Hepatitis E virus DNA vaccine elicits immunologic memory in mice.

Injection of an expression vector pJHEV containing hepatitis E virus (HEV) structural protein open reading frame 2 gene generates a strong antibody response in BALB/c mice that can bind to and agglutinate HEV. In this study, we tested for immunologic memory in immunized mice whose current levels of IgG to HEV were low or undetectable despite 3 doses of HEV DNA vaccine 18 months earlier. Mice previously vaccinated with vector alone were controls. All mice were administered a dose of HEV DNA vaccine to simulate an infectious challenge with HEV. The endpoint was IgG to HEV determined by ELISA. Ten days after the vaccine dose, 5 of 9 mice previously immunized with HEV DNA vaccine had a slight increase in IgG to HEV. By 40 days after the vaccine dose, the level of IgG to HEV had increased dramatically in all 9 mice (108-fold increase in geometric mean titer). In contrast, no control mice became seropositive. These results indicate that mice vaccinated with 3 doses of HEV DNA vaccine retain immunologic memory. In response to a small antigenic challenge delivered as DNA, possibly less than delivered by a human infective dose of virus, mice with memory were able to generate high levels of antibody in less time than the usual incubation period of hepatitis E. We speculate that this type of response could protect a human from overt disease.

An outbreak of hepatitis E in Northern Namibia, 1983.

In 1983 in Namibia's Kavango region, epidemic jaundice affected hundreds of people living in settlements lacking potable water and waste disposal facilities. Many were Angolan refugees. The disease, which after investigation was designated non-A non-B hepatitis, was most common in males (72%), in persons aged 15-39 years, and was usually mild except in pregnant women, who incurred 6 (86%) of the 7 fatal infections. Fifteen years later, archived outbreak-associated samples were analyzed. Hepatitis E virus (HEV) was detected by reverse transcription-polymerase chain reaction in feces from 9 of 16 patients tested. Total Ig and IgM to HEV were quantitated in serum from 24 residents of an affected settlement at the outbreak's end: 42% had IgM diagnostic of recent infection and 25% had elevated total Ig without IgM, consistent with past HEV infection. The Namibia outbreak was typical hepatitis E clinically and epidemiologically. This first report of hepatitis E confirmed by virus detection from southern Africa extends the known range of HEV and highlights its risk for refugees.

Seroprevalence of hepatitis E virus among United Nations Mission in Haiti (UNMIH) peacekeepers, 1995.

Information about the prevalence of hepatitis E virus (HEV) infection is sparse in many countries. Following the identification of four cases of acute HEV infection among Bangladeshi soldiers, a serologic survey was conducted to determine the prevalence of HEV infection among other peacekeepers from the United Nations Mission in Haiti (UNMIH) and Haitian civilians. Of the 981 participants in the survey, 876 were soldiers from eight UNMIH-participating countries representing Asia, Africa, and the Americas, and 105 were Haitian civilians. The prevalence of HEV infection by country (from highest to lowest) included Pakistan (62%), India (37%), Nepal (37%), Bangladesh (27%), Djibouti (13%), Honduras (6%), Guatemala (5%), Haiti (3%), and the United States (2%). More than 90% of those surveyed from Guatemala, Haiti, and Honduras, where prevalence data has been scarce, appeared susceptible to HEV infection. Future multinational missions like the UNMIH might also present unique opportunities to study health threats of widespread interest.

Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains.

We have employed a molecular genetic approach to characterize the nature of enteroinvasive Escherichia coli (EIEC) enterotoxic activity, as previously observed in Ussing chambers (A. Fasano, B.A. Kay, R.G. Russell, D.R. Maneval, Jr., and M.M. Levine, Infect. Immun. 58:3717-3723, 1990). The screening of TnphoA mutants of EIEC yielded a single insertion mutant which had significantly reduced levels of enterotoxic activity in the Ussing chamber assay. DNA flanking the insertion was used as a probe to screen for EIEC cosmid clones which conferred secretogenic activity. Such screening resulted in the identification of two overlapping cosmid clones which elicited significant changes in mucosal short-circuit current (Isc). Subcloning and nucleotide sequence analysis of a DNA fragment from one of the cosmid clones led to the identification of a single open reading frame which conferred this enterotoxic activity. By DNA hybridization, this gene (designated sen for shigella enterotoxin) was found in 75% of EIEC strains and 83% of Shigella strains and was localized to the inv plasmid of Shigella flexneri 2457T. By PCR, a sen gene with 99.7% nucleotide identity was cloned and sequenced from 2457T. A deletion in the EIEC sen gene was constructed by allelic exchange, resulting in significantly lower rises in Isc than were elicited by the wild-type parent; however, significant enterotoxic activity remained in the sen deletion mutant. To purify the Sen protein, the gene was cloned into the multiple cloning site of the expression vector pKK223-3. Purification of the sen gene product yielded a protein with a molecular mass of 63 kDa which elicited rises in Isc in the Ussing chamber. We believe that the sen gene product may constitute all or part of a novel enterotoxin in EIEC and Shigella spp.

