1. Introduction
The numerous studies that investigated the composition and the variation of gut microbiome in relation to healthy conditions and environmental factors for companion animals and livestock have attracted the scientific community and are growing exponentially [
1,
2]. The microbial population that inhabits the gastrointestinal tract of both humans and animals has been considered responsible of very important basic functions, from the regulation of metabolic activities to protection against pathogens and modulation of immune system and then physiologic functions [
3]. The advent of innovative technologies allows for a more frequent utilization of molecular methodologies to identify non-culturable bacteria within the canine gut, highlighting the high individual variability of microbial population, especially in dietary intervention studies [
4,
5]. Despite these evidences, it appears clear that some key bacterial species are consistently present in fecal samples of healthy subjects, suggesting the presence of a core fecal bacterial community [
6].
In a recent study on healthy dogs, the paramount role of diet and sex on the gut microbiome has been reported [
7], confirming the idea that the microbes inhabiting the gut can be considered as an individual fingerprint [
8]. Human microbiome can be split in enterotypes, meaning that individuals can be clustered on the basis of the abundance of microbial taxa of the gut [
9]. The categorization in enterotypes has not yet been applied to dogs, also because the contradictory results that can be found in the literature make the identification of a gut microbiome core in dogs difficult. Interestingly, Scarsella et al. (2020) [
7] showed that dogs fed with a homemade diet (H) cluster together with dogs fed with a raw-based diet supplemented with a complementary food (B.A.S.E.,
www.nutrigenefood.com., Italy), even though the composition of these two diets was different. On the opposite, dogs fed with a commercial extruded complete diet (K) and a commercial moist complete diet (W) formed two distinctly clusters. It is likely that the presence of raw meat in the former diets and the physical form of the diets had a similar impact on shaping gut microbiome. Similar results have been obtained analyzing the gut microbiome in relation to the sex factor.
The prevalence of specific taxa can be effective to identify dysbiosis events. The concept of “dysbiosis” refers to a change in the composition of symbiotic or commensal microbial communities [
10]. Considerable research has been dedicated to address the relationship between the gut microbiome and the health status of the subjects, both human and animals, but there is still a limited number of studies, only in the human field, that explored dysbiosis related to the blood microbiome and its potential role in pathogenesis. Blood has been traditionally considered to be a sterile environment, but some evidences in various domesticated mammals and birds [
11,
12] and in humans [
13,
14,
15] suggest that it is populated by microbes. The origin of these bacteria is mainly attributed to the translocation from the gastro-intestinal tract [
16], but it has been suggested that also a part of the microbial population of the oral cavity and the skin can diffuse into the blood [
17]. The hypothesis is that many bacteria found in healthy human blood may be in a dormant state [
18], or they are present in their L-forms [
19].
The present study has three principal aims: firstly, to confirm the results of our previous study regarding the role of diet on fecal microbiome; secondly, to investigate the presence of a microbial population in the bloodstream in heathy subjects’ and thirdly, to seek for a possible connection between the fecal and the blood microbiota.
4. Discussion
Several studies have pointed out that the gut microbiome is highly variable among healthy dogs [
23] and can represent an individual fingerprint [
8]. Several factors, other than different methodological approaches, affect the abundances of organisms in the gut microbiome, such as diet, sex, age and disease [
26,
27]. In humans, geographical variations, ethnicity, host genetic, immunity, lifestyle and dietary habits have been reported to affect gut microbiota [
28]. In healthy dogs, variations of microbiome with age, from weaning to adulthood, were reported [
27,
29], with a stabilization of the core gut microbiota at maturity. A strong similarity between human and dog microbiota has been reported, suggesting that the same factors affecting gut microbial community in the former can act also in the latter [
30].
Figure 2 showed a picture of the clustering between the three groups of dogs based on their diets. The more distinct clustering was appreciable for dogs fed with BARF diet, at phyla, family and genus level. Recently, a study on healthy dogs confirmed the role of diet on the gut microbiome [
7], but also several other studies indicated the influence of diet on intestinal microbiome [
2,
4,
23,
31]. Beyond this result, it is interesting to observe that some subjects did not fit in the cluster of their dietary group. This evidence could be due to an unpredictable and discontinuous administration of foods not foreseen in the daily diet of the dogs, since they were not recruited from a kennel or a shelter, but from local veterinary clinics. The owners could have fed the dogs with rewards or fresh foods but not necessarily every day. For instance, the subjects receiving a KIBBLE diet, marked with green squares, were fed also with some homemade or some raw-meat-based diet, and this could also be possible for some dogs fed with a BARF or a HOME diet, where kibble could have been fed.
