Although Pasteur in 1885 had expressed the opinion that germ-free life would be impossible, 10 years later Nutthal and Thierfelder produced the first germ-free guinea pig. Their ingenuously designed but complicated glass-based equipment was soon replaced by the much more simple metal isolator systems. Basically these consisted of a main compartment equipped with gloves to handle animals, presterilized food, and other necessities, with an attached two door entry port. Germ-free animals were obtained by caesarean section and entry into the sterile compartment either directly or via a germicidal bath. During the fifties plastic started to replace metal in the isolator systems, leading to the well-known systems used not only for germfree experimentation, but nowadays for protection of prematures and patients in need of special protection against environmental contamination.
I. INTRODUCTION
As early as 1885, Louis Pasteur had mentioned to his students the desirability of being able to study metabolism without the interference of an actively metabolizing intestinal microflora, mentioning at the same time that, in his opinion, life under those conditions would be impossible (Pasteur, 1885). However, before the end of that century, Nutthal and Thierfelder (1895) had built equipment that eventually enabled a newborn cesarean-derived guinea pig to survive (Fig. 46.1). Thus, the study of germ-free (GF) life had begun.
II. GERM-FREE ISOLATORS
Soon, the above contraption was replaced by simpler two-compartment steel autoclave-type equipment, in which one compartment served as a two-way entry port for sterilizing food and bedding, the other being the sterile compartment fitted with (nowadays) neoprene gloves, long enough for handling animals, cages, and equipment (Fig. 46.2). Gloves were often fitted with thin latex hand gloves to make necessary manipulations easier. Equipment was sterilized beforehand either by steam or, later, by chemical means. In another approach, lightweight steel units with all equipment in place were sterilized in a large autoclave. In the 1950s, plastic gradually replaced steel, although steel units are still in use in many institutions. While keeping the same two-compartment layout, the main compartment was now plastic, easily sterilized by chemical means, while food and other necessities were sterilized separately in round-bottom steel containers capped on one side with plastic. These were then connected to the main compartment via a plastic sleeve which, after chemical sterilization of the sleeve and removal of the plastic separators, allowed for transfer of the sterile material. Thus, the plastic isolator was born, now in use all over the world. Plastic isolators used for experimental purposes usually have an effective work area of 4__6_ or 4__8_. They resemble the well-known incubators used in hospitals to protect premature infants (Fig. 46.3). For colony breeding, much larger units are used, constructed to fit exigent requirements (Latuada, 1981). Caging requirements for GF animals generally resemble those of their CV counterparts. The versatility of the plastic material made it possible to adjust the isolators to requirements of experimentation and for industrial animal production. Production units are often larger, although the efficiency of larger units must be weighed against the potential loss due to contamination. Some very large units have been built for specific purposes, even one with a manhole in the supporting table with an attached “half-suit,” to make it possible for the caretaker to be within the unit and be able to reach any point necessary. The ultimate use has been the housing of a child with severe combined immunodeficiency (see Section V). In a different approach, GF mice were transferred in the isolator to a standard mouse shoebox cage, which was then capped with a tight-fitting bonnet, the top consisting of HEPA-like filter material. The cage could then be removed and placed in a laminar-flow bench outfitted with HEPAfilter. Properly protected personnel could now service the mice (Sedlacek et al., 1981).
