An ultrastructural study of HeLa cell invasion with Salmonella typhi GIFU 10007

Scanning electron micrograph of HeLa S3 monolayered cells, inoculated with viable bacteria of a Salmonella typhi strain GIFU 10007, revealed that the extended microvilli tangled the bacteria within 10 min after inoculation. The micrographs of HeLa cells, at 1 hr after inoculation, indicate the following: shortening of bacterium-attached microvilli, subsiding of tangled bacteria into microvilli bush, and then attachment of bacterial soma to cell surface making the cell membrane depressed. The transmission electron micrographs, at 1 hr after inoculation, demonstrated the findings of interaction between HeLa cell and S. typhi 10007, similar to those observed on scanning electron micrographs. Hair-like fine structures from the soma of challenge organisms were also observed. They were in contact with HeLa cell microvilli and cell membrane. The bacteria were first partially and then totally surrounded by the HeLa cell plasma membrane. One, two, or several bacteria with intact outer membrane were enclosed in intracytoplasmic membrane-bound vacuoles. Fragmented vacuolar membrane was still visible around the intracellularly accumulated bacteria at 24 hr after inoculation. The viable cells of S. typhi 10007 are regarded as internalizing into HeLa cells by a process of endocytosis and to multiply within the membrane-bound vacuoles.     

Apoptosis of human keratinocytes after bacterial invasion

In this study, we examined the invasive capacity of Staphylococcus aureus and Salmonella typhi in human keratinocytes and monitored the number of viable intracellular bacteria at different post-infection times. The strains tested entered keratinocytes; both S. typhi and S. aureus were internalized within 30 min to 2 h after infection. No intracellular multiplication was observed, but S. typhi and S. aureus remained viable 72 h after infection. We also demonstrated that keratinocyte death following S. typhi and S. aureus invasion occurs by apoptosis as shown by DNA fragmentation. After 24 h of infection with S. typhi, the number of cells undergoing apoptosis were higher compared to infection with S. aureus. For prolonged infection times (48 h, 72 h) with both bacteria, there was no significant change in the number of cells undergoing apoptosis. The results demonstrated that viable intracellular S. typhi and S. aureus induced apoptosis in keratinocyte cells.     

Campylobacter jejuni Actively Invades the Amoeba Acanthamoeba polyphaga and Survives within Non Digestive Vacuoles

The Gram-negative bacterium Campylobacter jejuni is able to enter, survive and multiply within the free living amoeba Acanthamoeba polyphaga, but the molecular mechanisms behind these events are still unclear. We have studied the uptake and intracellular trafficking of viable and heat killed bacterial cells of the C. jejuni strain 81–176 in A. polyphaga. We found that viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat killed bacteria. Furthermore, the kinetics of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were dramatically influenced by bacterial viability. Viable bacteria were internalized at a high rate already after 1 h of co-incubation and were observed in small vacuoles tightly surrounding the bacteria. In contrast, internalization of heat killed C. jejuni was low at early time points and did not peak until 96 h. These cells were gathered in large spacious vacuoles that were part of the degradative pathway as determined by the uptake of fluorescently labeled dextran. The amount of heat killed bacteria internalized by A. polyphaga did never reach the maximal amount of internalized viable bacteria. These results suggest that the uptake and intracellular survival of C. jejuni in A. polyphaga is bacterially induced.     

Non-opsonic phagocytosis of homologous non-toxigenic and toxigenic Corynebacterium diphtheriae strains by human U-937 macrophages

