Seasonal restructuring facilitates compositional convergence of gut microbiota in free-ranging rodents.

Gut microbes provide essential services to their host and shifts in their composition can impact host fitness. However, despite advances in our understanding of how microbes are assembled in the gut, we understand little about the stability of these communities within individuals, nor what factors influence its composition over the life of an animal. For this reason, we conducted a longitudinal survey of the gut microbial communities of individual free-ranging woodrats (Neotoma spp.) across a hybrid zone in the Mojave Desert, USA, using amplicon sequencing approaches to characterize gut microbial profiles and diet. We found that gut microbial communities were individualized and experienced compositional restructuring as a result of seasonal transitions and changes in diet. Turnover of gut microbiota was highest amongst bacterial subspecies and was much lower at the rank of Family, suggesting there may be selection for conservation of core microbial functions in the woodrat gut. Lastly, we identified an abundant core gut bacterial community that may aid woodrats in metabolizing a diet of plants and their specialized metabolites. These results demonstrate that the gut microbial communities of woodrats are highly dynamic and experience seasonal restructuring which may facilitate adaptive plasticity in response to changes in diet.

Analyses of Bloodmeal Hosts and Prevalence of Rickettsia parkeri in the Gulf Coast Tick Amblyomma maculatum (Acari: Ixodidae) From a Reconstructed Piedmont Prairie Ecosystem, North Carolina.

Host feeding patterns and the prevalence of infection with Rickettsia parkeri were determined for the primary vector, Amblyomma maculatum Koch as well as sympatric tick species A. americanum (Linnaeus) and Dermacentor variabilis (Say) collected from a reconstructed prairie in the Piedmont region of North Carolina during 2011 and 2012. The occurrence of R. parkeri among A. maculatum adults and nymphs was 36.9% (45/122) and 33.3% (2/6), respectively. Rickettsia parkeri was detected in a single male A. americanum 2.3% (1/43). A PCR-reverse line blot hybridization assay of a 12S rDNA fragment amplified from remnant larval and nymphal bloodmeals of host-seeking ticks was used to identify bloodmeal hosts. Of the tick samples tested, bloodmeal host identification was successful for 29.3% (12/41) of adult A. americanum and 39.2% (20/51) of adult D. variabilis. For A. maculatum, bloodmeal host identification was successful for 50% (61/122) of adults collected from vegetation and 100% (4/4) of nymphs removed from cotton rats (Sigmodon hispidus Say and Ord). The cotton rat was the most common bloodmeal host with 59.0% (36/61) identified for adult A. maculatum. No statistically significant association was observed, however, between bloodmeal host and pathogen prevalence for any tick species. While the cotton rat was an important bloodmeal host for A. maculatum nymphs, this vertebrate did not appear to be the primary source of R. parkeri infection for A. maculatum.

Microbiome stability and structure is governed by host phylogeny over diet and geography in woodrats (Neotoma spp.).

The microbiome is critical for host survival and fitness, but gaps remain in our understanding of how this symbiotic community is structured. Despite evidence that related hosts often harbor similar bacterial communities, it is unclear whether this pattern is due to genetic similarities between hosts or to common ecological selection pressures. Here, using herbivorous rodents in the genus Neotoma, we quantify how geography, diet, and host genetics, alongside neutral processes, influence microbiome structure and stability under natural and captive conditions. Using bacterial and plant metabarcoding, we first characterized dietary and microbiome compositions for animals from 25 populations, representing seven species from 19 sites across the southwestern United States. We then brought wild animals into captivity, reducing the influence of environmental variation. In nature, geography, diet, and phylogeny collectively explained ∼50% of observed microbiome variation. Diet and microbiome diversity were correlated, with different toxin-enriched diets selecting for distinct microbial symbionts. Although diet and geography influenced natural microbiome structure, the effects of host phylogeny were stronger for both wild and captive animals. In captivity, gut microbiomes were altered; however, responses were species specific, indicating again that host genetic background is the most significant predictor of microbiome composition and stability. In captivity, diet effects declined and the effects of host genetic similarity increased. By bridging a critical divide between studies in wild and captive animals, this work underscores the extent to which genetics shape microbiome structure and stability in closely related hosts.

Loss of function dysbiosis associated with antibiotics and high fat, high sugar diet.

