Search

A mutant (Ty21a) of Salmonella typhi, which lacks the enzyme uridine 5'-diphosphate-glucose-4-epimerase, was evaluated in volunteers for use as a live attenuated oral typhoid vaccine. Five to eight doses of vaccine (containing 3-10(10) viable organisms per dose) were given to 155 men without significant side effects. The rate of excretion of the vaccine strain in stools was low, and the majority of isolations occurred on day 1 after vaccination. Revertants able to fement galactose were not found in any of 958 stool isolates tested. The mutant, strain Ty21a, grown in brain-heart infusion broth (BHIB) with 0.1% galactose, produces more O side chain than the same vaccine strain cultivated without galactose. Volunteers vaccinated with strain Ty21a grown in galactose and then challenged with 10(5) virulen S. typhi were significantly protected from disease and also had decreased stool carriage of S. typhi as compared with controls. Strain Ty21a grown without galactose did not provide vaccinees significant protection nor decrease fecal excretion of S. typhi as compared with controls. Strain Ty21a, when grown in BHIB with 0.1% galactose, results in a safe, stable and protective oral vaccine that warrants further study in field trials.

A mathematical model was defined to estimate the degree of in vivo activity against Plasmodium falciparum sporozoites expressed by volunteers vaccinated with a synthetic peptide comprising the immunodominant epitope of the circumsporozoite protein. Relative to the course of infection in non-immunized controls, infections in vaccinated volunteers corresponded to the neutralization or delay of development of greater than 99% of challenge sporozoites.

Single doses (50, 100, or 200 chick cell-agglutinating [CCA] units) of split-product or whole-virus (50 or 100 CCA units), monovalent inactivated A/New Jersey/8/76 (Hsw1n1) virus vaccine or placebo were given to healthy children aged three to five years in Maryland. Split-product vaccine was nonreactogenic but also virtually nonimmunogenic. Lower doses of whole-virus vaccine (50 CCA units of Merck, Sharp and Dohme [West Point, Pa.] and 50 or 100 CCA units of Merrell-National Laboratories [Cincinnati, Ohio] vaccine) were mildly reactogenic (approximately 25% of the children had low-grade fevers of 100 F-101 F). These dosage levels of whole-virus vaccine stimulated titers of greater than or equal to 1:20 in 68% and titers of greater than or equal to 1:40 in 37% of the children. Two inoculations of whole-virus vaccine (primary immunization followed by a booster one month later) were tolerated and induced titers of greater than or equal to 1:80 in 100% of the children.

To characterize IgM and IgG antibody responses in Rocky Mountain spotted fever (RMSF), a microtiter enzyme-linked immunosorbent assay (ELISA) using density gradient-purified Rickettsia rickettsii as antigen was developed. Sera of vaccinated individuals and patients with RMSF were tested by ELISA and by indirect fluorescent antibody (IFA) tests. Diagnostic agreement between ELISA and the IFA test was 76% and 52% for IgG and IgM antibody, respectively. Diagnostic agreement between the ELISA for IgG antibody and the IFA test for total immunoglobulins was 84%. The ELISAs for IgM and IgG antibody were as specific (100%) and as sensitive (100%) as the IFA test (83%-100%) in detecting antibody increases in paired sera from persons with RMSF and were superior to the IFA test in detecting seroconversions in vaccinees. The ELISA also detected antibodies in a single convalescent-phase serum with sensitivity and reliability. The ELISA for IgG antibody is appropriate for seroepidemiology and serodiagnosis since it permits measurement of antibody at a single dilution of serum up to a year after illness.

Vibrio cholerae O1 A-B- vaccine strain JBK 70 and A-B+ CVD 101 prepared by recombinant DNA techniques from pathogenic EI Tor Inaba N16961 and classical Ogawa 395, respectively, were fed to 38 volunteers in single doses of 10(4) to 10(10). Although severe diarrhea did not occur in any vaccine, more than one-half developed mild diarrhea. These attenuated strains colonized well and elicited prominent vibriocidal and antitoxic (CVD 101) antibody responses. Recipients of a single dose of JBK 70 were significantly protected when challenged with 10(6) wild-type N16961. Diarrhea occurred in 7 of 8 controls but in only 1 of 10 vaccines (P less than 0.003, 89% vaccine efficacy), demonstrating the potency of immune mechanisms that do not involve cholera antitoxin. Further derivatives were prepared to explore the pathogenesis of the residual diarrhea, considering that either intestinal colonization by the vaccine itself or accessory toxins might be responsible. CVD 102, an auxotrophic mutant of CVD 101, did not cause diarrhea but colonized poorly and elicited feeble immune responses. Derivatives of JBK 70 and CVD 101 (CVD 104 and 105) deleted of genes encoding the EI Tor hemolysin still caused mild diarrhea. Genetically engineered strains can be colonizing, highly immunogenic, and protective single-dose oral vaccines, but they must be further attenuated before they can be considered for use as public health tools.

