The potential of the microbiome and inflammatory markers in the feces of dogs with gastrointestinal disease. However, an imbalance of the gut microbiota to the point of dysbiosis can cause both acute and over time chronic gastrointestinal disease. This study aims to investigate the importance of the gut microbiota and calprotectin in gastrointestinal disease.
The owners of the dogs have answered a survey that mainly focuses on general health, feed and possible sequelae and concomitant medication. 100% of dogs suffering from GI disorders had been treated at least once in the past year for vomiting and/or diarrhea compared to 8% of HC. Review currently available research on the importance of gut microbiota and calprotectin in GI disease.
Introduction
Studies have also implicated specific pathogenic bacteria and dysbiosis as important factors in the pathogenesis of GI disease (Suchodolski et al. More than 99% of bacterial phyla in the gut microbiota consist of Bacteroidetes, Fusobacteria, Firmicutes, Proteobacteria, and Actinoskibacteria (Suchodolskibacteria ) 2011a, Suchodolski 2011b Some bacteria in the digestive tract produce SCFAs through fermentation of non-digestible dietary fiber, carbohydrates and in some cases proteins (Minamoto et al. 2019).
They can modulate inflammation in the gastrointestinal tract by decreasing some pro-inflammatory cytokines and increasing some anti-inflammatory cytokines.
Literature Review
The canine gut microbiota
- The gut microbiota and Short-chain fatty acids (SCFAs)
- The effect of diet on the gut microbiota
The microbial profile varies from dog to dog, mainly at the bacterial species and strain level. The amount of bacteria and their diversity increases along the GI tract and can also differ from the lumen and the mucosa. The microbial communities vary along the GI tract and reflect their microenvironment, for example there are aerobic and facultative anaerobic bacteria in the small intestine and almost exclusively anaerobic bacteria in the colon (Pilla . & Suchodolski 2020).
The gastrointestinal tract is estimated to be inhabited by 1012 to 1014 microbes, which is about 10 times the number of host cells. Traditional extruded dry dog food contains both animal and vegetable products and is generally high in carbohydrates compared to meat. The ingredients' macronutrients seem to be more important than their kingdom of origin.
In a study by Bresciani et al. (2018), dogs were fed animal protein-free diets (APFD) but with a similar macronutrient composition to traditional extruded diets and found no changes in the fecal microbiota of healthy dogs. Raw meat diets differ significantly in macronutrient content compared to traditional extruded diets, including more protein and less fiber and carbohydrates (Pilla & Suchodolski 2020). Studies have shown that in dogs fed a raw diet, the total number of bacteria in the order Firmicutes and Bacteroidetes is reduced, most of the affected genera produce SCFA and digest dietary fiber.
Furthermore, bacteria from the phylum Proteobacteria Fusobacteria and two genera from the phylum Firmicutes (Lactinobacillus and Clostridium) were increased. Butyrate kinase (buck) genes have been associated with Clostridium perfringens and Clostridium dificille in dogs eating a carnivorous diet, suggesting that they can produce butyrate from protein sources essential in a carnivorous diet (Vital et al. 2015).
The gut microbiota and GI disease
- Chronic enteropathy (CE)
- Acute diarrhea (AD)
- Dysbiosis
- Calprotectin
A study with meta-analyses to identify biomarkers of GI functionality in dogs systematically reviewed 27 randomized controlled and case-controlled trials in which 815 healthy dogs and 786 with GI disease were included (Félix et al. 2022). Faecalibacterium prausnitzii, Blautia, Turicibacter and Fusobacterium are important in the gut microbiome, in part due to their ability to produce SCFAs (Pilla . & Suchodolski 2020, Ziese & Suchodolski 2021). They are clinically very similar, and no effective biomarkers have been found so far to distinguish between them (Alshawaqfeh et al. 2017).
Dogs with acute nonhemorrhagic diarrhea (NHD) and acute hemorrhagic diarrhea syndrome (AHDS) have dysbiosis (Suchodolski et al. 2012). Dogs with AHD also have an increased number of bacteria in the genera Suterella and Clostridium and the group Fusobacteria. The availability of oxygen in the intestinal lumen may be responsible for some microbial changes observed in dysbiosis (Pilla & Suchodolski 2020).
A study by Alshawaqfeh et al. 2017) developed a dysbiosis index (DI) to help differentiate between healthy dogs and dogs with CE. When released extracellularly, it functions as an endogenous danger signaling molecule (Heilmann et al. 2018) and causes inflammation by binding to TLRs (Foell et al. 2007). Fecal calprotectin is elevated in dogs with chronic diarrhea, especially in dogs with histological intestinal lesions related to HC (Grellet et al. 2013).
It can help distinguish between dogs with IRE and FRE or ARE, especially when combined with CRP and CCECAI scores (Heilmann et al. 2018). A study of dogs with AHDS found that they also had elevated levels of fecal calprotectin (Heilmann et al. 2017).
Material and Methods
Study population and sampling
Stool samples were collected from healthy dogs arriving at SLU Uppsala to donate blood, and from dogs with gastrointestinal disease at the University Animal Hospital (UDS). The stool was marked and a pea-sized sample was placed in a tube for microbial analysis. The tubes were stored at -20°C for 1-3 days and then moved to -80°C until microbial analyses.
