We recently investigated the applicability of antibiotic-treated recipient mice for transfer of different gut microbiota profiles. With this addendum we elaborate on perspectives and limitations of using antibiotics as an alternative to germ-free (GF) technology in microbial transplantation studies, and we speculate on the housing effect. It is possible to transfer host phenotypes via fecal transplantation to antibiotic-treated animals, but problems with reproducibility, baseline values, and antibiotic resistance genes should be considered. GF animals maintained in isolators still seem to be the best controlled models for long-term microbial transplantation, but antibiotic-treated recipients are also commonly utilized. We identify a need for systematic experiments investigating the stability of microbial transplantations by addressing 1) the recipient status as either GF, antibiotic-treated or specific pathogen free and 2) different levels of protected housing systems. In addition, the developmental effect of microbes on host physiological functions should be evaluated in the different scenarios.

Germ-free rodents colonized with microbiotas of interest are used for host-microbiota investigations and for testing microbiota-targeted therapeutic candidates. Traditionally, isolators are used for housing such gnotobiotic rodents due to optimal protection from the environment, but research groups focused on the microbiome are increasingly combining or substituting isolator housing with individually ventilated cage (IVC) systems. We compared the effect of housing systems on the gut microbiota composition of germ-free mice colonized with a complex microbiota and housed in either multiple IVC cages in an IVC facility or in multiple open-top cages in an isolator during three generations and five months. No increase in bacterial diversity as assessed by 16S rRNA gene sequencing was observed in the IVC cages, despite not applying completely aseptic cage changes. The donor bacterial community was equally represented in both housing systems. Time-dependent clustering between generations was observed in both systems, but was strongest in the IVC cages. Different relative abundance of a Rikenellaceae genus contributed to separate clustering of the isolator and IVC communities. Our data suggest that complex microbiotas are protected in IVC systems, but challenges related to temporal dynamics should be addressed.

Transplantation of germ-free (GF) mice with microbiota from mice or humans stimulates the intestinal immune system in disparate ways. We transplanted a human microbiota into GF C57BL/6 mice and a murine C57BL/6 microbiota into GF C57BL/6 mice and Swiss-Webster (SW) mice. Mice were bred to produce an offspring generation. 56% of the Operational Taxonomic Units (OTUs) present in the human donor microbiota established in the recipient mice, whereas 81% of the C57BL/6 OTUs established in the recipient C57BL/6 and SW mice. Anti-inflammatory bacteria such as Faecalibacterium and Bifidobacterium from humans were not transferred to mice. Expression of immune-related intestinal genes was lower in human microbiota-mice and not different between parent and offspring generation. Expression of intestinal barrier-related genes was slightly higher in human microbiota-mice. Cytokines and chemokines measured in plasma were differentially present in human and mouse microbiota-mice. Minor differences in microbiota and gene expression were found between transplanted mice of different genetics. It is concluded that important immune-regulating bacteria are lost when transplanting microbiota from humans to C57BL/6 mice, and that the established human microbiota is a weak stimulator of the murine immune system. The results are important for study design considerations in microbiota transplantation studies involving immunological parameters. The gut microbiota is an important component of human health. For studying its role in health and disease, aiming for the development of microbiota-targeting therapeutics, food products and ingredients, mice transplanted with human microbiotas (HMs) have been described and applied for several decades 1-6 , although concerns pertaining to limitations of this model system have been raised 7-10. Often such models are only studied on their phenotypic expression and the microbiota is not characterized, or the opposite is the case 8. Laboratory rodents are routinely raised in specific pathogen free (SPF) barrier facilities strictly protected from their wild conspecifics. For many years the microbial starting point for many rodent breeding colonies has been the Altered Schaedler Flora (ASF) or variants thereof. ASF consists of eight specific bacterial strains originating from conventional laboratory mice from the 1960's and 1970's, and it is therefore considered mouse-specific 11,12. In addition to this, laboratory rodents are exposed to microbes deriving from human staff in the facility, at least if bedding, food, and other materials are sterilized before introduced to the facility. Evolutionary adaptation to the host environment may drive formation of mouse-specific species and strains originally derived from humans, as

