Samuel B. Formal

Pathogenesis of Salmonellosis STUDIES OF FLUID SECRETION, MUCOSAL INVASION, AND MORPHOLOGIC REACTION IN THE RABBIT ILEUM

Strains of Salmonella typhimurium were studied in the ligated rabbit ileal loop model to gain insight into the mechanisms whereby bacteria which invade the gastrointestinal mucosa evoke fluid exsorption. The organisms employed differed in various biologic attributes including the ability to invade the ileal epithelium, multiply within the mucosa, elicit an acute inflammatory reaction, and disseminate across the intestinal wall. Some strains provoked small intestinal fluid exsorption although these did not elaborate enterotoxin. Only those strains which invaded the mucosa were accompanied by either mucosal inflammation or fluid exsorption. Noninvasive strains produced neither histologic abnormalities nor fluid secretion. While strains which invaded the mucosa caused an acute inflammatory reaction, not all such strains evoked fluid secretion. Furthermore, there was no correlation in ability of invasive organisms to evoke fluid secretion or in the intensity of mucosal inflammation, number of intramucosal salmonellae, or in ability to disseminate from the rabbit ileum. These observations suggest that, as is the case in shigellosis, mucosal invasion may be a necessary factor for the intestinal fluid loss in salmonellosis. A bacterial property or factor, in addition to invasion of the gastrointestinal mucosa, seems to be responsible for fluid exsorptin. However, it is unlikely that a salmonella enterotoxin comparable to that elaborated by Vibrio cholerae, toxigenic Escherichia coli, or Shigella dysenteriae 1 is related to fluid secretion in salmonellosis.

Studies in volunteers to evaluate candidate Shigella vaccines: further experience with a bivalent Salmonella typhi-Shigella sonnei vaccine and protection conferred by previous Shigella sonnei disease

A bivalent vaccine consisting of Salmonella typhi strain Ty21a containing the 120 MDa plasmid of Shigella sonnei and expressing both S. typhi and S. sonnei lipopolysaccharides (LPS) on its surface was previously shown to protect significantly against S. sonnei disease in experimental challenge studies. However, protective efficacy could not be reconfirmed in volunteers with five subsequent lots of vaccine. One vaccine lot which resembled the initial protective lots of vaccine in biochemical and serological tests, and by electron microscopy, was administered to 16 volunteers who ingested three doses of 10(9) organisms each. Antibody secreting cells (ASC) specific for S. sonnei LPS were detected in the blood of 100% of vaccines, but no protection of these vaccines was demonstrated during a S. sonnei challenge study. To assess the ability of the volunteer model to detect infection-derived immunity, six volunteers who had had clinical shigellosis due to S. sonnei two months earlier were rechallenged with wild-type S. sonnei, together with 12 controls. Prior infection provided 100% protection against febrile illness (p = 0.05) and diarrhea (p = 0.04), thereby validating the volunteer model for assessing Shigella vaccines.

Evaluation of the safety, immunogenicity, and efficacy in healthy adults of four doses of live oral hybrid Escherichia coli-Shigella flexneri 2a vaccine strain EcSf2a-2

In previous trials, live invasive Escherichia coli-Shigella flexneri 2a hybrid vaccine candidate EcSf2a-2, administered to adult volunteers as 3 doses of ca. 2 x 10(9) colony forming units (c.f.u.) spaced over one week, induced fever and/or diarrhea in 11% of subjects and provided only limited protection (36% efficacy) against illness following challenge with virulent S. flexneri 2a. We sought to improve the clinical safety of this vaccine by administering a lower inoculum, and to enhance protective immunity by administering additional booster doses at 2 weeks. Twenty-one healthy adults were immunized with ca. 7 x 10(8) c.f.u. of EcSf2a-2 on days 0, 3, 14, and 17. The vaccine consistently colonized the intestine without causing serious adverse reactions; mild diarrhea developed in one subject and low grade fever in another. Vaccination elicited an antibody secreting cell (ASC) response to lipopolysaccharide (LPS) in all subjects, which was highest on day 7 and notably diminished thereafter on days 10, 16, 21, and 24, suggesting that active mucosal immunity developed rapidly. The magnitude of the response was modest (geometric mean peak = 16 IgA ASC/10(6) peripheral blood mononuclear cells) and an IgG serological response to LPS was detected in only 19% of subjects. Following experimental challenge with virulent S. flexneri 2a administered with bicarbonate buffer, shigellosis (diarrhea, dysentery, or fever) developed in 10 of 16 vaccine recipients (63%) and in 12 of 14 unvaccinated controls (86%), resulting in a vaccine efficacy of 27% (95% confidence limits -197, 82, p = 0.15, 1-tailed).(ABSTRACT TRUNCATED AT 250 WORDS)