As interactions between bacteria and macrophages dictate the outcome of most infectious diseases, analyses of molecular mechanisms of non-opsonic phagocytosis should lead to new approaches for the prevention of diphtheria and systemic Corynebacterium diphtheriae infections. The present study aimed to evaluate human macrophage–bacteria interactions in the absence of opsonin antibodies and the influence of the tox gene on this process. Homologous C. diphtheriae tox + and tox – strains were evaluated for adhesion, entering and survival within U-937 human macrophages at different incubation periods. Higher numbers of viable bacteria associated with and internalized by macrophages were demonstrated for the tox + strain. However, viable intracellular bacteria were detected at T-24 hr only for the tox – strain. Cytoskeletal inhibitors, cytochalasin E, genistein and colchicine, inhibited intracellular viability of both strains at different levels. Bacterial replication was evidenced at T-24 hr in supernatants of monolayers infected with the tox – strain. Host cell death and nuclear alterations were evidenced by the Trypan blue exclusion assay and DAPI fluorescence microscopy. ELISA of histone-associated DNA fragments allowed detection of apoptosis and necrosis induced by tox + and tox – strains at T-1 hr and T-3 hr. In conclusion, human macrophages in the absence of opsonins may not be promptly effective at killing diphtheria bacilli. The presence of the tox gene influences the susceptibility of C. diphtheriae to human macrophages and the outcome of non-opsonic phagocytosis. C. diphtheriae strains exhibit strategies to survive within macrophages and to exert apoptosis and necrosis in human phagocytic cells, independent of the tox gene.     

Interactions Between Macrophages of Guinea Pigs and Salmonellae I. Fate of Salmonella typhimurium Within Macrophages of Normal Guinea Pigs

A suspended cell culture procedure was described for the cultivation of guinea pig macrophages infected with Salmonella typhimurium. The fate of the intracellular bacteria was assessed by quantitative recovery of viable bacteria with 0.5% solution of sodium desoxycholate. Two strains of S. typhimurium with different degrees of virulence for mice were compared. There was an initial destruction of intracellular bacteria of both strains; however, the extent of this destruction differed. Approximately 1% of the avirulent bacteria initially phagocytized survived at the end of 4 hr, whereas approximately 8% of the virulent bacteria survived at the end of 3 hr. After this initial killing, the intracellular bacteria began to multiply at a logarithmic rate between 3 and 21 hr after phagocytosis, and then a stationary phase was attained. The rate of this multiplication was comparable for both strains.     

Vi-Suppressed wild strain Salmonella typhi cultured in high osmolarity is hyperinvasive toward epithelial cells and destructive of Peyer's patches

Salmonella typhi GIFU10007-3 which lost a viaB locus on its chromosome became highly invasive in our previous study. To investigate the phenomenon, we controlled Vi expression in wild strain S. typhi GIFU10007, and studied the invasive phenotype both in vitro and in vivo. When the wild strain of S. typhi was cultured in 300 mM NaCl containing Luria-Bertani broth (LBH), the expression of Vi antigen was suppressed, but secretion of invasion proteins (SipC, SipB and SipA) was increased. In this condition, wild strain S. typhi became highly invasive toward both epithelial cells and M cells of rat Peyer's patches. When GIFU10007 was cultured under conditions of high osmolarity, the bacteria disrupted Peyer's patches and induced massive bleeding in these structures only 20 min after inoculation into the ileal loop. In contrast, Vi-encapsulated wild strain GIFU10007 cultured under low osmolarity was not destructive, even after 60 min. To understand the role of the type III secretion system under conditions of high osmolarity, we knocked out the invA and sipC genes of both GIFU10007 and GIFU10007-3. Neither invA nor sipC mutants could invade epithelial cells or M cells in a high osmolarity environment. Our data show that the highly invasive phenotype was only expressed when the wild strain S. typhi was cultured under high osmolarity, which induced a state of Vi suppression, and in the presence of the type III secretion system.     

HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O-antigenic polysaccharide

The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.     