The incidence of urinary stone disease (USD) has increased four-fold in 50 years. Oxalate, which is degraded exclusively by gut bacteria, is an important constituent in 80% of urinary stones. We quantified the effects of antibiotics and a high fat/high sugar (HFHS) diet on the microbial metabolism of oxalate in the gut. High and low oxalate-degrading mouse models were developed by administering fecal transplants from either the wild mammalian rodent Neotoma albigula or Swiss-Webster mice to Swiss-Webster mice, which produces a microbiota with or without the bacteria necessary for persistent oxalate metabolism, respectively. Antibiotics led to an acute loss of both transplant bacteria and associated oxalate metabolism. Transplant bacteria exhibited some recovery over time but oxalate metabolism did not. In contrast, a HFHS diet led to an acute loss of function coupled with a gradual loss of transplant bacteria, indicative of a shift in overall microbial metabolism. Thus, the effects of oral antibiotics on the microbiome form and function were greater than the effects of diet. Results indicate that both antibiotics and diet strongly influence microbial oxalate metabolism.

Metagenomic sequencing provides insights into microbial detoxification in the guts of small mammalian herbivores (Neotoma spp.).

Microbial detoxification of plant toxins influences the use of plants as food sources by herbivores. Stephen's woodrats (Neotoma stephensi) specialize on juniper, which is defended by oxalate, phenolics and monoterpenes, while closely related N. albigula specialize on cactus, which only contains oxalate. Woodrats maintain two gut chambers harboring dense microbial communities: a foregut chamber proximal to the major site of toxin absorption, and a cecal chamber in their hindgut. We performed several experiments to investigate the location and nature of microbial detoxification in the woodrat gut. First, we measured toxin concentrations across gut chambers of N. stephensi. Compared to food material, oxalate concentrations were immediately lower in the foregut, while concentrations of terpenes remained high in the foregut, and were lowest in the cecal chamber. We conducted metagenomic sequencing of the foregut chambers of both woodrat species and cecal chambers of N. stephensi to compare microbial functions. We found that most genes associated with detoxification were more abundant in the cecal chambers of N. stephensi. However, some genes associated with degradation of oxalate and phenolic compounds were more abundant in the foregut chambers. Thus, microbial detoxification may take place in various chambers depending on the class of chemical compound.

The rice rat Euryoryzomys russatus, a competent amplifying host of Rickettsia parkeri strain Atlantic rainforest for the tick Amblyomma ovale.

Rickettsia parkeri strain Atlantic rainforest (SAR) is the etiological agent of a spotted fever group rickettsiosis in Brazil, where it is transmitted to humans by the tick Amblyomma ovale. A previous study demonstrated that R. parkeri SAR was successfully maintained in A. ovale ticks by transstadial and transovarial passages; however, because this agent induced lower reproduction rates in A. ovale, the participation of a vertebrate amplifier host, yet to be determined, was speculated. Since the rice rat Euryoryzomys russatus was demonstrated to be the most important host for immature stages of A. ovale in a focus of R. parkeri SAR transmission, the present study evaluated the competence of rice rats to act as amplifying host of R. parkeri SAR for A. ovale ticks. Rice rats were infested with R. parkeri SAR-infected A. ovale nymphs, and four days later with uninfected A. ovale larvae. Rickettsial transmission to rats was confirmed by seroconversion to R. parkeri antigens. Detached engorged larvae were allowed to molt to nymphs, in which rickettsial DNA was detected in up to 60% (mean: 20%) of the specimens. When part of these nymphs was allowed to feed on susceptible rice rats, rickettsial transmission was confirmed by seroconversion, indicating that there was successful horizontal transmission of R. parkeri SAR from infected nymphs to uninfected larvae in the previous acquisition infestations. Because we used naïve, susceptible rats, we infer that this horizontal transmission occurred via a systemic infection (rickettsemia) in the rat. Our results, coupled with previous epidemiological studies, suggest that under natural conditions rice rats could be acting as amplifying hosts of R. parkeri SAR to A. ovale ticks.

New host species for Leptospira borgpetersenii and Leptospira interrogans serovar Copenhageni.

