The influence of dietary animal-derived proteins and carbohydrates on canine intestinal microbiota was investigated. The %G + C profiles, as well as order-level sequence distribution in fraction 5, between the DC and HC diet samples did not differ considerably, most likely indicating that the modulatory influence of the HC diet on canine fecal microbiota is smaller than that of the HP diet when compared with the DC diet phase of the trial. This result is not surprising since both DC and HC diets consisted mainly of carbohydrate-rich components.
The amount and type of fermentable carbohydrates reaching the colon are primary factors influencing the abundance and variety of the resident bacterial population. The bacteria that can most rapidly degrade and use the digesta will proliferate beyond the others . Corn starch, which was also included in our diets, has a high small intestinal digestibility and is therefore not expected to reach the large intestine in high amounts . However, the DGGE band patterns obtained in a previous study indicated that this carbohydrate affected the composition of faecal bacteria in rats . Therefore, the passage of corn starch into the large intestine might have been one of the reasons for sequence differences between DC and HP diet samples as well as between HC and HP diet samples.
The sequence diversity in the DC diet sample was generally higher than in the HC and HP diet samples, as the sequences were classified into five different orders, two of which, Lactobacillales and Bacteroidales, were completely absent in faecal samples from the HC and HP diets. The higher diversity was most likely due to the different ingredients of the DC diet sample, and the combination of corn and processing conditions might have resulted in a more versatile spectrum of fermentable substrates for various bacterial types .
In fraction 5 of the HP diet sample, the most abundant sequences belonged to the order Fusobacteriales and showed close similarity with the species F. varium and F. mortiferum. F. varium
F. nucleatum and F. equinum have been found to play roles in the pathogenesis of colonic, oropharyngeal, gingival, periodontal and other inflammatory processes, such as abscesses, pneumonia and sinusitis [25–27]. Given that the HP diet led to diarrhoea for all dogs in our study (data not shown), it could be hypothesized that species from the order Fusobacteriales could have caused the loss in faecal consistency, together with the high collagen concentration in the HP diet. The Greaves-meal diet, having a high digestibility, is known to soften the faeces, increase Clostridium perfringens levels and decrease bifidobacteria in dogs [28, 29]. To our knowledge, F. varium and F. mortiferum have not been previously detected in canine faecal samples. Further characterization of isolates of these species should clarify whether they are commensals or opportunistic pathogens, or both, which is the case in the human intestine .
The results obtained from fraction 10 again indicate that the increased sequence diversity with the DC diet relative to the HP and HC diets was most likely due to the more versatile nutrient composition. In the HC diet sample, the most abundant sequences belonged to the order Clostridiales, showing the closest similarity with Clostridium hiranonis, which has been discovered previously in the canine GI tract and is considered to belong to the normal canine intestinal microbiota .
In fraction 14, all sequences in faecal samples of all dietary groups belonged to the order Coriobacteriales, suggesting that members of Coriobacteriaceae may be indicators of a healthy GI microbiota. For instance, in humans a high abundance of Collinsella aerofaciens has been associated with a lowered risk of colon cancer and inflammatory bowel disease [30, 31]. To our knowledge, the presence of bacteria belonging to the order Coriobacteriales in canine faecal samples has been reported only in a recent 16 S rRNA gene sequencing study . The order Coriobacteriales within the phylum Actinobacteria was found to be more abundant than previously estimated with conventional sequencing studies also in human faecal samples . This is most likely due to the sequencing studies having been carried out without %G + C fractioning. It is evident that fractionating the total faecal DNA preparations minimizes PCR and cloning-derived bias, which is common in multi-template sequencing studies. In other words, fractionating facilitates the amplification and subsequent cloning of species with high G + C contents from diverse microbial communities [19, 20].
No bifidobacteria were found, consistent with an earlier study . However, contradictory data also exist, as many studies have found bifidobacteria in dogs [12, 32]. Possibly, bifidobacteria were not part of the predominant intestinal microbiota of the Beagle dogs participating in our study. Another potential explanation for this unanticipated result may be that the universal 16 S rRNA gene-targeted primer pair contained mismatches to many bifidobacterial species, which could have led to significant underestimation of bacteria belonging to this genus.
Clostridiales and Coriobacteriales were the most prevalent bacterial orders in the faecal samples of all dietary groups. Suchodolski and coworkers , by contrast, reported that Fusobacteriales and Bacteroidales were the most representative orders in the canine colon. It is noteworthy, however, that we analysed only three %G + C fractions, which showed the most pronounced alterations between the dietary groups. Our aim was not to obtain an overall picture of the canine faecal microbiota, but to elucidate the diet-derived effects on the microbial community structure in the lower intestine.