Germ-Free and Gnotobiotic

Germ-Free and Gnotobiotic

The environmentally exposed surfaces of mammals are colonized by a diverse ecosystem of microbes. Though many of these bacteria – particularly those of the distal gut – are considered symbiotic, the microbiome has the capacity to induce both pro- and anti-inflammatory responses. Accumulating evidence suggests that a properly balanced gut microbiome is crucial for a correctly functioning immune system, and that imbalances in the microbial community of the intestine are linked to a multitude of auto-inflammatory, auto-immune, and metabolic disease states. When studying such disease models with a microbiome component, germ-free and gnotobiotic mice provide an ideal model system.

Stats

•       14 Isolators

•       4-5 small or 1-2 large cages per isolator = 20-25 or 20-40 mice per isolator

•       Quality Control:

•       Autoclave loads validated by spore test

•       Three rounds of sterility conformation prior to animal arrival: isolator surface and mold trap

•       All animal manipulation throughout study occurs within GF isolator

•       Studies available with or without microbial monitoring

•       Biomodels’ anaerobic chamber allows for the culture and dosing of strict anaerobic bacteria without detrimental oxygen exposure.

 

Disease Areas of Interest

•       Inflammatory Bowel Disease

•       Graft versus Host Disease

•       Oncology

•       Cancer Supportive Care

•       Gastrointestinal Acute Radiation Syndrome

•       Experimental Autoimmune Encephalomyelitis/Multiple Sclerosis

•       Sepsis

•       Diet-Induced Obesity 

 

IBD and the Microbiome

A role for the intestinal microbiome has been demonstrated in multiple mouse models of IBD. Prevotellaceae species exacerbate chemically-induced colitis, and Helicobacter species drive colitis in IL10-/- mice. By contrast, Clostridium species confer resistance to colitis, and Polysaccharide A produced by Bacteroides fragilis confers protection from Helicobacter induced colitis in IL10-/- mice.

 

DSS-Induced Colitis

·       Colitis is induced by exposure to specific concentrations of DSS

·       DSS is administered in the drinking water between days 0 and 5

·       Conventional mice demonstrate:

o   Weight loss beginning on days 5-6

o   Peak weight loss observed on days 8-9, followed by a gradual recovery

o   Low mortality

·       Main endpoints include:

o   Survival

o   Weight loss

o   Endoscopy Score

o   Endoscopy Images

o   Histology (H&E – not shown)

o   Responds to treatment with anti-p40 mAb or anti-TNFα mAb

·       In GF mice, disease is exacerbated, with:

o   Higher mortality, beginning on day 6

o   Higher endoscopy scores

o   Reduced weight-loss as compared to conventional animals

Differential Response to DSS-Induced Colitis in Germ-Free vs Conventional Mice. Animals were treated with 2.5% or 3% DSS (drinking water) from days 0-5 and were euthanized on Day 10. GF animals were removed from the isolators for endoscopy and necropsy. Survival, Weight loss, Endoscopy Scores and Representative endoscopy images, n=5-10 per group. *p<0.05; **p<0.01
Microbial Monitoring

·       Sterility confirmation occurs throughout germ-free studies

·       Maintenance of continuing germ-free status:

o   1-2 times per week, or more often if requested

o   Includes:

§  Weekly swab and culture of surface, mold-trap and animal fur on TSA/5% sheep’s blood plates in aerobic and anaerobic conditions

§  Weekly culture of animal feces on TSA/5% sheep’s blood plates in aerobic and anaerobic conditions

§  Optional DNA isolation and qPCR amplification of fecal bacterial 16S rRNA gene (two primer pairs)

o   Studies are designed for minimal entries into isolators

Germ-free mice are shipped to Biomodels in Taconic Germ-Free shippers. The shippers are connected directly to the isolators, allowing for entry of animals to the isolator without compromise of germ-free status. Animals reside inside the GF isolators throughout the study.
Maintenance of GF status. Fecal samples were collected from animals at indicated time DNA was isolated and GF status was confirmed by qPCR amplification of the bacterial 16S rRNA gene. Blue: conventional; yellow: germ-free. n= 4 per group
Biomodels’ Anaerobic Chamber. Strict anaerobic bacterial species are of particular interest in microbiome-based studies. Bacterial culture and sample manipulation under anaerobic conditions maximizes the viability of Oxygen-sensitive strains.
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