Table Of ContentGermfree and Gnotobiotic
Animal Models
Background and Applications
Bernard S. Wostmann, D. Sc.
Lobund Laboratory
Notre Dame, Indiana
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Library of Congress Cataloging-in-Publication Data
Wostmann, Bernard S.
Germfree and gnotobiotic animal models : background and
applications / Bernard S. Wostmann.
p. cm.
Includes bibliographical references and index.
ISBN 0-8493-4008-X (alk. paper)
1. Laboratory animals. 2. Germfree animals. 3. Diseases—Animal
models. I. Title.
[DNLM: 1. Germ-Free Life. 2. Animals, Laboratory. QW 56 W935g
1996]
QL55.W67 1996
591'.0724—dc20
DNLM/DLC
for Library of Congress 96-4752
CIP
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F
o rew o rd
The desirability of having a truly germfree animal available for study
in the biomedical field had been expressed by Louis Pasteur as early as
the 1880s. Shortly thereafter, Nuttal and Thierfelder had been able to
produce a germfree guinea pig, albeit short-lived. However, it was only
in the late 1940s and early 1950s that it became possible to establish
breeding colonies of germfree rats and mice. Although this opened the
way for a multitude of studies in which that little-understood and uncon
trollable factor, the "normal" microbial flora, could be eliminated, obvi
ously the first question to be answered was "What kind of animal is this
germfree animal?" This question was especially pertinent since Pasteur
had not been certain that life under germfree conditions would even be
possible.
However, life under germfree conditions proved to be possible. After
colony production made available sufficient germfree rats and mice, and
later guinea pigs and rabbits, the first two decades beginning in 1950 were
largely devoted to the study of function and metabolism in the absence
of a microbial flora. Yet even in those early days, after it was recognized
that in the total absence of bacteria, e.g., dental caries did not develop,
studies were started which used the germfree rat to introduce specific
microorganisms to test their cariogenic potential and a beginning was
made in the study of the effects of intestinal microorganisms on cholesterol
and bile acid metabolism, obviously in support of the fight against car
diovascular disease.
Emphasis then gradually shifted to the application of the germfree
and the gnotobiotic animal in studies of specific problems in the biomed
ical field; but in the meantime a younger generation of workers had taken
over. Increasingly, they would need these animals in their studies; how
ever, they might not be quite familiar with the specific characteristics and
anomalies of this animal model, which could considerably affect the in
terpretation of their experimental data. It is for them that we have tried
to bring the most salient points of the earlier and more recent studies
together without going into too much detail, but giving references to the
pertinent literature as often as possible. We have also tried to interpret
earlier data against results of more recent studies.
Although the majority of the studies with germfree and gnotobiotic
animals have been carried out with rats and mice, the first animal to be
obtained germfree was the guinea pig, soon to be followed by the germfree
chicken. In both cases the fact that the newborn animal could survive
without maternal care explains these choices, whereas in the case of the
chicken the possibility of external sterilization of the egg added to its
attractiveness. Later experience with the difficulties of hand-feeding new
born Cesarian-derived rats and, particularly, newborn mice, would fully
justify these choices. This also explains why early studies, when absence
or control of the microflora was sought, were carried out with "first
generation" germfree chickens and guinea pigs. Then, once colony pro
duction of germfree rats and mice had been established, which had neces
sitated extensive experience with the hand-feeding of newborns, the germ
free rabbit seemed the next logical choice at the time because of its
importance for immunological studies. Although hand-feeding of the
newborn Cesarian-derived germfree rabbit posed no exceptional difficul
ties, problems posed by its very much distended cecum, in addition to
unexpected nutritional problems, took time to solve. By the time adequate
solutions had been found, the emphasis of most immunological studies
had shifted to the use of mice, and the germfree rabbit became an almost
forgotten research model. To an extent, the potential role of the germfree
rabbit in immunological studies has been taken over by the germfree and
gnotobiotic piglet in the extensive studies of Dr. Yoon Kim and co-workers
at the Department of Microbiology and Immunology of the Chicago Med
ical School. Of particular interest is the fact that they were able to rear
newborn germfree piglets on a diet of very low antigenicity.
The germfree gerbil has been of special interest to this author because,
as described in Chapters II and IV, its cholesterol and bile acid metabolism
are so close to those of the human. This model, especially as a controlled
polyassociate, may be of great help in solving problems related to cardio
vascular disease.
The GF dog, developed under the auspices of Dr. Charles Yale and
Dr. Jim Heneghan at the LSU School of Medicine, has served as an im
portant tool in the study of the pathology of intestinal strangulation.
