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Germ-free animal

From Wikipedia, the free encyclopedia
Germ-free mice are frequently used in scientific research

Germ-free organisms are multi-cellular organisms that have no microorganisms living in or on them. Such organisms are raised using various methods to control their exposure to viral, bacterial or parasitic agents.[1] When known microbiota are introduced to a germ-free organism, it usually is referred to as a gnotobiotic organism, however technically speaking, germ-free organisms are also gnotobiotic because the status of their microbial community is known.[2] Due to lacking a microbiome, many germ-free organisms exhibit health deficits such as defects in the immune system and difficulties with energy acquisition.[3][4] Typically germ-free organisms are used in the study of a microbiome where careful control of outside contaminants is required.[5]

Generation and cultivation

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Germ-free organisms are generated by a variety of different means, but a common practice shared by many of them is some form of sterilization step followed by seclusion from the surrounding environment to prevent contamination.  

Poultry

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Germ-free poultry typically undergo multiple sterilization steps while still at the egg life-stage. This can involve either washing with bleach or an antibiotic solution to surface sterilize the egg. The eggs are then transferred to a sterile incubator where they are grown until hatching. Once hatched, they are provided with sterilized water and a gamma-irradiated feed. This prevents introduction of foreign microbes into their intestinal tracts. The incubators and animals' waste products are continuously monitored for possible contamination. Typically, when being used in experiments, a known microbiome is introduced to the animals at a few days of age. Contamination is still monitored and controlled for after this point, but the presence of microbes is expected.[6][7][8]

Mice

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Mice undergo a slightly different process due to lacking an egg life-stage. To create a germ-free mouse, an embryo is created through in vitro fertilization and then transplanted into a germ-free mother. If this method is not available, a mouse can be born through cesarean birth, but this comes with a higher risk of contamination. This process uses a non-germ-free mother which is sacrificed and sterilized before the pups' birth. After the cesarean birth, the pups must then be transferred to a sterile incubator with a germ-free mother for feeding and growth.[9][10] These methods are only required for the generation of a germ-free mouse line. Once a line is generated, all progeny will be germ-free unless contaminated. These progeny can then be used for experimentation. Typically for experiments, each mouse is housed separately in a sterile isolator to prevent cross-contamination between mice. The mice are provided with sterilized food and water to prevent contamination. The sterilization methods can vary between experiments due to different diets or drugs the mice are exposed to. The isolators and waste products are continuously monitored for possible contamination to ensure complete sterility. As with poultry, a known microbiome can be introduced into the animals but contamination is still monitored for.[11][12][13]

Nematodes

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Nematodes can also be grown germ-free. Germ-free offspring of the nematode C. elegans, which is used in research, can be produced by rupturing adult worms to release eggs. The standard method for this is to introduce a population of adult worms to a bleach solution. This bleach solution ruptures the adult worms, breaking them down while simultaneously releasing and surface sterilizing any eggs. The sterilized eggs are washed and transferred to a plate of agar containing food for the worms. C. elegans consumes bacteria, so before the eggs can be transferred to the plate, the food must be killed by either heat or irradiation. This method for creating germ-free nematodes has the added benefit of age-synchronizing the worms, so that they are all of similar ages as they grow. Typically the worms will need to be transferred to a new plate as they consume all the food on the current plate, with each plate having been treated with heat or radiation as well. The plates can be protected from outside contamination by covering them and isolating them from possible contamination sources.[14]

Plants

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Seeds are surface sterilized with chemicals, such as ethanol or an antibiotic solution, to produce a germ-free plant. The seeds are then grown in water or other mediums until germination. After germination, the seeds are transferred to either sterile soil or soil with a specific microbiota for use in experiments. Seeds may also be transferred directly to soil and allowed to germinate. If the plants are transferred to sterile soil, typically there are two types of growth methods. The first is where the entire plant is kept sterile and in the other, only the root system is kept sterile. The method is chosen based on the requirements for the experiment. The plants are grown in isolators which are frequently checked for contamination along with the soil that the plants grow in.[15][16]

Fruit Flies

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Fruit flies, specifically the Drosophila species, have been one of the most used model organisms. Drosophila species has mostly been used in the field of genetics. There are two main methods for yielding gnotobiotic or axenic flies. One of them is to collect the eggs and dechorionate them. A chorion is the outer membrane around the embryo. In aseptic conditions, the eggs are washed twice for 2.5 minutes each, in 0.6% sodium hypochlorite solution. They are then placed with the specimens cup in 90 ml of bleach. Following this, they are washed thrice in sterile water. The dechorionated eggs are then placed in vials containing sterile diet.[17]

