The complex microorganisms that live symbiotically in the gastrointestinal tract (GI) are implicated in the onset and continuation of many common diseases of mammals, including diarrhoea, gastric discomfort, gastric inflammation, arthritis, allergies and food sensitivities.
New technologies such as high throughput and next-generation sequencing have made the investigation and identification of these bacterial communities relatively easy, non-invasive and cost-effective. As a result, there is an increasing number of studies, better knowledge and greater clarity on the relevance and implications of disturbances (dysbiosis) in the microbiome and how they are involved in the onset of medical conditions.
The gut microbiome and disease
The community of trillions of bacteria in the GI tract provides support to all aspects of the digestive system, such as breaking down nutrients, producing short-chain fatty acids, making vitamins and providing energy to the host and the other gut bacteria.
Some bacteria are specialists in breaking down and digesting fibre, releasing bioactive peptides during the process. The peptides then send signals through the nervous and endocrine systems via the brain, affecting mood and temperament.
Alterations in the microbiome can be caused by medication (especially antibiotics), stress and changes in diet. The resulting changes cause an imbalance between the good, health-supporting gut bacteria and the bad bacteria associated with diseases and infections, giving rise to a variety of initial clinical signs, including diarrhoea, constipation, vomiting, bloating, food sensitivities/allergies and weight gain or loss.
Changes [in the gut microbiome] cause an imbalance between the good, health-supporting gut bacteria and the bad bacteria associated with diseases and infections
The microbiota does not only influence the GI system; long-term changes in the bacterial community are linked to organs in other parts of the body and include:
- chronic kidney and heart disease (Summers et al., 2019)
- metabolic dysfunctions, including obesity (Bermudez Sanchez et al., 2020)
- neurological disorders such as meningoencephalomyelitis in dogs (Jeffery et al., 2017) and small cell lymphoma in cats (Marsilio et al., 2019)
Bacteria have very specific roles and benefits: some are known to have anti-inflammatory properties, while others help restore the gut wall barrier, regulate gut motility, modulate the immune system and regulate secondary bile acid production (Suchodolski, 2022).
Examples in the literature
A clear example of the links between gut bacteria and disease is described by Jeffery et al. (2017), who found that the abundance of two species of bacteria was associated with the common disorder meningoencephalomyelitis (MUO) in dogs. MUO is an immune-mediated condition, and gut bacteria are known to modulate responses, possibly increasing or decreasing susceptibility to the disease.
The study compared the abundance of two identified members of the microbiome, Faecalibacterium prausnitzii and Prevotellaceae,in 20 healthy dogs and 20 with MUO. They found that Prevotellaceae were significantly less abundant in MUO cases compared to the controls (p = 0.003). However, there was no difference in the abundance of F. prausnitzii, providing strong validation that a higher percentage of Prevotellaceae protects against the onset of immune-mediated brain disease.
Efficiency of new technology
Next-generation sequencing (NGS) of the 16S and ITS ribosomal RNA identifies strains of bacteria that might otherwise remain unidentified using traditional methods. This allows us to create a complete analysis of the gut bacteria community with a culture-free method from one faecal sample. With NGS, it is possible to do multiple samples on one run and achieve genus-level accuracy to identify fungi (mycobiome) and viruses (virome).
Next-generation sequencing of the 16S and ITS ribosomal RNA identifies strains of bacteria that might otherwise remain unidentified using traditional methods
The data produced by this technology is large and complex, and interpretation benefits from the use of other new technologies such as machine learning and artificial intelligence – both of which can interpret the results and convert the raw data into a user-friendly report covering health, diet and disease.
These tools can identify markers of disease and potential disease factors (infections/pathogens) and help develop nutritional strategies or other therapies (pro-/prebiotics to restore health to the microbiome).
Why is gut microbiome diversity important?
Measuring and scoring bacteria biodiversity can be a relatively simple way to assess the health status and homeostasis of the gastrointestinal tract, especially as the dog ages. Loss of biodiversity is an indicator of an unhealthy microbiome and is associated with imbalances, where the pathogenic or unfavourable opportunistic bacteria increase to above-average percentages, thus increasing the risk of other comorbidities (Deng et al., 2019). Having a good biodiversity score can also favour the species of good gut bacteria associated with a healthy ageing process, such as Fusobacterium perfoetens (You and Kim, 2021).
One study in dogs found that a higher body condition score has the same effect on the loss of biodiversity in the gut microbiota, repressing the good gut bacteria in dogs (with no other clinical signs) and increasing the percentage of opportunistic bacteria (You and Kim, 2021).
Measuring diversity – the Shannon index
Biodiversity is measured using the Shannon index (Figure 1), and having a good score is correlated to a healthy gut community and resilience against disease and allergies. Having a diverse and varied population will ensure healthy digestion with enough beneficial microbes to help absorb the essential nutrients and regulate the immune, metabolic and nervous systems. A high (in the green) Shannon index score means a healthier animal.
