C57BL/6NCrl (B6N) Germ-Free Mice

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1 Summary Germ-free rodents have been essential to microbiome research and the production of specific pathogen-free (SPF) rodent models. This document describes the background, uses, production, shipment, and microbiological monitoring of Charles River s C57BL/6NCrl (B6N) germ-free mice. RESEARCH MODELS AND SERVICES C57BL/6NCrl (B6N) Germ-Free Mice Background Soon after birth, the gastrointestinal tract and other body surfaces of mammals are colonized by complex communities of microorganisms, traditionally termed microflora; more recently, they have also been called microbiota and microbiomes, which some differentiate as referring to microbial taxa and genomes, respectively. The normal autochthonous (i.e., indigenous) mammalian gut microbiota consist largely of beneficial, or commensal, bacteria that synthesize vitamins essential to host nutrition and provide a barrier to infection by pathogens. Gut flora also include significant numbers of archaea, eukaryotes, and viruses (including bacteriophages). 1 Microbes are by far most numerous in the large intestines, with concentrations that can reach trillions of microbial cells per gram of feces in the colon and represent 1,000 different species. 2 In humans, the number of cells that compose the microbiota reportedly are equivalent to or 10-fold greater than the number of human somatic cells, depending on whether nonnucleated erythrocytes are counted. 3 Therefore, it is not surprising that the gut and other microbiota have been found to play a key role in the development and homeostasis of host anatomy, physiology, metabolism, and immunity, as evidenced by the many abnormalities, such as an underdeveloped immune system and a markedly enlarged cecum, that characterize axenic (germ-free) rodents demonstrably free of all foreign bacteria as well as fungi, protozoa, parasites, and viruses. 4 Research into the role of microbiota in health and disease has increased exponentially during the past decade, encouraged by advances in molecular genetics that have led to the development of numerous genetically engineered mutant animal models, as well as sophisticated, culture-independent molecular tools for analyzing the microbiome, notably massively parallel next-generation DNA sequencing. 5 This research has demonstrated that the constituents of the gut microflora can abrogate or accentuate the phenotypes of mutant models. 6,7,8 Clinical studies have linked dysbiosis, or imbalances of microbiota, and the loss of microbial diversity (in part caused by the overuse of antibiotics in agriculture and medicine) to spikes in the incidence of an array of human diseases, ranging from juvenile diabetes to autism. 9,10 EVERY STEP OF THE WAY

2 Furthermore, the composition of patients microflora has recently been reported to influence the efficacy of cancer immunotherapy. 11,12 Thus, studying and explicating the interaction between hosts and their microbiota is of critical importance to public health as well as animal research. Charles River s experience with germ-free technology goes back to the 1950s, when the veterinarian who founded Charles River Laboratories, Dr. Henry Foster, and his colleagues incorporated germ-free rederivation into the cesarean-originated barrier-sustained process they pioneered for the large-scale production of SPF mice and rats. 13 In this process, germ-free rodents are associated (i.e., colonized) with a defined cocktail of commensal bacteria to normalize their physiology and prime their immune systems. The cocktail most often used for this purpose is the altered Schaedler flora (ASF) developed by Roger Orcutt and colleagues at Charles River in the 1970s (Table 1). 14,15 In contrast to the original Schaedler flora 16 on which it was based, the ASF is fully anaerobic; moreover, half of the eight species of bacteria in the ASF are extremely oxygen-sensitive (EOS) fusiform anaerobes highly representative of the autochthonous microbiota. Germfree and defined flora-associated animals are classified as gnotobiotic, from the Greek roots gnostos ( known ) and bios ( life ). By contrast, barrier-maintained SPF rodents develop a complex microbiota that is defined only to the extent that it does not include a limited list of pathogens. Table 1. Compositive of Charles River Altered Schaedler Flora (ASF)* Designation In Original Schaedler Taxonomy Genbank Accession ASF 356 Clostridium species AQFQ ASF 360 Lactobacillus intestinalis AQFR ASF 361 Lactobacillus murinus AQFs ASF 457 Mucispirillum schaedleri AYGZ ASF 492 Eubacterium plexicaudatum AQFT ASF 500 Pseudoflavonifactor species AYJP ASF 502 Clostridium species AQFU ASF 519 Parabacteroides goldsteinii AQFV * The four ASF bacteria from the original Schaedler flora were isolated from the stomach and intestines of NCS mice in the 1960s by Russell W. Schaedler at Rockefeller University. The other ASF organisms were originally isolated from the large intestine of CD-1 mice in the 1960s at Charles River by Roger P. Orcutt (a graduate student of Schaedler s). Research Applications B6N germ-free mice may be used as embryo transfer recipients or foster dams for germ-free rederivation of mutant mouse models. In addition, they may be compared to SPF or Elite (opportunistic pathogen-free) B6N mice to generally assess the relationship between microbiota and phenotypes. Alternatively, the germ-free B6N mice may be associated with a single microbial species (monoassociated), defined microbiota like the ASF, or complex polymicrobial mixtures to measure and understand the effects of microbiota on phenotypes and experimental responses. 17,18,19 Germ-free mice have also been engrafted with human microbiota by fecal transfer or inoculation of defined microflora in order to investigate the contribution of microbe-host relationships to human diseases. 20 Production Rederivation The B6N strain was obtained by Charles River from the National Institutes of Health in The current colonies of germ-free B6N mice were rederived by sterile hysterectomy followed by fostering on germ-free dams provided by the Gnotobiotics and Microbiology Core at Boston Children s Hospital. Extensive testing by culture and cultureindependent methods described below has verified the germ-free status of the rederived B6N colonies. C57BL/6NCrl (B6N) Germ-Free Mice