The second method is by the use of antibiotics. Media, such as standard yeast-cornmeal diet, is supplemented with streptomycin or a combination of antibiotics. The concentration of the antibiotic is 400 μg/ml. Once the yeast-cornmeal diet has cooled, 4 ml of solution containing 10g of streptomycin dissolved in 100 ml of ethanol is added per litre of food. The media is then poured into vials and the freshly harvested eggs are transferred into the vials.[18]

Health effects on organism

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Due to lacking a healthy microbiome, many germ-free organisms exhibit major health deficits. The methods used to produce germ-free organisms can also have negative side effects on the organism. Decreased hatching rates were observed in chicken eggs incubated with mercuric chloride, while treatment with peracetic acid did not cause a significant effect on hatching rates.[8] The chickens also exhibited defects in small intestine growth and health.[6] Germ-free mice have been shown to have defects in their immune system and energy uptake due to lacking a healthy microbiome.[3][4] There is also strong evidence for interactions between the mouse microbiome and its brain development and health.[13][19][20] Germ-free plants exhibit severe growth defects due to lacking symbionts that provide necessary nutrients to them.[16][21]

Uses

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Microbiome research

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Germ-free animals are routinely used to establish causality in studies of the microbiome.[22][23] This is done by comparing animals with a standard commensal gut microbiome to germ free, or by colonising a germ free animal with an organism of interest. The gut microbiota can vary between research facilities which can be a confounder in experiments and be a cause of lack of reproducibility.[24] Several control microbiomes have been developed which correct the major health defects commonly present in germ free animals and can act as a reproducible control community.[25][26] Germ free animals have been used to demonstrate a causal role for the gut microbiome in varied settings such as neural development,[27] longevity,[28] cancer immunotherapy,[29] and numerous other health related contexts.[30]