Cytotoxicity and Calcium-Dependent Antigen of Yersinia

The relationship between invasiveness and calcium dependency was examined in various strains of Yersinia enterocolitica and Y. pseudotuberculosis by using established cell lines. Infection with calcium-dependent bacteria resulted in the formation of microvilli and the adherence of bacteria on the cell surface, and the adherent bacteria were ingested 1.5 hr after infection. Morphological changes in the cells became visible 2 to 3 hr after infection, and intracellular multiplication of the ingested bacteria was noted. When the cells were incubated with bacteria at 37 C for 1.5 hr and then at 25 C, however, the morphological changes in the infected cells were not observed. No isogenic strains that had lost calcium dependency for growth at 37 C were able to elicit the morphological changes in the cells, though they possessed the ability to adhere to and penetrate the cells. The antigen(s) supposedly related to cytotoxicity of the calcium-dependent Yersinia was sought by using antibodies prepared against calcium-dependent bacteria and then absorbed with calcium-independent bacteria and with calcium-independent bacterial cytosol. Double diffusion tests between the antisera and bacterial cytosol extracts revealed the presence of an antigen which was a cytoplasmic substance common to all calcium-dependent but not calcium-independent strains of Y. enterocolitica and Y. pseudotuberculosis.     

Differences in initial rate of intracellular killing of Salmonella typhimurium by granulocytes of Salmonella-susceptible C57BL/10 mice and Salmonella-resistant CBA mice

The contribution of granulocytes to differences in the innate susceptibility of mouse strains to infection by Salmonella typhimurium was assessed on the basis of the size and composition of the inflammatory exudate after i.p. injection of bacteria and the intracellular killing of the bacteria by exudate peritoneal cells and blood granulocytes of resistant CBA and susceptible C57BL/10 mice. The increase in the numbers of both peritoneal granulocytes and macrophages 24 hr after i.p. injection of various numbers of live S. typhimurium was two to four times higher in C57BL/10 mice (p less than 0.05) than in CBA mice. However, despite the larger number of phagocytes in the inflammatory exudate, the numbers of viable S. typhimurium in the peritoneal cavity 24 hr after injection was higher (p less than 0.01) in C57BL/10 mice than in CBA mice. Because the proportion of noningested bacteria was similar in the two mouse strains (less than 30%), these findings indicate a difference in the rate of intracellular killing of the bacteria by exudate peritoneal cells (greater than 75% granulocytes) of the two mouse strains. Subsequent determination of the initial rate of intracellular killing (Kk) of S. typhimurium revealed that after phagocytosis of the bacteria in vivo, exudate peritoneal granulocytes (harvested 24 hr after i.p. injection of 10(3) live S. typhimurium) of CBA mice killed S. typhimurium twice as efficiently (Kk = 0.014 min-1; p less than 0.01) as exudate granulocytes of C57BL/10 mice (Kk = 0.008 min-1) did. Similarly, the initial rate of intracellular killing of the ingested S. typhimurium by blood granulocytes of CBA mice (Kk = 0.017 min-1) was two times higher (p less than 0.01) than that of C57BL/10 mice (Kk = 0.007 min-1). These findings may be specific for S. typhimurium, because L. monocytogenes were killed with equal efficiency by exudate granulocytes and blood granulocytes of these mouse strains (p greater than 0.20). The results of the present study are relevant with respect to the innate resistance of mice to S. typhimurium, particularly during the initial phase of infection when the inflammatory exudate contains predominantly granulocytes.     

迟缓爱德华氏菌对Hep-2细胞的侵袭特性

用细胞裂解计数法及超薄切片电镜观察法分析了迟缓爱德华氏菌侵袭ＨＥｐ－２细胞的基本特性。在１５株来源各异的迟缓爱德华氏菌中,有６株细菌具有对ＨＥｐ－２细胞的侵袭能力。细菌侵入细胞后,主要位于空泡内。侵入细胞内的迟缓爱德华氏菌不仅可在细胞内增殖,而且可从细胞内释放出来。用细胞松弛素破坏微丝后可抑制其侵袭作用,而且表现出剂量依赖关系,而在秋水仙素破坏微管后不影响其侵袭力。这表明在迟缓爱德华氏菌对ＨＥｐ－     

Cytopathogenic effect of Salmonella typhi GIFU 10007 on M cells of murine ileal Peyer's patches in ligated ileal loops: an ultrastructural study

An electron microscopic study revealed that, within 30 min after inoculation into the ligated ileal loop of anesthetized mice, cells of Salmonella typhi GIFU 10007 adhered to the M cell surface of Peyer's patch lymphoid follicle epithelium, and induced almost complete destruction of M cells. The M cell cytoplasms were pinched off and extruded from the epithelial lining into the luminal space together with the lymphoid cells primarily enfolded into the corresponding M cells. When two or more M cells were destroyed, a large defect in the epithelial lining was apparent, and a number of bacteria appeared near the basal lamina of the epithelial lining. These findings suggest, as far as anesthetized murine ileal loops and strain 10007 are concerned, that ileal M cells are the target cell at an early stage of S. typhi infection and the infection may further progress to deeper tissues and to the general circulation.     