To examine whether prior immunity against a carrier protein modulates the serological response to injected peptide haptens attached to the same carrier in man, baseline tetanus antitoxin levels in volunteers who received a malaria sporozoite peptide-tetanus toxoid conjugate vaccine were compared with post-vaccination IgM and IgG antibody titres against the sporozoite antigen. In tetanus-vaccinated North American recipients of low doses of conjugate vaccine there were significant dose-dependent negative correlations between these variables, which suggests that epitopic suppression may occur in man. In contrast, Venezuelans living in non-malarious areas and mostly naive to tetanus toxoid showed a notable IgM response to the sporozoite antigen. The findings indicate that epitopic suppression and immune enhancement occur in man, and that the specific immunological responses to conjugate peptide vaccines may be difficult to predict.

Enterotoxigenic Escherichia coli fimbriae are immunogenic and play a key role in intestinal colonization. Native colonization factor antigen I, coli surface antigen 1, and coli surface antigen 3 fimbriae were purified by a common method involving shearing, differential centrifugation, gel filtration, and density gradient ultracentrifugation. The compositions and N-terminal sequences were determined. Coli surface antigen 3 possesses two N-terminal isoforms, one of which matches the published DNA sequence, except for the previously proposed signal sequence cleavage point.

To explore the relationship between neuraminidase immunity and the degree of attenuatíon of live influenza A virus vaccines, a comparative evaluation of three Victoria/75-ts-1[E] (Vic/75-ts-1[E]) recombinant viruses in serum hemagglutination-inhibiting-negative (titer,

Four volunteers were rechallenged with Vibrio cholerae (10(6) classical Ogawa 395 organisms) 33-36 months after their initial induced cholera infection; none of the four veterans and four of five control volunteers developed diarrhea (P = 0.04). All control subjects, but only one veteran, had positive coprocultures. Three of the four veterans had significant levels of serum IgG antitoxin before challenge, but none had measurable intestinal levels of secretory IgA antitoxin. Significant rises in levels of serum vibriocidal and antitoxic antibody occurred in all control subjects and in two veterans, who also manifested rises in levels of intestinal secretory IgA antitoxin. The impressive duration of infection-derived immunity suggests that the most promising approach to development of cholera vaccines may be to mimic natural immunity with orally administered, attenuated strains of V. cholerae.

Purified cholera toxoid is antigenic when given enterally and orally. Purified toxoid fails to provide protection against experimental challenge. Clinical cholera confers formidable protection against homologous or heterologous rechallenge. Failure to culture vibrios from intestinal fluid or stool of re-challenge volunteers suggests that the predominant immune mechanism is antibacterial rather than antitoxic.

Two strains of Campylobacter jejuni ingested by 111 adult volunteers, in doses ranging from 8 x 10(2) to 2 x 10(9) organisms, caused diarrheal illnesses. Rates of infection increased with dose, but development of illness did not show a clear dose relation. Resulting illnesses with strain A3249 ranged from a few loose stools to dysentery, with an average of five diarrheal stools and a volume of 509 mL. Infection with strain 81-176 was more likely to cause illness, and these illnesses were more severe, with an average of 15 stools and 1484 mL of total stool volume. All patients had fecal leukocytes. The dysenteric nature of the illness indicates that the pathogenesis of C. jejuni infection includes tissue inflammation. Ill volunteers developed a serum antibody response to the C. jejuni group antigen and were protected from subsequent illness but not infection with the same strain.

Oral rehydration of infants with diarrhoea is an effective therapy that is becoming increasingly available in developing countries. To formulate judicious recommendations for preparation and storage of such solutions, we assessed the capability of recognized bacterial enteropathogens to survive and proliferate in solutions made either with sterile distilled or river water collected in two developing countries. Shigella flexneri, an enteropathogen typically transmitted by faecal/oral contact rather than by water or food, survived very poorly. In contrast, Vibrio cholerae and enterotoxigenic Escherichia coli, pathogens classically associated with transmission by food and water, reached concentrations of 103-104 per ml by 12 h and 104-106 by 24 h after inoculation of solutions made with river water and somewhat lower concentrations in distilled water. This potential exposure to bacteria must be considered in the context of the field situation where children are already ingesting high levels of bacteria in drinking water and food and where the oral rehydration solution would probably add little to their exposure. Although it is probably wise to prepare solutions fresh each day with water as free from faecal pollution as possible, in situations where lack of fuel to boil water or scarce supply of glucose/electrolyte packets preclude compliance with these recommendations prompt administration of oral rehydration solutions to infants with diarrhoea should nevertheless proceed.