See also

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References

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  1. ^ "Germ Free Mouse Facility". University of Michigan. Archived from the original on 30 October 2015.
  2. ^ Reyniers JA (1959). "Germfree Vertebrates: Present Status". Annals of the New York Academy of Sciences. 78 (1): 3. doi:10.1111/j.1749-6632.1959.tb53091.x. S2CID 84048961.
  3. ^ a b Boulangé CL, Neves AL, Chilloux J, Nicholson JK, Dumas ME (April 2016). "Impact of the gut microbiota on inflammation, obesity, and metabolic disease". Genome Medicine. 8 (1): 42. doi:10.1186/s13073-016-0303-2. PMC 4839080. PMID 27098727.
  4. ^ a b Round JL, Mazmanian SK (May 2009). "The gut microbiota shapes intestinal immune responses during health and disease". Nature Reviews. Immunology. 9 (5): 313–23. doi:10.1038/nri2515. PMC 4095778. PMID 19343057.
  5. ^ Armbrecht J (2 August 2000). "Of Probiotics and Possibilities". Department of Bacteriology, University of Wisconsin-Madison. Archived from the original on 11 March 2007.
  6. ^ a b Cheled-Shoval, S. L.; Gamage, N. S. Withana; Amit-Romach, E.; Forder, R.; Marshal, J.; Van Kessel, A.; Uni, Z. (2014-03-01). "Differences in intestinal mucin dynamics between germ-free and conventionally reared chickens after mannan-oligosaccharide supplementation". Poultry Science. 93 (3): 636–644. doi:10.3382/ps.2013-03362. ISSN 0032-5791. PMID 24604857.
  7. ^ Thomas, Milton; Wongkuna, Supapit; Ghimire, Sudeep; Kumar, Roshan; Antony, Linto; Doerner, Kinchel C.; Singery, Aaron; Nelson, Eric; Woyengo, Tofuko; Chankhamhaengdecha, Surang; Janvilisri, Tavan (2019-04-24). "Gut Microbial Dynamics during Conventionalization of Germfree Chicken". mSphere. 4 (2). doi:10.1128/mSphere.00035-19. ISSN 2379-5042. PMC 6437271. PMID 30918057.
  8. ^ a b Harrison, G. F. (April 1969). "Production of germ-free chicks: a comparison of the hatchability of eggs sterilized externally by different methods". Laboratory Animals. 3 (1): 51–59. doi:10.1258/002367769781071871. ISSN 0023-6772.
  9. ^ Biosciences, Taconic. "What Are Germ-Free Mice and How Are They Sourced?". www.taconic.com. Retrieved 2019-11-29.
  10. ^ Arvidsson, Carina & Hallén, Anna & Bäckhed, Fredrik. (2012). Generating and Analyzing Germ-Free Mice. 10.1002/9780470942390.mo120064.
  11. ^ Cash, Heather L.; Whitham, Cecilia V.; Behrendt, Cassie L.; Hooper, Lora V. (2006-08-25). "Symbiotic Bacteria Direct Expression of an Intestinal Bactericidal Lectin". Science. 313 (5790): 1126–1130. Bibcode:2006Sci...313.1126C. doi:10.1126/science.1127119. ISSN 0036-8075. PMC 2716667. PMID 16931762.
  12. ^ Duerkop, Breck A.; Clements, Charmaine V.; Rollins, Darcy; Rodrigues, Jorge L. M.; Hooper, Lora V. (2012-10-23). "A composite bacteriophage alters colonization by an intestinal commensal bacterium". Proceedings of the National Academy of Sciences. 109 (43): 17621–17626. Bibcode:2012PNAS..10917621D. doi:10.1073/pnas.1206136109. ISSN 0027-8424. PMC 3491505. PMID 23045666.
  13. ^ a b Heijtz, Rochellys Diaz; Wang, Shugui; Anuar, Farhana; Qian, Yu; Björkholm, Britta; Samuelsson, Annika; Hibberd, Martin L.; Forssberg, Hans; Pettersson, Sven (2011-02-15). "Normal gut microbiota modulates brain development and behavior". Proceedings of the National Academy of Sciences. 108 (7): 3047–3052. Bibcode:2011PNAS..108.3047H. doi:10.1073/pnas.1010529108. ISSN 0027-8424. PMC 3041077. PMID 21282636.
  14. ^ Stiernagle, T. Maintenance of C. elegans (February 11, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.101.1, http://www.wormbook.org.
  15. ^ Niu, Ben; Paulson, Joseph Nathaniel; Zheng, Xiaoqi; Kolter, Roberto (2017-03-21). "Simplified and representative bacterial community of maize roots". Proceedings of the National Academy of Sciences. 114 (12): E2450–E2459. Bibcode:2017PNAS..114E2450N. doi:10.1073/pnas.1616148114. ISSN 0027-8424. PMC 5373366. PMID 28275097.
  16. ^ a b Strissel, Jerry Fred, "Bacteria-free soybean plants " (1970). Retrospective Theses and Dissertations. 4800. https://lib.dr.iastate.edu/rtd/4800
  17. ^ Koyle, Melinda L.; Veloz, Madeline; Judd, Alec M.; Wong, Adam C.-N.; Newell, Peter D.; Douglas, Angela E.; Chaston, John M. (2016-07-30). "Rearing the Fruit Fly Drosophila melanogaster Under Axenic and Gnotobiotic Conditions". Journal of Visualized Experiments (113): 54219. doi:10.3791/54219. ISSN 1940-087X. PMC 5091700. PMID 27500374.
