Rabbits, as compared to other mammals, have a distinctly different gastrointestinal (GI) microbiome as a result of their hindgut-fermenting physiology in combination with cecotrophy as part of their normal feeding behavior. The normal gut microbiome of the rabbit contains billions of species of bacteria and archaea as well as yeast.1–4 The rabbit GI tract is highly specialized, with even the different organs (stomach, cecum, intestines) containing varying types of bacteria and yeast.2,3,5–7 When comparing the GI microbiome of rabbits to other hindgut fermenters, such as guinea pigs, major differences have been noted.8 Interestingly, varying breeds of rabbits have been found to have differences in their normal GI microbiome, though this was not assessed in this study.9,10
Gastrointestinal motility disorders are 1 of the most common disease processes in pet rabbits. Ileus, commonly referred to as rabbit GI syndrome (RGIS), can be caused by a variety of underlying illnesses.1 Historically, the primary cause of RGIS was believed to be dietary; however, recent research has shown that any disorder that causes pain or systemic stress can lead to RGIS.1 Diarrhea can also be life threatening for rabbits as it leads to dehydration, which can in turn facilitate the development of GI obstructions from both foreign and normal ingesta that would normally be able to be moved with a healthy GI.1 Several specific bacteria have been isolated as causes of diarrhea of varying severity in rabbits, including Clostridium spp, Spiroforme spp, Escherichia coli, Lawsonia intracellularis, Campylobacter spp, and Mycobacterium spp.11–13 It is unclear at this time whether these organisms can be part of the normal microbiome of the rabbit GI tract and whether they cause disease when the GI environment changes, leading to those species exceeding a certain threshold.
Next-generation DNA sequencing (NGS) technology is superior to traditional agar plate culture given its ability to identify organisms that do not grow under standard aerobic, anaerobic, and fungal culture conditions, such as clostridial organisms, Aspergillus spp, and Mycobacterium spp; it has allowed for recent advances in our understanding of the normal microbiome of many companion animal species, including cats, dogs, rabbits, and horses.14–17 However, there is limited research using NGS to evaluate the fecal microbiome in rabbits, and few studies11,12 are currently published comparing the fecal microbiome of healthy rabbits to those with GI disease. In a recent study, NGS was used to compare the microbiome of the rabbit external ear canal with what is normally expected based on traditional agar plate cultures; this study showed that there are many organisms that are not commonly cultured using traditional methods that can be identified using NGS.14 Such a breakthrough in our understanding of the normal microbiome of rabbit ears suggests that the same technology can be used to broaden our knowledge of the microbiome of the rabbit GI tract.
The purpose of this study was to elaborate on the expected normal fecal microbiome in healthy rabbits using a technology that is accessible to veterinarians and to compare it to the fecal microbiome of rabbits with GI disease. It also aimed to highlight a potential hole in our understanding of what constitutes a “normal” fecal microbiome in rabbits using NGS technology.
Methods
Ethics statement
All rabbits were privately owned, and samples were collected during their visit to an exotic pet specialty veterinary clinic. Rabbits were presented for either normal wellness exams or for GI disease. Samples were collected and analyzed with owner consent.
Rabbits included and study design
This study included a total of 50 fecal samples: 25 fecal samples from rabbits free of clinical signs and 25 fecal samples from rabbits with RGIS. Rabbits’ age in the study ranged from 2 months to 9 years old. All fecal samples were composed of at least 2 fecal pellets or approximately 1 to 3 g of loose fecal material. A variety of phenotypic breeds, including Rex, New Zealand White, Dutch, Hotot, Lionhead, and mixed breeds, such as Dwarf and Lop rabbits, were included but were not analyzed as breeds could not be confidently confirmed. Additional information regarding the study population and diet specifications for each individual can be found in Supplementary Table S1. Rabbits in the study were divided into 2 groups: healthy or diseased GI. Rabbits in the diseased GI group were symptomatic for 24 to 48 hours and were assessed for acute-onset GI disease. No rabbit in the study was administered oral or injectable antibiotics or antiparasitics for at least 2 weeks prior to sample collection.
Sample collection, DNA extraction, library preparation, and sequencing
Fecal samples were collected by the clinicians using a sterile, dry, DNA-free HydraFlock swab (HydraFlock; Puritan catalog [Cat] No. 25–3406-H).