We investigated the presence of infection by Leptospira spp. in an assembly of Sigmodontinae rodents from the Paraná Delta, Argentina. Rodents were captured in places with natural grassland, implanted forest, with and without raising cattle and in sites prone and not prone to flooding. The DNA was amplified from cultured isolates by PCR and Leptospira spp. strains were genotyped using Multiple - Locus Variable Number Tandem Repeat Analysis (MLVA). We isolated Leptospira interrogans serovar Copenhageni from Oligoryzomys nigripes, Leptospira borgpetersenii from Scapteromys aquaticus and Leptospira interrogans serovar Icterohaemorrhagiae from Akodon azarae. The zoonotic Leptospira isolated and genotyped from O. nigripes and S. aquaticus are the first reports from these species. The geographic range of these rodent species include, in addition to Argentina, the countries of Paraguay, Uruguay and Brazil, suggesting that these rodents might be involved in the transmission of spirochetes in other regions. Human and animal health care professionals should be alert to the potential occurrence of leptospirosis in areas where these rodent species are present.

Microbial Community Transplant Results in Increased and Long-Term Oxalate Degradation.

Gut microbes are essential for the degradation of dietary oxalate, and this function may play a role in decreasing the incidence of kidney stones. However, many oxalate-degrading bacteria are susceptible to antibiotics and the use of oxalate-degrading probiotics has only led to an ephemeral reduction in urinary oxalate. The objective of the current study was to determine the efficacy of using whole-community microbial transplants from a wild mammalian herbivore, Neotoma albigula, to increase oxalate degradation over the long term in the laboratory rat, Rattus norvegicus. We quantified the change in total oxalate degradation in lab rats immediately after microbial transplants and at 2- and 9-month intervals following microbial transplants. Additionally, we tracked the fecal microbiota of the lab rats, with and without microbial transplants, using high-throughput Illumina sequencing of a hyper-variable region of the 16S rRNA gene. Microbial transplants resulted in a significant increase in oxalate degradation, an effect that persisted 9 months after the initial transplants. Functional persistence was corroborated by the transfer, and persistence of a group of bacteria previously correlated with oxalate consumption in N. albigula, including an anaerobic bacterium from the genus Oxalobacter known for its ability to use oxalate as a sole carbon source. The results of this study indicate that whole-community microbial transplants are an effective means for the persistent colonization of oxalate-degrading bacteria in the mammalian gut.

Effect of Dietary Oxalate on the Gut Microbiota of the Mammalian Herbivore Neotoma albigula.

Diet is one of the primary drivers that sculpts the form and function of the mammalian gut microbiota. However, the enormous taxonomic and metabolic diversity held within the gut microbiota makes it difficult to isolate specific diet-microbe interactions. The objective of the current study was to elucidate interactions between the gut microbiota of the mammalian herbivore Neotoma albigula and dietary oxalate, a plant secondary compound (PSC) degraded exclusively by the gut microbiota. We quantified oxalate degradation in N. albigula fed increasing amounts of oxalate over time and tracked the response of the fecal microbiota using high-throughput sequencing. The amount of oxalate degraded in vivo was linearly correlated with the amount of oxalate consumed. The addition of dietary oxalate was found to impact microbial species diversity by increasing the representation of certain taxa, some of which are known to be capable of degrading oxalate (e.g., Oxalobacter spp.). Furthermore, the relative abundances of 117 operational taxonomic units (OTU) exhibited a significant correlation with oxalate consumption. The results of this study indicate that dietary oxalate induces complex interactions within the gut microbiota that include an increase in the relative abundance of a community of bacteria that may contribute either directly or indirectly to oxalate degradation in mammalian herbivores.

Inoculation of tannin-degrading bacteria into novel hosts increases performance on tannin-rich diets.

It has been hypothesized that herbivores host tannin-degrading bacteria (TDB) to overcome the toxic challenges posed by plant tannins. While TDB have been isolated from the guts of numerous mammals, their functional significance to their hosts has never been explicitly tested. We introduced TDB into lab rats, which do not host TDB, and measured host performance on tannin-rich diets. We first isolated three species of TDB, Escherichia coli, Bacillus subtilis and Enterococcus faecalis, from the guts of the desert woodrat (Neotoma lepida), which regularly feeds on tannin-rich plants. Then, we inoculated isolated TDB, as well as full woodrat microbial communities into laboratory rats. A control group was inoculated with sterilized woodrat faeces. Recipient lab rats were fed increasing concentrations of tannic acid, and we monitored tannic acid intake, body mass and liver damage as measured by serum alanine aminotransferase activity. Lab rats given TDB as isolates or full communities exhibited increased tannic acid intake, higher maintenance of body mass and lower indicators of liver damage compared with control animals. These differences were maintained when the trial was repeated after 6 weeks of feeding on tannin-free diets. Our results are the first to demonstrate that TDB significantly increase host performance on tannin-rich diets.