  18. ^ Heys, Chloe; Lizé, Anne; Blow, Frances; White, Lewis; Darby, Alistair; Lewis, Zenobia J. (2018-03-26). "The effect of gut microbiota elimination in Drosophila melanogaster: A how‐to guide for host–microbiota studies". Ecology and Evolution. 8 (8): 4150–4161. doi:10.1002/ece3.3991. ISSN 2045-7758. PMC 5916298. PMID 29721287.
  19. ^ Park, A J; Collins, J; Blennerhassett, P A; Ghia, J E; Verdu, E F; Bercik, P; Collins, S M (September 2013). "Altered colonic function and microbiota profile in a mouse model of chronic depression". Neurogastroenterology and Motility. 25 (9): 733–e575. doi:10.1111/nmo.12153. ISSN 1350-1925. PMC 3912902. PMID 23773726.
  20. ^ Mayer, Emeran A.; Tillisch, Kirsten; Gupta, Arpana (2015-03-02). "Gut/brain axis and the microbiota". The Journal of Clinical Investigation. 125 (3): 926–938. doi:10.1172/JCI76304. ISSN 0021-9738. PMC 4362231. PMID 25689247.
  21. ^ Kutschera, Ulrich; Khanna, Rajnish (2016-12-01). "Plant gnotobiology: Epiphytic microbes and sustainable agriculture". Plant Signaling & Behavior. 11 (12): e1256529. doi:10.1080/15592324.2016.1256529. PMC 5225935. PMID 27830978.
  22. ^ Gérard, Philippe (November 2017). "Gut Microbiome and Obesity. How to Prove Causality?". Annals of the American Thoracic Society. 14 (Supplement_5): S354–S356. doi:10.1513/AnnalsATS.201702-117AW. ISSN 2325-6621. PMID 29161082.
  23. ^ Fritz, Joëlle V.; Desai, Mahesh S.; Shah, Pranjul; Schneider, Jochen G.; Wilmes, Paul (2013-05-03). "From meta-omics to causality: experimental models for human microbiome research". Microbiome. 1 (1): 14. doi:10.1186/2049-2618-1-14. ISSN 2049-2618. PMC 3971605. PMID 24450613.
  24. ^ Rausch, Philipp; Basic, Marijana; Batra, Arvind; Bischoff, Stephan C.; Blaut, Michael; Clavel, Thomas; Gläsner, Joachim; Gopalakrishnan, Shreya; Grassl, Guntram A.; Günther, Claudia; Haller, Dirk (August 2016). "Analysis of factors contributing to variation in the C57BL/6J fecal microbiota across German animal facilities". International Journal of Medical Microbiology. 306 (5): 343–355. doi:10.1016/j.ijmm.2016.03.004. hdl:11858/00-001M-0000-002B-7B28-4. ISSN 1618-0607. PMID 27053239. S2CID 36454231.
  25. ^ Eberl, Claudia; Ring, Diana; Münch, Philipp C.; Beutler, Markus; Basic, Marijana; Slack, Emma Caroline; Schwarzer, Martin; Srutkova, Dagmar; Lange, Anna; Frick, Julia S.; Bleich, André (2020-01-10). "Reproducible Colonization of Germ-Free Mice With the Oligo-Mouse-Microbiota in Different Animal Facilities". Frontiers in Microbiology. 10: 2999. doi:10.3389/fmicb.2019.02999. ISSN 1664-302X. PMC 6965490. PMID 31998276.
  26. ^ Darnaud, Marion; De Vadder, Filipe; Bogeat, Pascaline; Boucinha, Lilia; Bulteau, Anne-Laure; Bunescu, Andrei; Couturier, Céline; Delgado, Ana; Dugua, Hélène; Elie, Céline; Mathieu, Alban (2021-11-18). "A standardized gnotobiotic mouse model harboring a minimal 15-member mouse gut microbiota recapitulates SOPF/SPF phenotypes". Nature Communications. 12 (1): 6686. Bibcode:2021NatCo..12.6686D. doi:10.1038/s41467-021-26963-9. ISSN 2041-1723. PMC 8602333. PMID 34795236.
  27. ^ Hoban, A E; Stilling, R M; Ryan, F J; Shanahan, F; Dinan, T G; Claesson, M J; Clarke, G; Cryan, J F (April 2016). "Regulation of prefrontal cortex myelination by the microbiota". Translational Psychiatry. 6 (4): e774. doi:10.1038/tp.2016.42. ISSN 2158-3188. PMC 4872400. PMID 27045844.
  28. ^ Lynn, Miriam A.; Eden, Georgina; Ryan, Feargal J.; Bensalem, Julien; Wang, Xuemin; Blake, Stephen J.; Choo, Jocelyn M.; Chern, Yee Tee; Sribnaia, Anastasia; James, Jane; Benson, Saoirse C. (2021-08-24). "The composition of the gut microbiota following early-life antibiotic exposure affects host health and longevity in later life". Cell Reports. 36 (8): 109564. doi:10.1016/j.celrep.2021.109564. ISSN 2211-1247. PMID 34433065. S2CID 237306510.
  29. ^ Blake, Stephen J.; James, Jane; Ryan, Feargal J.; Caparros-Martin, Jose; Eden, Georgina L.; Tee, Yee C.; Salamon, John R.; Benson, Saoirse C.; Tumes, Damon J.; Sribnaia, Anastasia; Stevens, Natalie E. (2021-12-21). "The immunotoxicity, but not anti-tumor efficacy, of anti-CD40 and anti-CD137 immunotherapies is dependent on the gut microbiota". Cell Reports. Medicine. 2 (12): 100464. doi:10.1016/j.xcrm.2021.100464. ISSN 2666-3791. PMC 8714857. PMID 35028606.
  30. ^ Round, June L.; Palm, Noah W. (2018-02-09). "Causal effects of the microbiota on immune-mediated diseases". Science Immunology. 3 (20): eaao1603. doi:10.1126/sciimmunol.aao1603. ISSN 2470-9468. PMID 29440265. S2CID 3268605.