Canine microbiome dysbiosis

Introduction

The intestinal microbiome is the name given to the collective genome of all microbes (i.e., bacteria, viruses, fungi, and protozoa) in the gastrointestinal (GI) tract, with bacteria being the most abundant constituent. The microbiome can be seen as both a component of the immune system and as a metabolic entity, as the bacteria produce metabolites that affect both the GI tract and other body organs. Dysbiosis is the name given to changes that occur in the microbiome during disease, and encompasses a reduction in microbiome diversity (i.e., number of different bacteria), changes in quantities of bacteria, and functional changes (e.g., altered production of bacteria-derived metabolites). Dysbiosis often occurs secondary to underlying pathologies within the intestine and will contribute to the clinical signs in some patients 1; because of this it is an additional marker for intestinal disease and should be assessed along with a patient’s overall history and clinical presentation. Therapy for dysbiosis should aim to address the underlying pathology, with dietary manipulation as the first-line treatment.

Microbiome function

Bacteria either directly produce (vitamins) or convert dietary (fiber, protein, fat) or host (bile acids) molecules into bacteria-derived metabolites, and therefore the microbiota exerts many beneficial effects on the host. Important metabolites include short chain fatty acids (SCFA), indoles and secondary bile acids; these have various effects, including anti-inflammatory actions, modulation of intestinal motility, inhibition of enteropathogens, improvement of gut barrier function, and increased mucin production 2. Dysbiosis, which is often secondary to various luminal factors (Box 1), leads to altered microbiota function which then contributes to clinical signs 1. Of particular interest to the regulation of microbiota are intestinal bile acids (BA). Briefly, primary BAs (cholic and chenodeoxycholic acids) are released into the small intestine after a meal to aid in fat digestion. Up to 95% of BA are reabsorbed in the ileum for enterohepatic circulation 3, with the remainder reaching the colon where they are converted by bacteria (mainly Clostridium hiranonis in dogs and cats) to secondary BAs 4. This conversion has important health consequences, as secondary BAs, in the correct quantity, have beneficial effects. They act as signaling agonists for various receptors across multiple organs, inducing anti-inflammatory and glucose-lowering effects and suppression of enteropathogens 5.

Box 1. Conditions and factors associated with intestinal dysbiosis.

Assessment of the microbiome

There are various options for assessing a dog’s microbiome, but some are more effective than others.

Bacterial culture

While still used by many veterinarians for the diagnosis of dysbiosis, bacterial culture of feces is not useful for microbiome assessment, as the majority of intestinal bacteria are strict anaerobes requiring special growth media (Figure 1). Consequently, only a small percentage of bacterial species can be cultured by diagnostic laboratories. In a recent study, different fecal aliquots from healthy dogs and dogs with chronic diarrhea were submitted to three veterinary reference laboratories for the evaluation of dysbiosis 6. There was no agreement in the culture results between laboratories, and dysbiosis was actually more frequently reported amongst the healthy dog group. This study demonstrates that bacterial culture should not be used for microbiota assessment in dogs with chronic diarrhea, except for specific pathogens such as Salmonella spp. 

Molecular sequencing of 16S rRNA genes

Molecular techniques based on sequencing of 16S rRNA genes provides comprehensive information on the microbial composition of a fecal sample, and are used in research settings. Various companies offer sequencing for microbiome assessment on a commercial basis for individual animals, but there are currently no standardized methods (e.g., DNA extraction, PCR primers used) between these laboratories. Because no reference intervals are defined for animals, and because each company has a different report, result interpretation is difficult. Furthermore, inter-assay variation is common, and no analytical validation data have been reported for these assays, therefore sequencing-based microbiome assessment is not currently recommended for individual patients.

Canine microbiota dysbiosis index (DI)

The dysbiosis index (DI) is a quantitative PCR based test that is currently commercially available in North America and Europe and is now used in many clinical studies 4,7 as it is the only validated assay to assess canine microbiome dysbiosis *. The DI measures the levels of seven intestinal bacteria (Box 2) which are commonly altered in dogs with chronic enteropathies (CE) or after broad-spectrum antibiotic use (e.g., tylosin, metronidazole) 8,9. The assay provides reference intervals for these bacterial groups and combines data into a single number that expresses the extent of the dysbiosis (Figure 2); a DI between 0 and 2 represents a moderate shift in the microbiota, whilst a DI > 2 indicates a major shift. The sensitivity and specificity of the method is shown in Box 3.

* https://tx.ag/DysbiosisGI

Box 2. The seven bacterial groups included in the canine Dysbiosis Index and how their levels alter in dysbiosis.

Box 3. The sensitivity and specificity of the Dysbiosis Index (DI) for chronic enteropathies; a DI between 0-2 represents a moderate shift in the microbiota, whilst above 2 indicates a major shift.

