Morris Wagner

Use of the Germfree Animal Technic in the Study of Experimental Dental Caries I. Basic Observations on Rats Reared Free of All Microorganisms

pESPITE widespread interest in dental caries, and the consequent accumulJation of numerous well-planned experimental investigations in search of the causes of this disease, a clear understanding of its etiology and pathogenesis is definitely lacking. There is evidence, however, of many factors both systemic and local, modifying or contributory to the various process. Study of the interrelationships of these factors has given origin to a few attractive theories, but the validity of current theories still depends on the discovery of final proof of etiology. The bacterial flora of the mouth, and more specifically of the various lesion, is so complex that any potential cariogenic effects of microorganisms are constantly subject to modifying influences arising from the simultaneous action of some or all of the numerous species and varieties commonly present in this localized area. These modifying effects could be either additive or symbiotic, they could be neutralizing, or they could be either inhibitory or antibiotic. Even if the cause of dental caries is to be found in phenomena other than those associated with the metabolic activities of the local bacterial flora, as has been claimed by a few investigators, the objective demonstration of such phenomena is made exceedingly difficult, if not impossible, by the ubiquitous nature of oral microorganisms, some of which have the capacity, in vitro, to bring about a dissolution of tooth substance closely resembling in some respects certain stages in the dental caries process. In view of the foregoing considerations, we have undertaken a series of investigations with animals which are initially free of all bacteria, but to which single or multiple strains of known bacteria can be introduced. Only by this means does it seem possible to demonstrate clearly the bacterial effect on the teeth of animals fed a diet which in normal animals induces abundant tooth decay.

DETERMINATION OF GERMFREE STATUS

The determination of the microbiological status of animals maintained by germfree techniques presents a complex and challenging problem to the microbiologist. Germfree technology, by the implications of the term, must separate the animal from a variety of microbial forms found in close association with the host during its existence in nature. Bacteria, yeasts, molds, actinomycetes, rickettsia, viruses, protozoa, worms, and arthropods are all known to establish varying degrees of association with the host, ranging from obligate parasitism to only seemingly casual association. The ability of cesarotomy to isolate the host completely from intimately associated forms of life presents an important problem. The determination of germfree status requires microscopic or culture methods or both for the detection of the previously mentioned forms. The complexity of the test procedures varies with the organism sought. Routine bacteriological and mycological culture methods employing artificial culture media are used to detect the presence of the more common and more readily cultivated bacterial and mycological forms. The addition of body fluids or other supplements to artificial media aids in the cultivation of more fastidious organisms that require enrichment for growth. The detection of organisms that are difficult to cultivate or whose growth is obligately dependent on other living cells calls for still more complex methods involving animal inoculation or tissue culture. Finally, there are those organisms that have resisted all cfforts a t cultivation to date. It is necessary to depend solely on microscopic observation to detect their presence provided, of course, that they are visible under the microscope and are present in sufficient numbers to detect and recognize. Organisms that do not meet any of these requirements and produce no detectable effect upon the host must be considered inapparent contaminants. Animals that harbor only such “silent” contaminants will pass as germfree until such time as: (1) new techniques of detection are developed or (2) a shift in the equilibrium of the host-contaminant relationship in favor of the microorganism allows detection by some recognizable means. FIGURE 1 presents a general scheme for determining the germfree status of an animal. The culture methods shown in the upper two quadrants of. the scheme generally meet the nutritional, oxygen, and temperature requirements for growth of most bacterial and mycologic forms that have the greatest probability of gaining entry to the germfree apparatus through a fault in the mechan* The work reported in this paper was supported in part by Contract NR103-067 between the Office of Naval Research, Department of the Navy, Washington, D. C., and the University of Notre Dame.