EXPERIMENTAL MODELS IN THE INVESTIGATION OF THE VIRULENCE OF DYSENTERY BACILLI AND ESCHERICHIA COLI

The knowledge gained from experiments in laboratory models has been very useful in helping us to reach our present understanding of the pathogenesis of some diarrheal diseases of human beings. This is especially true of bacillary dysentery. Dysentery may be defined as a syndrome in which blood, inflammatory cells, and mucus are present in the watery stool. A cardinal feature of classical shigellosis is an ulcerative lesion of the colonic mucosa, for it is through this defect in the epithelial barrier that the red blood cells reach the intestinal lumen. Thus, in order to gain some insight into at least one aspect of the pathogenesis of classical bacillary dysentery, one must understand how the ulcerative lesion evolves. The previous concept of the evolution of the colonic ulcer envisioned consecutive waves of absorption and excretion of heat-stable toxin across the intestinal wall, resulting in hypoxia and death of the epithe1ium.l For a variety of reasons2 we rejected this hypothesis and sought an alternate explanation. We started by using a virulent Shigella flexneri 2a strain and an avirulent mutant derived from it. As far as could be ascertained, the two strains were identical, with the exception that the parent strain caused disease when fed to starved, opiated guinea pigs or to rhesus monkeys, while the mutant failed to do so. When animals infected with either of the strains were studied using the fluorescent antibody technique, a distinctly different pattern of distribution of the organisms in the intestine was observed. The avirulent mutant was seen only in the lumen of the bowel. On the other hand the virulent bacteria were present in the epithelial cells of the intestine, and also both free and within phagocytic cells in the lamina propria. Rarely were the organisms viewed in the submucosa or in the mesenteric lymph nodes.2 From the results of these experiments we concluded that an essential step in the pathogenesis of bacillary dysentery is the penetration of the intestinal epithelial cell by the pathogen. If the organism is unable to enter the epithelium for one reason or another, few, if any, signs of disease are observed. Similar observations have been made independently by Voino-Yasenetsky and Khavkin and confirmed in extensive studies by Ogawa and colleague^.^ Other procedures are available to test for the ability of dysentery bacilli to

Failure of Parenteral Vaccines to Protect Monkeys Against Experimental Shigellosis

Summary Monkeys injected subcutane-ously with either a combination of heat-killed or acetone-killed S. flexneri 2a vaccines or with vaccines made from living, virulent S. flexneri are not rendered resistant to experimental oral challenge with the homologous organism.

Experimental Shigella Infections. III. Sensitivity of Normal, Starved and Carbon Tetrachloride Treated Guinea Pigs to Endotoxin

Summary Pretreatment of Hartley strain guinea pigs either by subcutaneous injection with carbon tetrachloride or by 4 day period of starvation renders them more susceptible to intravenously administered endotoxin. The LD50 for normal animals is in excess of 1300 μg; for starved animals 52.8 μg and for carbon tetrachloride treated animals 4.2 μg. Distribution of Cr51 label of Cr51 tagged endotoxin in organs of normal animals which survive intravenous dose of 52 μg of endotoxin is the same as that in carbon tetrachloride treated animals which succumb following this challenge.