The DI also predicts, by assessing the concentration of C. hiranonis, the ability of the intestinal microbiota to convert primary BAs to secondary BAs 4. Normal amounts of secondary bile acids are antimicrobial and suppress potential enteropathogens such as C. difficile, C. perfringens, and E. coli 10, so reduced levels of C. hiranonis and decreased conversion of bile acids is strongly associated with intestinal dysbiosis and overgrowth of enteropathogens in dogs (Figure 2) 4,7,8,11. Identification of some or all of these enteropathogens in a dog with diarrhea will suggest overgrowth due to an underlying dysbiosis secondary to chronic enteropathy, rather than a primary infection. Up to 60% of dogs with a chronic enteropathy (CE) have decreased levels of C. hiranonis, and therefore decreased secondary BA 12.

The microbiome in disease

Table 1 summarizes the various ways in which intestinal bacteria can contribute to disease, although the underlying pathologies will vary between individual patients depending on the location and severity of intestinal damage. The microbiota is in contact with the mucus layer of the gut, the immune system, and luminal substrates, and changes in one or more of these will affect microbiota composition, so dysbiosis is often an early marker of an abnormal gut environment in disease (Figure 3).

Table 1. Mechanisms by which bacteria contribute to GI disease.

A dysbiosis restricted mainly to the gut lumen is often present in patients with exocrine pancreatic insufficiency (EPI) 13, after broad-spectrum antibiotic treatment 8,9, or in younger animals due to an immature immune system. Chronic enteropathies are accompanied by inflammation and destruction of the mucus layer and mucosal structure, leading to more oxygen at the mucosal surface, increased numbers of aerobic bacteria (E. coli), and a decrease in normal anaerobic flora. The loss of mucosal architecture that develops with CE leads to a lack of transporters for carbohydrates, amino acids, fatty acids and bile acids, resulting in malabsorption of these compounds 14. Increased amounts of these substrates in the GI lumen can directly lead to osmotic or secretory diarrhea, as well as bacterial overgrowth.

Due to disruption of the mucus layer covering the epithelium, dogs with CE have often increased number of mucosa-adherent bacteria 15. This is linked to a reduction in C. hiranonis and therefore abnormal bile acid conversion, allowing a secondary overgrowth of C. difficile and C. perfringens which can lead to increased pro-inflammatory host responses.

A diagnostic approach to dysbiosis

Since dysbiosis commonly develops secondary to a changed gut environment with intestinal disease and/or altered environmental factors, it should be assessed along with a patient’s medication history and the clinical presentation. Interpretation of the DI result should be done alongside the levels of the individual bacterial taxa, and especially C. hiranonis, as a decrease in the latter is a major contributor to an abnormal microbiome. A DI above 2 indicates dysbiosis with high specificity, while a DI in the equivocal range indicates a minor shift in the fecal microbiome. Some dogs with CE can have a DI < 0 but with some bacterial taxa outside the reference intervals, and this represents a minor form of dysbiosis. In general, an abnormal DI suggests underlying intestinal disease, and a workup for CE is therefore indicated.

Note that some drugs can influence the DI. For example, omeprazole can lead to a transient increase, but with normal levels of C. hiranonis, and the DI normalizes 1-2 weeks after therapy finishes. Broad-spectrum antibiotics (e.g., metronidazole and tylosin) can induce severe fecal dysbiosis (Figure 4), but again the microbiota typically normalizes within 2-4 weeks after administration ends in most dogs, although some individuals may have a persistent dysbiosis with lack of C. hiranonis for several months 8,11.

Compositional microbiota changes in the small intestine will often lead to detectable changes in the fecal microbiome as assessed by the DI. However, in some patients, an increased number of bacteria in the small intestine may cause disease. Small intestinal dysbiosis is suggestive if serum concentrations of folate are increased and serum cobalamin are decreased on a GI panel, but note that both markers have low sensitivity and specificity.

Therapy for dysbiosis 

Dysbiosis is often just one component of intestinal disease, and multi-modal therapy addressing the underlying cause is usually required. In some cases, such as in animals with EPI, treatment with pancreatic enzyme supplementation leads to improvement in clinical signs, and often the intestinal microbiome will normalize after several weeks 13, but in dogs with CE there are no markers that predict which treatment is best for an individual patient, so stepwise treatment trials are often necessary 16. Therapies for dysbiosis include dietary modulation, pre- and probiotics, antimicrobials, and fecal microbiota transplantation (FMT), with each approach addressing a different mechanism (Table 2); a combination of treatments will often offer the best success. 

Table 2. Treatment options for dysbiosis. 

Dietary changes should always be the first treatment option in stable patients. Various studies have shown that between 50-70% of dogs with CE are food-responsive 16, and highly digestible diets containing hydrolyzed or novel proteins are most commonly used. Most of these diets are hypoallergenic and reduce undigested nutrients in the GI lumen, decreasing the potential for bacterial overgrowth. In most cases of food-responsive enteropathy, the dietary change alone is sufficient to achieve clinical remission, leading to gradual improvement of intestinal inflammation and dysbiosis over several months 10,17. 

