COLONIZATION OF THE MOUSE INTESTINE WITH ESCHERICHIA COLI

Young albino Swiss mice, of the NCS and NCS-D colonies, proved highly susceptible to the establishment of intestinal infection with an enteropathogenic strain of E. coli administered per os or by stomach tube. The period of highest susceptibility was rather short, extending from the day of birth to approximately 2 weeks of age. Adult NCS and NCS-D mice failed to become experimentally colonized with E. coli, even when large doses were administered per os on 3 consecutive days. The extent of colonization of the various parts of the gastrointestinal tract was related to the size of the infective dose. Many of the young mice died within 2 to 3 days following per os infection with large doses of enteropathogenic E. coli. However, practically all the animals which survived cleared their intestinal infection at approximately the same age. For example, in mice infected with 23 x 106 bacteria, colonization of the intestinal tract usually came to an abrupt end when the animals were 24 to 28 days old, irrespective of the age at which they had been infected. There is suggestive evidence that the acquisition of resistance with age, and the ability of adult animals to control the intestinal infection, are related to the development in the gastrointestinal tract of a microbiota which is antagonistic to E. coli

LASTING BIOLOGICAL EFFECTS OF EARLY ENVIRONMENTAL INFLUENCES : IV. NOTES ON THE PHYSICOCHEMICAL AND IMMUNOLOGICAL CHARACTERISTICS OF AN ENTEROVIRUS THAT DEPRESSES THE GROWTH OF MICE

Physicochemical and immunological techniques have been used in an attempt to characterize a filterable agent, separated from the intestines of mice raised under ordinary conditions of husbandry, which produces a lasting depression of weight in specific pathogen-free (SPF) mice when administered to them orally shortly after birth. Although this agent has not yet been identified, it will be tentatively designated here as enterovirus. The mouse enterovirus can be readily sedimented by ultracentrifugation and by precipitation at pH 4.3; it does not pass through cellophane membranes. Its infective power is completely destroyed by ultraviolet radiation, but is resistant to heating at 56°C, exposure to ether, treatment with trypsin, ribonuclease, and deoxyribonuclease. Dialysis and treatment with ether and nucleases greatly increase the infective activity of the intestinal filtrates containing the enterovirus, a finding which suggests that these procedures eliminate or destroy some inhibitory substance(s). The mouse enterovirus causes hemagglutination of mouse red blood cells. When injected into rabbits, it elicits in them an immune response that renders their serum capable of neutralizing its weight-depressing activity. As measured by inhibition of hemagglutination or complement fixation, the sera of infected mice do not exhibit any significant activity against usual mouse viruses. Centrifugation of the mouse enterovirus in 50%–20% sucrose gradient gave almost complete recovery of the infectivity and of hemagglutinating activity in the same fraction. In contrast, the protein content of the material was distributed through the various fractions. Consequently, this procedure resulted in a marked increase of specific activity

EFFECT OF NUTRITION ON THE RESISTANCE OF MICE TO ENDOTOXIN AND ON THE BACTERICIDAL POWER OF THEIR TISSUES

Mice on inadequate nutritional regimens were found to be more susceptible to various bacterial diseases than mice fed a complete diet containing 15 to 20 per cent casein. The tests of susceptibility included: (a) infections with virulent bacteria; (b) injection of large doses of avirulent coagulase-negative staphylococci; and (c) lethal effects of bacterial endotoxins. The infection-enhancing effect of nutritional deficiencies could be rendered even more striking by administering the infective inoculum simultaneously with a sublethal dose of endotoxin. Despite their great susceptibility to infection, malnourished animals retained much of their ability to eliminate bacteria from the blood, liver, spleen, kidneys, and lungs, at least during the early phases of the infectious processes. This was true even when the animals had received endotoxin simultaneously with the infective dose. The results suggest that under the conditions of the present study, the nutritional state influenced the outcome of infection not primarily by affecting the fate of the pathogens in vivo, but rather by modifying the ability of the host to resist their toxic effects

