-brain barrier and interferes with memory, cognition and mood. 

Inflammation is a major component of the gut-brain connection, but not the only one.

Neurotransmitters and hormones

Horses and humans use neurotransmitters to communicate between nerve cells. Brains and their attendant nerve bundles constitute a sophisticated network, which makes it somewhat alarming that microbes also produce neurotransmitters. Microbes use neurotransmitters to converse with each other, but also to converse with their host. The entire gut is enmeshed in nerve cells that are gathered up into the vagus nerve that travels to the brain. Microbial neurotransmitters including serotonin and dopamine thus allow certain microbes to communicate directly with the brain via the vagus nerve. We know this happens with specific bacteria, including Lactobacillus species, because when the vagus is severed, their psychobiotic effects disappear. 

As well as neurotransmitters, hormones are involved in gut-brain communications. The hypothalamus-pituitary-adrenal (HPA) axis controls the stress response in animals. The hypothalamus is located low in the brain and responds to stressors – such as a lurking predator – by producing hormones that stimulate the neighbouring pituitary, which then triggers the adrenal gland to produce cortisol, the stress hormone. Cortisol acts as a threat warning and causes the horse to ramp up glucose production, supplying the energy needed to escape a predator. This is the same hormonal circuit that trainers exploit for racing.

The HPA axis produces cortisol in response to stress. Cortisol inhibits the immune system, which in combination with a leaky gut allows pathogens to enter the bloodstream. Susequent systemic inflammation and vagal feedback lead to stereotypies.

The production of these hormones redirects energy to the heart, lungs and muscles at the expense of the immune system. From an evolutionary point of view, the tradeoff makes sense: first escape the predator and deal with infections later. After the danger has passed, cortisol causes the HPA to return to normal – the calm after the storm. 

However, continued stress disrupts that cycle, causing anxiety and diminishing the brain’s ability to store memories. This can dramatically interfere with training. Stress can also induce the release of norepinephrine, which promotes the growth of pathogenic bacteria including Campylobacter jejuni, Listeria, Helicobacter pylori, and Salmonella. Prolonged high cortisol levels can increase gut leakiness, potentially leading to infection and further compounding the situation. In the long term, continued stress leads to systemic inflammation, which is a precursor to problematic behaviours.

Short-chain fatty acids

When microbes consume proteins and fibre, they break them down into their constituent molecules, such as amino acids, fatty acids and sugars. These are the metabolites of the microbes. As well as neurotransmitters and hormones, the gut-brain conversation is mediated by metabolites like butyrate, an important short-chain fatty acid which plays multiple roles in the body. 

In the gut, butyrate serves as a preferred nutrient for the cell lining. It encourages the differentiation of stem cells to replenish gut cells that are routinely sloughed off or damaged. It plays an important role in the production of mucus – an essential part of gut protection – which coats the gut from mouth to anus. In the muscles, butyrate boosts the growth of skeletal muscle, crucial to athletic performance, as well as inducing the production of glucose, the primary muscle fuel. One-quarter of systemic glucose is driven by butyrate. In its gut-brain role, butyrate passes through the blood-brain barrier, where it nourishes and enhances the growth of new brain cells. 

These factors make butyrate a star player in the gut-brain connection. They also highlight the benefits of prebiotic fibre, especially when high-energy, low-fibre feeds are provided.

Starting a microbiota

We’ve explored the major pathways of the gut-brain connection: inflammation, neurotransmitters, hormones and fatty acids. Some of these pathways are at odds with each other. How does such a complicated system come together?

As mentioned, the microbiota is an animal’s first line of defence against pathogens, attacking and killing them often before the immune system is even aware of them. That means a healthy microbiota is an essential part of the immune system. However, the immune system is designed to attack foreign cells, which includes bacteria. For the microbiota to survive, the immune system must therefore learn to accept beneficial microbes. This lesson in tolerance needs to take place early in the foal’s development, or its immune system may forever fight its microbiota.

There are multiple ways nature ensures that foals get a good start on a microbiota that can peacefully coexist with the immune system. The first contribution to a protective microbiota comes from vaginal secretions that coat the foal during birth. After birth, microbes are included in the mare’s milk. These microbes are specially curated from the mare’s gut and transported to the milk glands by the lymphatic system. Mare’s milk also includes immune factors including immunoglobulins that help the foal to distinguish between microbial friends and foes. An additional way to enhance the microbiota is through coprophagia, the consumption of manure. Far from an aberration, foals eat their mother’s manure to buttress their microbiota. 

Microbes affect the growth and shape of neurons in various brain sites as the foal develops – a remarkable illustration of the importance of a healthy early gut microbiota. 

The cooperation between the immune system and the microbiota is inevitably complex. Certain commensal bacteria, including Clostridiales and Verrucomicrobia, may be able to pacify the immune system, thus inhibiting inflammation. This is a case where microbes manage the immune system, not the other way around. These convoluted immune-microbial interactions affect the mental state – and consequently the behaviour – of the horse, starting at birth.

Stereotypies

A 2020 study of 185 performance horses conducted by French researchers Léa Lansade and Núria Mach found that the microbiota, via the gut-brain connection, is more important to performance than genetics. They found that microbial differences contributed significantly to behavioural traits, both good and bad. A diversified and resilient microbiota can help horses better handle stressors including stalling, training, and trailering. A weakened or dysbiotic microbiota contributes to bad behaviours (stereotypies) and poor performance. 

The horses in this study were all carefully managed performance horses, yet the rates of stereotypies were surprisingly high. A kind of anxiety called hypervigilance was observed in three-quarters of the horses, and almost half displayed aggressive behaviour like kicking or biting. 