During studies of diarrhoea due to Escherichia coli in 316 adult volunteers, ABO and Rh blood group determinations were done to look for differences in the occurrence or severity of illness in association with certain blood groups. In studies using heat-labile enterotoxin-producing E. coli, volunteers with O blood group had a significantly higher attack rate for diarrhoea than persons with other blood groups. In contrast, in studies with enteropathogenic or heat-stable enterotoxin-producing E. coli, no association was found between occurrence of diarrhoea and ABO group. These studies, and previous studies finding a similarly increased susceptibility to cholera in persons with O blood group, suggest that the mechanism of increased risk involves an interaction between blood group substances and the similar enterotoxin produced by E. coli and Vibrio cholerae.

A soluble hemagglutinin/protease from Vibrio cholerae has been suggested to be a putative virulence factor and protective antigen. However, clinical cholera infection gave rise to detectable serum antibody responses to soluble hemagglutinin in only 2 of 10 Bangladeshi patients or 1 of 17 cholera-infected North American volunteers. A gut mucosal immunoglobulin A antibody response to soluble hemagglutinin was seen in 4 of 8 Bangladeshi patients, but in 0 of 10 North American volunteers. These responses were much weaker than those to cholera toxin or lipopolysaccharide.

During a 2-year prospective study of gastroenteritis in children less than 2 years of age, the role of enteric adenovirus as a cause of infantile diarrhea was examined in three clinical settings in a case-control fashion. Using a monoclonal antibody-based enzyme-linked immunosorbent assay with specificity for adenovirus serotypes 40 and 41, enteric adenovirus was identified in 10 of 246 episodes of diarrhea in outpatients (4.1%), 13 of 211 children admitted to the hospital with diarrhea (6.2%), and 5 of 81 children in whom nosocomial diarrhea developed (6.2%), making this agent the third most commonly identified etiologic agent of diarrheal disease. Asymptomatic infections were uncommon (5 of 372 control subjects, or 1.3%) and were seen most frequently in the nosocomial setting. Cases occurred in every calendar month except March and April of each year. A syndrome of watery diarrhea of longer duration compared with other patients with diarrhea (mean 5.4 vs 3.8 days, P = .01), associated with vomiting and dehydration, was present in most cases. Compared with patients with rotavirus, patients were as likely to experience fever and dehydration and more likely to vomit. Household contact with gastroenteritis, often with a child 2 to 5 years of age, was a predisposing factor. It was concluded that enteric adenovirus is an important cause of infantile diarrhea in Baltimore children. Although far less common than rotavirus, this agent was associated with diarrheal illnesses that were at least as severe as those seen with rotavirus.

The usefulness of a single Widal test to diagnose typhoid fever in endemic areas was investigated. Reciprocal Salmonella typhi O and H titers greater than or equal to 40 and greater than or equal to 80, respectively, occurred in approximately 90% of 42 Mexican patients with bacteriologically-confirmed typhoid fever at the time of presentation to hospital and, by day 4 to 5 of clinical illness, in 70% of U.S. adult volunteers who developed typhoid fever in the course of vaccine efficacy trials but in only 0.7% (O) to 3% (H) of 275 healthy individuals from a non-endemic area. Healthy Peruvians from areas endemic for typhoid fever commonly had antibody which was age-related. Peak prevalence was found in 15- to 19-yr-olds in whom 29% had O titers greater than or equal to 40 and 76% had H titers greater than or equal to 80. A single Widal test in an unvaccinated individual showing elevated O and H titers is strongly suggestive of typhoid fever if the person comes from a non-endemic area or is a child less than 10 yr of age in an endemic area. Because of the high prevalence of antibody amongst healthy invididuals over 10 yr of age in endemic, areas, a single Widal test offers virtually no diagnostic assistance in adolescents and adults.

In recent years notable advances have been made in the development of improved vaccines to prevent cholera. These new vaccines are administered orally to maximally stimulate intestinal secretory immunity. Killed vibrios, given in conjunction with purified B subunit or administered alone, in three spaced doses, caused no adverse reactions and have conferred significant protection in volunteer challenge studies and in field trials. Two attenuated mutants of V. cholerae, prepared by recombinant DNA techniques, CVD 103 and CVD 103-HgR are well-tolerated and elicit prominent immune responses and protective immunity after ingestion of a single oral dose. Other modern approaches being pursued include the development of auxotrophic strains and of modifying attenuated S. typhi strain Ty21a to express V. cholerae Inaba and Ogawa LPS antigens.