Feces were immediately mixed with a DNA preservation buffer (DNA/RNA Shield; Zymo Research Corp) that lyses all cells upon contact. Therefore, no growth or decay of any microbial cells would occur to avoid any microbial ‘bloom’ in collected fecal samples as previously described by Amir et al.18 Utility and preservation capacity of the buffer has been previously demonstrated.19 Samples were shipped for processing to the NGS technology diagnostic center for processing. Genomic DNA was purified using the ZymoBIOMICSTM-96 DNA kit (Cat No. 79 D4304; Zymo Research Corp). Sample library preparation and data analysis for bacterial profiling were performed by MiDOG LLC using a Quick-16S NGS Library Prep Kit (Cat No. D6400; Zymo Research Corp) with minor modifications. Next-generation DNA sequencing testing in a clinical diagnostic setting requires a quality control system designed to ensure immaculate sample preparation and analysis with the highest standard of precision, data accuracy, and controls as previously highlighted.20
As internal controls to ensure the accuracy and cleanliness of the data generated, and to control for any potential contamination of the equipment, sequencing buffers, and other material, several negative controls were run for both the extraction process and the library preparation. These included an “extraction negative control,” which was the storage buffer (DNA/RNA Shield; Cat No. R1100-50) and lysed, extracted, library prepped, and sequenced in parallel with experimental samples. Further, a “library preparation negative control” and a “no-template control” for the library preparation were run. The workflow was automated using a Hamilton Star liquid handling robot (Hamilton Company) to reduce human errors during sample processing.
To control for contamination, and as positive controls, both cellular and DNA mock communities were used as positive controls (ZymoBIOMICS microbial community standard, Cat Nos. D6300 and D6305; Zymo Research Corp) to account for any bias in the workflow starting from the DNA extraction process. ZymoBIOMICS Microbial Community Standard (Zymo Research Corporation) was used as a positive control to monitor the performance of all steps of the NGS workflow, including the bioinformatic analysis.
Primer sequences targeted the 16S rRNA V1–V3 region for the bacteriome and the ITS-2 region for the mycobiome analysis as previously described.15 Libraries were sequenced using an Illumina HiSeq 1500 sequencer for a sequencing depth of 7 to 8 million reads, generating at least 10,000 reads per sample. Reads were filtered through Dada2 (R package, version 3.4). Taxonomy prediction was performed with the Centrifuge tool combined with a custom reference database (Zymo Research, version 24) curated, in part, from draft or complete genomic sequences available from the National Center for Biotechnology Information GenBank. To assign a species identification, a Y factor of ≥ 97% was used.21
Phylotypes were computed as percentage of proportions based on the total number of sequences in each sample. Species-level resolution of the sequencing approach used here has previously been confirmed by shotgun sequencing comparison of the same clinical samples.15 Absolute microbial quantification was achieved using real-time PCR targeting the 16S rRNA V1–V3 and ITS-2 regions.
Statistical analyses
Unless otherwise stated, results were expressed as average values with SD and the corresponding 95% CI shown in brackets. Measurements of α-diversity and evenness were calculated using the number of observed species. α-Diversity measurements are commonly used in microbial ecology and assess the microbial diversity within a sample. This data was not normally distributed and therefore log-transformed before it was analyzed using ANOVA. β-Diversity was calculated using Bray-Curtis distance using the species taxonomic level. β-Diversity is a measurement of “differentiation diversity” between samples and commonly used to understand how variation in community composition is correlated with environmental or clinical variables.22 Statistical significance between groups and 95% CIs were calculated for β-diversity data using the Adonis function in the vegan package (version 2.4-2) of R, version 3.6.l. Composition visualization, α-diversity, and β diversity analyses were performed with (Qiime [version 1.9.123; Qiime]), and (Prism [version 9.2.0; GraphPad Software]).23 The principal coordinates analysis plots were generated using the ggplot package of R, version 3.6.l.24 The distance matrix and the principal coordinate analysis were performed using the vegan package in R. Linear discriminant analysis effect size was used to identify taxa that were significantly enriched in each group using the default settings.25 A P value of .05 was considered significant.
Results
Fecal samples from 25 clinically asymptomatic and 25 symptomatic rabbits were collected for this study and compared for both their bacterial and fungal microbiome composition. On average, 250.8 ± 110 (188.0 to 293.0) different bacterial species were detected in the asymptomatic healthy rabbit group and 243.3 ± 108 (198.0 to 270.0) species in the symptomatic GI disease group, which was not statistically significantly different (P = .87). The fungal profile per sample was less diverse than the bacterial profile, with 12.68 ± 7 (8.0 to 15.0) different fungal species in the healthy group and 20.54 ± 26 (8.0 to 21.0) species in the GI disease group, which was also not statistically different (P = .46) (Figure 1).