Inability to sustain intraphagolysosomal killing of Staphylococcus aureus predisposes to bacterial persistence in macrophages

Macrophages are critical effectors of the early innate response to bacteria in tissues. Phagocytosis and killing of bacteria are interrelated functions essential for bacterial clearance but the rate‐limiting step when macrophages are challenged with large numbers of the major medical pathogen Staphylococcus aureus is unknown. We show that macrophages have a finite capacity for intracellular killing and fail to match sustained phagocytosis with sustained microbial killing when exposed to large inocula of S. aureus (Newman, SH1000 and USA300 strains). S. aureus ingestion by macrophages is associated with a rapid decline in bacterial viability immediately after phagocytosis. However, not all bacteria are killed in the phagolysosome, and we demonstrate reduced acidification of the phagolysosome, associated with failure of phagolysosomal maturation and reduced activation of cathepsin D. This results in accumulation of viable intracellular bacteria in macrophages. We show macrophages fail to engage apoptosis‐associated bacterial killing. Ultittop mately macrophages with viable bacteria undergo cell lysis, and viable bacteria are released and can be internalized by other macrophages. We show that cycles of lysis and reuptake maintain a pool of viable intracellular bacteria over time when killing is overwhelmed and demonstrate intracellular persistence in alveolar macrophages in the lungs in a murine model.     

Campylobacter fetus adheres to and enters INT 407 cells

Campylobacter fetus is a Gram-negative bacterial pathogen of humans and ungulates and is normally transmitted via ingestion of contaminated food or water with infection resulting in mild to severe enteritis. However, despite clinical evidence that C. fetus infection often involves transient bacteremic states from which systemic infection may develop and the frequent isolation of C. fetus from extra-intestinal sites, this organism displays very poor invasiveness in in vitro models of infection. In this study, immunofluorescence microscopy and gentamicin protection assays were used to investigate the ability of six clinical isolates and one reference strain of C. fetus to adhere to and invade the human intestinal epithelial cell line, INT 407. During an initial 4-h infection period, all C. fetus strains were detected intracellularly using both techniques, though adherence and internalization levels were very low when determined from gentamicin protection assays. Microscopy results indicated that during a 4-h infection period, four of the five clinical strains tested were adherent to 41.3-87.3% of INT 407 cells observed and that 25.2-34.6% of INT 407 cells contained intracellular C. fetus. The C. fetus reference strain displayed the lowest levels of adherence and internalization. A modified infection assay revealed that C. fetus adherence did not necessarily culminate in internalization. Despite the large percentage of INT 407 cells with adherent bacteria, the percentage of INT 407 cells with intracellular bacteria remained unchanged when incubation was extended from 4 h to 20 h. However, microscopy of INT 407 cells 24 h postinfection (p.i.) revealed that infected host cells contained clusters of densely packed C. fetus cells. Gentamicin protection assays revealed that intracellular C. fetus cells were not only viable 24 h p.i. but also that C. fetus had increased in number approximately three- to fourfold between 4 and 24 h p.i., indicative of intracellular replication. Investigation of the role of the host cell cytoskeleton revealed that pretreatment of host cells with cytochalasin D, colchicine, vinblastine, taxol, or dimethyl sulfoxide (DMSO) did not impact upon C. fetus adherence or internalization of INT 407 cells. Microscopy indicated neither rearrangement nor colocalization of either microtubules or microfilaments in INT 407 cells in response to C. fetus adherence or internalization. Together, these data indicate that clinical isolates of C. fetus are capable of adhering, entering, and surviving within the nonphagocytic epithelial cell line, INT 407.     