III. PRODUCTION OF GERMFREE ANIMALS
Germ-free animals are obtained via cesarean section. In one approach, a specially constructed steel autoclave-type unit (as has been described) is used, which is divided into two compartments by a horizontal separator with a circular opening that is closed with a sheet of plastic. After autoclaving the units with the surgical equipment in the upper part, the lower part is opened to the environment. This (now nonsterile) part contains a small platform on which the anesthetized pregnant female can be raised so that the belly of the animal is tight against the plastic. The babies are then removed by hysterectomy. With a cauterizing knife, a cut is made through the plastic and abdominal wall, heat-sealing the abdominal cavity to the GF compartment. The babies can now be removed and placed in a separate isolator for cleaning and feeding. In this way, GF mice, rats, guinea pigs, and rabbits have been produced. After birth, the newborn had to be fed a specifically adjusted formula, using a nipple attached to a suitable syringe-type container. Germfree guinea pigs had to be fed for only a few days because of their advanced development at birth. Other species, however, had to be fed in this way for several weeks. This turned out to present no special problems, except for the feeding of newborn GF mice. After many trials, a pair of baby C3H mice were obtained that grew to maturity, bred, and produced offspring. Thereafter, newborn mice of any strain could be foster-nursed by the GF mice already available. All of the species mentioned above reproduced well under GF conditions, rabbits only after an anomaly in Fe absorption was recognized, which needed some dietary adjustment. Germ-free gerbils have been obtained by foster nursing on GF mice that were 2–3 days postpartum. However, these animals did not reproduce because of the size of the cecum (Bartizal et al., 1982). All GF rodents and leporidae have significantly enlarged ceca. In GF rats, the distension starts about two weeks after birth. Depending on diet, the GF cecum could be from 4% to 8% of body weight, versus around 1% in the CV rat, and more in GF rabbit and guinea pigs. The enlarged cecum is caused by the absence of a microflora that normally will digest the intestinal mucus which would otherwise accumulate in the cecum. This accumulation of acidic mucus changes the local mineral balance, leaving insufficient sodium ions necessary for water removal (Gordon and Wostmann, 1973). As a result, the water content of the cecal material resembles that of the ilium. Although comparable absorption of water occurs in the colon, water content of rat GF feces is more than 20%, against about 4% in its CV counterpart. While the GF rat excretes the larger part of its water intake with the feces, the CV rats excretes most in the urine. The rather fluid feces of the GF animal may create a substantial housekeeping problem. As mentioned earlier, GF gerbils had to be associated with a microflora consisting of six well-defined microbial species before reproduction would occur (the hexaflora). Association with this hexaflora has been used for cecal reduction, the major abnormality of the above-mentioned GF species.
The hexaflora (or other multiflora) components were introduced by bringing sterile monocultures into the isolator after sterilization the outside of the usual test tubes. Germ-free animals have also been obtained via Cesarean section in a clean, minimally contaminated environment, subsequently passing the newborn via a germicidal trap into the GF unit. This method had been originally used for rats and has later been used to produce larger GF animals like pigs, lambs, and calves (Miniats, 1984). Depending on the requirements of the study, either noninbred or inbred strains have been used to produce the GF animal. When noninbred strains were used, regular introduction of GF animals derived from the CV stock was used to assure genetic comparability between GF and CV animals. In case inbred strains were used, it would seem that this might be unnecessary but, presumably, still advisable after prolonged periods to prevent genetic drift from occurring. Germ-free chickens have been produced from fertilized eggs that had been incubated for at least 18 days. The eggs are then placed in the entry port of a GF isolator, chemically sterilized, and taken inside. They are spread out and temperature is kept at 37–38 _C, humidity between 70% and 80%. After hatching, the birds are kept at 37 _C for a few days. They rapidly learn to eat and drink. Thereafter, the temperature can be gradually lowered to normal room temperature (Coates, 1984).
IV. DIETS FOR GERM-FREE ANIMALS
Diets for GF animals are nowadays well established. They fall into three categories: natural ingredient diets fortified for the losses that occur during sterilization (Table 46.1); diets with minimal antigenicity, used mainly for immunological studies; and the chemically defined antigen-free diets, used for immunological and other studies where absolute definition of environment and dietary intake is required, again used mainly in immunology. For more defined work in the first category, diets based on rice starch, a well-defined source of protein like casein or soy protein, some fat, generally corn oil, and well-defined vitamin and mineral mixes have been developed (Table 46.2) and are commercially available. Diets of minimal antigenicity have been developed for the study of the very early phases of immune response in young colostrum-deprived gnotobiotic (GN) piglets (Kim and Watson, 1969). Extreme definition was obtained with the development of the GF mouse reared on a totally chemically defined, water-soluble antigen-free diet (CD diet), consisting of dextrose, amino acids, water-soluble vitamins, minerals, and a fat supplement, sterilized by ultrafiltration (Table 46.3) (Pleasants et al., 1986).