Shiga-like-toxin-producing Escherichia coli in retail meats and cattle in Thailand.

Specific DNA probes were used to identify Shiga-like toxin I (SLT I)- and SLT II-producing Escherichia coli in vegetables, meats, cattle, and farm animals in Thailand. SLT-producing E. coli was isolated from 9% of market beef specimens, from 8 to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal specimens from cattle. Animals were frequently infected with several different SLT-producing E. coli types that hybridized with either the SLT I, SLT II, or both SLT probes. Of 119 SLT-producing E. coli isolates, 24% hybridized with the SLT I probe, 31% hybridized with the SLT II probe, and 44% hybridized with both SLT probes. The enterohemorrhagic E. coli plasmid probe hybridized with 64% (68 of 106) of SLT-producing E. coli isolates from food and cattle and with 8% (17 of 201) of E. coli isolates from pigs. No SLT-producing E. coli was detected in pigs. Seventy-six percent (26 of 34) of E. coli isolates that hybridized with the SLT II probe were cytotoxic to Vero but not to HeLa cells, suggesting that they produced the variant of SLT II. The high prevalence of SLT-producing E. coli in beef-producing animals suggests that exposure to animals and eating beef may pose a health risk for acquiring enterohemorrhagic E. coli infections in Thailand.

Nationwide surveillance program to identify diarrhea-causing Escherichia coli in children in Thailand.

Escherichia coli strains isolated from children with diarrhea were collected from 16 hospitals in different districts in Thailand during 1985 and 1986 and submitted to the National Reference Laboratory. Isolates were identified by serogrouping or as enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC) adhesin factor (EAF) E. coli, or Shiga-like-toxin (SLT)-producing E. coli by DNA hybridization. EPEC strains of known serogroups were isolated from 10%, ETEC strains were isolated from 6%, EAF E. coli strains were isolated from 4%, EIEC strains were isolated from less than 1%, and SLT-producing E. coli strains were isolated from none of 393 children with diarrhea. Among 278 children whose ages were recorded, the highest rate of isolation of EAF E. coli was 11% (9 of 85) from children less than 6 months old. ETEC was isolated from 5% (4 of 85) of children less than 6 months old, from 10% (12 of 118) of children 6 to 23 months old, and from 1% (1 of 75) of children greater than 23 months old. EPEC strains of known serogroups were isolated from 18% (15 of 85) of children less than 6 months old, from 11% (13 of 118) of children 6 to 23 months old, and from 9% (7 of 75) of children greater than 23 months old. E. coli strains that hybridized with the EIEC probe were isolated from three children who were 20, 36, and 48 months old. Examining E. coli for hybridization with DNA probes for virulence determinants is a practical way of conducting nationwide surveillance of diarrhea-causing E. coli. Since only 33% (13 of 39) of EPEC serogroups hybridized with the EAF probe and none hybridized with the SLT probes, identification of EPEC by serogroups analysis, followed by serotyping, should continue to be used in the identification of EPEC.

An epidemic of Vibrio cholerae el tor Inaba resistant to several antibiotics with a conjugative group C plasmid coding for type II dihydrofolate reductase in Thailand.

Between June and October 1982, Vibrio cholerae el tor Inaba phage type Russian 13, resistant to ampicillin (Ap), chloramphenicol (Cm), colistin, neomycin (Nm), kanamycin (Km), gentamicin (Gm), trimethoprim sulfamethoxazole (TMP-SMZ), and tetracycline (Tc), was isolated from 31 children with diarrhea at a hospital in Samutsakorn, Thailand. Thirty of these children were less than 2 years of age and were admitted to a single pediatric ward. Seventeen of the cases, infected with V. cholerae (MARV) resistant to several antibiotics, were admitted to the hospital for non-gastrointestinal illnesses; these children developed diarrhea and positive cultures for MARV 1-greater than 10 days after admission. The majority of cases occurred in September, when the attack rate in the patient population in 1 pediatric ward was 11.5%. During this period, MARV with the same characteristics was isolated from water used for bathing in a reservoir on the pediatric ward where most of the cases occurred. MARV was not isolated from adults with diarrhea at the hospital. No further MARV infections occurred at the hospital after the water reservoir had been drained and disinfected. V. cholerae isolates from children and water contained a conjugative incompatibility group C plasmid of 100 megadaltons (mDa) encoding resistance to Ap, Cm, Nm, Km, Gm, TMP-SMZ, and Tc. This plasmid hybridized with a DNA probe for genes encoding Type II dihydrofolate reductase (DHFR). As far as we know, this is the first report of MARV with V. cholerae that contained genes coding for Type II DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of colonies and stool blots for detection of enteropathogens by DNA hybridization with eight DNA probes.