Molecular dissection of internalization of Porphyromonas gingivalis by cells using fluorescent beads coated with bacterial membrane vesicle

Porphyromonas gingivalis is one of the causative agents of adult periodontitis, and has been reported to be internalized by nonphagocytic epithelial cells. However, the mechanism for the internalization remains unclear. In the present study, we addressed this issue using fluorescent beads coated with bacterial membrane vesicles (MVs) that retain surface components of P. gingivalis. We established an assay system in which we could easily quantify the bead internalization to cells. MVs-coated beads were internalized by HeLa cells in kinetics similar to that of living bacteria. The internalization depended on dynamin but not clathrin. The beads were internalized through the actin-mediated pathway that is controlled by phosphatidylinositol (PI) 3-kinase. The dynamics of microtubule assembly and disassembly was also required. Further, the treatment of cells with cholesterol-binding reagents significantly inhibited bead internalization, and the internalized beads were apparently colocalized with ganglioside GM1 and caveolin-1, which suggest the involvement of the lipid raft in the process. These results suggest that P. gingivalis accomplishes its internalization utilizing membrane lipid raft and cytoskeletal functions of the target cells.     

Penetration of the coral-bleaching bacterium Vibrio shiloi into Oculina patagonica

Inoculation of the coral-bleaching bacterium Vibrio shiloi into seawater containing its host Oculina patagonica led to adhesion of the bacteria to the coral surface via a beta-D-galactose receptor, followed by penetration of the bacteria into the coral tissue. The internalized V. shiloi cells were observed inside the exodermal layer of the coral by electron microscopy and fluorescence microscopy using specific anti-V. shiloi antibodies to stain the intracellular bacteria. At 29 degrees C, 80% of the bacteria bound to the coral within 8 h. Penetration, measured by the viable count (gentamicin invasion assay) inside the coral tissue, was 5.6, 20.9, and 21.7% of the initial inoculum at 8, 12, and 24 h, respectively. The viable count in the coral tissue decreased to 5.3% at 48 h, and none could be detected at 72 h. Determination of V. shiloi total counts (using the anti-V. shiloi antibodies) in the coral tissue showed results similar to viable counts for the first 12 h of infection. After 12 h, however, the total count more than doubled from 12 to 24 h and continued to rise, reaching a value 6 times that of the initial inoculum at 72 h. Thus, the intracellular V. shiloi organisms were transformed into a form that could multiply inside the coral tissue but did not form colonies on agar medium. Internalization of the bacteria was accompanied by the production of high concentrations of V. shiloi toxin P activity in the coral tissue. Internalization and multiplication of V. shiloi are discussed in terms of the mechanism of bacterial bleaching of corals.     

Effect of Cookery and Holding on Hams and Turkey Rolls Contaminated with Clostridium perfringens

Canned hams, turkey rolls, and ground-beef casseroles were inoculated with a mixture of vegetative cells and spores of selected strains of Clostridium perfringens, in approximately known numbers. After cooking and holding at different temperatures for various times, samples of the food were plated directly on sulfadiazine-polymixin-sulfite-agar. In all cases, small but measurable percentages of the organisms survived cookery. The number of cells viable after cookery of the ham or turkey was influenced by the position of the slice of meat in the roast as well as by the final temperature to which the product was heated. Plate counts for turkey or beef casserole held at temperatures in the range of 5 to 10 C for 48 hr indicated stabilization of the population or a tendency to decrease. At 24 C, the multiplication of cells was apparent in 4 hr and rapid in 6 hr. When the food was maintained at 68 C, populations remained viable for 6 hr and the counts did not change markedly. In turkey maintained at 37 C, the number of cells increased sharply within 4 hr.     

Invasion of HeLa cells by beta-hemolytic group G streptococci

The invasion of HeLa cells by beta-hemolytic Lancefield group G streptococci was studied by measuring the number of bacterial cells that survive exposure to gentamicin. Approximately 50% of bacteria introduced to the HeLa cell monolayer survived gentamicin treatment, suggesting that they were intracellular. Electron microscopy of these preparations showed intracellular bacteria in the cytoplasm, not surrounded by host cell membranes. Trypsinized bacteria incubated with HeLa cells were all killed by gentamicin. It appears that the beta-hemolytic group G streptococci have mechanisms for entry into human epithelial cells which may have importance in the virulence of the organisms.     

