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Two of the main features of human cholera-induction of diarrhoea and confinement of the infection to the lumen of the intestinal tract-may be reproduced in three experimental models: (a) the streptomycin-treated, starved guinea-pig; (b) the intestinal loop in the adult rabbit; and (c) the suckling rabbit. In this paper the author compares the two last-mentioned models with his earlier work in guinea-pigs.Intestinal antibody (coproantibody) was highly protective, while circulating antibody had little or no effect, in all three models. The protective coproantibody was specific for the heat-stable vibrio antigens. It did not affect the growth of vibrios in the intestine, and its function may possibly be regarded as antitoxic rather than antibacterial. Oral vaccination protected adult rabbits against challenge by the loop technique. Heat-killed vaccine was as effective in this respect as live vaccine.The author feels that the present uncertainty concerning the protective value of cholera vaccination may be due to the fact that conventional vaccine is designed to induce high serum titres only. He considers that since oral vaccine has been shown to induce and maintain the production of coproantibody in human volunteers, a field trial should be carried out to determine whether coproantibody is as protective in man as it has proved to be in the experimental models.

A method has been devised for inhibiting the normal enteric flora, permitting long term asymptomatic enteric infections of mice and guinea pigs with streptomycin-resistant strains of Shigella flexneri or Vibrio cholerae. Introduction of a streptomycin-resistant strain of E. coli into the intestinal tract of experimental animals resulted in a rapid elimination of the enteric pathogens studied. No in vitro production of antibiotic substances by this coli strain could be demonstrated. Active and oral passive immunization did not noticeably influence the number of Shigella or Vibrio organisms recoverable from the feces of infected animals.

An anaerobic glove box constructed of clear flexible vinyl plastic is described. It is sufficiently inexpensive and simple in operation to be used not only in research but also in a clinical laboratory by technicians without special training. Conventional bacteriological techniques may be used inside the glove box for culturing and transferring anaerobic bacteria. The box may be heated to 37 C and thus serve as an anaerobic incubator as well, permitting inspection of cultures at any time. Media may be prepared and agar plates may be poured on the laboratory bench in the conventional manner. An overlay of trace amounts of palladium black catalyst over plated agar media reduces the medium to an oxidation-reduction (O-R) potential of - 300 mv within 2 days after introduction into the glove box. In spite of its greater simplicity, the system matched or excelled the roll tube method with respect to all parameters tested, including O-R potential obtainable in the media, O(2) concentration in the gas phase, and efficiency in isolating anaerobic bacteria from the mouse cecum. Comparative studies indicate that the conventional anaerobic jar method was inadequate for the isolation of strict anaerobes from human gingival specimens and from the mouse cecum. This was due to the exposure of specimens and media to air during plating on the open laboratory bench. Anaerobic jars were adequate for maintaining the proper conditions for growth of anaerobic bacteria once these had been established in the glove box.

Earlier work had shown that a collection of anaerobic bacteria, in conjunction with facultative anaerobes, may be implanted into germfree mice, thereby rendering the animals "normal" with respect to a variety of parameters tested. The present experiments indicate that a different collection of anaerobic bacteria, isolated from the cecum of normal mice, was necessary to convert germfree mice to the "normal" state when the animals were fed a crude diet, rather than the refined food which had been used in the earlier work. The nature and level of short-chain fatty acids associated with various natural or synthetic "normal" floras in the ceca of mice did not always correlate with the Escherichia coli population present, indicating that fatty acids were not the sole agents inhibiting bacterial populations in the intestine. Experiments are reported which indicate that intestinal anaerobes may under certain circumstances be sufficient to control the populations of other intestinal bacteria such as E. coli. In other instances, such as control of Shigella populations in the mouse intestine, intestinal anaerobes appeared to act synergistically with an E. coli strain, in spite of the fact that the population of the latter was itself suppressed by the anaerobes.

The results presented indicate that blocking curves of decreased slope are obtained with the Talmage test when the labelled antibody eluate contains a portion of antibody which is not represented in the unknown serum. The blocking curves of late antisera had steeper slopes than those of early antisera when tested against labelled antibody eluates prepared from late antisera. This situation was reversed when labelled eluates from early antisera were used in the Talmage test. The findings were interpreted to indicate that early antisera did not have a full complement of antibodies, directed against all surface antigens of the bacterial cell.

Freter, Rolf (Jefferson Medical College, Philadelphia, Pa.), and Atsushi Ozawa. Explanation for limitation of populations of Escherichia coli in broth cultures. J. Bacteriol. 86:904-910. 1963.-Veal Infusion Broth surrounding a cellophane bag containing a 24-hr culture of Escherichia coli was inhibitory to an inoculum of the same strain when kept under an atmosphere of nitrogen plus CO(2). The inhibition could be abolished by addition of glucose or by aeration. When a small inoculum of a dulcitol-positive E. coli strain was introduced into a fully grown (24 hr) static Veal Infusion Broth culture of a dulcitol-negative E. coli, no multiplication occurred. However, the inoculum did grow in the presence of a carbon source (dulcitol) which could not be utilized by the static population. The logarithmic growth rate attained by the dulcitol-positive inoculum under these conditions was independent of the dulcitol concentration. In contrast, the maximal population size was a function of the amount of dulcitol present. Similar results were obtained when sucrose or salicin was substituted for the dulcitol. All strains grew well in filtrates of 24-hr broth cultures with growth rates and maximal populations approximating those attained in fresh broth. It was thus concluded that populations of E. coli in broth cultures were limited by exhaustion of all those carbon and energy sources which could be utilized under the prevailing highly reduced conditions. No evidence of inhibitory metabolic end products was found except in broth supplemented with 1% glucose. Partial inhibition of multiplication was noted in the latter medium even when the pH was maintained between 6.9 and 7.2.

