The Dysbiosis Index (DI) is a PCR based assay that quantifies the abundances of 7 bacterial groups and total bacteria and summarizes them in one single number [1].
The DI allows veterinarians to assess whether a dog has a normal or abnormal fecal microbiota composition. This assay has been used in more than 30 clinical studies, and we have established reference intervals based on healthy dogs from various countries worldwide (Table 1).
As a secondary interpretation, based on the abundance of Clostridium hiranonis, the assay can predict normal or abnormal conversion of bile acids in the intestine (i.e., predicts a lack of conversion of primary to secondary bile acids), as the lack of secondary BA is a major contributor to an abnormal microbiota.
Table 1. Reference intervals
Normal abundance
Change observed in dysbiosis
Faecalibacterium
3.4 – 8.0
decreased
Turicibacter
4.6 – 8.1
decreased
Streptococcus
1.9 – 8.0
increased
E. coli
0.9 – 8.0
increased
Blautia
9.5 – 11.0
decreased
Fusobacterium
7.0 – 10.3
decreased
C. hiranonis
5.1 – 7.1
decreased
Dysbiosis Index
< 0 normal
0-2 equivocal
> 2 dysbiosis
Data expressed log DNA/gram of feces
Sample requirements
Approximately 1 gram of feces (approximately the size of one grape) is needed. Samples must remain cold until they reach the lab. Ship samples by overnight courier with frozen gel ice packs. Samples can be stored in the refrigerator over the weekend if you cannot ship them by Thursday (lab personnel are not here at the weekend to receive samples). Results will typically be reported within 2 days.
The microbiota is an important immune and metabolic contributor to health. Changes in the microbiota have been associated with various intestinal disorders. The conversion of primary to secondary bile acids (BA) by intestinal bacteria appears to be of particular importance. In the canine colon, primary BA are converted by C. hiranonis into secondary BA. These secondary BA have local and systemic anti-inflammatory properties, activate various receptors in other organ systems, and play a crucial role in suppression of potential enteropathogens such as C. difficile, C. perfringens, and E. coli [2]. Consequently, a decreased abundance of C. hiranonis, and therefore decreased conversion of primary to secondary BA, is strongly associated with intestinal dysbiosis in dogs (Figures 1 and 2) [3; 4; 5].
Increased Dysbiosis Index
The DI should always be interpreted together with the abundance of C. hiranonis. A DI below 0 indicates a normal microbiota. A DI between 0 and 2 is equivocal, as some animals may have individual bacterial taxa outside the reference intervals, indicating a minor shift in the microbiota. In such cases, assessment of a follow-up sample a few weeks later can be considered. A DI >2 indicates microbiota dysbiosis. Most of these dogs will have a decreased abundance of C. hiranonis, indicating abnormal conversion of primary to secondary BA, and the lack of secondary BA is a major contributor to an abnormal microbiota.
An increased DI together with decreased abundance of C. hiranonis is commonly seen in dogs with digestive disease such as exocrine pancreatic insufficiency (EPI) and chronic enteropathies (CE) (Figure 2). Some dogs with acute diarrhea will also show a transient increase in the DI, but the microbiota typically normalizes within a couple of weeks. A persistent increase in the DI may suggest presence of an underlying intestinal disease, and a workup for CE may be indicated.
Some environmental factors have been shown to induce intestinal dysbiosis. Proton-pump inhibitors (e.g., omeprazole) lead to an increase in the DI. However, dogs on omeprazole do typically have a normal abundance of C. hiranonis [6]. The DI decreases within 14 days after the cessation of proton-pump inhibitor therapy. Similarly, some dogs fed bones and raw food (BARF) diets may show an increased DI, but normal abundance of C. hiranonis, especially if those diets contain high protein and high fat but low fiber, which induces increases in E. coli and reductions in short-chain fatty acid producing bacteria such as Faecalibacterium [7].
Recent administration of broad-spectrum antibiotics (e.g., metronidazole or tylosin) also induces major shifts in the microbiota and an increase in the DI, with a decrease in C. hiranonis. While the microbiota typically normalizes within several weeks after end of administration, some dogs may have a persistent dysbiosis with lack of C. hiranonis [3; 4; 8].