This successful colony was started with inbred GF BALB/cAnN mice obtained from the GF colony maintained at the University of Wisconsin. Pregnant GF mice of this colony originally were fed natural ingredient diet L-485 (Table 46.1), then transferred to another isolator and fed CD diet. Their offspring, never having been in contact with other than the CD diet, were housed in plastic isolators in shoebox-type mouse cages, with lids modified to hold the inverted diet and water bottles. The plastic bottoms of the cages had been replaced with stainless steel wire mesh above removable drip pans. Fortified soyderived triglycerides provided essential fatty acids plus readily available calories. Purified vitamins A, D, E, and K were added. Again, this mixture was ultrafiltered. It was then fed in stainless steel planchets that were welded to the stainless steel dividers in the cages (Fig. 46.4). Whatman Ashless filter paper provided indigestible fiber while also serving as bedding and nesting material. This was autoclaved for 25 min. at 121 _C or irradiated at 4.5 Mrad before being taken into the isolator. Here, the original purpose had been the establishment of nutritional requirements in the absence of a metabolizing intestinal microflora. But again, without any original exposure to antigen or microflora, this proved an ideal tool for the study of the early and later development of the immune system. For these studies, the inbred GF BALB/c mouse proved to be the animal of choice, because of excellent breeding results through at least 6 generations. This was in contrast to earlier studies with inbred C3H mice, which did not reproduce beyond the second generation. While, obviously, the GF animal maintained on a chemically defined diet is the animal of choice to study the various aspects of immunology, the GF animal maintained under less rigorous conditions will be of absence of a microflora on function and metabolism. Comparing GF and CV rats, it was found that the stress of a metabolizing microflora reduces life span to an extent that otherwise can only be achieved by an involuntary reduction of dietary intake by 30% (Fig. 46.5).
V. APPLICATION OF GERM-FREE TECHNIQUES: GNOTOBIOTES
A. The boy in the bubble In general, most controlled studies have been carried out with GF and GN rats and mice. The GF animal technique has the potential for selective association of the originally GF animal with a specific microflora element, including parasites. Mentioned earlier is the potential of bringing its distended cecum back within acceptable limits by association with a well-defined cocktail of microbial species. These are introduced by bringing monocultures grown in test tube into the main unit after chemical exterior sterilization in the entry compartment. In this way, studies have become possible of what is called the gnotobiote, the animal that harbors a desired but defined combination of microflora elements. However, the term gnotobiote per se also includes the GF animal. Once the GF animal had been produced, the logical extension was the study of function and metabolism of this gnotobiote. Apart from anomalies mentioned earlier, the absence of a metabolizing microflora resulted in the absence of certain nutrients normally produced by the flora (e.g., vitamin K) and certain flora-produced stimuli (e.g., LPS). Because of this, diet had to be adjusted, apart from adjustment necessitated by heat or radiation sterilization. The effects of the GF state on immune function are substantial, although all GF species proved to be eventually immunologically competent. Upon antigenic stimulation, the GF animal generally shows a delayed, but eventually adequate, immune response.
However, antibacterial response may be inadequate, due to slow response of macrophages, which originally are defective in chemotaxis and destructive and lytic capacity, leading to a delayed presentation of antigenic material to other elements of the immune system (Wostmann, 1996). The possibility of the production of gnotobiotes with a stable and defined microflora opened many possibilities and can be regarded as the originally GF animal’s major potential. It soon became obvious that GF rats did not develop that scourge of our society, dental caries, thereby indicating its microbial origin. GF rats were then associated with a number of different bacteria. This resulted in the recognition of Streptococcus mutans as the major originator of caries and put the acid-producing lactobacilli in second place (Orland et al., 1955). Similarly, it was shown that GF guinea pigs inoculated with the parasite Entomoebia histolytica, the cause of a potentially lethal intestinal infection, do not develop any symptoms. The animals retained the amoeba for only a few days. A microflora is obviously needed to change intestinal conditions to the point where the infection could take hold (Phillips, 1964). On the other hand, it was found that a CV microflora, by its stimulation of the immune system, may affect a certain amount of protection against Schistosomiasis (Bezerra et al., 1985). Studies of the various factors involved in the establishment of the nematode Trichinella spiralis were carried out by Przyjalkowski (Przyjalkowski et al., 1983) and Despommier (Despommier, 1984). In the pork industry, GF pigs have been used to solve problems of bacterial and viral infection. Gnotobiotes are important to the study of colonization resistance, which seeks to determine which microflora elements may be important for the flora’s stability and which for its potential to resist pathogens. In a similar way, gnotobiotes enable the study of microbial translation to determine what microflora composition will enhance or inhibit certain of its members to pass the intestinal barrier and possibly cause disease.
Recently, it has become possible to establish a “normal” human microflora in originally GF mice. After the actual composition of this flora has been established, and its stability ascertained, this could open the door to a multitude of studies pertaining to human health and disease. The plastic isolator, already in general use to protect premature infants, found its culmination in its use to protect David, the “Boy in the Bubble.” Before birth diagnosed as having SCID (severe combined immune deficiency), he was born via Cesarean section and placed in an isolator, which eventually grew to a fourroom apartment. Over the years, he accumulated a number of non-life-threatening organisms, but death came at the age of about 121 2 years, apparently caused by the sequela of an unsuccessful bone marrow transplant (Bealmear et al., 1985).
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