To determine the role of Escherichia coli heat-stable enterotoxin (ST) as a virulence factor in human diarrhea, a strain that elaborates only ST (E. coli 214-4) was fed to free-living volunteers in doses of 10(6), 10(8), and 10(10) organisms. Short-lived (1 day) mild illness consisting of abdominal cramps with vomiting or diarrhea occurred in three of five individuals fed 10(8). Typical travelers' diarrhea (loose stools, abdominal cramps, and low-grade fever for 2 to 3 days) was seen in four of five volunteers given 10(10); two had brief cholera-like purging of rice-water stools. Despite fever, there was no evidence of mucosal invasion. E. coli 214-4 became the predominant coliform in stools; coproculture isolates were uniformly negative for heat-labile enterotoxin (LT), whereas most produced ST. Ten of 13 individuals developed rises in antibody to somatic E. coli antigen, and none had rises in LT antitoxin. E. coli that elaborate only ST can cause diarrheal disease in adults.

We genetically modified attenuated Salmonella typhi strain Ty21a to express the form I O polysaccharide antigen of Shigella sonnei. Three doses of this bivalent, live oral vaccine strain (1-8 X 10(9) organisms/dose) were given to young adults who, along with unvaccinated controls, were challenged one month later with pathogenic S. sonnei. The vaccinees had 40% protection against diarrhea and 56% against Hematest-positive diarrhea. Two of three vaccine lots provided higher levels of protection (53% against diarrhea and 71% against Hematest-positive diarrhea), but the third lot, prepared for a large-scale field trial, demonstrated no protective efficacy. Vaccinees had serum and local intestinal immune responses to S. sonnei lipopolysaccharide, and the presence of specific serum IgA or IgG antibody before challenge with pathogenic S. sonnei was correlated with protection from illness. Some lots of this bivalent vaccine strain provide significant protection against S. sonnei disease, but the problem of lot-to-lot variability must be overcome.

To determine risk factors for cholera in infants, a retrospective matched-pair study of 42 cases and their controls was undertaken during an outbreak of El Tor cholera in Bahrain in the autumn of 1978. The highest attack-rate of cholera (125/10 000) occurred in infants in the 6--11 month age-group, which corresponds to the weaning age in this community. Significantly more cases than controls were principally bottle fed (greater than 50% milk intake by bottle) than principally breast fed during the week before onset of illness (p=0.004). Analysis of various patterns of breast and bottle feeding did not determine whether the protection afforded by breast feeding was a negative effect (due to the lack of exposure to contaminated bottle feedings for breast fed infants) or a positive effect (due to protective functions of constituents of human breast milk). Cholera infection (with or without symptoms) among mothers of either case or control infants was uncommon (case mothers 3, control mothers 5), and mean serum vibriocidal and antitoxic antibody levels were similar for the two groups of mothers. These observations suggest that maternal infection did not affect the relative risk of infants having symptomatic cholera.

Three strains of enteropathogenic Escherichia coli (EPEC), originally isolated from humans and previously shown to cause diarrhea in human volunteers by unknown mechanisms, and one rabbit EPEC strain were shown to attach intimately to and efface microvilli and cytoplasm from intestinal epithelial cells in both the pig and rabbit intestine. The attaching and effacing activities of these EPEC were demonstrable by light microscopic examination of routine histological sections and by transmission electron microscopy. It was suggested that intact colostrum-deprived newborn pigs and ligated intestinal loops in pigs and rabbits may be useful systems to detect EPEC that have attaching and effacing activities and for studying the pathogenesis of such infections. The lesions (attachment and effacement) produced by EPEC in these systems were multifocal, with considerable animal-to-animal variation in response to the same strain of EPEC. The EPEC strains also varied in the frequency and extent of lesion production. For example, three human EPEC strains usually caused extensive lesions in rabbit intestinal loops, whereas two other human EPEC strains usually did not produce lesions in this system.