Moreover, the gut microbiome ecosystem has strong interactions with the environment and the genetics of the host. Vazquez-Baeza et al. (2016) [
32] reported that the diversity and structure of microbial community, more than the variation of single taxon, could be used as a signature of the fecal microbiota to separate dogs with IBD from healthy dogs. The better characterization of the gut microbiome has been obtained, in the past, thanks to studies with subjects that lived in the same controlled environment, such as a shelter, kennel or university facilities, where the dogs received the same diet under strict control, either for a short or long period of time [
5,
29,
31]. In this study, although the potential bias due to the effect of the different environments and of the owners can be claimed, it was still possible to separate dogs based on their diet. Moreover, the characterization of gut microbiome with a multivariate approach allowed the identification of those subjects for which diet was mixed in terms of administered type.
Another aim of the study was to investigate if gut microbes can translocate to the blood crossing the gut wall. The so called “leaky gut” condition is widely studied in humans and dogs and often is related to inflammatory bowel diseases and related enteropathies [
33,
34,
35,
36], but this functional deficiency should not happen in healthy animals. In the present study, we analyzed the presence of bacterial DNA in blood of healthy animals, and, surprisingly, several taxa were detected. Blood has been traditionally considered to be a sterile environment, but some evidences for bacterial presence in various domesticated mammals and birds [
11,
12] and in humans [
13,
14,
15] do exist. Despite the difficulties to cultivate blood bacteria, several studies reported the successful growth of numerous bacteria from blood of healthy individuals confirmed by microscopic observation [
18,
37]. Even if the majority of taxa annotated in blood was present at a very low abundances, they were detectable. One of the hypotheses is that the bacteria populating the blood are in a “dormient phase” [
38], and this would explain why there is a microbial population even in healthy subjects and also why this condition is not pathogenic. The hypothesis is that, occasionally, some of these bacteria “wake up” and reproduce, becoming active again and being transported to various tissues and organs of the body, thus inducing a state of chronic disease [
39]. To the best of our knowledge, this is the first study characterizing canine blood microbiome. External factors, such as contamination of reagents and blood with external bacteria during the sampling, could have led to an artifactual appearance of microbiome into the blood. For what the reagents and the sequencing pipeline is concerned, a mock bacterial community was used as internal standard to validate the methodology, and the results confirmed the lack of contamination.
Interestingly, the number of bacterial taxa annotated for blood samples was much higher compared to fecal samples, especially at a family level, with 92 taxa annotated in blood and 22 in feces (
Figure 1). The blood bacterial taxa found in this study are in line with the literature found for humans [
16]. The most detected bacteria in blood (
Table 1) were Proteobacteria and Actinobacteria, which differed consistently from the predominant phyla of the gut microbiome, that were Firmicutes and Bacteroidetes [
2,
40]. This difference between blood and feces could be explained by the role of filter played by intestinal and immune cells, which could have limited the translocation of certain bacteria. The recognition of “good” or “bad” microorganisms is due to the presence of dendritic cells in the gut [
41], although environmental factors can shift commensal bacteria to pathogenic bacteria, causing a disruption at a variable extent of tight junction and a leaky gut. Furthermore, tissues and organs, such as skin, oral cavity and nasal or vaginal mucosa, can probably contribute to the bacterial DNA present in the blood [
42]. Although the microbial composition in the feces was not equal to those in the blood, in terms of abundances and presence of certain taxa, the clustering of dogs in the three diets was comparable. The PCA multivariate analysis allowed to separate the dogs by using the gut microbiome (
Figure 2), and almost the same results were obtained by using the blood microbial community (
Figure 3).
5. Conclusions
This study confirmed that diet is a factor driving the shift of gut microbial population in dogs, and researches in this direction are still needed to better clarify the association between this factor and gut microbiome.
A limited number of research studies have been published on blood microbiota in humans, and this is the first evidence of the presence of a bacterial population in the blood of healthy dogs. From our results, we can speculate that blood microbiome, or at least a part of it, can derive from the translocation of some gut bacteria, and it consequently could be associated with a shift of the intestinal microbial population. These preliminary results deserve further studies, including also dogs suffering of gastrointestinal diseases; however, if confirmed, the results pave the way for the use of blood microbiome for diagnostic purposes.