Role of internalization in the pathogenicity of Shiga toxin-producing Escherichia coli infection in a gnotobiotic murine model

We investigated the role of bacterial internalization in the killing caused by Shiga toxin-producing Escherichia coli (STEC) infection using a gnotobiotic murine model. A high number of internalized STEC was found in the colonic epithelial cells of STEC-infected mice by both an ex vivo assay and transmission electron microscopy. Most of these mice were killed within 10 days after infection. However, the implantation of lactic acid bacteria in such mice before infection markedly decreased the number of internalized STECs and also completely protected these hosts from killing by a STEC infection. The inhibition of such internalization by immunoglobulin also prevented the hosts from being killed. The Shiga toxin levels in these hosts indicated an inhibition of the penetration of Shiga toxins produced in the colon to the underlying tissue. These results suggested that the internalization plays an important role in the pathogenicity caused by STEC infection in a gnotobiotic murine model.     

Streptolysin O and its Co-Toxin NAD-glycohydrolase Protect Group A Streptococcus from Xenophagic Killing

Group A Streptococcus (Streptococcus pyogenes or GAS) causes pharyngitis, severe invasive infections, and the post-infectious syndromes of glomerulonephritis and rheumatic fever. GAS can be internalized and killed by epithelial cells in vitro, a process that may contribute to local innate defense against pharyngeal infection. Secretion of the pore-forming toxin streptolysin O (SLO) by GAS has been reported to stimulate targeted autophagy (xenophagy) upon internalization of the bacteria by epithelial cells. Whereas this process was associated with killing of GAS in HeLa cells, studies in human keratinocytes found SLO production enhanced intracellular survival. To reconcile these conflicting observations, we now report in-depth investigation of xenophagy in response to GAS infection of human oropharyngeal keratinocytes, the predominant cell type of the pharyngeal epithelium. We found that SLO expression was associated with prolonged intracellular survival; unexpectedly, expression of the co-toxin NADase was required for this effect. Enhanced intracellular survival was lost upon deletion of NADase or inactivation of its enzymatic activity. Shortly after internalization of GAS by keratinocytes, SLO-mediated damage to the bacteria-containing vacuole resulted in exposure to the cytosol, ubiquitination of GAS and/or associated vacuolar membrane remnants, and engulfment of GAS in LC3-positive vacuoles. We also found that production of streptolysin S could mediate targeting of GAS to autophagosomes in the absence of SLO, a process accompanied by galectin 8 binding to damaged GAS-containing endosomes. Maturation of GAS-containing autophagosome-like vacuoles to degradative autolysosomes was prevented by SLO pore-formation and by SLO-mediated translocation of enzymatically active NADase into the keratinocyte cytosol. We conclude that SLO stimulates xenophagy in pharyngeal keratinocytes, but the coordinated action of SLO and NADase prevent maturation of GAS-containing autophagosomes, thereby prolonging GAS intracellular survival. This novel activity of NADase to block autophagic killing of GAS in pharyngeal cells may contribute to pharyngitis treatment failure, relapse, and chronic carriage.     

Salmonella typhi vaccine strain in vitro; low infectivity in human cell line U937

Salmonella typhi strain Ty21a has been used for live oral vaccine. The infectivity of Ty21a, in comparison with S. typhi Ty2, was evaluated using the human monocyte-macrophage cell line U937. Assays were performed by quantitative microscopy and viable count technique. Ty2 infected approximately 100% of the cells, multiplied extensively within these cells and caused cell death. The same dose of Ty21a infected only about 15% of the cells, resulting in a low number of intracellular bacilli and cell survival. The use of gentamicin in the test confirmed intracellular multiplication of Ty2 but not Ty21a. The system described may be suitable as a test system for characterization of the degree of virulence of Ty21a and other live, oral typhoid vaccines.