This index is a mathematical calculation of the number of different species in the sample – ranging from 600 to 900 for cats and dogs and 800 to 1,500 for horses – together with an equation to measure the proportion or richness of the species. The “richness” of the species refers to how many of each different species there are and how many there are in comparison to other bacteria. For example, if one species of bacteria is present in high numbers (perhaps accounting for 80 percent of the microbiome with a total of 800 species), the rest would be present at very low percentages (eg 0.001 percent of the biome). The result would be a lower biodiversity score (Figure 1A). On the other hand, a high Shannon index score (Figure 1B) would indicate a greater number of individual species and a more even representation.
Measuring diversity – the dysbiosis index
Sequencing is a reliable method of identifying the complex microbial communities that exist in the ears, lungs, skin, conjunctiva, respiratory tract, genitourinary tract and gastrointestinal tract of animals. Recently, sequencing has been used by clinicians to identify resistant pathogens, zoonotic pathogens and infection control in humans.
In 2017, a dysbiosis index was developed for cats and dogs with chronic inflammatory enteropathies (CIE) and used in clinical studies (Al Shawaqfeh et al., 2017). This index focuses on seven species of bacteria that are routinely altered in chronic gastric inflammation, enteropathies and during/following broad-spectrum antibiotic use. The dysbiosis index includes a diversity score and uses quantitative PCR assays rather than genomic sequencing. Each species of bacteria is given a reference interval to indicate the normal average range, based on a canine population from Texas, USA, which ate a variety of diets.
Results may differ depending on the geographical location (Porras et al., 2021) of the dog population being tested. For example, pro-inflammatory Escherichia coli, one of the seven bacteria identified by the index that increases in dogs with chronic enteropathy, has an average range (reference interval) of 0.9 to 8.00. However, the average from 500 normal healthy dogs in a UK-based population is much lower (0.05 to 0.40). The reference interval range for E. coli in the cat is also lower. These geographical differences are likely to alter the dog’s susceptibility to developing disease or enteropathy.
The health of the microbiome relies upon the contribution of good gut bacteria, one of which is Clostridium hiranonis. C. hiranonis is thought to be a major benefactor of gastric health, and a reduction of this species in the gut microbiome is thought to contribute the most to a major shift in the other six species in the dysbiosis group. C. hiranonis converts primary bile acid into secondary bile acid which has antimicrobial properties for reducing and controlling enteropathic bacteria such as Clostridium difficile, Clostridium perfringens and E. coli. A reduction of C. hiranonis is often the result of antibiotic use.
How does the gut microbiome protect against pathogenic infection and inflammation?
The gut bacteria population provides the first line of defence against the onset of gastric infections and inflammation (Wiertsema et al., 2021), which is a common occurrence in cats and dogs (Marks et al., 2011). In order to cause pathogenic infection, there are three barriers that pathogens must overcome: the intestinal microbiota, the epithelium and the mucosal immune system.
The intestinal microbiota
Microbes have evolved alongside the host, are inherited from the dam and contribute to the protection of the host from pathogenic infection or overgrowth. If the commensal bacterial community is disturbed in any way, pathogens and opportunistic bacteria can thrive and grow.
Anxiety, stress, antibiotics, [NSAIDs] and agrichemicals cause the most damage to the intestinal microbiota because they allow pathogens to colonise, overgrow and persevere
Anxiety, stress, antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs) and agrichemicals cause the most damage to the intestinal microbiota because they allow pathogens to colonise, overgrow and persevere. The pathogens and harmful biofilms compete against the commensal bacteria for nutrients and space in the biome, causing the host’s health to reduce immediately as they no longer have access to vital nutrients.
The good gut bacteria and the host mount a response and use a mechanism called quorum sensing to continuously scan the microbial community. When increasing levels of pathogens are sensed, quorum sensing works to alter the phenotype of bacteria, causing an increase in protective compounds to help restore homeostasis. However, long-term medication will eventually overwhelm this process, causing the pathogens to overcome the first barrier, triggering low-grade inflammation, reduced resistance to colonisation of the gut and an increased susceptibility to infection.
The epithelium
The epithelium is a physical barrier that protects against disease by preventing pathogens from crossing the gut wall to access, infect and damage internal systems. This barrier uses a set of gates called tight junctions as a prevention mechanism. The dynamics of the tight junctions can, however, be disrupted by an increase in toxin-secreting pathogens as they overcome the intestinal bacteria.
The mucosal immune system
The mucosal immune system is the lining of the epithelium, and its role is to prevent pathogenic bacteria from interacting with the physical barrier. Seventy to eighty percent of the immune system cells live in the gut. This is for good reason, because most disease starts there. The immune cells live in the gut wall barrier and protect against an invasion of pathogens.