SEROLOGIC ASPECTS OF GERMFREE LIFE

Since the latter part of the Nineteenth Century numerous investigators have concerned themselves with the study of so-called natural antibodies. These antibodies have been called natural because they can be demonstrated in the sera of normal animals that have not been subjected to any injection or treatment with a specific antigenic material by the investigator. In the case of the natural antibacterial antibodies against specific pathogens, these antibodies can frequently be demonstrated in animals not known to have suffered any clinical symptoms of the disease. In many cases there has been no evidence of any contact between pathogen and host, so that even subclinical infection could not always explain the presence of antibacterial antibody for a specific organism. However, an accumulation of knowledge regarding the antigenic components of plants, animals, and microorganisms has shown that they may all share common or closely related antigenic substances. It is therefore no longer necessary to assume that each antibody entering into in vitro serologic reactions with a particular organism has been specifically produced in response to that organism. Wiener1 has presented a similar argument for the origin of the natural human isoantibodies anti-A and anti-€3. He argues that, while production of the human blood group substances A and B is controlled genetically from within, the stimulus for the production of the reciprocal isohemagglutinins arises from without through exposure to microorganisms that contain substances similar to human blood groups. Historically, studies on the occurrence of natural antibodies have been repeated as improvements in animal care, isolation, sanitation, and nutrition have been made. The availability of animals reared free of known viable microbial life poses the question of whether natural antibodies make their appearance under conditions of germfree life. However, it should be kept in mind that the sterile diets used in this work are not free of antigens. This is a refinement that has not yet been attempted. Observations were made on the occurrence of heterohemagglutinins and antibacterial agglutinins in the sera of chickens and rats under the following four conditions : (1) germfree animals undergoing no specific antigenic treatment (the normal germfree animal); (2) antibody response of germfree animals to parenteral injection of nonviable antigens; (3) antibody response of germfree animals to dead bacteria purposely incorporated in the diet; and (4) antibody response of “germfree” animals to monocontamination with pure cultures of viable bacteria. Limited observations also were made on properdin and complement titers in germfree rats. Since this monograph on germfree vertebrates includes consideration of the * The work reported in this paper was supported in part by Contract NR103-067 between the Office of Naval Research, Department of the Navy, Washington, D. C., and the University of Notre Dame.

SERUM PROTEIN FRACTIONS AND ANTIBODY STUDIES IN GNOTOBIOTIC ANIMALS REARED GERMFREE OR MONOCONTAMINATED

The germfree animal has been described as differing in various aspects from animals reared under conventional animal room conditions.’ These differences have for the most part been ascribed to the respective absence or presence of an associated microbial flora. The role played by specific members of the normal flora in establishing the “physiologic state” of conventional life is obscured by the complex nature of the normal flora. This paper describes some observations made on the serum protein fractions and antibacterial agglutinins found in animals maintained under less complex conditions; that is, either entirely germfree or in association with only single selected strains of intestinal microorganisms (monocontaminated). In another paper,

The size pH, and redox potential of the cecum in mice associated with various microbial floras.

the response of the germfree rat to a purified antigen, bovine serum albumin, is described. Earlier work on the comparison of serum proteins of germfree and conventional rats has characterized the germfree rat as having lower but nevertheless detectable levels of alphaz , beta, and gamma-globulin fraction~.~J A similar comparison in chickens showed the principal difference to be a decreased gamma globulin level under germfree conditions? The characterization of other germfree-reared species from this standpoint, for example, mouse, guinea pig, rabbit, sheep, and goat have been reported and reviewed by Wostmann?J In general, all germfree-reared animal species studied to date have shown varying degrees of hypogammaglobulinemia, depending on the species, age and, possibly, dietary factors. Germfree rat and chicken serum have been found to be devoid of many, but not necessarily all, antibacterial agglutinins demonstrable in the serum of conventional animals.4s6 The agglutinins that have been demonstrated in germfree animals are believed to be formed in response to dead bacteria and probably other antigenic substances indigenous to the autoclaved diet. These dietary antigens may thus be responsible, a t least in part, for the levels of the probable antibody-carrying serum protein fractions seen in germfree animals. Exposure of young adult germfree rats to the normal intestinal flora of conventional rats resulted in a gradual increase in various globulin fractions.6 The increase occurred first in the alphaz fraction at one week post-contamination followed by an increase in beta globulin by the second week. Gamma globulin stayed at the low germfree level and only started to rise after a two to

Experimental caries in germfree rats inoculated with enterococci

Summary Cecal size and in situ redox potential and pH of cecal contents were determined in conventionally reared mice and mice reared under a variety of gnotobiotic conditions: germfree, monoassociated with a cecal Clostridium sp., hexaflora-associated and thermoduric polyflora-associated. The mean Eh was approximately +200 mV in germfree and −200 mV in conventional mice. The Eh was close to zero in the monoassociated mice, thus occupying a position intermediate between the germfree and conventional mice. The potentials observed in the hexaflora and the thermoduric flora groups were indistinguishable from those of conventional animals. The degree of normalization was more advanced with respect to the redox potential than to the cecal size in the various gnotobiotic groups. In the thermoduric polyflora-associated group, normalization was observed in both cecal size and redox potential. This demonstrates that normalization can be accomplished with a relatively simplified microflora, at least with regard to the parameters studied.