The developmental stages in gut microbiota, bile acids, and clostridial species in healthy puppies | VETgirl Veterinary Continuing Education Podcasts

In today’s VETgirl online veterinary CE podcast, we discuss the gastrointestinal (GI) microbiome in puppies. Lately it seems that microbiome talk is all the rage! We are inundated with commercials for probiotics, catchy news articles about the microbiome, and news of novel medical treatments like fecal microbiota transplants (FMTs). In parallel to the newsworthy buzzwords, we are also seeing a tsunami of research being released about the microbiome, ranging across a broad spectrum of animal species, environmental ecosystems, and states of health and disease. Small animal veterinarians have joined this conversation as well, with plenty of exciting research being published regarding various aspects of canine and feline microbial communities. However, the development of the gut microbiome in puppies after birth and before doggy adulthood is one area where our research is lacking. So, Blake et al wanted to evaluate this in a study entitled Developmental stages in microbiota, bile acids, and clostridial species in healthy puppies to examine the composition of the fecal microbiome and fecal bile acid profiles in puppies, young adult dogs, and adult dogs.

This was a prospective, observational study. Healthy puppies were recruited from the Guide Dogs for the Blind, Inc in San Rafael, California. Not only are these puppies helping the blind, but they are contributing to research as well! At the time of enrollment, all puppies were 10 weeks of age or younger and in good health. Fecal samples were voided naturally for collection, and the samples were divided into 5 groups based on the puppy’s age (1-2 weeks, 3-4 weeks, 5-6 weeks, 7-9 weeks, and 10-16 weeks). At 8-10 weeks of age, the puppies ventured out into the world (ie, the world outside of their facility), after which the researchers were unable to gather additional samples. Therefore, the study also recruited 33 client-owned, healthy dogs that were 8 weeks or older. These pups also had naturally voided fecal samples collected, and this group was divided into 4 age groups (7-9 weeks, 10-16 weeks, 20-48 weeks, and > 52 weeks). This allowed the investigators to expand the age range they were examining. Note that of all the Guide Dogs for the Blind puppies, there were 5 dogs represented in 2 age groups, and of this latter group, there were 2 dogs represented in 2 age groups. The puppies from the whelping facility were fed Purina Pro Plan Large Breed Puppy Chicken and Rice formula with water, and they were all fully weaned by 6 weeks of age. As expected, the client-owned dogs consumed variable diets, but they were all indoor dogs in good clinical health. Dogs with a history of antibiotic usage within 6 months of study enrollment were excluded.

Okay, so now that we know who these dogs are, let’s hear about what the investigators were actually testing! First, microbial DNA was extracted from the feces, and was then subjected to various tests. Specifically, a quantitative polymerase chain reaction (qPCR) was performed to assess both total bacteria counts and examine specific bacterial taxa, which included Faecalibacterium spp., Turicibacter spp., Streptococcus spp., Escherichia coli, Blautia spp., Fusobacterium spp., and Clostridium hiranonis. The investigators also tested for the Clostridium perfringens NetF toxin gene. Additionally, qPCR assays were performed for Clostridium difficile, Clostridium perfringens enterotoxin gene (CPE), Salmonella spp., and Campylobacter jejuni. If dogs tested positive for C. difficile, and if a sufficient fecal sample remained after initial testing, additional screening for C. difficile glutamate dehydrogenase antigen and toxins A and B were performed. Similarly, if positive for CPE, additional ELISA testing was performed. The authors tested for unconjugated fecal bile acids on these fecal samples as well.

A fecal dysbiosis index (DI) was also performed in all dogs of this study. Now some of you may already be familiar with this test, but others may have less experience. To give a brief explanation, the fecal DI is a commercially-available assay that uses PCR technology to determine the abundance of both total bacteria and the abundances of 7 specific bacterial taxa, all of which are relevant to canine GI health. The assay uses a mathematical algorithm that incorporates the abundances of these taxa to produce a single numeric value (AlShawaqfeh). This single value can then be used as a marker to indicate whether the dog’s GI microbiome is normal or abnormal. In adult dogs, a DI value less than zero suggests a healthy microbiota, but remember that here we are looking at puppies, not adults!

Clearly, these investigators sought to learn as much as possible from these pups’ fecal samples, so let’s hear some results! First of all, in this population, the authors found that the DI was significantly higher in puppies between 1-6 weeks of age than in dogs greater than 1 year of age. In other words, the DI was higher in young puppies than in adults. Recall that a negative value is indicative of health in adults, so knowing that a higher value is present in clinically healthy puppies is valuable information for clinicians interpreting DI results. When examining specific bacterial taxa, the investigators found a number of differences between age groups. For instance, Faecalibacterium, Blautia, and Fusobacterrium were lower in puppies of certain age groups as compared to adults, whereas Streptococcus and E. coli were increased in several age groups of puppies. Overall total abundance of bacteria was actually higher in the 3-6 week old puppies as compared to adults.

