Summary Germ-free have been essential to microbiome research and the production of specific pathogen-free (SPF) models. This document describes the background, uses, production, shipment, and microbiological monitoring of Charles River’s C57BL/6NCrl (B6N) germ-free mice.

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C57BL/6NCrl (B6N) Germ-Free Mice

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

EVERY STEP OF THE WAY www.criver.com Furthermore, the composition of patients’ microflora has immune systems. The cocktail most often used for this recently been reported to influence the efficacy of cancer purpose is the altered Schaedler flora (ASF) developed by immunotherapy.11,12 Thus, studying and explicating the Roger Orcutt and colleagues at Charles River in the 1970s interaction between hosts and their microbiota is of critical (Table 1).14,15 In contrast to the original Schaedler flora16 on importance to public health as well as animal research. which it was based, the ASF is fully anaerobic; moreover,

Charles River’s experience with germ-free technology half of the eight species of bacteria in the ASF are extremely goes back to the 1950s, when the veterinarian who oxygen-sensitive (EOS) fusiform anaerobes highly founded Charles River Laboratories, Dr. Henry Foster, and representative of the autochthonous microbiota. Germ- his colleagues incorporated germ-free rederivation into free and defined flora-associated animals are classified as the “cesarean-originated barrier-sustained” process they gnotobiotic, from the Greek roots gnostos (“known”) and pioneered for the large-scale production of SPF mice and bios (“life”). By contrast, barrier-maintained SPF rodents rats.13 In this process, germ-free rodents are associated develop a complex microbiota that is defined only to the (i.e., colonized) with a defined cocktail of commensal extent that it does not include a limited list of pathogens. bacteria to normalize their physiology and prime their

Table 1. Compositive of Charles River Altered Schaedler Flora (ASF)* Designation In Original Schaedler Genbank Accession ASF 356 X Clostridium species AQFQ00000000.1 ASF 360 X Lactobacillus intestinalis AQFR00000000.1 ASF 361 X Lactobacillus murinus AQFs00000000.1 ASF 457 schaedleri AYGZ00000000.1 ASF 492 Eubacterium plexicaudatum AQFT00000000.1 ASF 500 Pseudoflavonifactor species AYJP00000000.1 ASF 502 Clostridium species AQFU00000000.1 ASF 519 X Parabacteroides goldsteinii AQFV00000000.1 * 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 Production B6N germ-free mice may be used as embryo transfer Rederivation recipients or foster dams for germ-free rederivation of The B6N strain was obtained by Charles River from the mutant mouse models. In addition, they may be compared National Institutes of Health in 1974. The current colonies of to SPF or Elite (opportunistic pathogen-free) B6N mice germ-free B6N mice were rederived by sterile hysterectomy to generally assess the relationship between microbiota followed by fostering on germ-free dams provided by the and phenotypes. Alternatively, the germ-free B6N mice Gnotobiotics and Core at Boston Children’s may be associated with a single microbial species (mono- Hospital. Extensive testing by culture and culture- associated), defined microbiota like the ASF, or complex independent methods described below has verified the polymicrobial mixtures to measure and understand the germ-free status of the rederived B6N colonies. 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

C57BL/6NCrl (B6N) Germ-Free Mice Husbandry An example of the plastic isolators in which the germ-free isolator is confirmed by culturing swabs of surfaces, mice are housed is shown in Figure 1. Before being used, caging, and supplies collected from the isolator over several an isolator is tested for leaks, chemically sterilized with 2% weeks. peracetic acid, and ventilated. Sterilization of the ventilated

Figure 1. Plastic isolator for germ-free husbandry

Once in use, an isolator is kept sterile by being ventilated printouts are examined to confirm that the appropriate with HEPA-filtered air under positive pressure. Supplies, cycle was chosen and ran without error. Temperature such as food, bedding, water, and caging, are autoclaved indicators are evaluated and the bioindicators, including in transfer cylinders. To assure sterilization, supplies are those retrieved from inside the test cylinder, are incubated arranged in cylinders following standard configurations (at 55-60 oC). Cylinders are released for use only if all (Figure 2). Self-contained bioindicators containing heat- bioindicators read negative. stable bacterial spores (e.g., EZTest® Steam, Mesa Labs) and temperature indicators are placed throughout each autoclave run, including inside a test cylinder. Autoclave

[email protected] • www.criver.com 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., CLIDOX-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 Microbiological Monitoring culture media, then incubated aerobically and anaerobically Germ-free colonies are monitored for extraneous bacteria in a dedicated anaerobic workstation. Because microbial and fungi, and for pathogens. Table 2 shows the test contaminants may be fastidious or non-cultivable on methods, samples, and frequencies. Testing for extraneous cell-free media (like much of the indigenous microbiota), microbes is conducted frequently, based on the potentially culture-independent methods are employed. Wet mounts high incidence of this type of contamination and its of the slurries collected each week are examined by significant consequences to customers. Weekly, a slurry phase microscopy for motile organisms. In addition, feces from each isolator (consisting of feces and environmental collected quarterly from mice in each isolator are assayed swabs in animal drinking water) is inoculated onto various by PCR for the bacterial 16S ribosomal RNA (rRNA) gene.