The samples were sent to the National Veterinary Institute (SVA) for bacterial culture and calprotectin analysis. The same study will be used in a sister study where stool samples will be collected from dogs before and 3 weeks after the start of cytostatic treatment.
Analysis of stool
- Analyses of microbiota
- Analyses of calprotectin
Paired sequence reads were merged using FLASH (version 1.2.7) UPARSE software (version 7.0.1001) was used to cluster the remaining sequences into operational taxonomic units (OTUs), with ≥ 97%. For annotation, the SSU rRNA database SILVA (Quast et al. 2013) was used (licensed under Creative Commons Attribution 4.0 (CC-BY 4.0). In a later stage of this study, calprotectin will be analyzed using a dog calprotectin ELISA- kit from Abbexa.
Literature search
As previously mentioned, gut microbiota and calprotectin analyzes will be performed later. Of the 22 sampled dogs, 12 were healthy control dogs, 2 had acute gastroenteritis and 8 had chronic gastrointestinal disorders. 100% of dogs with gastrointestinal disorders and 8% of HC were treated at least once in the past year for vomiting and/or diarrhea.
50% of owners of dogs with GI disorders and 8% of owners of HC perceived their dog to have a mildly affected activity level. 40% of owners of dogs with GI disorders perceived their dog to have a slightly reduced quality of life compared to 0% of HC. 90% of dogs with GI disorders and 67% of HC were fed traditional dry and/or wet dog food as their main diet.
10% of dogs with GI disorders and 8% of HC were fed exclusively raw meat as their main diet. 0% of dogs with GI disorders were fed raw meat food in combination with extruded food as their main diet compared to 25% of HC.
Results
Survey
The original aim of this study was to collect faecal samples for the analysis of the gut microbiota and calprotectin. Consequently, this study will focus on the potential of the gut microbiota and calprotectin in gastrointestinal disorders. The dysbiosis index is an example of a way to use the gut microbiota as a diagnostic tool.
As previously mentioned, the qPCR panel consists of total bacteria, Faecalibacterium, Turicibacter, Escherichia coli, Streptococcus, Blautia, Fusobacterium and Clostridium hiranonis, all of which have been shown to be affected in CE (Alshawaqfeh et al. 2017). A meta-analysis study by Félix et al. 2022) observed a significant difference in the dysbiosis index between HC and dogs with GI disorders, with HC having a lower DI. However, they did not observe a significant difference between dogs with GI disorders before and after treatment.
They did, however, have only two studies in their meta-analysis examining DI before and after treatment, with a total of 38 dogs, eight of which had acute clinical signs. An interesting approach for a future study would be to evaluate how effective DI is in differentiating between HC and dogs with acute disorders, as it was specifically developed for dogs with CE (Alshawaqfeh et al. 2017). In addition to the dysbiosis index, the identification and measurement of certain bacteria sensitive to gut homeostasis changes may be a valuable diagnostic for monitoring gastrointestinal functionality.
Discussion
- The diagnostic value of the gut microbiota
- Therapeutic approaches to dysbiosis
- The diagnostic value of calprotectin
- Problems and limitations
- The survey
- Population size
- Sampling
In a study by Heilmann et al. 2018), they found that calprotectin levels are higher in the feces of dogs with IRE than in dogs with FRE and ARE, but significance was not reached, possibly due to the low number of patients. They also found that faecal calprotectin levels were significantly higher in dogs with chronic diarrhea and histological lesions than in dogs with only chronic diarrhea. They found that calprotectin levels were significantly higher in dogs with gastrointestinal disease compared to healthy dogs.
A dysbiosis index to assess microbial changes in faecal samples from dogs with chronic inflammatory enteropathy. 2018) Effect of an extruded animal protein-free diet on faecal microbiota in dogs with food-reactive enteropathy. Faecal markers of inflammation, protein loss and microbial changes in dogs with acute hemorrhagic diarrhea syndrome (AHDS), Journal of Veterinary Emergency and Critical Care.
Faecal short-chain fatty acid concentrations and dysbiosis in dogs with chronic enteropathy, Journal of Veterinary Internal Medicine, 33 (4). Clostridium perfringens netE and netF toxin genes in feces from dogs with acute hemorrhagic diarrhea syndrome, Journal of Veterinary Internal Medicine, 33 (1). Molecular analysis of the bacterial microbiota in duodenal biopsies from dogs with idiopathic inflammatory bowel disease.
Faecal microbiome in dogs with acute diarrhea and idiopathic inflammatory bowel disease, PLOS ONE, 7 (12) e51907. Molecular-phylogenetic characterization of microbial community imbalances in the small intestine of dogs with inflammatory bowel disease. In this study, we collected faecal samples from both healthy dogs and dogs with gastrointestinal disorders to investigate any correlation between changes in microbiota, inflammatory markers, and what disorders the dogs were suffering from.
However, due to unforeseen events, the analysis of the feces will only be carried out at a later stage of this study.