Figure 1. The Dysbiosis Index (DI) in clinically healthy dogs. A small subset of clinically healthy dogs has an increased DI and most of these dogs have a decreased abundance of C. hiranonis (in red). Some of these dogs may have persistent dysbiosis due to prior antibiotic exposure, as broad-spectrum antimicrobials (e.g., metronidazole, tylosin) have been shown to decrease C. hiranonis for weeks to months.
Figure 2. The DI in dogs with chronic enteropathy (CE), exocrine pancreatic insufficiency (EPI), and healthy dogs on omeprazole and fed bones and raw food (BARF) diet. Some dogs with gastrointestinal disease have an increased DI which is highly associated with decreased abundance of C. hiranonis (red dots). In contrast, healthy dogs on omeprazole and BARF diets have a normal abundance of C. hiranonis.
Therapeutic approach to microbiota dysbiosis
History and clinical presentation are important for a proper therapeutic approach to dysbiosis. Medications such as antimicrobials and proton-pump inhibitors can explain an increased DI in dogs that have otherwise no signs of intestinal disease. In most dogs the microbiota will normalize within weeks after end of therapy.
Dogs with positive results for C. difficile on an enteropathogen panel often have an increased DI and decreased abundance of C. hiranonis, as the lack of secondary BA allows the proliferation of the former. In such cases, dietary modulation with a highly digestible diet, and addition of probiotics or prebiotics (fermentable fibers) may help modulate the microbiota. In cases of persistent dysbiosis, a fecal microbiota transplant (FMT) can be considered (see below) [9].
In dogs with chronic enteropathies, intestinal dysbiosis is often a secondary component of the disease. Therefore, in these patients treatment should be primarily aimed to treat the underlying inflammatory enteropathy – please click here for more information about folate and small intestinal dysbiosis – https://vetmed.tamu.edu/gilab/research/folate-information/
Fecal microbiota transplantation (FMT)
FMT is the transfer of stool from a healthy donor into the gut of a recipient with the goal of treating intestinal dysbiosis. It can be done via oral capsules, nasogastric tube, endoscopy, colonoscopy, or enema.
FMT has a very high success rate to treat humans with C. difficile infections, and works in patients refractory to antibiotic therapy [10]. The therapeutic success is in part due to restoration of the normal microbiota and restoration of normal intestinal bile acid (BA) metabolism.
FMT is an emerging therapy in dogs, and consequently there are only a few published studies. The therapeutic success of FMT appears to depend on the underlying disease process. Recent data in dogs has shown that FMT may help in restoring bile acid metabolism by restoring C. hiranonis, the main BA-converting bacterial species in dogs (Figure 3) [3]. Therefore, FMT may be particular useful in dogs with abnormal BA conversion with associated overgrowth of enteropathogens such as C. difficile or C. perfringens as an alternative to antibiotic use [3; 9]. FMT has also been used as an adjunct to standard antimicrobial treatment in puppies with parvovirus infection, which showed faster resolution of diarrhea compared to those receiving only standard therapy [11].
As mentioned above, in dogs with chronic enteropathy, dysbiosis is often a secondary effect of the intestinal inflammation, and based on our own unpublished data, FMT has a very variable success rate. In dogs with CE, treatment of the underlying disease process is required (see above), and FMT should be currently recommended only as adjunct treatment.
Figure 3. A dog with persistent dysbiosis and recurrent C. difficile infection. After FMT, the Dysbiosis Index normalized and the abundance of C. hiranonis increased. The dog was subsequently negative for C. difficile.
Suggested FMT protocol [3]
Selection of donor dog – the donor should be healthy, with no history of gastrointestinal disease and recent antibiotic exposure, and should have no signs of systemic disease. The donor feces should be screened for parasites and enteropathogens. Because some clinically healthy dogs lack C. hiranonis, which is necessary for proper BA conversion, the feces should also be evaluated using the DI
Storage of donor stool – stool can be fresh or stored at 4 C for up to one week in plastic bags. When feces needs to be frozen for longer storage, mixing the stool with glycerol before freezing may better preserve bacteria. Protocol: 10 grams of stool, 35 mls of saline and 5 mls of glycerol, freeze in 50 ml aliquots.
FMT as enema – donor feces (2.5 grams per kg of recipient BW) is blended in 60 ml of 0.9% NaCl until liquefied. The stool is then transferred into the proximal colon of the recipient via a 60 ml catheter tip syringe, with an attached 12 or 14 inch French red rubber catheter. The recipient dog does not need to be sedated. If possible, do not feed and restrict the recipient dog’s activity for 4-6 hours after the transplant to lessen the chances of a premature bowel movement.