The microbial community structure of the cotton rat nose.

The cotton rat nose is commonly used as a model for Staphylococcus aureus colonization, as it is both physiologically and anatomically comparable to the human nares and can be easily colonized by this organism. However, while the colonization of the human anterior nares has been extensively studied, the microbial community structure of cotton rat noses has not been reported so far. We describe here the microbial community structure of the cotton rat (Sigmodon hispidus) nose through next-generation sequencing of 16S rRNA gene amplicons covering the V1-V2 region and the analysis of nearly full length 16S rRNA genes of the major phylotypes. Roughly half of the microbial community was composed of two undescribed species of the genus Campylobacter, with phylotypes belonging to the genera Catonella, Acholeplasma, Streptobacillus and Capnocytophaga constituting the predominant community members. Thus, the nasal community of the cotton rat is uniquely composed of several novel bacterial species and may not reflect the complex interactions that occur in human anterior nares. Mammalian airway microbiota may, however, be a rich source of hitherto unknown microbes.

Gut microbes of mammalian herbivores facilitate intake of plant toxins.

The foraging ecology of mammalian herbivores is strongly shaped by plant secondary compounds (PSCs) that defend plants against herbivory. Conventional wisdom holds that gut microbes facilitate the ingestion of toxic plants; however, this notion lacks empirical evidence. We investigated the gut microbiota of desert woodrats (Neotoma lepida), some populations of which specialise on highly toxic creosote bush (Larrea tridentata). Here, we demonstrate that gut microbes are crucial in allowing herbivores to consume toxic plants. Creosote toxins altered the population structure of the gut microbiome to facilitate an increase in abundance of genes that metabolise toxic compounds. In addition, woodrats were unable to consume creosote toxins after the microbiota was disrupted with antibiotics. Last, ingestion of toxins by naïve hosts was increased through microbial transplants from experienced donors. These results demonstrate that microbes can enhance the ability of hosts to consume PSCs and therefore expand the dietary niche breadth of mammalian herbivores.

Wild-caught rodents retain a majority of their natural gut microbiota upon entrance into captivity.

Experiments conducted on captive animals allow scientists to control many variables; however, these settings are highly unnatural. Previous research has documented a large difference in microbial communities between wild animals and captive-bred individuals. However, wild-caught animals brought into captivity might retain their natural microbiota and thus provide a better study system in which to investigate the ecology of the gut microbiome. We collected individuals of the desert woodrat (Neotoma lepida) from nature and investigated changes in the microbial community over 6 months in captivity. Additionally, we inventoried potential environmental sources of microbes (food, bedding) from the wild and captivity. We found that environmental sources do not make large contributions to the woodrat gut microbial community. We documented a slight decrease in several biodiversity metrics over 6 months in captivity, yet the magnitude of change was small compared with other studies. Wild and captive animals shared 64% of their microbial species, almost twice that observed in other studies of wild and captive-bred individuals (≤ 37% shared). We conclude that wild-caught animals brought into captivity retain a substantial proportion of their natural microbiota and represent an acceptable system in which to study the gut microbiome.

The gastrointestinal tract of the white-throated Woodrat (Neotoma albigula) harbors distinct consortia of oxalate-degrading bacteria.

The microbiota inhabiting the mammalian gut is a functional organ that provides a number of services for the host. One factor that may regulate the composition and function of gut microbial communities is dietary toxins. Oxalate is a toxic plant secondary compound (PSC) produced in all major taxa of vascular plants and is consumed by a variety of animals. The mammalian herbivore Neotoma albigula is capable of consuming and degrading large quantities of dietary oxalate. We isolated and characterized oxalate-degrading bacteria from the gut contents of wild-caught animals and used high-throughput sequencing to determine the distribution of potential oxalate-degrading taxa along the gastrointestinal tract. Isolates spanned three genera: Lactobacillus, Clostridium, and Enterococcus. Over half of the isolates exhibited significant oxalate degradation in vitro, and all Lactobacillus isolates contained the oxc gene, one of the genes responsible for oxalate degradation. Although diverse potential oxalate-degrading genera were distributed throughout the gastrointestinal tract, they were most concentrated in the foregut, where dietary oxalate first enters the gastrointestinal tract. We hypothesize that unique environmental conditions present in each gut region provide diverse niches that select for particular functional taxa and communities.