Another line of research led to the production of germfree and gnotobiotic
pigs at the University of Guelph, until recently under the supervision of
Dr. Paul Miniats. Here the emphasis was on the production of a pathogen-
free animal which, as such, could be introduced into the swine population
for commercial purposes. Soon gnotobiotic pigs would also be used ex
tensively to study the pathology of the bacterial diseases that plague
commercial pork production (see Chapter XI). Last but not least, the great
amount of work which went into the various endeavors to save children
born with severe combined immune deficiency (SCID) should be men
tioned. In this country this led to the story of "The Boy in the Bubble",
which is described in the Proceedings of the VIII International Symposium
on Germfree Research held at the University of Notre Dame in 1984.
This monograph has been limited mainly to germfree and gnotobiotic
rats, mice, and gerbils, all of which we have been closely associated with.
As mentioned earlier, the vast majority of the more critical studies have
been carried out with these animal models. Whenever pertinent, results
obtained with germfree and/or gnotobiotic chickens, guinea pigs, rabbits,
dogs, and pigs have been introduced. Chapters I to IX concentrate mostly
on the anomalies which may occur as a result of the germfree and of
certain gnotobiotic states. Chapters X, XI, and XII describe the application
of these animal models to a variety of problems as they occur in the
biomedical sciences.
We are acutely aware of the fact that some work that should have
been included may have been overlooked. For this we apologize; no
personal files are ever complete. For completeness we often had to rely
on abstracts from Index Medicus. Our sincere thanks are due to Dr. Morris
Pollard and Dr. Paul Weinstein, who were willing to give much needed
help in fields we were least familiar with.
T A
he uthor
Bernard S. Wostmann, D.Sc, received a M.Sc degree in biological
chemistry from the University of Amsterdam, The Netherlands in 1945.
He received his D.Sc degree in 1948 while working in the Department of
Physiological Chemistry of the Medical School of Amsterdam University,
where he joined the staff as a lecturer in Physiological Chemistry. In 1950
to 1951 he was a Rockefeller Research Fellow at the California Institute
of Technology, working under Dr. Linus Pauling and Dr. Dan H. Campbell
on antigen-antibody combining ratios. In 1951 to 1955 he served as As
sistant Professor in above department, while also being a member of the
staff of The Netherlands Institute of Nutrition. In 1955 he became Assistant
Research Professor at the Lobund Laboratory of the University of Notre
Dame in charge of its Division of Biochemistry and Nutrition. In 1958 he
became an Associate and from 1965 to 1988, Professor, first in the Depart
ment of Microbiology, later in the Department of Biological Sciences. He
taught Gnotobiology, Nutrition, and the chemical background of antibi
otics to undergraduate and graduate students. In 1988 he retired as an
active member of the department, while retaining his connection with the
Lobund Laboratory. His research has been supported by grants from the
National Institutes of Health, the National Science Foundation, the Nu
trition Foundation, the University of Notre Dame, Erasmus University,
Rotterdam, The Netherlands, and various smaller foundations.
He has written approximately 190 scientific papers, has edited one
book, and has lectured in the U.S., Canada, Mexico, Brazil, Europe, Japan,
and China. Memberships in scientific societies include the American As
sociation for the Advancement of Science, the Association for Gnotobiot-
ics, the International Association for Gnotobiotics, the American Institute
of Nutrition, the Society for Experimental Biology and Medicine, the
Indiana Academy of Science, the Society of Sigma Xi, and the International
Committee for Laboratory Animal Science (ICLAS).
T C
able of ontents
Chapter I
Introduction......................................................................................................................................1
Chapter II
Early Growth, Body Weight, Reproduction, and Life Span........................................9
General Aspects........................................................................................................................9
Growth.........................................................................................................................................9
Body Weight of the Mature Animal..............................................................................10
Reproduction............................................................................................................................10
Life Span....................................................................................................................................11
Conclusions..............................................................................................................................14
Chapter III
Anatomy, Morphology, and Function of the Gastrointestinal System...................19
General Aspects......................................................................................................................19
Stomach.....................................................................................................................................20
Small Intestine........................................................................................................................21
Cecum.........................................................................................................................................29
Large Intestine........................................................................................................................32
Conclusions..............................................................................................................................33
Chapter IV
Morphology and Physiology, Endocrinology and Biochemistry.............................43
General Aspects......................................................................................................................43
Energy Metabolism...............................................................................................................46
Liver Function.........................................................................................................................50
Cholesterol and Bile Acid Metabolism.........................................................................54
Rats and Mice....................................................................................................................54
Gerbils..................................................................................................................................57
Dogs and Pigs..................................................................................................................58
Other Indicators of Function............................................................................................59
Water Balance and Kidney Function.............................................................................60
Mineral Metabolism..............................................................................................................61
Endocrine System..................................................................................................................62
Conclusions..............................................................................................................................64
Chapter V
Nutrition..........................................................................................................................................71