As mentioned, C. hiranonis is one of the bacterial species these authors specifically examined, and this bacteria is also assessed as a component of the DI test. While many of us may hear Clostridium and immediately think pathogen, C. hiranonis requires a different perspective. This bacteria actually plays an important role in converting primary to secondary bile acids, which is a good thing! In the puppies in the 1-6 week old age groups, C. hiranonis was significantly lower than in adults. Secondary bile acids were positively correlated with C. hiranonis, which makes sense knowing that this organism helps convert primary into secondary bile acids, and this finding held true across all samples. Interestingly, C. difficile was negatively correlated with both C. hiranonis and secondary bile acids, and C. difficile abundance was higher in puppies from 1-6 weeks of age. So as C. hiranonis and secondary bile acids increased with time, C. difficile decreased! The authors note that C. difficile’s growth and spore germination is actually impaired by secondary bile acids, so perhaps this explains part of this negative correlation. C. difficile is higher in these young puppies, yet by 7-9 weeks of age the investigators observe increasing C. hiranonis and secondary bile acids mirrored by decreasing C. difficile. This VETgirl finds this relationship between bile acids and these Clostridial species fascinating, and this clearly represents an important component of the puppy gut microbiome development!

Let’s not forget about our other Clostridium of interest, C. perfringens. C. perfringens abundance was higher in puppies compared to adults, but importantly, no fecal samples contained the NetF toxin gene. The authors note that this finding is not terribly unexpected given that these are healthy pups and that this toxin is often associated with acute hemorrhagic diarrhea syndrome (Mehdizadeh). The authors also tested for CPE via qPCR in 87 samples, of which 8 were positive. The authors further tested 6 of those 8 samples via ELISA, and none tested positive. Given limitations in the fecal sample sizes, Salmonella and C. jejuni assays were performed only in adults and in puppies less than 9 weeks of age, which totaled 70 samples overall. All samples were negative for C. jejuni, but 4 puppy samples between 5-9 weeks of age were positive for Salmonella. When looking at the overall prevalence of Salmonella-positive fecal samples in this study, this still falls within the range that has been previously reported for healthy dogs.

Lastly, the authors evaluated fecal bile acids in the majority of these pups, though there were 7 dogs that did not have sufficient samples for bile acid analysis. Recall that bile acids influence gut health in several ways. For instance, we already mentioned that secondary bile acids may impair C. difficile growth, but this is also true for other bacterial species, including E. coli and C. perfringens! Also, disruptions in the balance of primary and secondary bile acids or in bile acid absorption can cause diarrhea in itself. In this study, the total concentrations of primary bile acids were higher in puppies in the 3-9 week old age groups as compared to adults, and total secondary bile acids were lower in young puppies (1-6 weeks of age) compared to adults. Recall that secondary bile acids positively correlated with C. hiranonis abundance and negatively correlated with C. difficile abundance. Young puppies in the 1-2 week age group also had lower overall concentrations of fecal bile acids compared to adults.

So what can we take away from this VETgirl podcast? Well, this study provides valuable insight into the gastrointestinal microbial populations in healthy puppy and adult dogs, and it demonstrates that the fecal microbiome of puppies is different from adults! For instance, the DI values of young puppies were significantly higher than adults in this population, which is important to remember when interpreting DI results in the future. The authors also observed specific microbial shifts, such as higher E. Coli in puppies. Interestingly, decreasing abundance of C. difficile in growing puppies paralleled increasing abundance of secondary bile acids and abundance of C. hiranonis, which is a bacteria important in converting primary to secondary bile acids. The authors discuss a number of limitations for this project, such as the small sample sizes in certain age groups, lack of follow-up for many dogs, variable enrollment locations and living environments, and the lack of testing for Giardia or Cryptosporidium. It is also important to remember that this study included healthy puppies and adults, so more investigation is needed to extrapolate what occurs to the gut microbiome of puppies with gastrointestinal pathology. That said, this study provides incredibly important information on the characteristics and development of the healthy gut microbiome in puppies!

Blake A, Cigarroa A, Klein H, et al. Developmental stages in microbiota, bile acids, and clostridial species in healthy puppies. J Vet Intern Med 2020;1-12.

AlShawaqfeh MK, Wajid B, Minamoto Y, et al. A dysbiosis index to assess microbial changes in fecal samples of dogs with chronic inflammatory enteropathy. FEMS Microbiol Ecol 2017:1;93(11).

Mehdizadeh GI, Parreira VR, Nowell VJ, et al. A novel pore- forming toxin in type A Clostridium perfringens is associated with both fatal canine hemorrhagic gastroenteritis and fatal foal necrotizing enterocolitis. PLoS One. 2015;10:e0122684.

CPE – Clostridium perfringens enterotoxin
DI – Dysbiosis index
FMT – Fecal microbiota transplant
GI – Gastrointestinal
qPCR – Quantitative polymerase chain reaction

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