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contaminants are environmental bacteria or fungi;
however, human skin commensals can enter isolators
through holes in the gloves. Therefore, samples from
the animals' environment are highly relevant for hygienic monitoring. Microbiological monitoring of gnotobiotic animals is performed on collected bedding, food,
swabs from various isolator positions as well as faecal
samples.21 Gnotobiotic mice should also be monitored
for specific pathogens, for example those listed by the
Federation of Laboratory Animal Science Associations
(FELASA).21,24 Viruses, except those included in the
FELASA recommendations, are not specifically
excluded from germ-free animals. Therefore, it is recommended that the gnotobiotic colony be tested whenever a new virus has been discovered.21 Furthermore,
the probability that the gnotobiotic colony will be
infected with pathogenic bacteria or fungi is very low.
Based on the current published recommendations for
monitoring germ-free rodents, bedding, food and fresh
faecal samples should be collected and tested by culture
every 4 weeks, and animals should be tested by full
necropsy (including testing for the pathogens from the
FELASA recommendation list) every 3-6 months.21
Some facilities report that they monitor their germfree colonies every 2 weeks or even weekly. The diagnostic techniques include macroscopic and microscopic
examination as well as culture-based and molecular
methods.21 The presence of metazoan and protozoan
parasites can be assessed by analysing cecal content
or by using a tape test when animals are available for
necropsy. For successful microbial monitoring, it is crucial that the samples are obtained under sterile conditions to prevent false-positive results. Detailed
procedures and protocols for the microbiological monitoring of gnotobiotic rodent colonies can be found
elsewhere.21,22

Rederivation and characteristics of
gnotobiotic animals
Germ-free animals are rederived from existent rodent
colonies. Once a new strain is rederived germ-free, the
colony can be bred and maintained in the isolator.
Furthermore, this colony can be used as a source of
new pathogen-free colonies or be transferred to isolators or microisolator cages dedicated to experiments
(Figure 2). Today, germ-free rodents are rederived by
hysterectomy/Caesarean section or by embryo transfer.
Detailed descriptions of these methods can be found
elsewhere.22,25 In short, the hysterectomy method
includes timed mating of the donor and recipient
strain. During the procedure, the gravid uterus of the
donor strain is removed and transferred into the isolator under sterile conditions using a dip-tank filled with
disinfectants, for example iodine or 10% bleach.22

Laboratory Animals 53(3)
In the isolator, the neonates are fostered by a germfree foster mother until weaning.22 During the
embryo transfer method, two-cell embryos of the
target strain are implanted into the oviduct of a pseudopregnant, germ-free female.25 For this technique we
recommend the use of a dedicated sterilized laminar
flow surgical area that is connected to an isolator to
ensure a fully sterile work process.
Life in the germ-free environment induces marked
anatomical and physiological changes in rodent
models. Germ-free animals have smaller hearts, lungs
and livers.26 However, the most prominent characteristic of germ-free animals is the cecum size, which can be
up to five times larger than that of SPF animals. This
cecal swelling occurs due to undegraded mucopolysaccharides, which attract water, and intestinal atonia.26
Even though an enlarged cecum could affect the reproduction of germ-free mice, in our experience, the litter
size of mice bred under germ-free conditions is similar
to that of mice kept under SPF conditions.
Furthermore, various mucosal parameters such as epithelial cell renewal, bowel motility and mucosa thickness are decreased in the absence of microbiota. The
faecal pellets of germ-free animals contain more water
and are softer. Moreover, the intestinal microbiota
plays a role in the metabolism of bile acids and in the
production of SCFA and vitamins.26 The absence of
microbial stimulation also has a prominent impact on
the development of the immune system.27 Germ-free
mice display reduced numbers of gut-associated lymphoid tissues, poorly formed T-cell and B-cell zones in the
germinal centres, reduced numbers of intestinal T cells
and decreased IgA production.27-29 Detailed characteristics of germ-free immunological phenotypes are
reviewed elsewhere.27-30

Gnotobiotic animals as a tool in
microbiome research
Gnotobiotic animal models have become a powerful
tool for investigating the functional effects of hostmicrobe interactions at the level of single, defined, or
complex
microbial
communities
(Figure
3).
Furthermore, these strategies allow mechanistic
approaches under highly standardized conditions. The
protective or adverse nature of host-microbe interactions depends on the respective bacteria and the
host susceptibility. Additionally, the disease phenotypes of various animal models may depend on the
composition of their microbiota.31,32 Moreover,
immense interfacility differences in microbiota abundance and composition have been reported.33
Therefore, researchers should be aware that differences
in the microbiota composition can impact the outcomes
of their studies or modify disease pathologies.31,34



Laboratory Animals - June Issue

Table of Contents for the Digital Edition of Laboratory Animals - June Issue

Contents
Laboratory Animals - June Issue - Cover1
Laboratory Animals - June Issue - Cover2
Laboratory Animals - June Issue - Contents
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