We compared three methods for detecting enteropathogens in 416 children with diarrhea: (i) examination of 10 lactose-fermenting and all non-lactose-fermenting Escherichia coli (colony blots); (ii) examination of 300 colonies (replicate blots); and (iii) determination of the total bacterial growth of stools (stool blots). All specimens were spotted onto Whatman 541 filters and hybridized with specific radiolabeled DNA probes. Enterotoxigenic E. coli was detected in 38 patients by examining colony blots, in 52 patients by examining replicate blots, and in 45 patients by examining stool blots. Enteropathogenic E. coli adhesin factor was detected in 12 patients by colony blots, in 25 patients by replicate blots, and in 16 patients by stool blots. E. coli that hybridized with the enterohemorrhagic E. coli probe was detected in 2 patients by colony blots, in 11 patients by replicate blots, and in 0 patients by stool blots. Shiga-like toxin-producing E. coli was detected in 0 patients by colony blots, in 12 patients by replicate blots, and in 0 patients by stool blots. Shigella spp. were identified by standard bacteriological methods in 82 patients, and enteroinvasive E. coli was identified by colony blots in 11 patients (total, 93), by replicate blots in 56 patients, and by stool blots in 35 patients. Of 82 culture-confirmed Shigella infections, 45 were identified by examining replicate blots with the 17-kilobase-pair probe and 36 were identified by examination with the Ipa probe (P less than 0.05). Examining replicate blots with specific probes identified more enterotoxigenic E.coli (P < 0.005), enteropathogenic E.coli adhesion factor-producing E.coli (P < 0.001), and Shiga-like toxin-producing E.coli (P < 0.005) infections than examining colony blots. More Shigella and enteroinvasive E.coli infections were identified by standard bacteriological methods and examining colony blots with a specific probe than by examining replicate and stool blots (P < 0.001).

Determination by DNA hybridization of Shiga-like-toxin-producing Escherichia coli in children with diarrhea in Thailand.

Specific DNA probes were used to identify Shiga-like-toxin (SLT) I- and II-producing Escherichia coli from children less than 5 years of age with bloody diarrhea, in infants with diarrhea, and in controls of the same age without diarrhea in Thailand. At one hospital, SLT-producing E. coli was identified in 4 (7%) of 54 children with bloody diarrhea from whom other enteric pathogens were not identified and from 3 (6%) of 50 children without diarrhea. In the positive specimens, SLT-producing E. coli constituted only 0.3 to 4% of the 100 to 300 colonies on the replica blots. Non-toxin-encoding 933J and 933W bacteriophagelike DNA sequences were detected by colony hybridization with E. coli isolates from 18 (33%) of 54 children with bloody diarrhea and 23 (46%) of 50 controls. At another hospital, SLT-producing E. coli was not identified in 115 infants with diarrhea and 119 controls without diarrhea. One infant with diarrhea was infected with E. coli O76:H7 that hybridized with the enterohemorrhagic E. coli probe but not with the SLT probes. E. coli producing SLT I or SLT II was isolated in small numbers from a similar proportion of Thai children with bloody diarrhea in whom no other enteric pathogen was identified and from controls without diarrhea.

DNA probes to identify Shiga-like toxin I- and II-producing enteric bacterial pathogens isolated from patients with diarrhea in Thailand.

When Shigella species, Escherichia coli, and five other bacterial enteric pathogens isolated from children with diarrhea in Thailand were tested for hybridization under stringent conditions with probes for Shiga-like toxins I and II, only 30 Shigella dysenteriae 1 hybridized with the Shiga-like toxin I probe.

Foods as a source of enteropathogens causing childhood diarrhea in Thailand.