Herbivorous rodents (Neotoma spp.) harbour abundant and active foregut microbiota.

Symbiotic gut microbes have facilitated the success of herbivorous mammals, which are generally grouped into foregut- and hindgut-fermenters. However, rodents are primarily herbivorous and exhibit a variety of gastrointestinal anatomies. Most rodents house microbes in hindgut chambers, such as the caecum and colon. Some rodents also exhibit stomach segmentation with a foregut chamber proximal to the stomach. For over a century, scientists have hypothesized that this foregut chamber houses a microbial community, yet this has never been explicitly examined. We investigated the capacity of each of the gut regions to house microbes by measuring size, pH, bacterial cell density, concentrations of microbial metabolites and digesta transit time in woodrats (Neotoma spp.). We also compared microbial communities across gut chambers, as well as faeces, using 16S rRNA sequencing. This allowed us to test the appropriateness of using faeces as a proxy for microbial communities of other gut chambers. We found that woodrats house foregut microbial communities with similar density and volatile fatty acid concentrations to rumen ecosystems. Resident microbial communities varied between gut chambers, and faecal bacterial communities were significantly different from caecal and colonic communities. The foregut microbiota may provide a number of physiological services to the host.

The rare ospC allele L of Borrelia burgdorferi sensu stricto, commonly found among samples collected in a coastal plain area of the southeastern United States, is associated with ixodes affinis ticks and local rodent hosts Peromyscus gossypinus and Sigmodon hispidus.

The rare ospC allele L was detected in 30% of Borrelia burgdorferi sensu stricto strains cultured from a tick species, Ixodes affinis, and two rodent host species, Peromyscus gossypinus and Sigmodon hispidus, collected in a coastal plain area of Georgia and South Carolina, in the southeastern United States.

Antigen variability in Anaplasma phagocytophilum during chronic infection of a reservoir host.

Anaplasma phagocytophilum is an obligately intracellular, tick-transmitted, bacterial pathogen of humans and other animals. In order to evade host immunity during the course of infection, A. phagocytophilum utilizes gene conversion to shuffle approximately 100 functional pseudogenes into a single expression cassette of the msp2(p44) gene, which encodes the major surface antigen, major surface protein 2 (Msp2). The role and extent of msp2(p44) recombination in a reservoir host for A. phagocytophilum have not been evaluated. In the current study, we explored patterns of recombination and expression site variability of the msp2(p44) gene in three chronically infected woodrats, a reservoir for the disease in the Western USA. All three woodrats developed persistent infection of at least 6 months duration; two of them maintained active infection for at least 8 months. In total, we detected the emergence of 60 unique msp2(p44) expression site variants with no common temporal patterns of expression site recombination among the three A. phagocytophilum populations. Both the strength of infection (i.e. pathogen load) and the genetic diversity of pseudogenes detected at the msp2(p44) expression site fluctuated periodically during the course of infection. An analysis of the genomic pseudogene exhaustion rate showed that the repertoire of pseudogenes available to the A. phagocytophilum population could in theory become depleted within a year. However, the apparent emergence of variant pseudogenes suggests that the pathogen could potentially evade host immunity indefinitely. Our findings suggest a tightly co-evolved relationship between A. phagocytophilum and woodrats in which the pathogen perpetually evades host immunity yet causes no detectable disease.

Borrelia carolinensis sp. nov., a novel species of the Borrelia burgdorferi sensu lato complex isolated from rodents and a tick from the south-eastern USA.

A group of 16 isolates with genotypic characteristics different from those of known species of the Borrelia burgdorferi sensu lato complex were cultured from ear biopsies of the rodents Peromyscus gossypinus and Neotoma floridana trapped at five localities in South Carolina, USA, and from the tick Ixodes minor feeding on N. floridana. Multilocus sequence analysis of members of the novel species, involving the 16S rRNA gene, the 5S-23S (rrf-rrl) intergenic spacer region and the flagellin, ospA and p66 genes, was conducted and published previously and was used to clarify the taxonomic status of the novel group of B. burgdorferi sensu lato isolates. Phylogenetic analysis based on concatenated sequences of the five analysed genomic loci showed that the 16 isolates clustered together but separately from other species in the B. burgdorferi sensu lato complex. The analysed group therefore represents a novel species, formally described here as Borrelia carolinensis sp. nov., with the type strain SCW-22(T) (=ATCC BAA-1773(T) =DSM 22119(T)).