Previously described GM1 ganglioside enzyme-linked immunosorbent assays (GM1-ELISA) for the detection of Escherichia coli heat-labile enterotoxin (LT) showed sensitivity equal to the Y-1 adrenal cell assay when anti-LT (a reagent not commercially available) was used. However, when antitoxin to immunologically related (commercially available) cholera toxin was substituted, a marked loss in sensitivity occurred. We modified the GM1-ELISA that employed anti-cholera toxin to make it comparable in sensitivity to the Y-1 adrenal cell assay. When five media commonly used for LT production were compared, Mundell's Casamino Acids medium was shown to be significantly superior. Lincomycin (45 micrograms/ml) added to E. coli cultures significantly increased net optical densities in the GM1-ELISA, a direct measure of the amount of LT. Treatment of broth cultures or bacterial cell pellets with polymyxin B or extension of culture time to 48 h also significantly increased net optical density by allowing enhanced release of periplasmic LT. A major innovation involved the direct culture of E. coli strains in GM1-coated wells of microtiter plates followed by ELISA. This direct culture method GM1-ELISA (DCM-GM1-ELISA) saved not only assay time, but also materials and reagents. The net optical densities that result from this assay allow the test to be read visually without a spectrophotometer. Three independent observers read plates with E. coli tested by DCM-GM1-ELISA. Thirty-four of 35 adrenal cell-positive strains (97% sensitivity) and 30 of 30 LT-negative control E. coli strains (100% specificity) were identified by all three observers reading coded plates. The DCM-GM1-ELISA provides a simple, practical and efficient assay for LT for less sophisticated laboratories.

A series of O:H serotypes of E. coli were incriminated by epidemiologic studies in the period 1945-1960 as a cause of epidemic diarrhea in infant nurseries as well as a major cause of sporadic infant diarrhea in the community. The term enteropathogenic E. coli was coined to refer to these infant diarrhea-associated serotypes. In the early 1970s, with the advent of laboratory tests to assess heat-labile and heat-stable enterotoxin production and enteroinvasiveness of E. coli, the classic serotype enteropathogenic E. coli strains were found to lack those particular properties. These observations led some to question their pathogenicity. However, since 1978, enteropathogenic E. coli have come to be appreciated anew as a separate class of diarrheagenic E. coli that cause diarrhea by distinct pathogenic mechanisms. The pathogenesis of these strains, which have been shown to cause diarrhea in volunteers, appears to involve both an enteroadhesiveness step and production of a toxin identical to Shigella toxin. A 55- to 65-Mdalton plasmid is involved in the attachment of enteropathogenic E. coli to intestinal mucosa which results in a pathognomonic histopathologic lesion visualized by electron microscopy. The lesion involves dissolution of enterocyte microvilli by the bacteria, effacement of the enterocyte outer membrane, and formation of a pedestal around the bacterium at point of contact with the outer membrane of the enterocyte. Case-control epidemiologic studies carried out since 1975 document that enteropathogenic E. coli remain an important cause of sporadic infant diarrhea in the community with up to 30 per cent of cases of acute diarrhea in young infants in Brazil and South Africa being attributed to these pathogens. Although nursery epidemics of enteropathogenic E. coli diarrhea have virtually disappeared from industrialized countries, some sporadic enteropathogenic E. coli diarrhea in infants in the community continues to occur. The relative importance of enteropathogenic E. coli as a cause of sporadic diarrhea in both industrialized and developing countries needs to be reassessed. New diagnostic techniques are awaited to simplify this task.

The widely available heat-phenol-inactivated whole cell typhoid vaccine, which provides approximately 65% protection, has limited usefulness because of the adverse reactions it evokes. In contrast, several new typhoid vaccines promise protection without reactogenicity. Attenuated oral vaccine Ty21a has been evaluated in three field trials of efficacy in Santiago, Chile, involving 530,000 schoolchildren. Three doses of Ty21a in an enteric-coated formulation given within one week provided 69% efficacy for at least four years. Fewer doses conferred less protection, while adding a fourth dose significantly enhanced protection; increasing the interval between doses did not improve protection. Large-scale vaccination with Ty21a appeared to cause a herd-immunity effect. Ty21a has reached the stage of being a practical tool for public health. With respect to other vaccines, the safety and immunogenicity of an auxotrophic (Aro-,Pur-) Salmonella typhi mutant (strain 541Ty) has recently been evaluated. Lastly, parenteral purified Vi polysaccharide of S. typhi was safe and immunogenic and provided 64%-72% protection (for at least 17-21 months) in controlled field trials in Nepal and South Africa.

A DNA probe to detect genes conferring localized adherence of enteropathogenic Escherichia coli (EPEC) to Hep-2 cells was evaluated by using E. coli isolates from the stools of Peruvian infants with and without diarrhea. The probe was both sensitive and specific and revealed that Hep-2 adherence (because of the EPEC adherence factor [EAF] was more frequent in some O serogroups of EPEC than in others. Those serogroups in which EAF is almost always found have been designated class I EPEC; serogroups in which EAF is rarely found have been designated class II. Both class I (EAF-positive) and class II EPEC are associated with diarrheal disease.

