The objective of the study was to assess whether genotypic characterization by means of DNA-fingerprinting pattern (DFP) and multilocus enzyme electrophoresis (MEE) profile as compared to phenotypic characterization would improve the differentiation of Neisseria meningitidis strains associated with outbreaks from strains associated with sporadic cases of meningococcal disease. In addition, the differentiation of serogroup C carrier strains from those associated with an outbreak of serogroup C meningococcal disease was investigated.

A total of 118 N. meningitidis strains were available for the study: 59 from patients involved in outbreaks of meningococcal disease (2 serogroup B and 2 serogroup C), 37 from patients considered to be sporadic cases and 22 serogroup C carrier strains.

Among the 59 strains from patients involved in outbreaks the 4 strains isolated from the patient registered as the first in each outbreak were designated the index strains. Among the remaining 55 outbreak strains 52 were either DFP-identical or DFP-indistinguishable when compared with the one relevant out of the 4 index strains. This was only the case for 17 of the 37 strains isolated from sporadic cases caused by the same serogroup of meningococci during the outbreak periods, and 5 of the 22 meningococcal strains isolated from healthy carriers. Among the 56 (52 + 4) DFP-identical or DFP-indistinguishable outbreak strains 5 different electrophoretic types were identified by MEE.

Among 59 assumed outbreak strains a total of 4 were identified as genotypically distinct. Among the 37 mainly DFP-indistinguishable or DFP-different strains from sporadic cases 17 different ETs were identified, and among the 22 mainly DFP-different carrier strains 13 different ETs were identified. Two strains among those selected from sporadic cases were identical to the outbreak strain. None of the local serogroup C carrier strains isolated during the outbreak of serogroup C disease were identical to the outbreak strain.

Both DNA-fingerprinting and MEE improved the differentiation of meningococci when compared with phenotypic characterization. The results indicate that tracing a virulent strain within an open group of contacts is irrelevant.

This study determined the prevalence of the eaeA gene and its relationship to serotype and type of verotoxin produced in a collection of 432 verotoxigenic Escherichia coli (VTEC) obtained from the faeces of healthy cows and calves in a systematic random survey involving 80 dairy farms in Southwest Ontario. A PCR amplification procedure involving primer pairs which target the conserved central region of the O157:H7 eaeA. gene showed that 151 (35·2%) strains were positive for the eaeA gene. All isolates (9–21 for each O group) of O groups 5, 26, 69, 84, 103, 111, 145 and 157 were positive, whereas all isolates (7–34 for each O group) of O groups 113, 132, and 153 and serotype O156:NM (38 isolates) were negative for eaeA. Seventy-three percent of 130 isolates of eaeA-positive serotypes produced VT1 only compared with 20% of 253 isolates of eaeA-negative serotypes. We conclude that there is a strong association between certain O groups and the eaeA gene, that serotypes of eaeA-positive and eaeA-negative VTEC implicated in human and cattle disease are present at high frequency in the faeces of healthy cattle, that VT1 is more frequently associated with eaeA-positive than with eaeA-negative serogroups, and that the eaeA gene is more frequently found in VTEC from calves compared with VTEC from adult cattle.

To improve both safety and stability of the oral vaccines used in the field to vaccinate foxes against rabies, a recombinant vaccinia virus, which expresses the immunizing G protein of rabies virus has been developed by inserting the cDNA which codes for the immunogenic glycoprotein of rabies virus into the thymidine kinase (TK) gene of the Copenhagen strain of vaccinia virus. The efficacy of this vaccine was tested by the oral route, primarily in foxes. The immunity conferred, a minimum of 12 months in cubs and 18 months in adult animals, corresponds to the duration of the protection required for vaccination of foxes in the field. Innocuity was tested in foxes, domestic animals, and in numerous European wild animal species that could compete with the red fox for the vaccine bait. No clinical signs or lesions were observed in any of the vaccinated animals during a minimum of 28 days post vaccination. Moreover, no transmission of immunizing doses of the recombinant occurred between foxes or other species tested. To study the stability of the vaccine strain, baits containing the vaccine were placed in the field. Despite considerable variations of environmental temperatures, the vaccine remained stable for at least one month. Because bait is taken within one month, it can be assumed that most animals taking the baits are effectively vaccinated. To test the field efficacy of the recombinant vaccine, large-scale campaigns of fox vaccination were set up in a 2200 km2 region of southern Belgium, were rabies was prevalent. A dramatic decrease in the incidence of rabies was noted after the campaigns. The recombinant is presently used to control wildlife rabies in the field both in several European countries and in the United States.

In February 1992, an outbreak of cholera occurred among persons who had flown on a commercial airline flight from South America to Los Angeles. This study was conducted to determine the magnitude and the cause of the outbreak. Passengers were interviewed and laboratory specimens were collected to determine the magnitude of the outbreak. A case-control study was performed to determine the vehicle of infection. Seventy-five of the 336 passengers in the United States had cholera; 10 were hospitalized and one died. Cold seafood salad, served between Lima, Peru and Los Angeles, California, was the vehicle of infection (odds ratio, 11·6; 95% confidence interval, 3·3–44·5). This was the largest airline-associated outbreak of cholera ever reported and demonstrates the potential for airline-associated spread of cholera from epidemic areas to other parts of the world. Physicians should obtain a travel history and consider cholera in patients with diarrhoea who have travelled from cholera-affected countries. This outbreak also highlights the risks associated with eating cold foods prepared in cholera-affected countries.