Foods obtained in markets in Bangkok were cultured for bacterial enteric pathogens and examined for their similarity to strains isolated from children under 5 years of age in Bangkok in 1986. Salmonella was isolated from 17%, Campylobacter from 12%, and enterotoxigenic Escherichia coli (ETEC) from 3% of 510 foods examined. Campylobacter was isolated from 13.5%, ETEC from 13%, and Salmonella from 12% of 1,230 children under 5 years of age with diarrhea. Eighty-eight percent of children infected with Salmonella were infected with serotypes isolated from foods of animal origin. Six percent of children with Salmonella were infected with the same serotype containing plasmids with identical endonuclease restriction patterns as isolates from food. Eighty-seven percent of children with Campylobacter were infected with the same serotypes and biotypes found in food of animal origin. Thirty-one percent of heat-labile enterotoxin (LT) producing ETEC from foods containing genes coding for LT II, but LT II ETEC was not isolated from children. Twenty-one percent of ETEC isolated from foods vs. 53% isolated from children were resistant to 2 or more antibiotics (P less than 0.01). Salmonella and Campylobacter, but not ETEC, isolated from foods were similar to strains isolated from children. Foods of animal origin are an important source of Salmonella and Campylobacter in Thailand.

Influence of strain characteristics and immunity on the epidemiology of Campylobacter infections in Thailand.

To determine how strain differences and immunity affect the clinical expression of Campylobacter infections, we conducted a study of acute diarrheal disease in Thailand in which specimens from children with Campylobacter infections were cultured weekly for up to 12 weeks to determine the serotype-specific length of time of convalescent-phase excretion and rate of reinfection. Levels of immunoglobulin G to cell-surface antigens of C. jejuni were determined in another population of healthy children who were closely related by age and location to the children in the diarrheal disease study. Campylobacter species were initially isolated from 18% of 586 children under 5 years old with diarrhea; most isolates in Thailand belonged to serotypes commonly found in developed countries. C. coli was significantly less often associated with symptomatic infections and with bloody diarrhea than C. jejuni (P less than 0.001 and P = 0.045, respectively). The peak age of isolation and the peak level of immunoglobulin G to Campylobacter species occurred before 2 years of age. The mean duration of convalescent-phase excretion was 14 +/- 2 (standard error of the mean) days for children less than 1 year old and 8 +/- 2 days for children 1 to 5 years old (P = 0.02, t test). Infection with another Campylobacter serotype was found in 34% of 105 children during the 12-week follow-up period. The rate of reinfection in these children was 15% (range, 8 to 22%) each week. Hyperendemic exposure to Campylobacter species in Thailand confers immunity to infection that is associated with an early peak in specific serum antibodies and an age-related decrease in the case-to-infection ratio and duration of convalescent-phase excretion but does not prevent asymptomatic infections.

Type II heat-labile enterotoxin-producing Escherichia coli isolated from animals and humans.

Heat-labile enterotoxin (LT)-producing Escherichia coli strains, as identified by the Y1 adrenal cell assay, were examined with a DNA probe coding for type I and type II LTs. Of 236 LT-producing E. coli isolates, 60% hybridized with LT-I, 17% hybridized with LT-II, and 23% did not hybridize with either probe and no longer produced LT as determined by the Y1 adrenal cell assay. These isolates presumably lost plasmids coding for LT-I during storage. A total of 75% of LT-producing E. coli isolates (27 of 36) from cows, 64% of LT-producing E. coli isolates (7 of 11) from buffalo, 31% of LT-producing E. coli isolates (4 of 13) from beef obtained in markets, and 2% of LT-producing E. coli isolates (3 of 168) from humans contained genes coding for LT-II. Genes coding for LT-II were not found in 50 LT-I-producing and heat-stable enterotoxin-producing E. coli isolates from 11 children with diarrhea and 44 LT-nonproducing and heat-stable enterotoxin-producing E. coli isolates from 12 other children with diarrhea. A total of 9% of LT-II-producing E. coli isolates (3 of 34) from cows and buffalo hybridized with DNA probes for genes coding for verocytotoxin 2 (VT2), and 18% (6 of 34) hybridized with a DNA probe coding for enterohemorrhagic E. coli (EHEC) adhesin fimbriae. E. coli SA-53, the original isolate in which LT-II was found, contained genes coding for VT2 and EHEC adhesin fimbriae. Five VT-producing, LT-II-producing E. coli isolates that hybridized with the EHEC probe did not contain DNA sequences coding for VT1 or VT2. LT-II-producing E. coli strains were frequently isolated from cattle and buffalo but were rarely isolated from humans.

Application of DNA hybridization techniques in the assessment of diarrheal disease among refugees in Thailand.