Monovalent influenza A/Victoria/3/75 whole-virus vaccines prepared by Merck Sharp and Dohme (West Point, Pa.) and Merrell-National Laboratories (Cincinnati, Ohio) and split-virus vaccines prepared by Parke, Davis and Company (Detroit, Mich.) and Wyeth Laboratories (Philadelphia, Pa.) containing 200, 400, and 800 chick cell-agglutinating units per dose were compared with a placebo in double-blind trials in which 208 adults participated. Titers of hemagglutination-inhibiting antibody of greater than or equal to 1:20 were found in greater than 80% of the volunteers 21 days after vaccination. Seroconversion, defined as a fourfold or greater increase in antibody titer, occurred more frequently among seronegative volunteers than among seropositive volunteers. The geometric mean titers obtained with the whole-virus or split-virus vaccines were not significantly different. Reaction rates had no relation to seroconversion, nor did seronegative subjects have more reactions than seropositive subjects. Local reactions from all vaccines increased with increasing dose. Significantly more overall reactions, "bothersome" reactions, and febrile reactions occurred in the recipients of whole-virus vaccine. Of nine volunteers who reported temperatures of greater than 100 F, one had received split-virus vaccine, seven had received whole-virus vaccine, and one had received the placebo. Most systemic reactions were mild, and all were self-limited.

The increased ability to leukocytes to reduce nitroblue tetrazolium (NBT) has been used to detect the presence of systemic bacterial infection. This test has been utilized to evaluate infections and leukocyte dysfunction in children, but has not been extensively applied to traumatized patients or infected volunteers. Moreover, the technic as originally described presented methodologic difficulties. In this study of 889 such patients, a modified NBT test provided excellent differentiation of 63 systemic bacterial infections (NBT score greater than or equal to 10%) from non-infectious fevers, local enteric diseases, and certain viral and plasmodial infections (NBT score less than or equal to 9%). Splenectomy was associated with a transient false-positive score and clinical typhoid fever with a false-negative response

Enterohemorrhagic Escherichia coli (EHEC) of serotype O157:H7 has two putative virulence factors: (i) a fimbrial adhesin, specified by a 60-megadalton (MDa) plasmid, and (ii) bacteriophage-specified cytotoxin(s), known as Shiga-like toxin (SLT) or verotoxin. The contribution of these factors to the pathogenesis of EHEC-induced disease in gnotobiotic piglets was examined. The bacterial strains included the following: two EHEC strains and their corresponding plasmid-cured derivatives; another EHEC isolate and its derivative which had spontaneously lost the ability to produce SLT; one E. coli K-12 transconjugatant containing a 60-MDa plasmid from an EHEC strain; two K-12 strains into which an SLT-producing phage had been transduced (one of these strains also carried a 60-MDa EHEC-derived plasmid); and the parent K-12 strain. Each strain was fed to four piglets, which were observed for diarrhea and examined for development of characteristic mucosal lesions 3 or 5 days after inoculation. All 24 piglets inoculated with the three EHEC strains and their respective derivatives (two plasmid cured and one SLT negative) showed the typical mucosal lesions of bacterial attachment: effacement of microvillous border and cell membrane dissolution culminating in destruction of surface and glandular epithelium in the cecum and colon. No such lesions were observed in 12 piglets inoculated with three strains of E. coli K-12, including the strain which carried both the 60-MDa plasmid and a phage which specified production of SLT. Moderate to severe diarrhea was observed in 16 piglets inoculated with two EHEC strains and their derivatives (one plasmid cured and one SLT negative). The third EHEC strain and its plasmid-cured derivative produced fewer typical mucosal lesions and no diarrhea. The reason for the reduced virulence of this strain was not clear. These results demonstrate that neither the 60-MDa plasmid nor the capacity to produce SLT is essential for expression of virulence by E. coli O157:H7 in gnotobiotic piglets.

The immune response of 114 volunteers with diarrhea after experimental challenge with four strains of Vibrio cholerae O1 was characterized in a microtiter enzyme-linked immunosorbent assay antibody detection system by using a partially purified outer membrane preparation (OMP) from these strains as an antigen. Analysis of paired sera from 29 persons with noncholera diarrhea (negative control population), demonstrated that a rise in net optical density greater than 0.10 was significant. A total of 50% of the 79 cholera volunteers challenged with El Tor biotype and 54% of the 35 volunteers challenged with classical biotype had significant rises in immunoglobulin G anti-OMP. Paired sera that showed significant rises when tested against homologous OMP all manifested significant rises when also tested against a serotype-heterologous OMP. Immunoblotting techniques showed that the antigens to which antibodies were reacting were mainly protein in nature, not lipopolysaccharide. Furthermore, absorption with lipopolysaccharide decreased the optical density by a mean of only 12% (0 to 30%), corroborating that antibody was mainly directed against OMP and not lipopolysaccharide. This study indicates that there is a human immunoglobulin G response to OMP during clinical cholera infection and that this response is constant among bio- and serotypes.