STUDIES ON FRACTIONS OF METHANOL EXTRACTS OF TUBERCLE BACILLI : I. FRACTIONS WHICH INCREASE RESISTANCE TO INFECTION

Fractionation procedures yielding partially purified vaccine preparations from a 60°C. methanol extract of tubercle bacilli have been described. Some of the preparations have the characteristics of lipopolysaccharides. Certain ones have been found capable of increasing resistance to experimental tuberculosis in albino mice of the Rockefeller Swiss strain. The levels of resistance elicited by these preparations are equivalent to those following vaccination with BCG (Phipps) in this strain of mice as reported by other authors. The admixture of two of the crude fractions in amounts as small as 0.05 mg. each per dose per mouse affords an even greater increase in resistance. Neither of these substances alone in larger doses can approach this degree of efficacy in mouse protection experiments. The protective activity appears to involve the stimulation of two supplementary mechanisms, one providing a peak resistance between 1 and 3 weeks post vaccination but falling off to a lower level thereafter, the other not responding fully until approximately 6 weeks but continuing undiminished through a 12 week post-vaccination period. The first of these peaks corresponds to an increase in resistance against staphylococci as well as tubercle bacilli. The possibility that the term "broad specificity," rather than "non-specificity," might best describe this phenomenon permits the implication of classical immune mechanisms

THE EFFECT OF BACTERIAL ENDOTOXINS ON THE WATER INTAKE AND BODY WEIGHT OF MICE

Injection of endotoxin of Gram-negative bacilli into NCS mice caused an immediate reduction or interruption of water intake by these animals, with a resultant loss of body weight. Endotoxins prepared by three different techniques from four different cultures of Gram-negative bacilli yielded products having approximately the same activity in inhibiting water intake. The minimum effective dose was 0.1 µg. or less. With all toxin preparations tested, the duration of the effect was directly related to the dose injected. The heat-killed cells of Esch. coli proved approximately as effective as the endotoxins prepared from Gram-negative bacilli. In contrast, heat-killed cells of Mycobacterium tuberculosis (BCG) were much less active, and heat-killed cells of Staphylococcus aureus were essentially inactive. Mice previously treated with endotoxin exhibited a marked degree of tolerance to the inhibition of water intake caused in normal animals by a subsequent treatment with the same material. Tolerance could also be induced by vaccination with heat-killed Gram-negative bacilli. Tolerance overlapped from one bacterial species to another but was more pronounced toward the endotoxin prepared from the bacterial culture with which the animal was vaccinated. The duration of the inhibitory effect of endotoxin on water intake was much shorter with mice fed a complete diet than with mice fed a deficient diet (corn). It took approximately the same dose of endotoxin (0.1 µg.) to inhibit water intake, reduce the influx of polymorphonuclear leucocytes, and enhance staphylococcal infection

THE SUSCEPTIBILITY OF MICE TO BACTERIAL ENDOTOXINS

Albino mice (Rockefeller NCS strain) raised and maintained free of ordinary bacterial pathogens, as well as of intestinal Escherichia coli and of Proteus bacilli, were found to be highly resistant to the lethal effect of bacterial endotoxins. When newborn mice of this NCS colony were nursed by foster mothers from another colony raised under ordinary conditions (SS colony from which the NCS colony was derived), they acquired the intestinal flora of the latter animals and became susceptible to the lethal effects of endotoxins. NCS adult mice could be rendered susceptible to the lethal effect of endotoxins by vaccination with heat killed Gram-negative bacilli. The susceptibility thus induced exhibited a certain degree of specificity for the bacterial strain used in vaccination. Although untreated NCS mice were resistant to the lethal effect of endotoxins, they proved exquisitively susceptible to the infection-enhancing effect of these materials. For example, 1 µg. or less of endotoxin was found sufficient to help establish a rapidly fatal septicemia with Staphylococcus aureus. Small amounts of endotoxin (1 µg. or less), administered alone, caused a marked but transient loss of weight. Vaccination with heat-killed Gram-negative bacilli or with killed BCG increased the resistance of NCS mice to the infection-enhancing effect of small amounts of endotoxin. This protective effect exhibited a certain degree of specificity for the bacterial strain from which the toxin used in the infection-enhancing test was derived. These various findings can be explained by assuming that the pathological effects of endotoxins involve at least two unrelated mechanisms; (a) a primary toxicity illustrated in this study by the loss of weight and enhancement of infection resulting from the injection of small doses of toxin; (b) an immunological reaction with lethal consequences which became manifest only in animals sensitized to the endotoxin by prior exposure to Gram-negative bacilli

