The Gnotobiotic Animal As a Tool in the Study of Host Microbial Relationships HELMUT A
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BACTERIOLOGICAL REvEws, Dec. 1971, p. 390-429 Vol. 35, No. 4 Copyright © 1972 American Society for Microbiology Printed in U.S.A. The Gnotobiotic Animal as a Tool in the Study of Host Microbial Relationships HELMUT A. GORDON AND LASZLO PESTI1 Department ofPharmacology, College ofMedicine, University ofKentucky, Lexington, Kentucky 40506 INTRODUCTION.......................................................... 390 GNOTOBIOTIC TERMINOLOGY AND CRITERIA............................ 392 THE MICROBIAL ASSOCIATES ............................................. 393 Development and Characteristics of the Normal Flora............................. 393 Attempts to Establish a Microbial Flora in Germ-Free Animals .................... 395 Dietary Effects ......................................................... 396 Effects of Closed Environment................................................. 397 Factors Affecting Microbial Passage into the Host ............................... 397 THE ANIMAL HOST: GERM-FREE OR MODIFIED BY MICROBES..... .... 398 Nutrition, Digestion, and Metabolism .......................................... 398 Rats and mice.......................................................... 398 Other animal species....................................................... 400 Structure and Function of Various Organ Systems ............................... 401 Cardiovascular systems..................................................... 401 Gastrointestinal tract (upper segments) ........... ............................ 402 Enlarged cecum of germ-free rodents......................................... 402 Other autocoid substances of the germ-free intestine ............................ 403 Water absorption inhibition in the germ-free lower bowel ....................... 403 Other organ systems....................................................... 404 Body Defenses.......................................................... 404 Cells, tissues, and organs associated with defense functions ...... .................. 405 Humoral defensive elements................................................. 406 Immune responses ......................................................... 407 Tissue Repair.......................................................... 410 Survival and Aging .......................................................... 410 THE GNOTOBIOTIC ANIMAL IN THE STUDY OF DISEASE................ 411 Shock, Intestinal Obstruction, and Thermal Trauma.............................. 411 Wasting Syndrome .......................................................... 412 Oral Diseases.......................................................... 413 Dental caries .......................................................... 413 Periodontal disease........................................................ 414 Enteric Infections .......................................................... 414 Ulcerative Colitis.......................................................... 416 Uremia.................................................................... 416 Mycoplasmosis .......................................................... 417 Tuberculosis................................................................ 417 Yeast Infections.......................................................... 418 Protozoal Infections ......................................................... 418 Amebiasis ........................................................... 418 Histomoniasis............................................................. 419 CONCLUSION.............................................................. 419 LITERATURE CITED........................................................ 420 INTRODUCTION offers considerable potential as a tool in the of host microbial because Gnotobiotic animals, living either in the ab study relationships (i) it portrays the host either when free from sence of or in association with known viable germs and left to its own resources or when heterologous agents, represenwthrepresent -anowxensviaean extension oof modified by known microbial or other associates, the microbiologist's pure culture concept to all (ii) it permits the study of interflora relationships biological forms. The gnotobiotic experiment within the host organism, and (iii) it may be used ' On leave of absence from the Veterinary Medical Research In- in the study of any external or endogenous factor stitute of the Hungarian Academy of Sciences, Budapest. (e.g., in nutrition, immune reactions, responses 390 VOL. 35, 1971 THE GNOTOBIOTIC ANIMAL 391 to various forms of injury) where the pure distinct types of response became apparent in actions of such factors, affected or unaffected by this form of life. The underdevelopment of some associates in the host, are of interest. In the latter elements of the cellular and humoral defensive sense, the gnotobiotic experiment represents system (see below) seemed to be a plausible con- microbiological standardization of the animal sequence of the greatly reduced exposure to stock in the same fashion as it is commonly prac- antigens. By the same token, the germ-free ticed in terms of animal strain, diet, housing, animal's increased resistance to some stressor and various conditions of the physical environ- agents [e.g., X irradiation as reported by Wilson ment. and Piacsek (344)] or the indication of longer Historically, the concept of gnotobiotic ex- life span (see below) have been attributed to perimentation is credited to Pasteur's efforts in relief from the conventional microbial burden. 1885 (223). This is unquestionably correct in the In these instances, the contribution of the flora microbial sense of our definition. However, the appeared as a complicating or as an outright recognition of the need to work with pure sys- detrimental factor in the conventional host's life. tems in biological experimentation, from which On the other hand, it could be contended, the Pasteur appears to have drawn his analogy presence or absence of the flora can not be too [quoted by Ducluzeau (71)], can be traced far reaching in its overall effects on the host in further back to the experiments of Boussingault view of the normal growth, development, and (33) on nitrogen fixation of plants grown in a reproduction which were observed in germ-free "sterile" soil which were carried out during the animals when compared to their conventional first half of the past century. Various reviews on controls. This reassurance came particularly gnotobiotic experimentation (e.g., 164, 178) have after the conditions of the germ-free experiment commented in detail on Pasteur's keynotes of could be standardized and a plentiful supply of our subject. It is less well known that Schottelius, normally born, mother-suckled, adult germ-free a pioneer in the area of gnotobiotics who in his rats and mice became available for investigation. younger years spent some time in Pasteur's In addition, many morphological and functional laboratory, has in one of his papers (290) ex- details, originating mainly from parts of the body panded the master's scanty published remarks. that are not in immediate contact with microbes, In addition to outlining explicitly the actual proved similarity between germ-free and conven- gnotobiotic experiment, Pasteur speculated that tional animals. Finally, there were observations, on elimination of microbial associates, as is the some of them quite old, which suggested that case in the germ-free experiment, life of the there is something basically different, or perhaps animal host would become impossible. Appar- even wrong, with germ-free animals. Although ently, he based this idea on the concept of the ultimately caused by the absence of bacteria, "survival of the fittest" in evolution. Following these differences could not be readily explained this principle, he assumed that in the course of in terms of the lack of microbial stimulation and phylogenesis microbial associates have become a resultant tissue underdevelopment, as was the synergists which are indispensable in the life of case in the defensive system. There is an expand- the host. At the same time, the opposite view ing group of observations indicative of more or was held by Nencki (209) and later Metchnikoff less pronounced departures from conventional (190, 191), who considered microbes to be an- standards in structure, chemical composition, tagonists to the well being of the host. Definite and function of various organs in germ-free ani- proof that normal life of higher organisms is mals. Some of these differences did not appear possible in the absence of germs was offered first to influence particularly the economy of the host, by Reyniers and his co-workers at Lobund e.g., the ones resulting from the sparing effect of Laboratories, University of Notre Dame, in the germ-free life on intestinal degradation of diges- 1940's, who reared rats (267) and chickens (268) tive enzymes (see below). Other changes seemed in the germ-free state to successive generations. patently detrimental to the animal's health, e.g., This event, besides settling unequivocally the the greatly enlarged cecum and its consequences. original question, made the gnotobiotic animal These observations have indicated that the animal available as a practical tool for research for the host left to its own resources is not entirely self- first time. The breeding of germ-free animal sustained and that the normal microbial flora or colonies (mainly rats and mice) was successfully some of its components or products are needed initiated in the 50's by the groups