Strictly anaerobic and facultatively anaerobic bacteria were isolated from the intestinal tract of normal mice. Germ-free mice were associated with mixtures of varying complexity of pure cultures of these bacteria. The development of normal features in these animals was then determined on the basis of the following criteria: (i) size of the cecum, (ii) size of the Escherichia coli population in the cecum, (iii) histology of the intestinal tract, and (iv) development of a mucosa-associated flora in stomach and large intestine. Germ-free mice contaminated with cecal contents from conventional mice were used as controls to establish normal values for these parameters. Some strictly anaerobic bacteria could be implanted into germ-free mice only after prior implantation of an E. coli strain. E. coli was found in large numbers in stomach and cecum of mice monocontaminated with this organism. Use of restraining devices indicated that the E. coli population in the stomach was maintained by coprophagy and did not contribute significantly to the size of the cecal population. A mixture of 50 strictly anaerobic bacteria plus 80 facultative anaerobes rendered recipient animals normal with respect to the criteria tested. Other, less complex bacterial mixtures reduced the cecal size and the intestinal E. coli population to levels intermediate between those found in normal and germ-free mice. With all bacterial mixtures tested, the intestinal E. coli population decreased, if at all, within a period of about 10 days after introduction of other bacteria, and remained stable thereafter. This suggests that the intestinal E. coli population is controlled by a mechanism which reduces population size without affecting the growth rate.

The present studies are concerned with the parameters which control the appearance of locally synthesized or serum-derived antibodies in the intestine. The data show that intestinal antibody may be found in rabbits as well as in conventional or germfree mice after active immunization with Vibrio cholerae. However, a large fraction of the intestinal antibody in rabbits and conventional mice originated from the serum as indicated by (i) analysis of correlation between serum and intestinal antibody titers, and (ii) the occurrence of intestinal antibody after parenteral administration of antiserum. In contrast, only locally synthesized 11S immunoglobulin A antibody was detected in the intestine of actively immunized germfree mice. No intestinal antibody was demonstrable in germfree mice after parenteral injection of V. cholerae antiserum. With respect to the appearance of serum antibody in the intestine, the response of conventionalized (ex-germfree) mice was intermediate between that of rabbits or conventional mice and germfree mice. The availability of serum-derived coproantibody in germfree and conventional mice was related to the rates of intestinal degradation of serum antibody. When enzymes were removed by prior washing of intestinal segments, serum antibodies entered the intestine of germfree or conventional mice at similar rates. Rates of entry of serum antibodies into the lumen were comparable at different levels of the small intestine. The presence of a normal enteric flora appeared to protect intestinal antibody from degradation by lowering the concentration or activity of intestinal enzymes. The results are discussed in relation to the question of whether antibacterial immunity to cholera involves local or systemic mechanisms.

Passive oral immunization with O-antiserum was found to protect guinea pigs against fatal enteric cholera. The number of vibrios recoverable from the intestines of infected animals was not noticeably reduced by active and oral passive immunization. Passive immunization by the intraperitoneal route was not protective even though high titers of circulating antibody were obtained shortly after injection. Oral administration of H-antiserum also had no protective effect. Introduction of E. coli into the intestinal tract increased the resistance of guinea pigs to fatal enteric cholera.

Studies of the relative combining capacities of different antisera to Vibrio cholerae and Shigella flexneri were carried out using the Talmage test. In this test the absorption of an I(131) labelled antibody preparation by antigen is blocked by the addition of unlabelled unknown serum. Sera from rabbits in the early stages of immunization ("early antisera") had a high agglutinin titer, but low combining capacity. Sera from hyperimmune rabbits ("late antisera") had higher combining capacities than early antisera, but similar or lower agglutinin titers. More antigen was needed to absorb the agglutinins from late antisera than from early ones of the same agglutinin titer, indicating that late antisera contained relatively more antibody. At high concentrations, sera from hyperimmune rabbits agglutinated the homologous antigen more rapidly than did early antisera, even though early sera had similar or higher agglutinin titers. Sera drawn after the anamnestic response to bacterial antigen had the characteristics of late sera; i.e., their combining capacities were high. The possible significance of these findings for the in vitro estimation of protective antibody is discussed.

Earlier studies have shown that intestinal antibody (coproantibody) decreases the adsorption of vibrios to the intestinal wall of rabbits. The mechanism underlying this phenomenon was studied by means of an in vitro model in which vibrios were grown on slices of rabbit ileum in a moist chamber. Antibody prevented the adsorption of vibrios onto such slices in the same manner as it did in vivo. Studies of the in vitro growth rate of vibrios on slices of ileum indicated an antibody-dependent antibacterial mechanism on the mucosal surface. This mechanism appeared to require the presence of viable mucosal cells, as it was not present when filter paper was substituted for the tissue slices and it could not be demonstrated in fresh scrapings from the intestinal mucosa. Antigen-antibody mixtures were added to the surface of slices of rabbit ileum or were introduced into the lumen of intestinal loops. Presence of these mixtures did not inhibit the antibody-dependent antibacterial mechanism on the mucosa in either the in vivo or the in vitro system. The amount of antigen-antibody mixture used in vivo was at least 12 times that required to neutralize the complement released by homogenization from an entire intestinal loop. The results obtained support the hypothesis that coproantibody protects by decreasing the adsorption of vibrios on the intestinal mucosa. The mechanism responsible appears to be an antibacterial effect on the mucosal surface which requires antibody plus some additional factor(s) supplied by viable mucosal cells. The postulated factor does not appear to be complement.