α-Diversity analysis of clinically healthy (CH) and diarrhea cases as measured by the number of observed bacterial (A) and fungal (B) species per sample. Shown are the means and SDs. GI = Gastrointestinal.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.07.0193
In the clinically healthy group, the most dominant bacterial species were members of the phyla Bacteroidetes, Proteobacteria, Verrumicrobioa, Synergistetes, Firmicutes, and Tenericutes (Figure 2). Specifically, the most abundant species detected were a species within the order of Clostridiales (17.51 ± 6.6%) (which could not be closer identified based on the 16S rRNA sequence), a species within the family of Ruminococcaceae (8.41 ± 4.9%), and Akkermansia sp (6.01 ± 4.4%) (Supplementary Table S2). There was considerable variation between individuals within the same group (Figure 3). Especially sample CH6 was very different from the rest of the healthy fecal samples as it was the only one that presented without any Clostridiales sp present, whereas that species was present in all other healthy samples. Further testing on this individual to evaluate for underlying immunosuppressive disease conditions that could have contributed to this difference, such as Encephalitozoon cuniculi or Treponema spp, were not permitted due to owner financial constraints.
Microbiome analysis based on the sample type for CH versus GI disease cases. Shown are the average microbiome profiles per group for bacteria (A) and fungi (B). Shown and listed are the top 25 most abundant species as well as those species identified to be significantly different between groups according to the LEfSe (Linear discriminant analysis Effect Size) analysis. Those species significantly different between groups are highlighted with an asterisk next to the species name. Samples are color coded by health status, with green background color for clinically healthy samples and blue background color for the GI disease group.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.07.0193
The individual microbiome profile for each individual sample for the bacterial (A) and fungal profile (B) at the species level. Samples are color coded by health status, with green background color for clinically healthy samples and blue background color for the GI disease group.
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.07.0193
In the symptomatic RGIS group, the most abundant bacterial species were the same Clostridiales species as in the healthy group (14.44 ± 8.3%), the same species within the family of Ruminococcaceae (10.84 ± 8.0%), and a species within the family of Lachnospiraceae (5.95 ± 7.6%). Interestingly, there was 1 individual that had 82.7% of its bacteriome dominated by a single species, an Enterobacter cloaca (D6).
Eleven species were significantly more abundant in the healthy group than in the RGIS group and 4 species in the GI disease group (Figure 4; Supplementary Table S2). At the family level, 3 families were significantly enriched in the healthy group. Specifically Prevotellaceae (P = .01), a family within the phylum of Melainabacteria (P = .02), and 1 within the phylum Saccharibacteria (P = .02).
LEfSe analysis summary of bacterial (A) and fungal (B) species that were significantly different between groups. Bar graphs show the relative abundance of a given species per group for clinically healthy samples in green and GI disease samples in blue (left, y-axis). Symbols show the frequency on how often a specific species was found in the dataset (right, y-axis). The color fill of the graph indicates if the taxa are enriched in the CH group (green background color) or the GI disease group (blue background color).
Citation: American Journal of Veterinary Research 86, 1; 10.2460/ajvr.24.07.0193
Only 1 species of Lactobacillus was detected in this study, Lactobacillus iners, with an average abundance of 0.002 ± 0.01% in the healthy and no positive results in the GI disease group.
The fungal profile showed less diversity per individual; 23 of 25 individuals in the healthy group had 1 fungal species dominating the sample with over 95% relative abundances, and 19 of 25 individuals in the GI disease group showed 1 fungal species dominating the sample similarly (Figure 3). Only 3 samples in the GI disease group showed a higher fungal diversity than all other samples with more than 10 different fungal species present (D25, D6, and D7). Fungi from the phyla Ascomycota and Basidiomycota were detected, with Cyniclomyces guttulatus being the most abundant species in the healthy and GI disease groups, with a relative abundance of 95.17 ± 18.3% and 86.76 ± 30.1%, respectively (Figure 2). Only 1 fungal species was significantly different between the groups and significantly enriched in the GI disease group, Malassezia restricta. It is noteworthy that this species was present at only 0.011% relative abundance in the GI disease group, so in very low numbers. However, it was never present in the healthy group (Figure 4; Supplementary Table S2).