The epidemiology and etiology of acute diarrheal disease were determined in a Hmong refugee camp on the Thai-Laotian border from April 11 to May 14, 1985. DNA hybridization techniques were used to detect Shigella species, enteroinvasive Escherichia coli, and enterotoxigenic E. coli. A monoclonal enzyme-linked immunosorbent assay was used to detect rotavirus, and standard microbiology was used to detect other enteropathogens. The age-specific diarrheal disease rates were 47 episodes per month per 1,000 children less than five years old and 113 episodes per month per 1,000 children less than one year old. Rotavirus, enterotoxigenic E. coli, Campylobacter, and Cryptosporidium were the predominant pathogens in children less than two years old. The DNA probe hybridized with 94% of 31 specimens identified as enterotoxigenic E. coli by the standard assays and with none of the specimens in which the standard assays were negative. The probe for Shigella and enteroinvasive E. coli hybridized in eight of 10 stools that contained Shigella and four of 314 stools from which Shigella and enteroinvasive E. coli were not isolated. The use of DNA probes allows specimens to be collected in remote areas with a minimum amount of equipment and technical expertise so that they can be easily transported to a central laboratory for further processing.

Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes.

Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates.

Trimethoprim-resistant Shigella and enterotoxigenic Escherichia coli strains in children in Thailand.

The percentage of Shigella and enterotoxigenic Escherichia coli (ETEC) strains resistant to trimethoprim (TMP)-sulfamethoxazole isolated from children with diarrhea at the outpatient department of the Children's Hospital in Bangkok increased from 3 and 0%, respectively, in 1982 to 29% and 25% in 1986. One hundred thirty-nine Shigella and 22 ETEC strains resistant to greater than 1024 micrograms/ml of trimethoprim (TMPr) isolated from children with diarrhea in Bangkok in 1984 and 1985 were analyzed for the presence of type I, II and III plasmid-specific dihydrofolate reductase (DHFR) genes. Thirty-two percent (45 of 139) of TMPR Shigella had genes encoding type II and 9% (13 of 139) had genes encoding type I DHFR genes. Fifty percent (11 of 22) of TMPR ETEC had type II and 14% (3 of 22) had type I DHFR genes. Plasmids encoding DHFR were identified by the Southern technique in 24% (14 of 58) of Shigella and 1 of 14 ETEC that contained genes encoding DHFR. Plasmids coding for type II DHFR were transferred to E. coli K12 by conjugation from 13 of 14 Shigella and a plasmid coding for type I DHFR was transferred from the single ETEC containing a plasmid coding for type I DHFR. Genes coding for DHFR were presumably situated on the chromosome in 76% (44 of 58) of Shigella and 93% (13 of 14) of ETEC that contained genes encoding DHFR. Since 58% (81 of 139) of TMPR Shigella and 36% (8 of 22) of TMPR ETEC strains examined did not contain genes encoding type I, II or III DHFR, high level TMP resistance was presumably caused by other types of DHFR genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative study of synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes to identify enterotoxigenic Escherichia coli.

Escherichia coli isolated from 2,126 children in Thailand and the Philippines was examined for enterotoxin production and for DNA hybridization with synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes. A total of 233 infections with E. coli that were detected by one or more of these assays were identified. Of the infections, 75% (164/233) were identified by all three methods. An additional 18% (43/233) were identified by two of three methods. Isolates from 10% (19/183) of infections with E. coli that hybridized with both the oligonucleotide and cloned enterotoxin gene probes were nontoxigenic, as determined by the Y1 adrenal cell and suckling mouse assays. Although synthetic oligonucleotide probes to detect enterotoxigenic E. coli are more uniform and easier to use than cloned enterotoxin gene probes, the heat-labile toxin oligo probe used in this study did not identify 13% (11/87) of infections with E. coli that produced heat-labile toxin, as identified with the Y1 adrenal cell assay and the cloned enterotoxin gene probe. Synthetic oligonucleotide probes enable laboratories with only minimal equipment to use DNA hybridization assays to identify enterotoxigenic E. coli.

Potential sources of enterotoxigenic Escherichia coli in homes of children with diarrhoea in Thailand.

PIP: A year-long study was performed to identify potential sources of enterotoxigenic Escherichia coli (ETEC) within the homes of children with diarrhea in Bangkok. ETEC was identified in 8% (10 of 130) of the inhabitants of 42 homes with children with ETEC diarrhea and 6% (8 of 137) of their neighbors, but in only 2% (49 of 3077) of those individuals living in 866 homes not associated with children with ETEC diarrhea. While 46% (13 of 28) of the children under age 2 infected with ETEC were identified on home visits as having had a recent history of diarrhea, only 13% (5 of 39) of those over age 2 presented such a history. ETEC was isolated from 14% of the mothers' hands, 13% of the children's hands, and 7% of jars containing bath water that was used for washing the children after defecation. Drinking water was identified as a probable source of infection in 1 of 42 cases. Further studies are needed to determine whether ETEC from water stored in the home can spread and cause secondary infections.^ieng