Isolates of the most common O serogroups of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea (designated class I) adhere to Hep-2 cells; the genes for this adhesin, termed EPEC adherence factor (EAF), are located on plasmids 50-70 MDa in size. Volunteers ingested 10(10) organisms of an O127:H6 Hep-2-adhesive class I strain (E2348/69) or its plasmid-minus, nonadhesive derivative. Diarrhea occurred in nine of 10 volunteers who ingested the parent strain (mean, 1,178 ml) but in only two of nine who took the plasmid-minus variant (mean, 433 ml; P less than .006). All volunteers ill from strain E2348/69 mounted serum IgA and IgG responses to a 94-kDa plasmid-associated outer membrane protein of E2348/69; this protein was found in other class I EPEC but not in enterotoxigenic or meningitic strains. The 50-70-MDa EAF plasmid seems necessary for full expression of pathogenicity in EPEC that exhibit Hep-2 adhesiveness. EPEC isolates of certain other, less common, O serogroups (O44, O86, and O114) are rarely Hep-2 adhesive. These EPEC, designated class II, possess distinct 50-70 MDa plasmids lacking EAF genes. Diarrhea was caused by 10(8) or 10(10) organisms of an O114:H2 class II EPEC strain (mean, 1,156 ml) in six of 11 volunteers. This result confirmed that class II EPEC are pathogenic by a mechanism not involving Hep-2 adhesiveness.

Enterotoxigenic Escherichia coli is a common cause of traveler's diarrhea. Prophylaxis against traveler's diarrhea has been associated with side effects from bismuth subsalicylate and the development of resistance to antimicrobial agents. We undertook a double-blind controlled trial in which a bovine milk immunoglobulin concentrate with high titers of antibodies against enterotoxigenic E. coli was used as prophylaxis against E. coli challenge in volunteers. Lyophilized milk immunoglobulins were prepared from the colostrum of cows immunized with several enterotoxigenic E. coli serotypes and fimbria types, E. coli heat-labile enterotoxin, and cholera toxin. As a control, an immunoglobulin concentrate with no anti-E. coli activity was prepared. Ten volunteers received buffered immunoglobulin concentrate against enterotoxigenic E. coli, and 10 received the control immunoglobulin concentrate, dissolved in water, three times a day. No side effects were observed. On the third day of immunoglobulin prophylaxis, the volunteers were given 10(9) colony-forming units of enterotoxigenic E. coli H10407 (O78:H11). This strain produces colonization factor antigen I and heat-labile and heat-stable enterotoxins. None of the 10 volunteers receiving the immunoglobulin concentrate against E. coli had diarrhea, but 9 of the 10 controls did (P less than 0.0001). All volunteers excreted E. coli H10407. We conclude from these preliminary results that milk immunoglobulin concentrate may be an effective prophylaxis against traveler's diarrhea.

We obtained clinical, epidemiological, and laboratory data (including three stool cultures) from 155 (96%) of 161 household contacts of 24 patients less than 16 years old with culture-confirmed typhoid fever; these 24 patients represented approximately 40% of such patients seen in three hospitals in Santiago during a 12-week period. A chronic typhoid carrier was identified in only one household, with concurrent or secondary cases seen in two other households. When index cases were matched with household members nearest in age, no specific risk factors for illness could be identified. There was evidence of generalized exposure to enteric pathogens within these households, with nine persons from seven different households culture-positive for non-typhoidal Salmonella, and nine, from eight different households, culture-positive for Shigella; transmission of these pathogens within households did not appear to be common since no household had more than one family member with the same serotype or species of either pathogen.

Incubation of Vibrio cholerae of O-group serotype 1 with chitin particles resulted in adsorption of vibrios onto chitin; chitin-adsorbed V. cholerae survived exposure to acid better than nonadsorbed vibrios. V. cholerae multiplied in dialyzed chitin suspended in 4.2% NaCl, suggesting that adherence to ingested chitin of crustacea might be of epidemiological significance by providing a substrate for vibrio multiplication as well as protection from gastric acid during stomach transit.