Discussion
Rabbits are particularly susceptible to GI diseases due to their highly specialized hindgut fermentation and cecotrophy; therefore, an understanding of the normal GI microbiome of rabbits is essential for clinicians attempting to treat their disorders. Recent research has suggested that the most common organisms present in the GI tract of rabbits include bacteria from the families Ruminococcaceae and Lachnospiraceae as well as bacteria from the phylum Bacteroidetes.7,10 However, when analyzed separately the microbiome of cecotropes mimics the microbiome of the cecum and differs dramatically from that of normal fecal pellets, suggesting more complexity to the GI microbiome in rabbits than has previously been discussed.5–7,26
Our study was intended to more specifically classify the microbiome of the feces of healthy and diseased rabbits based on the organisms present in their standard fecal pellets. This was accomplished using NGS in order to rule out the potential for some bacteria to overgrow or outcompete more fastidious species on traditional agar plate cultures. Similarly, by using NGS to classify all of the bacteria and fungi in the samples, the need for more species- or genus-specific quantitative PCR primers, which could easily fail to identify large groups of organisms, is overcome. While collecting data via broad NGS does produce a large volume of information for each sample, comparing the microbiomes of healthy rabbits against unhealthy rabbits allowed for a better understanding of which organisms are more prominent in each subset of samples.
Historically, organisms from the phylum Bacteroidetes have been identified in abundance in the fecal microbiome of rabbits.8,9,27 Recent studies7,26 have suggested that when comparing the microbiome of the rabbit cecum with that of the actual fecal material, there are major differences. Specifically, Bacteroidetes have historically been mainly characterized in the cecal microbiome but not in the fecal microbiome.7 In comparison, the healthy rabbit feces in our study contained many organisms from the Bacteroidetes phylum, suggesting historical misinterpretation of the represented species due to the inaccuracies of agar plating techniques. Specifically, Bacteroidales sp, Odoribacter sp, and Paraprevotella xylaniphila were noted to be more prevalent in the feces of healthy rabbits as compared to rabbits with GI disease. Only Bacteroides uniformis was found in higher abundance in unhealthy rabbit feces as compared to healthy. This suggests that Bacteroidetes are not a clear indicator of GI health or disease unless present in overwhelming majority.
Previous research has suggested that compared to other mammals, rabbits lack Lactobacillus in their gut microbiome but do normally carry Clostridium species.2,3 This is important to consider when selecting probiotic supplements for rabbits as the majority made for mammals include Lactobacillus colonies or other organisms within the same family.28–30 In our study, only 1 organism from the Lactobacillales order was identified above the minimal threshold of abundance (Enterococcus gallinarum-saccharolyticus); however, this organism was not present in significantly different numbers when comparing healthy and diseased rabbits. In comparison, many species within the Clostridiales order were present in the feces of both healthy and unhealthy rabbits. Specifically, Lachnospiraceae sp, Papillibacter sp, and Ruminococcus sp were present in higher abundance in healthy rabbits. Comparatively, Lachnoclostridium sp, Anaerotruncus sp, and Subdoligranulum sp were found in greater abundance in rabbits with GI disease. In addition, only 1 rabbit in the healthy group was found to lack Clostridiales sp organisms completely in its fecal sample. While individual variation in the microbiome does occur normally, this could also be explained by the small sample size of the study and/or undetected underlying diseases leading to immunosuppression.
In a recent study31 on weanling rabbits, Lachnoclostridium sp was noted to be positively associated with lower weaning weights in meat rabbits, and organisms from the family Ruminococcaceae were associated with higher weaning weights. On the contrary, in our study Ruminococcaceae spp were found in abundance in both healthy and unhealthy rabbit feces, with no statistically significant difference in the groups. Our results were in agreement for Lachnoclostridium sp to be associated with a disease-state GI tract as stated previously. We suspect the disparity in our findings compared to Fang et al31 was a result of different microbial DNA extraction techniques; specifically, the technique used by Fang et al31 uses chemical lysis and heat, which can bias the results toward easier-to-lyse microorganisms. The DNA extraction method employed in this study uses chemical lysis as well as standard mock communities (cell mock communities and extracted DNA mock communities), which are used as a positive control to account and detect any bias in the NGS workflow. Another study32 in weanling rabbits suggested that as more solid food is incorporated into the diet, proportions of Bacteroidetes and Proteobacteria decrease and Firmicutes increase. Similar results were found in Combes et al3; the GI microbiome of rabbits is dominated by organisms from the Bacteroidetes phylum at a young age but shifts to contain more Firmicutes, Lachnospiraceae, and Ruminococcaceae as rabbits mature.3 In our study, at the family level only Prevotellaceae were significantly more abundant in healthy rabbit samples. These differences could be explained by the fact that our study population contains a wide range of ages and the fact that all the rabbits in our study were above weaning age.
Studies comparing the microbiome of the different sections of the rabbit GI tract have identified Akkermansia sp as a common bacteria in the healthy cecum of rabbits as well as in the stomach and intestines of rabbits suffering from epizootic rabbit enteropathy (ERE).7,12,13,33 In our study, Akkermansia sp were significantly more common in the feces of healthy rabbits than rabbits with GI disease. None of the rabbits in our study were diagnosed with ERE. This suggests that Akkermansia sp may normally be an indicator of GI health but can become an opportunistic pathogen in cases of ERE where the normal GI microbiome is disrupted.