VIRULENCE PHENOTYPE AND GENETIC CHARACTERISTICS OF THE T32-ISTRATI SHIGELLA FLEXNERI 2A VACCINE STRAIN

The T32-ISTRATI strain, which has been used as an oral attenuated Shigella flexneri 2a vaccine, has lost the invasive phenotype due to a spontaneous deletion in the shigella virulence plasmid. This deletion has eliminated three plasmid loci (ipaBCDA, invA and virG) that are necessary for production of a positive Sereny test by Shigella species. Virulence in the Sereny test was reconstituted in the T32-ISTRATI strain by the conjugal transfer of an intact 140 M Da virulence plasmid from S. flexneri 5. The T32-ISTRATI vaccine is safe when given orally in multiple doses of 50-100 x 10(9) organisms, and both homologous and heterologous protection has been reported in large Romanian and Chinese field trials. Although the protective antigen(s) in this vaccine have not been identified, the potential use of non-invasive plasmid deletion mutants as living shigella vaccines is illustrated by the T32-ISTRATI vaccine.

Experimental Diarrhea: I. Intestinal water and electrolyte transport in rat salmonella enterocolitis

A model for studying the pathophysiology of diarrhea has been developed by inducing salmonella enterocolitis in rats. In vivo intestinal net water and electrolyte transport rates were determined in infected rats with and without diarrhea and were compared with control animals. The only significant alteration in net water and electrolyte transport between control animals and infected animals without diarrhea was a diminution of ileal absorption and a reversal of ileal HCO, transport from secretion to absorption. In the infected animals with diarrhea, jejunal and large intestinal transport was not significantly different from that in infected animals without diarrhea. However, in all animals with diarrhea there was ileal secretion of H2O, Na, K, and Cl. Thus ileal secretion appeared to be a major physiological determinant of diarrhea in this disease model. Among the possible mechanisms to explain the net blood to lumen transport, the most likely are either (1) a passive transudation of fluid and electrolyte secondary to increased hydrostatic pressure in the lamina propria or (2) active electrolyte secretion by the mucosa.

Genotypic and phenotypic characterization of an aroD deletion-attenuated Escherichia coli K12-Shigella flexneri hybrid vaccine expressing S. flexneri 2a somatic antigen

The construction and characterization of EcSf2a-2, an aroD-deleted Escherichia coli-Shigella hybrid vaccine carrying chromosomal and plasmid genes from Shigella flexneri and expressing S. flexneri 2a somatic antigen in association with E. coli K12 core are described. Expression of hybrid lipopolysaccharide and deletion of aroD resulted in the attenuation of phenotypic characteristics associated with pathogenicity. The addition of an aroD deletion results in a requirement for an aromatic precursor of para-aminobenzoic acid (PABA), an essential bacterial metabolite not present in mammalian tissues. The biosynthesis of hybrid somatic antigen prevents expression of a Sereny-positive reaction by invasive bacteria capable of expressing a plaque-positive phenotype. A functional kcpA gene is required for expression of the plaque-positive phenotype. The presence of an aroD deletion does not interfere with expression of an invasive phenotype; however, in bacteria containing a functional kcpA gene, replication and spread by invading bacteria are limited, preventing development of the plaque-positive phenotype.

Ion Transport Across Isolated Ileal Mucosa Invaded by Salmonella

Abstract The effects of two different invasive strains (TML and SL 1027) of Salmonella tryphimurium on Na and Cl transport in the absence of an electrochemical gradient were measured across rabbit ileal mucosa in vitro. Only strain TML elicits ileal fluid secretion in vivo. Net Na transport is negligible and net Cl secretion occurs across mucosae invaded by TML. In contrast, mucosae invaded by SL 1027 show absorption of both Na and Cl, as occurs in normal ileum. The luminal addition of glucose stimulates Na absorption without affecting Cl transport across ileum invaded by either strain. Theophylline stimulates Na transport across TML-invaded mucosa, causing net secretion, and enhances Cl secretion. Across SL 1027-invaded tissues, theophylline reverses spontaneous ion absorption, causing secretion of both Na and Cl. The data suggest that stimulation of active Cl secretion combined with inhibition of spontaneous Na absorption may account for the diarrhea of salmonella enteritis. However, invasion of the mucosa alone by S. typhimurium is not necessarily associated with major alterations in ion transport, suggesting that a factor, in addition to invasion, is necessary for fluid secretion. The effects of theophylline are compatible with strain TML exerting submaximal stimulation of cyclic adenosine monophosphate activity or involving another metabolic pathway which also inhibits spontaneous Na absorption and stimulates Cl secretion.