Mucus has a reservoir for antimicrobial agents produced by the host. The production of antimicrobial agents is regulated by the host through a sensing system that recognises the patterns and metabolites released by bacteria.
There is continuous interaction between the gut bacteria and the epithelium, with continuous signalling between the two to ensure gut homeostasis. Inflammation and infection are the result when this system is overwhelmed or disrupted by increased pathogens or the growth of a biofilm, which takes over the space reserved for the good gut bacteria.
How can we separate true pathogens from beneficial ones?
One of the biggest benefits of using full genomic sequencing technology is that multiple pathogenic species can be not only identified but quantified as a percentage of the microbiome. Figure 2A indicates the averages calculated between population data (Petbiome, n.d.) from healthy dogs (light blue) and dogs affected by gastric discomfort (dark blue) to determine the number of “true pathogens”.
Animals have very individual and complex microbial profiles and being able to identify all the pathogenic species in one analysis is of greater benefit than identifying a smaller set of bacteria (PCR). Determining the averages from a large population set of healthy dogs is also crucial as pathogens exist in the microbiome of all animals and are thought to contribute to health when present in small numbers (Figure 2B) by priming the immune system (Mogensen, 2009).
There are many other pathogenic species and emerging zoonotic species that can now be identified with genomic sequencing, including known toxin-producing and tissue-destroying species from the genus Clostridium. An individual dog can harbour 10 to 70 different species of Clostridium (Petbiome, n.d.), and a high percentage of them are beneficial; however, others produce toxic metabolites (Popoff and Brüggemann, 2022) that have a negative effect on the health and vitality of the host (Figure 2C).
The importance of the microbe–gut–brain axis
There is bidirectional communication between the gut microbiota and the brain, which is mediated by endocrine (cortisol), immune (cytokines) and neural (vagus nerve and enteric nervous system) pathways (Figure 3). These pathways involve (Guo et al., 2019):
- Acetylcholine
- Adrenocorticotropic hormone
- Circular muscle
- Corticotropin-releasing factor
- Longitudinal muscle
- The myenteric plexus
- Short-chain fatty acids
- The submucosal plexus
Symptoms of widespread muscular/joint pain, anxiety, nervousness and depression are common in dogs. But new research has indicated that the onset of these symptoms may originate from an imbalanced microbial community in the gut causing changes through the microbe–gut–brain axis. This axis is crucial for creating and maintaining the health of the entire body. If the balance of the gut bacteria community is altered (through diet, environment and/or stress) the result can be an increased production of chemicals that affect the nervous system, promoting pain and sensitivity (Malatji et al., 2017).
What happens when there are imbalances in the gut microbiome?
Gut bacteria can produce secondary metabolites that either support good health or are detrimental to good health. Having too many bacteria that produce toxic/harmful chemicals will alter the homeostasis of the animal and cause symptoms to appear in other organs of the body away from the gut as they react to higher levels of chemicals that are not good for them.
Though all the bacteria listed below are part of the core family of microbes present in most animals, an increase of any can cause an imbalance that can have devastating effects on the homeostasis of the gut and the rest of the body.
- Eubacterium spp, Faecalibacterium prausnitzii, Ruminococcus spp, Clostridium spp andActinomycetaceae: these bacteria reside predominantly in the small intestine and produce hippuric acid. While hippuric acid production is a normal and important part of metabolism, an excess (which can be measured in urine) is an indication of an unnatural (detoxification) process by the gut
- High levels of lactic acid-producing bacteria such as Lactobacillus. Lactic acid production is part of many metabolic and biochemical processes in all mammals, but increases in production are linked to illness, trauma or poor diet
- Higher percentages of Fusobacterium nucleatum and Faecalibacterium spp cause an increase in 2-hydroxyisobutyric acid (the most damaging of the three chemicals described here), which is chemically similar to lactic acid. Higher levels of fusobacteria only occur in dogs under stress. A high percentage of fusobacteria is also linked to inflammatory bowel disease
A rise in lactic or 2-hydroxyisobutyric acid sets up a process to restore a normal pH away from the acidic environment, but in the process causes the release and increase of chemicals that irritate the nervous system, triggering pain and fatigue.
If the gut microbiome and chemical imbalance is not rectified then the persistent, chronic noxious stimulant produced by the metabolites mentioned above can sensitise the nervous system
If the gut microbiome and chemical imbalance is not rectified then the persistent, chronic noxious stimulant produced by the metabolites mentioned above can sensitise the nervous system and eventually make three long-term changes that cause chronic pain to perpetuate. These are:
- The growth of extra nerves
- Nerves that increase their area of innervation (creating oversensitivity to touch)
- Nerves that become more sensitive to pain stimuli