Other species of interest in this study include Melainabacteria sp and Saccharibacteria sp. Our results suggest that these organisms are more prevalent in the GI tract of healthy rabbits as opposed to unhealthy rabbits and could therefore be used as predictors of a healthy microbiome. Melainabacteria sp are related to cyanobacteria and have previously been identified in ground water samples as well as symbiotically in the human GI tract.34 Melainabacteria sp have not been previously reported in the GI microbiome of rabbits. Saccharibacteria sp are parasitic bacteria that inhabit other bacterial organisms. They have been identified in the human oral microbiome, and some species within this genus have been associated with oral mucosal infectious diseases.35 Saccharibacteria sp have been reported in the healthy microbiome of rabbits, though it should be noted that they were 1 of the least abundant bacterial phyla recognized in that particular study.12 This may also be a reflection of the region as this study was limited to 1 private practice; future studies would benefit from a wider sample range collected from other regions in the country or different countries.
Of note, commonly pathogenic bacteria, such as E coli and L intracellularis, were not isolated in significant numbers from either the healthy or diseased samples in this study. Historically, organisms from the family Enterobacteriaceae, such as E coli, have been associated with disease in the GI tract.36 L intracellularis is the causative organism of proliferative enteropathy in rabbits.37,38 Based on our study, such known pathologic organisms should never be considered a normal finding in the rabbit GI tract, though again relatively small sample size and small sample region are considerations for further study of these organisms.
The normal GI tract of the rabbit contains C guttulatus, a commensal yeast. This organism is commonly isolated from the stomach lining.3,39 It has been found in moderate numbers in healthy rabbits but has been studied as a potential opportunistic pathogen in cases of rabbit diarrhea.39 C guttulatus has also been isolated from fecal samples of dogs with diarrhea but has not been defined as the cause of clinical disease.40,41 C guttulatus was found in abundance in both healthy and diseased samples in our study, though without statistically significant differences. Further studies are required to determine the function of this organism in the rabbit GI tract and its importance or relevance to overall GI health.
While M restricta is a common fungal organism that often causes cutaneous disease in mammalian species, it is an uncommon finding in the fecal material of domestic mammals. In humans, M restricta has been isolated from fecal samples of patients afflicted with other primary GI disease, such as inflammatory bowel disease, as well as in patients with hepatocellular carcinoma.42–46 There are no reports of M restricta associated with GI disease in rabbits. In our study, M restricta was not identified above the minimum threshold in any healthy fecal samples; however, it was isolated in small numbers (but statistically significant numbers) from rabbits with GI disease. M restricta could be contributing to GI disease; however, it should be investigated further as a marker for an unhealthy GI microbiome in rabbits.
A potential limitation of this study is its descriptive nature and the lack of specific correlation between certain organisms and clinical disease. Further research would be required to determine whether specific organisms could be markers of impending ileus and dysbiosis. This study also did not evaluate other organisms outside of bacteria and fungi, such as parasites or viruses, or their impact on the overall GI microbiome. Clinically, this study is important as it gives a baseline of what could be considered normal organisms present in the fecal material of healthy rabbits; in the future, if clinicians continue to use NGS to evaluate the fecal microbiome of sick rabbits, it is important to differentiate which organisms are always present in the microbiome and which can be potentially pathogenic and require more specific treatment.
This study assessed the prominent bacterial and fungal families and specific species present in the feces of healthy rabbits, as well as those with GI disease, using NGS as a diagnostic tool to describe the microbiome in detail. Based on the differences noted in this study, organisms that could be considered specific markers of healthy GI microbiome include Akkermansia sp, Melainabacteria sp, and Saccharibacteria sp. Organisms that should be studied more closely and could be potential markers of a diseased GI microbiome include B uniformis and, interestingly, M restricta. It is also important to note that at no point did our study find large numbers of Lactobacillus organisms if any at all; for this reason, clinicians should use caution when selecting appropriate probiotic supplements, many of which marketed for rabbits and mammals are composed primarily of Lactobacillus colonies or other organisms that are not present in the normal rabbit GI microbiome.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
None reported.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
The authors would like to acknowledge the MiDOG LLC group for their funding of the DNA analysis for this study.
ORCID
G. Vecere https://orcid.org/0000-0002-6116-5103
J. A. Krumbeck https://orcid.org/0000-0002-4865-7170
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