The study found that oral stereotypies like biting and cribbing were positively correlated with Acinetobacter and Solibacillus bacteria and negatively correlated with Cellulosilyticum and Terrisporobacter. Aggressive behaviour was positively correlated with Pseudomonas and negatively correlated with Anaeroplasma. 

Some of these behaviours can be corrected by certain Lactobacillus and Bacteroides species, making them psychobiotics. That these personality traits are correlated to gut microbes is truly remarkable. 

Intriguingly, the breed of a horse has very little impact on the makeup of its microbiota. Instead, the main contributor to the composition of the microbiota is diet. Feeding and supplements are thus key drivers of the horse’s mental state and performance. 

The gut-brain connection and training

How might the gut-brain connection affect your training practices? Here are some of the unexpected areas where the gut affects the brain and vice-versa:

High-energy feed. Horses evolved to subsist on low-energy, high-fibre forage and thus have the appropriate gut microbes to deal with it. A high-energy diet is absorbed quickly in the gut and can lead to a bloom in lactic acid-producing bacteria, which can negatively impact the colonic microbiota. High-energy feeds are designed to improve athletic output, but over time, too much grain can make a horse antisocial, anxious and easily spooked. This can damage performance—the very thing it is trying to enhance. Supplementary prebiotics may help to rebalance the microbiota on a high-starch regimen.

Changing feed regimens quickly. When you change feed, certain microbes will benefit, and others will suffer. If you do this too quickly, the microbiota can become unbalanced or dysbiotic. Slowly introducing new feeds helps to prevent overgrowth and allows a balanced collection of microbes to acclimate to a new regimen. 

Stress. Training, travelling and racing all contribute to stress in the horse. A balanced microbiota is resilient and can tolerate moderate amounts of stress. However, excessive stress can lead, via the HPA axis, to a leaky gut. Over time, it can result in systemic inflammation, stereotypies and poor performance.

Overuse of antibiotics. Antibiotics are lifesavers but are not without side effects. Oral antibiotics can kill beneficial gut microbes. This can lead to diarrhoea, adversely affecting performance. The effects of antibiotics on the microbiota can last for weeks and may contribute to depression and anxiety. 

Exercise and training. Exercise has a beneficial effect on the gut microbiota, up to a point. But too much exercise can promote gut permeability and inflammation, partly due to a lack of blood flow to the gut and consequent leakiness of the intestinal lining. Thus, overtraining can lead to depression and reduced performance.

Knowing how training affects the gut and how the gut affects the brain can improve outcomes. With a proper diet including sufficient prebiotic fibre to optimise microbiota health, a poor doer can be turned into a model athlete. 

References

Mach, Núria, Alice Ruet, Allison Clark, David Bars-Cortina, Yuliaxis Ramayo-Caldas, Elisa Crisci, Samuel Pennarun, et al. “Priming for Welfare: Gut Microbiota Is Associated with Equitation Conditions and Behavior in Horse Athletes.” Scientific Reports 10, no. 1 (May 20, 2020): 8311.

Bulmer, Louise S., Jo-Anne Murray, Neil M. Burns, Anna Garber, Francoise Wemelsfelder, Neil R. McEwan, and Peter M. Hastie. “High-Starch Diets Alter Equine Faecal Microbiota and Increase Behavioural Reactivity.” Scientific Reports 9, no. 1 (December 9, 2019): 18621. https://doi.org/10.1038/s41598-019-54039-8.

Lindenberg, F., L. Krych, W. Kot, J. Fielden, H. Frøkiær, G. van Galen, D. S. Nielsen, and A. K. Hansen. “Development of the Equine Gut Microbiota.” Scientific Reports 9, no. 1 (October 8, 2019): 14427.

Lindenberg, F., L. Krych, J. Fielden, W. Kot, H. Frøkiær, G. van Galen, D. S. Nielsen, and A. K. Hansen. “Expression of Immune Regulatory Genes Correlate with the Abundance of Specific Clostridiales and Verrucomicrobia Species in the Equine Ileum and Cecum.” Scientific Reports 9, no. 1 (September 3, 2019): 12674. 

Daniels, S. P., J. Leng, J. R. Swann, and C. J. Proudman. “Bugs and Drugs: A Systems Biology Approach to Characterising the Effect of Moxidectin on the Horse’s Faecal Microbiome.” Animal Microbiome 2, no. 1 (October 14, 2020): 38.

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 diarrhoea, 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 faeces 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 diarrhoea 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 faecal 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 organisational structure of the bacterial populations can take many years to re-establish the original structure map that was laid out in the gut pre-antibiotic treatment. 

The equine studies certainly show similarities to the 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 7-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 harbouring 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 unfavourable 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 minimised 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 diarrhoea 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 defence 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), nor may they not be the best course (presence of Extended-spectrum -β-lactamase producing (ESBL) organisms) nor 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 faecal microbiome 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 Organisation (FAO) and the World Health Organisation (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 diarrhoea—to prevent diarrhoea (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 is now being recognised 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 colonisation 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 restabilising the commensal gut microbiota; 3) protecting the intestinal epithelial barrier; 4) by inducing an immune response.

It is known that there is 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 consequence 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 large gastrointestinal populations.

Regulation is lacking regarding labelling 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 minimise the introduction of antibiotic-resistant bacterial strains in the gut, and in turn, protect future antibiotic efficacy. 

No foot no horse - the influence of diet on the hoof

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First published in European Trainer issue 57 - April '17 - June '17