References
[1] M.K. AlShawaqfeh, B. Wajid, Y. Minamoto, M. Markel, J.A. Lidbury, J.M. Steiner, E. Serpedin, and J.S. Suchodolski, A dysbiosis index to assess microbial changes in fecal samples of dogs with chronic inflammatory enteropathy. FEMS Microbiol Ecol (2017) 1;93(11).
[2] S. Wang, R. Martins, M.C. Sullivan, E.S. Friedman, A.M. Misic, A. El-Fahmawi, E.C.P. De Martinis, K. O’Brien, Y. Chen, C. Bradley, G. Zhang, A.S.F. Berry, C.A. Hunter, R.N. Baldassano, M.P. Rondeau, and D.P. Beiting, Diet-induced remission in chronic enteropathy is associated with altered microbial community structure and synthesis of secondary bile acids. Microbiome 7 (2019) 31;7:126.
[3] J. Chaitman, A.L. Ziese, R. Pilla, Y. Minamoto, A.B. Blake, B.C. Guard, A. Isaiah, J.A. Lidbury, J.M. Steiner, S. Unterer, and J.S. Suchodolski, Fecal microbial and metabolic profiles in dogs with acute diarrhea receiving either fecal microbiota transplantation or oral metronidazole. Front Vet Sci 7 (2020) 16;7:192.
[4] R. Pilla, F.P. Gaschen, J.W. Barr, E. Olson, J.B. Honneffer, B.C. Guard, A.B. Blake, D. Villanueva, M.R. Khattab, M. Alshawaqfeh, J.A. Lidbury, J.M. Steiner, and J.S. Suchodolski, Effects of metronidazole on the fecal microbiome and metabolome in healthy dogs. J Vet Intern Med in press (2020).
[5] P.R. Giaretta, R.R. Rech, B.C. Guard, A.B. Blake, A.K. Blick, J.M. Steiner, J.A. Lidbury, A.K. Cook, M. Hanifeh, T. Spillmann, S. Kilpinen, P. Syrja, and J.S. Suchodolski, Comparison of intestinal expression of the apical sodium-dependent bile acid transporter between dogs with and without chronic inflammatory enteropathy. J Vet Intern Med 32 (2018) 1918-1926.
[6] J.F. Garcia-Mazcorro, J.S. Suchodolski, K.R. Jones, S.C. Clark-Price, S.E. Dowd, Y. Minamoto, M. Markel, J.M. Steiner, and O. Dossin, Effect of the proton pump inhibitor omeprazole on the gastrointestinal bacterial microbiota of healthy dogs. FEMS Microbiol Ecol 80 (2012) 624-36.
[7] M. Schmidt, S. Unterer, J.S. Suchodolski, J.B. Honneffer, B.C. Guard, J.A. Lidbury, J.M. Steiner, J. Fritz, and P. Kolle, The fecal microbiome and metabolome differs between dogs fed Bones and Raw Food (BARF) diets and dogs fed commercial diets. PloS One (2018) 15;13(8):e0201279.
[8] A.C. Manchester, C.B. Webb, A.B. Blake, F. Sarwar, J.A. Lidbury, J.M. Steiner, and J.S. Suchodolski, Long-term impact of tylosin on fecal microbiota and fecal bile acids of healthy dogs. J Vet Intern Med 33 (2019) 2605-2617.
[9] K. Sugita, N. Yanuma, H. Ohno, K. Takahashi, K. Kawano, H. Morita, and K. Ohmori, Oral faecal microbiota transplantation for the treatment of Clostridium difficile-associated diarrhoea in a dog: a case report. BMC Vet Research 15 (2019) 11.
[10] A.R. Weingarden, C. Chen, A. Bobr, D. Yao, Y. Lu, V.M. Nelson, M.J. Sadowsky, and A. Khoruts, Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile infection. American journal of physiology. Gastrointestinal and liver physiology 306 (2014) G310-9.
[11] G.Q. Pereira, L.A. Gomes, I.S. Santos, A.F. Alfieri, J.S. Weese, and M.C. Costa, Fecal microbiota transplantation in puppies with canine parvovirus infection. J Vet Intern Med 32 (2018) 707-711.
