Gnotobiotics and the Germ-Free Animal

The development of the art of gnotobiotics has facilitated the production of germfree animals and has encouraged their use as research tools. The eventual elimination of the anomalies of germfree life by the reintroduction of the essential microflora will produce a controllable experimental animal. vestigator can purchase an isolator, complete with germfree animals, bedding, diet, and water. With a knowledge of aseptic procedures and gnotobiotic techniques, the investigator may readily incorporate these "living test tubes" into his studies. The variety of available isolators have been described in detail (Symposium, 1959, J. A. Reyniers, consult. ed.). Published techniques are available for the rearing of germfree chicks, turkeys, quail, rats, mice, guinea pigs, rabbits, pigs, and dogs (Reyniers et al., 1960; Proceedings, 1963; Lev, 1963). Once the sterile barrier has been established, eggs may be treated with germicide and passed through traps into the isolator for hatching. Mammals are usually obtained by caesarian section in one isolator and the young are aseptically transferred to a second isolator, where they are hand-fed (Pleasants, et al., 1963) until weaning. If the animals and their environment are found to have attained the germfree state (Wagner, 1959), they are fed a semisynthetic or practical diet which is designed to yield an adequate supply of nutrients after steam sterilization (Wostmann, 1959). Rats and mice have reproduced at Lobund through 20 successive generations in the germfree state (Teah, 1964). The use of the term "germfree" has been criticized because of the paucity of reports that such animals are PPLOand virus-free. Recently, Pollard stated "Thus far, PPLO has not been detected in the germfree rats and mice of Lobund Laboratory . . ." (1963), and that "Viral agents have been looked for in germfree rats and mice by means of serology, tissue culture, challenge inoculations with standard viruses, histology, and electron microscopy. Thus far no viruses have been isolated from germfree animals. While none has as yet b en detected by conventional procedures . . . we could not disregard the possibility that some viruses are transmitted congenitally . . ., as is indeed described in connection with leukemia. . . . Demonstration of the occurrence of radiogenic leukemia in germfree mice provides the first suggestion that a virus exists in these mice" (1964). Characteristics of the germfree animal Early workers observed that the germfree animal differs from its conventional BioScience March 1965 187 I This content downloaded from 157.55.39.102 on Mon, 03 Oct 2016 06:18:24 UTC All use subject to http://about.jstor.org/terms counterpart not only by the lack of an associated flora but also by alterations in the host which apparently resulted from the absence of microorganisms. "Defense mechanisms" are underdeveloped in the germfree animal (Gordon, 1960). The weight of lymphoidal tissue and lymphocyte counts in organs normally in contact with bacteria are reduced in the germfree animal. The germfree chicken has a significantly lower number of reticuloendothelial cells in the wall of the ileum. The serum of germfree animals contains low levels of gamma globulins, which is probably a direct reflection of the deficiency in the production of bacterial antibody. The detection of antibodies to dead bacteria (Wagner, 1959) and to proteins in the sterilized diet has resulted in the development of an "antigen-free," filter-sterilized, liquid-type diet (Pleasants, et al., 1963; Proceedings, 1964). Extensive coverage of the altered morphological, biochemical, physiological, and serological characteristics of the germfree animal is presented by Luckey (1963). The enlarged cecum, with its more liquid contents is the most pronounced anomaly in the germfree rodent. The reduction in the volume of the cecal contents and weight of the cecal sack is brought about by the introduction of the "normal flora" (conventional cecal content) (Gordon, 1960) and occasionally by single species of bacteria (Skelley et al., 1962). Recently we reported that the elevated level of a bioactive substance in germfree rodent cecal content decreases, as does the cecal size, when these animals are contaminated with Salmonella typhimurium (Wiseman and Gordon, 1964, 1965). The germfree animal as a research tool The extensive use of the germfree animal in nutritional studies has been reviewed by Mickelson (1962) and by Levenson and Tennant (1963). It is probable that in this area the germfree animal will play a major role, since it is ge erally accepted that the microflora is an essential contributor of nutrients to the host. For example, Wostmann et al. (1963) noted that when graded amounts of radiation sterilized thiamine were added to B1 deficient diets, a comparison of the growth of germfree and conventional rats showed that only those on the lowest levels benefited by the presence of the flora. The effect was lost when higher levels were fed, indicating that the biosynthethized thiamine does not alter the dietary requirement for the vitamin. The germfree animal has also been introduced into dental research. It has been reported that carious teeth do not occur in rats when there is a complete FIG. 1. Germfree mice and chicks in; pre-sterilized flexible plastic isolators. Isolators may be attached to autoclave (right) by means of steel lock (lower isolator) for the transfer of supplies. Air is sterilized and circulated through the isolators via filters (right and left front, upper isolator). absence of the oral flora, but that calculus formation has been observed on the teeth of germfree rodents (Fitzgerald, 1963). In an attempt to distinguish between the effects of thermal injury from those due to superimposed infection, Levenson and his associates (1963) have begun a series of studies on wound healing in the presence and absence of bacteria. They have related the slowed healing in the germfree guinea pigs to the impaired production of leukocytes which are necessary in the healing