Whereas a significant role for intestinal microbiota in affecting the pathogenesis and progression of chronic hepatic diseases is well documented, the contribution of the intestinal flora to acute liver injury has not been extensively addressed. Elucidating the influence of the intestinal microbiota on acute liver inflammation would be important for better understanding the transition from acute injury to chronic liver disease. Using the Concanavalin A (ConA)-induced liver injury model in laboratory mice, we show that the severity of acute hepatic damage varies greatly among genetically identical mice raised in different environments and harboring distinct microbiota. Through reconstitution of germ-free (GF) mice, and the co-housing of conventional mice, we provide direct evidence that manipulation of the intestinal flora alters susceptibility to ConA-induced liver injury. Through deep sequencing of the fecal microbiome, we observe that the relative abundance of Ruminococcaceae, a Gram(+) family within the class Clostridia, but distinct from segmented filamentous bacteria, is positively associated with the degree of liver damage. Searching for the underlying mechanism(s) that regulate susceptibility to ConA, we provide evidence that the extent of liver injury following triggering of the death receptor Fas varies greatly as a function of the microbiota. We demonstrate that the extent of Fas induced liver injury increases in GF mice after microbiota reconstitution, and decreases in conventionally raised mice following reduction of intestinal bacterial load, by antibiotic treatment. We also show that the regulation of sensitivity to Fas induced liver injury is dependent upon the Toll-Like Receptor signaling molecule MyD88.ConclusionThe status and composition of the intestinal microbiota determine the susceptibility to ConA-induced acute liver injury. The microbiota acts as a rheostat, actively modulating the extent of liver damage in response to Fas triggering.

Rats with implanted telemetry transponders were blood sampled by jugular puncture, periorbital puncture or tail vein puncture, or sampled by jugular puncture in carbon dioxide (CO2), isoflurane or without anaesthesia in a crossover design. Heart rate, blood pressure and body temperature were registered for three days after sampling. Initially blood pressure increased, but shortly after sampling it decreased, which led to increased heart rate. Sampling induced rapid fluctuations in body temperature, and an increase in body temperature. Generally, rats recovered from sampling within 2-3 h, except for rats sampled from the tail vein, which showed fluctuations in body temperature in excess of 30 h after sampling. Increases in heart rate and blood pressure within the first hours after sampling indicated that periorbital puncture was the method that had the largest acute impact on the rats and that it might take an extra hour to recover from it. CO2 anaesthesia seemed unable to prevent the increase in blood pressure and the fluctuations in body temperature induced by blood sampling, and up to 10 h after sampling, the rats were still affected by CO2 anaesthesia. Rats anaesthetized with isoflurane showed lower increases in blood pressure after, and fewer fluctuations in body temperature during sampling, and the post-anaesthetic effects of isoflurane, if any, seemed to disappear immediately after sampling. It is, therefore, concluded that blood sampling in rats by jugular puncture seems to be the method from which rats most rapidly recover when compared with periorbital puncture and tail vein puncture, and that for anaesthesia, isoflurane is recommended in preference to CO2.

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Laboratory mice show poor translation to humans within a range of pre-clinical fields, which might be due to their specific pathogen free (SPF) status. SPF mice have CD8+ effector memory T cell levels more comparable to newborn than to adult humans or pet shop mice. However, reintroducing pathogens would re-introduce diseases, and, therefore, research facilities should look for safer alternatives. We inactivated mouse adenovirus type 1, minute virus of mice, mouse hepatitis virus, Sendai virus, Theiler’s encephalomyelitis virus (GD 7), and Mycoplasma pulmonis by ultraviolet irradiation, and mixed them with the adjuvant Addavax®. A subcutaneous injection twice with two weeks interval with 10 µg of each pathogen generated CD8+ effector memory T cell quantities significantly higher than untreated mice, and 82.3% of the pre-immunized mice were comparable with pet shop mice, while the overall quantities and percentages of T cells were un-affected. None of the pathogens were transferred from the pre-immunized mice to co-housed non-immunized mice. Pre-immunization with inactivated murine pathogens, therefore, may be used to safely create an immunological memory in mice.