Table 2. Microbiological Surveillance of Germ-Free Mouse Isolators Sample Type: Slurry Feces Animals Methodology Tests Frequency: Weekly Quarterly Annually Culture X X Microbiology Phase microscopy X X Bacterial 16S rRNA X PCR Rodent Pathogens X Necropsy X Gross and Microscopic Exams Parasitology X Serology MFIA/IFA X

Comprehensive health monitoring for pathogens is colonial and cellular morphology and by MALDI-TOF mass performed annually on mice from each isolator. This spectrometry, and, if necessary, by PCR. In addition, swabs comparatively low testing frequency is justified by the of the skin, oral cavity, and feces are tested by PCR for historically negligible incidence of pathogens infecting pathogens of all types. isolator-housed colonies. Animal organs and tissues The confirmed detection of bacterial, viral, parasitic, or are grossly examined, and histopathology is carried out fungal agents in germ-free mice or isolators would result if lesions suggesting an infectious disease process are in immediate cessation of shipment from the isolator and observed. Specimens from the gut and skin are examined immediate elimination of the isolator colony. Charles River microscopically for endo- and ectoparasites. Blood considers each isolator to be a microbiologic unit and samples are screened for pathogen-specific antibodies by will not test and cull individual cages within an isolator. ® the multiplexed fluorometric immunoassay (MFIA ), with It remains our policy to inform our customers in a timely corroboration of unexpected (or indeterminate) findings manner of any breaches in animal health or genetic integrity, mostly by indirect immunofluorescence assays (IFA). providing urgent colony health information via email or other For cultural isolation of bacteria and fungi, various method. For assistance regarding specific information on microbiologic culture media are inoculated with Charles River monitoring procedures, additional data on respiratory and gut samples and incubated aerobically animals, or interpretation of the monitoring information, and anaerobically. Samples for anaerobic culture are please direct inquiries to Charles River Technical Services collected from euthanized mice dissected in an anaerobic (877-274-8371) or email [email protected]. workstation. Isolates are identified both according to their

[email protected] • www.criver.com References 11. Kinross, J. M., A. W. Darzi and J. K. Nicholson (2011). “Gut 1. Sommer, F. and F. Backhed (2013). “The gut microbiota--masters microbiome-host interactions in health and disease.” Genome Med of host development and physiology.” Nat Rev Microbiol 11(4): 3(3): 14. 227-238. 12. Gopalakrishnan, V., C. N. Spencer, L. Nezi, A. Reuben, 2. Rajilic-Stojanovic, M. and W. M. de Vos (2014). “The first M. C. Andrews, T. V. Karpinets, P. A. Prieto, et al. “Gut microbiome 1000 cultured species of the human gastrointestinal microbiota.” modulates response to anti-PD-1 immunotherapy in melanoma FEMS Microbiol Rev 38(5): 996-1047. patients.” Science 359(6371): 97-103.

3. Sender, R., S. Fuchs and R. Milo (2016). “Revised Estimates for 13. Foster, H. L., S. J. Foster and E. S. Pfau (1963). “The Large Scale the Number of Human and Bacteria Cells in the Body.” PLoS Biol Production of Caesarean-Originated, Barrier-Sustained Mice.” Lab 14(8): e1002533. Anim Care 13: 711-718.

4. Nicklas, W., L. Keubler and A. Bleich (2015). “Maintaining and 14. Dewhirst, F. E., C. C. Chien, B. J. Paster, R. L. Ericson, Monitoring the Defined Microbiota Status of Gnotobiotic Rodents.” R. P. Orcutt, D. B. Schauer and J. G. Fox (1999). “Phylogeny of the ILAR J 56(2): 241-249. defined murine microbiota: altered Schaedler flora.” Appl Environ Microbiol 65(8): 3287-3292. 5. Shreiner, A. B., J. Y. Kao and V. B. Young (2015). “The gut microbiome in health and in disease.” Curr Opin Gastroenterol 15. Wymore Brand, M., M. J. Wannemuehler, G. J. Phillips, 31(1): 69-75. A. Proctor, A. M. Overstreet, A. E. Jergens, R. P. Orcutt and J. G. Fox (2015). “The Altered Schaedler Flora: Continued Applications 6. Bleich, A. and J. G. Fox (2015). “The Mammalian Microbiome and of a Defined Murine Microbial Community.”ILAR J 56(2): 169-178. Its Importance in Laboratory Animal Research.” ILAR J 56(2): 153-158. 16. Schaedler, R. W., R. Dubos and R. Costello (1965). “The Development of the Bacterial Flora in the Gastrointestinal Tract of 7. Hansen, A. K., L. Krych, D. S. Nielsen and C. H. Hansen (2015). “A Mice.” J Exp Med 122: 59-66. Review of Applied Aspects of Dealing with Gut Microbiota Impact on Rodent Models.” ILAR J 56(2): 250-264. 17. Smith, K., K. D. McCoy and A. J. Macpherson (2007). “Use of axenic animals in studying the adaptation of mammals to their 8. Hormannsperger, G., M. Schaubeck and D. Haller (2015). commensal intestinal microbiota.” Semin Immunol 19(2): 59-69. “Intestinal Microbiota in Animal Models of Inflammatory Diseases.” ILAR J 56(2): 179-191. 18. Al-Asmakh, M. and F. Zadjali (2015). “Use of Germ-Free Animal Models in Microbiota-Related Research.” J Microbiol Biotechnol 9. Paun, A., C. Yau and J. S. Danska (2017). “The Influence of the 25(10): 1583-1588. Microbiome on Type 1 Diabetes.” J Immunol 198(2): 590-595. 19. Ericsson, A. C. and C. L. Franklin (2015). “Manipulating the Gut Microbiota: Methods and Challenges.” ILAR J 56(2): 10. Strati, F., D. Cavalieri, D. Albanese, C. De Felice, C. Donati, J. 205-217. Hayek, O. Jousson, et al. (2017). “New evidences on the altered gut microbiota in autism spectrum disorders.” Microbiome 5(1): 20. Arrieta, M. C., J. Walter and B. B. Finlay (2016). “Human 24. Microbiota-Associated Mice: A Model with Challenges.” Cell Host Microbe 19(5): 575-578.

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