Probiotics can be administered alone in mild cases, or together with dietary modulation. Because the number of bacteria administered in any probiotic is small when compared to the existing gut microbiota, they have a minor direct impact on microbiota composition. However, they attach to the mucosa and can exert beneficial effects, including shortening the duration of acute diarrhea and reducing antibiotic-associated GI side effects such as vomiting or diarrhea 18. High-potency multi-strain probiotics have been shown to reduce C. perfringens in dogs with acute hemorrhagic diarrhea 19 and strengthen the intestinal barrier in dogs with CE 20. However, because many commercial products lack proper quality controls, it is important to choose a preparation that has shown efficacy in a published clinical study. 

Prebiotics are indigestible carbohydrates that promote growth of beneficial micro-organisms, and can be divided into soluble/insoluble and fermentable/non-fermentable fibers. Fermentable prebiotics are converted by colonic bacteria to SCFA. Most commercial GI diets contain prebiotics, but for some diseases (e.g., colitis) high-fiber diets can be beneficial. Addition of psyllium husk, a soluble fiber, to the diet at 0.5-1 g/kg bodyweight daily can improve stool quality in animals with large bowel disease. The product should be introduced at lower doses and titrated up to achieve the desired stool consistency. 

Antibiotics such as tylosin or metronidazole have been traditionally recommended for treatment of CE, but their first-line use is now debated 16. Although they can lead to an improvement in clinical signs, presumably due to a reduction in bacterial load, patients will often relapse after treatment as the bacteria regrow, as antibiotics rarely resolve the underlying disease process 15,21,22. Commonly used options include metronidazole (10-15 mg/kg q12h) and tylosin (25 mg/kg q12h) for 4-6 weeks, but as noted above, both drugs have been shown to induce large intestinal dysbiosis that can sometimes last for months 8,9,11. Studies report that metronidazole has promoted lasting dysbiosis in dogs with acute diarrhea 11, while amoxicillin-clavulanic acid can encourage an increase in resistant E. coli 23. Antibiotics are generally not recommended as first-line treatment in CE for a variety of reasons – only 10-16% of CE dogs are antibiotic-responsive, most cases relapse after treatment is withdrawn, and the drugs have negative effects on the microbiome. Antibiosis should, however, be considered after failed dietary and anti-inflammatory trials, or for patients with signs of systemic inflammation 16 and invasion and persistence of bacteria in the intestinal mucosa (e. g., E. Coli associated granulomatous colitis). A small subset of dogs with CE may respond to no other treatment, in which case long-term administration may be necessary, with the dosage tapered to the lowest effective point.

Fecal microbiota transplantation (FMT) can help restore the normal microbiota and improve clinical signs 11 in some cases of dysbiosis. The technique involves the transfer of stool from a healthy donor into the gut of a recipient via oral capsules, endoscopy or enema (Figures 5 and 6). In humans, FMT has a high success rate (> 90%) with infectious and recurrent C. difficile infection, but has more limited success for inflammatory bowel disease due to the chronic underlying intestinal inflammation.

FMT in animals is still an emerging therapy. A simple protocol is shown in Box 4, although to date only a few case series have been reported, with success apparently dependent on the underlying disease 24. The technique helps to restore bile acid metabolism by promoting levels of C. hiranonis (Figure 7), so it may be useful in dogs with abnormal BA conversion with associated overgrowth of enteropathogens such as C. difficile or C. perfringens and/or animals with antibiotic-induced dysbiosis and minor underlying damage of the intestinal mucosa. It has also been shown to improve fecal scores in cases of acute diarrhea and when used as an adjunct to standard antimicrobial therapy in puppies with parvovirus infection, and for young dogs with chronic diarrhea due to confirmed C. difficile infection 25.

Box 4. FMT protocol via enema (based on 24).

In dogs with CE, dysbiosis is often a secondary effect of the intestinal inflammation and structural damage, and recurrence of dysbiosis and clinical signs will occur if the underlying pathology is not eradicated. FMT therefore has a very variable success rate in CE, and anecdotal reports suggest many dogs with CE will have improved fecal scores within 2-3 days of treatment, but will relapse and develop recurrent diarrhea a few weeks later. Therefore, in these patients appropriate dietary and anti-inflammatory treatment of the underlying disease process is required (see above), and FMT can be considered as adjunctive treatment for patients that show a suboptimal response (e.g., continuing soft stools) despite standard therapies.

Conclusion

The intestinal microbiome plays a crucial role in host health and many animals with gastrointestinal disease will develop dysbiosis, resulting in abnormal microbial function which can contribute to clinical signs. The dysbiosis index is a useful diagnostic tool for many cases, but as there can be various underlying causes, a multi-modal and often long-term therapeutic approach is necessary to improve microbiota composition.