3 Husbandry An example of the plastic isolators in which the germ-free mice are housed is shown in Figure 1. Before being used, an isolator is tested for leaks, chemically sterilized with 2% peracetic acid, and ventilated. Sterilization of the ventilated isolator is confirmed by culturing swabs of surfaces, caging, and supplies collected from the isolator over several weeks. Figure 1. Plastic isolator for germ-free husbandry Once in use, an isolator is kept sterile by being ventilated with HEPA-filtered air under positive pressure. Supplies, such as food, bedding, water, and caging, are autoclaved in transfer cylinders. To assure sterilization, supplies are arranged in cylinders following standard configurations (Figure 2). Self-contained bioindicators containing heatstable bacterial spores (e.g., EZTest Steam, Mesa Labs) and temperature indicators are placed throughout each autoclave run, including inside a test cylinder. Autoclave printouts are examined to confirm that the appropriate cycle was chosen and ran without error. Temperature indicators are evaluated and the bioindicators, including those retrieved from inside the test cylinder, are incubated (at o C). Cylinders are released for use only if all bioindicators read negative. askcharlesriver@criver.com

4 Figure 2. Cylinder for autoclaving supplies View of a drum filled with cages, the inside of the drum showing underlying perforations in the steel structure required for sterilizing steam to reach internal materials during the autoclave cycle. To antiseptically import the supplies, the cylinder is attached to the isolator port with a plastic sleeve (Figure 3); the supplies are transferred into the isolator through a double-door lock system that has been disinfected by spraying with 2% peracetic acid or a sterilizing level of chlorine dioxide (e.g., CLIDO-S diluted 1:3:1). The double-door lock system is also used to transfer animals, samples, and other materials in and out of the isolator. All manipulations of mice and supplies inside the isolator are through gloves and sleeves attached to the isolator walls. Figure 3. Autoclaved cylinder attached to isolator Shipping To preserve their germ-free status during shipment, ethylene oxide gas. Upon receipt of a lightweight shipper, mice are transferred from the isolator in which they are the shipper should be attached to the port of the isolator housed through the disinfected double-door lock system and the mice antiseptically transferred. into cages within a germ-free shipper sterilized with C57BL/6NCrl (B6N) Germ-Free Mice

5 Microbiological Monitoring Germ-free colonies are monitored for extraneous bacteria and fungi, and for pathogens. Table 2 shows the test methods, samples, and frequencies. Testing for extraneous microbes is conducted frequently, based on the potentially high incidence of this type of contamination and its significant consequences to customers. Weekly, a slurry from each isolator (consisting of feces and environmental swabs in animal drinking water) is inoculated onto various culture media, then incubated aerobically and anaerobically in a dedicated anaerobic workstation. Because microbial contaminants may be fastidious or non-cultivable on cell-free media (like much of the indigenous microbiota), culture-independent methods are employed. Wet mounts of the slurries collected each week are examined by phase microscopy for motile organisms. In addition, feces collected quarterly from mice in each isolator are assayed by PCR for the bacterial 16S ribosomal RNA (rrna) gene. Table 2. Microbiological Surveillance of Germ-Free Mouse Isolators Methodology Microbiology PCR Gross and Microscopic Exams Tests Sample Type: Frequency: Culture Phase microscopy Bacterial 16S rrna Rodent Pathogens Necropsy Parasitology Slurry Weekly Feces Quarterly Serology MFIA/IFA Animals Annually Comprehensive health monitoring for pathogens is performed annually on mice from each isolator. This comparatively low testing frequency is justified by the historically negligible incidence of pathogens infecting isolator-housed colonies. Animal organs and tissues are grossly examined, and histopathology is carried out if lesions suggesting an infectious disease process are observed. Specimens from the gut and skin are examined microscopically for endo- and ectoparasites. Blood samples are screened for pathogen-specific antibodies by the multiplexed fluorometric immunoassay (MFIA ), with corroboration of unexpected (or indeterminate) findings mostly by indirect immunofluorescence assays (IFA). For cultural isolation of bacteria and fungi, various microbiologic culture media are inoculated with respiratory and gut samples and incubated aerobically and anaerobically. Samples for anaerobic culture are collected from euthanized mice dissected in an anaerobic workstation. Isolates are identified both according to their colonial and cellular morphology and by MALDI-TOF mass spectrometry, and, if necessary, by PCR. In addition, swabs of the skin, oral cavity, and feces are tested by PCR for pathogens of all types. The confirmed detection of bacterial, viral, parasitic, or fungal agents in germ-free mice or isolators would result in immediate cessation of shipment from the isolator and immediate elimination of the isolator colony. Charles River considers each isolator to be a microbiologic unit and will not test and cull individual cages within an isolator. It remains our policy to inform our customers in a timely manner of any breaches in animal health or genetic integrity, providing urgent colony health information via or other method. For assistance regarding specific information on Charles River monitoring procedures, additional data on animals, or interpretation of the monitoring information, please direct inquiries to Charles River Technical Services ( ) or AskCharlesRiver@crl.com. askcharlesriver@criver.com

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