THE EFFECT OF ANTIBACTERIAL DRUGS ON THE FECAL FLORA OF MICE

Oral administration of penicillin, terramycin, or chloramphenicol to NCS mice rapidly brought about profound changes in their fecal flora. The lactobacilli disappeared completely, whereas the numbers of enterococci and Gram-negative bacilli reached very high levels. In contrast, no effect on the fecal flora could be detected following administration of isoniazid in any amount. The intensity and duration of the effects on the fecal flora were related to the type of drug and to the amount of it administered. Chloramphenicol produced disturbances which were less profound and of shorter duration than those produced by penicillin or terramycin. The duration of the disturbances in the fecal flora produced by antibacterial drugs was markedly conditioned by the nutritional regimen. The fecal flora returned to its pretreatment state (large numbers of lactobacilli, few enterococci, and few Gram-negative bacilli) within less than 4 weeks after discontinuing the drug when the mice were fed a complex diet of ill defined composition (commercial pellets). Contrariwise, the fecal flora remained markedly different from that of control mice when the animals were fed semisynthetic diets containing as source of protein either 15 per cent casein or 15 per cent wheat gluten (both supplemented with cystine); or 15 per cent wheat gluten supplemented with lysine, threonine, and cystine. The fecal flora of mice treated with penicillin contained large numbers of lactose-fermenting Gram-negative bacilli, not found in the untreated animals. These lactose fermenters persisted for several months after discontinuance of the drug in mice fed either the casein or gluten diets, but they disappeared rapidly from mice fed pellets. Similar results, although less striking, were obtained with Swiss mice from colonies maintained under usual conditions, and therefore having a fecal flora more complex than that of NCS mice

LASTING BIOLOGICAL EFFECTS OF EARLY ENVIRONMENTAL INFLUENCES : V. VIABILITY, GROWTH, AND LONGEVITY

The effects of neonatal influences on the growth and longevity of mice were studied by using animals derived from a highly inbred germfree colony that had been reassociated with a microbial flora free of known pathogens. The size of the animals at weaning time could be conditioned predictably by manipulating the diet of their mothers during gestation and lactation or by shortening or lengthening the period of lactation. A deficient diet during gestation or during lactation decreased the metabolic efficiency of the adult animal, even if it was fed an optimum diet after weaning. The effect was greatest when malnutrition occurred during both pregnancy and lactation. In contrast, an optimum diet during gestation and lactation rendered the animal less susceptible to the depressing effects of nutritional deficiency during adult life. A marked and lasting growth depression could be reproducibly achieved by contaminating newborn mice orally with an unidentified enterovirus. But neonatal infection with enterobacteria or mycobacteria even though severe, did not significantly alter the growth rate. Regardless of its initial cause, the depression of the growth rate during the preweaning period persisted throughout the whole life span of the animals, even when they were placed under optimum sanitary and nutritional conditions after weaning. Agencies (nutritional or infectious) which brought about a depression of whole body weight also affected the absolute and relative sizes of the various organs, especially of the brain. By manipulating neonatal influences, it was possible to produce at will in a given colony of highly inbred mice a family of strikingly different growth curves. This could be done without causing the death of any animal or affecting longevity

MEASUREMENT OF RESISTANCE TO EXPERIMENTAL TUBERCULOSIS IN MICE : THE HYPERACUTE PHASE

NCS mice were found to be susceptible to a hyperacute form of disease induced by injection of relatively large doses of Mycobacterium tuberculosis. Susceptibility to this type of disease was conditioned by the sex, age, and dietary history of the animals. Unlike the more usual response of mice to mycobacterial disease, these conditions affected primarily the relative occurrence of early-acute deaths rather than average survival time. Resistance to this form of disease was increased by previous exposure of the animals to Gram-negative bacteria, to their endotoxin, or to various preparations of killed mycobacterial cells

THE EFFECT OF ANTIBACTERIAL DRUGS ON THE WEIGHT OF MICE

NCS mice gained weight rapidly when fed a gluten diet deficient in several amino acids, but their weight gain on the same regimen was very much retarded if they were given antibacterial drugs, even for a short period of time. This retardation of growth could not be entirely corrected by supplementing the gluten diet with lysine and threonine. The decrease in growth rate brought about by antibacterial drugs could probably be traced to the alteration in intestinal flora resulting from drug treatment. The intensity and duration of both types of changes were related to the dose of drug administered, and to the length of the treatment period. Whatever the nutritional regimen, treatment with penicillin caused a retardation of weight gain in NCS mice. The retardation was more pronounced, and longer lasting, when the animals were fed semisynthetic regimens (containing casein or gluten) than when they were fed crude diets (pellets) containing natural materials of ill defined composition. These differences probably had their origin in the fact that the changes in fecal flora induced by the drugs were profoundly influenced by the composition of the diet. Antibacterial drugs which retarded weight gain of Swiss NCS mice, in contrast increased weight gain in ordinary Swiss mice raised under usual conditions. It is probable that this difference in response to the antibacterial drugs resulted from the fact that ordinary Swiss mice have a much more complex intestinal flora than NCS animals