Laboratory Animals - June Issue - 233

Basic and Bleich
An interest in developing a detailed understanding of
host-microbe interactions and their impact on host
health and disease established a focus on gnotobiotic
techniques and methods within the scientific community. Gnotobiotics or gnotobiology explores organisms,
whose microbial state is strictly defined and controlled.
The term is derived from the Greek words for 'know'
(gnotos) and 'life' (bios). Over the past years gnotobiotic
animal models have become a key tool for studying
complex host-microbiota interactions.9,10 Gnotobiotic
animal models include germ-free animals, which are
free of all foreign organisms, and animals colonized
with single or multiple defined microorganisms.
However, this term needs to be distinguished from specified pathogen-free (SPF), as such animals are free of
particular pathogens, but their endogenous microbiota
is complex and undefined.

The rise of gnotobiotics
The development of gnotobiotics started in the late
19th century. In 1885 and 1886 life scientists were confronted with two opposing hypotheses proposed by
Louis Pasteur and Marceli Nencki. While Pasteur
hypothesized that life is impossible without commensal
bacteria, Nencki suggested that the absence of germs
would prolong life and health.11 To resolve this controversy, scientists started to develop techniques and
equipment to isolate living animals as much as possible
from their inner and outer environments.
Pioneering work was done in the period from 1885 to
1896 by George H. F. Nuttall and Hans Thierfelder at
the University of Berlin. They delivered germ-free guinea
pigs by Caesarean section and fed them sterile milk. This
accomplishment demonstrated that life without bacteria
is possible. However, when sterile vegetables were used
as a diet, these animals gained less weight than the controls, indicating the importance of bacteria for digestion.12-14 Several years later, Ernst G. F. Kuester
designed the first isolator prototype, in which he maintained germ-free goats for over a month.15
In the middle of the 20th century several institutions
dedicated to gnotobiotic work were opened. At the
University of Lund in Sweden, Go¨sta Glimstedt established a gnotobiotic department and worked with germfree guinea pigs. In the USA, James A. Reyniers
founded The Laboratories of Bacteriology at the
University of Notre Dame (LOBUND) and, together
with Philip C. Trexler, became the world's leader in
germ-free research. Furthermore, a gnotobiotic centre
was also established by Masasumi Miyakawa in Japan
several years after those in the USA and Europe.11,14
Gnotobiotic institutions focused not only on the technical aspects of long-term maintenance and housing of
germ-free animals but also on the phenotypic

233
characterization of germ-free animals. Although many
species such as chickens, rabbits and non-human primates were successfully raised to be germ-free, germfree rodent colonies were still not established at that
time due to difficulties in hand feeding.11
In the 1940s, Bengt E. Gustafsson joined Glimstedt at
the University of Lund and started to develop germ-free
rat colonies. He first reported on hand-reared and
weaned germ-free rats in 1946.16 Later in 1946
Reyniers published the first detailed report on the technique used for rearing germ-free rodents.17,18 Hand feeding germ-free mice was even more challenging than hand
feeding germ-free rats. Finally, germ-free mice were successfully weaned and have been available as reproducing
colonies at the LOBUND institute since 1954.18,19 After
germ-free rodent colonies were established, hand feeding
was replaced with cross-fostering of neonates by germfree foster mothers, which made the rearing of germ-free
rodents easier and more accessible for scientists.
The first germ-free rodents showed growth retardation and premature death due to lack of appropriate
nutrition. These phenotypes prompted studies addressing the cause of the nutritional deficiencies in germ-free
animals. After years of research, which improved
knowledge of important nutritional components, the
diets of germ-free animals were fortified with supplements such as vitamins K and B, resulting in the capability of providing long-term housing under germ-free
conditions.11 By overcoming these obstacles, gnotobiotics pursued two important goals: providing a
nucleus of pathogen-free animals for biomedical
research and studying anatomical and physiological
particularities in germ-free animals.
With advances in microbiome research, the interest
in gnotobiotics has once again increased. Since 2000,
the number of publications obtained using the PubMed
search engine including terms microbiome, microbiota
or microflora exponentially increased. The number of
publications including these terms increased from five
in the year 2000 up to more than 2000 published yearly
since 2015. Together with methodological progress and
the availability of various mouse models, gnotobiotics
provides insights into the host processes regulated by
the presence and composition of the intestinal microbiota. Furthermore, gnotobiotics allows the establishment of standardized conditions in which the
functionality and causality of host-microbe-host interactions can be addressed to reveal important genetic,
microbial and environmental factors shaping homeostasis or pathological conditions.

Gnotobiotic rodent husbandry
Long-term housing of gnotobiotic rodents is possible in
positive pressure isolators (Figure 1(a)). Before plastics



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
Laboratory Animals - June Issue - 214
Laboratory Animals - June Issue - 215
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