Typhoid fever is an important endemic health problem in Santiago, Chile. Its incidence has more than doubled in recent years, during which access to potable water and sewage disposal in the home became almost universal in the city. A matched case-control study was carried out to identify risk factors and vehicles of transmission of paediatric typhoid fever; 81 children in the 3-14-years age group with typhoid fever were compared with controls, matched with respect to age, sex, and neighbourhood. It was found that case children more frequently bought lunch at school and shared food with classmates. Also, case children more often consumed flavoured ices bought outside the home; none of 41 other food items considered in the study was associated with a higher risk of typhoid fever. Only two food handlers for cases and one for controls were positive for Salmonella typhi, indicating that persons preparing food solely for their own family were not the main source of S. typhi infection. Rather, the risk factors identified in this study are consistent with the hypothesis that paediatric endemic typhoid fever in Santiago is largely spread by consumption of food-stuffs that are prepared outside the individual's home and are shared with or sold to children.

Epidemiologic evidence suggests that Plesiomonas shigelloides is an enteric pathogen. We conducted in vitro, animal, and volunteer studies on P. shigelloides isolates from patients with diarrhea. Five strains gave a negative keratoconjunctivitis reaction in guinea pigs and did not invade HeLa cells. Genetic probes for heat-stable enterotoxins related to those of enterotoxigenic Escherichia coli and for gene sequences common to the invasiveness plasmids of Shigella spp. and enteroinvasive E. coli were negative. Heat-labile enterotoxins were not found when a modified GM1-enzyme-linked immunosorbent assay was used. Rabbits did not develop diarrhea but were transiently colonized when inoculated with up to 10(11) P. shigelloides CFU using the reversible intestinal tie adult rabbit diarrhea model. A very large plasmid (between 118 and 312 megadaltons) was found in all isolates. Strain P012 was cured of its plasmid by novobiocin. This strain, but not its cured derivative, invaded the mucosa of the distal ileum of gnotobiotic piglets given 10(10) CFU. At a lower inoculum (10(9) CFU), strain P012 induced inflammation of the colonic mucosa and diarrhea at day 6. The same isolate was fed to 33 healthy volunteers in doses of 1 X 10(3) to 4 X 10(9) CFU. Thirty-six percent of the volunteers shed the organism, but none became ill. These data are only weakly supportive of a role for P. shigelloides in diarrheal illness and suggest the need for more studies with other strains to better understand its pathogenicity.

Colonization of the small intestine is an essential step in the pathogenesis of Vibrio cholerae diarrhea. At least one type of fimbriae, known as TcpA are required for the colonization process. This paper reports electron microscopic evidence that V. cholerae strains can express at least two other fimbrial types. Classical strains express three types: TcpA fimbriae are 5-6 nm in diameter and form bundles of parallel undulating filaments up to 15 micron long; Type B are 3 nm wide and of wavy morphology, and Type C are rigid, isolated filaments 5-6 nm wide and 180-800 nm long. El Tor strains express fimbriae resembling Types B and C. Types B and C were also found on a tcpA- isogenic mutant of V. cholerae 395 N1, and are thus encoded by genetically distinct loci. TcpA fimbriae, but not Types B or C, were labeled with gold-conjugated anti-TcpA antibody. Four El Tor strains, including two environmental isolates that poorly colonize humans, expressed fimbriae resembling Types B and C, but did not express TcpA. Multiple types of fimbriae may represent colonization factors for surfaces present in the environment and in the human gut. Characterization of the role of fimbriae in immunogen presentation and immunity could facilitate the improvement of cholera vaccines.

PCF8775 is a putative colonization factor complex present on the surface of 10 to 20% of enterotoxigenic Escherichia coli strains and has been reported to be composed of antigen CS6 (morphology undefined) expressed alone or together with either of the rigid fimbrial antigens CS4 and CS5. To better define the individual components of this complex and the determinants of their expression, we prepared antiserum to the PCF8775 complex as it was expressed on prototype strain E8775 and then used the antiserum to identify the subunit structure of the antigens, to study their morphology, and to detect expression of individual components of the complex after transfer of plasmids into laboratory strain HB101. CS4 was purified from strain E8775, confirmed to be fimbrial by electron microscopy, and found to be composed of a 22-kilodalton protein subunit whose N-terminal amino acid sequence (1 to 20) was similar to that of colonization factor antigen I. Transconjugants that express CS6 but not CS4 were obtained by mating prototype strain E8775 with HB101. CS6 expression was mediated by a 61-megadalton plasmid. Expression of CS6 in the transconjugants correlates with expression of a 16-kilodalton cell surface protein. The CS6 antigen was confirmed to be present on the cell surface by immunogold labeling, but its morphology was beyond the limits of resolution by electron microscopy.