A total of 125 strains of Neisseria meningitidis recovered in the course of outbreaks from patients with systemic disease in 11 African countries between 1989 and 1994 were analysed by serogrouping, serotyping and multilocus enzyme electrophoresis. Of the 125 patient strains 115 (92%) belonged to the clone-complex of serogroup A meningococci, designated subgroup III. Among the remaining strains, 4 were also serogroup A, but belonged to the clonal groups I and IV-1 (2 strains each), whilst 6 strains (4 serogroup C and 2 serogroup W135) represented clones of the ET-37 complex. Our results indicated that the second pandemic caused by clones of subgroup III is still spreading in Africa. Towards the West it has reached Niger, Mali, Guinea and The Gambia, and towards the South, the Central African Republic, Uganda, Rwanda, Burundi, Tanzania and Zambia.

Neisseria gonorrhoeae were collected from men attending out-patient clinics in Dubai. The susceptibility to a range of therapeutic antibiotics and their auxotype and serotype was determined. The plasmid content of all penicillinase-producing strains was also analysed. Thirty-six strains of N. gonorrhoeae were isolated from specimens collected from 79 patients over a 24-day period. Of the 36 isolates, 9 (25%) were penicillinase-producing N. gonorrhoeae (PPNG) and 15 (42%) were chromosomally resistant N. gonorrhoeae (CMRNG). CMRNG exhibited higher levels of resistance to cefuroxime, chloramphenicol, tetracycline and erythromycin than PPNG. All isolates were susceptible to ceftriaxone and spectinomycin. Three (8%) isolates showed reduced susceptibility (MIC, ≥ 0·25 mg/1) to ciprofloxacin. Six isolates of PPNG carried the 4·4 MD and three the 3·2 MD penicillinase encoding plasmid. The total gonococcal population was phenotypically diverse, with 12 serovars, 6 auxotypes and 21 A/S classes. Gonorrhoea was found to be a major cause of urethritis in Dubai and the strains exhibited high levels of resistance to penicillin.

This review presents experimental, preclinical and clinical data illustrating the multiple uses of recombinant non-replicating virus vectors in the fields of immunoprophylaxis and gene therapy.

Epidemic cholera reached Guatemala in July 1991. By mid-1993, Guatemala ranked third in the hemisphere in reported cases of cholera. We conducted a case-control study with two age-, sex-, and neighbourhood-matched controls per patient in periurban Guatemala City. Twenty-six patients hospitalized for cholera and 52 controls were enrolled. Seven (47%) of 15 stool cultures obtained after admission yielded toxigenic Vibrio cholerae Ol. All seven were resistant to furazolidone, sulfisoxazole, and streptomycin, and differed substantially by pulsed-field gel electrophoresis from the Latin American epidemic strain dominant in the hemisphere since 1991. In univariate analysis, illness was associated with consumption of left-over rice (odds ratio [OR] = 7·0, 95% confidence interval [CI] = 1·4–36), flavored ices (‘helados’) (OR = 3·6, CI = 1·1–12), and street-vended non-carbonated beverages (OR = 3·8, CI = 1·2–12) and food items (OR = 11·0, CI = 2·3–54). Street-vended food items remained significantly associated with illness in multivariate analysis (OR = 6·5, CI = 1·4–31). Illness was not associated with drinking municipal tap water. Maintaining water safety is important, but slowing the epidemic in Guatemala City and elsewhere may also require improvement in street vendor food handling and hygiene.

Two common forms of invasive disease due to Haemophilus influenzae type b (Hib) are epiglottitis and meningitis. It is not known why some children develop epiglottitis and others meningitis. To examine the hypothesis that epiglottitis occurs in children who may have been previously exposed to Hib, and who would therefore exhibit a more vigorous antibody response in convalescence, we measured levels of antibody to Hib capsule in 92 children. Geometric mean convalescent-phase IgG, IgA, IgM and total antibody levels were significantly higher in 45 children with epiglottitis than in 47 with meningitis, even after adjustment for age differences (mean total antibody, 95% confidence intervals: meningitis 0·38, 0·34–0·43; epiglottitis: 2·25, 2·0–2·54 μg/ml). However, contrary to previous reports, a poor antibody response was only observed in a minority of children with meningitis; the antibody response of the majority was indistinguishable from that observed in children with epiglottitis.

This report summarizes studies on 1699 foodborne outbreaks, in Italy, reported to the Istituto Superiore di Sanità (ISS) (the National Institute of Health of Italy, Rome) during the period 1991–4. The most frequently reported foodborne outbreaks were caused by salmonellae (81%), in particular by Salmonella enteritidis and non-serotyped group D salmonella (34% and 33% of the total salmonella outbreaks, respectively). A vehicle was implicated in 69% of the salmonella outbreaks; eggs were implicated in 77% of the outbreaks for which a vehicle was identified or suspected. Salmonella strains isolated in 54 outbreaks were studied for phenotypic and genotypic characteristics. The isolates belonged to S. enteritidis (50 outbreaks), S. typhimurium (three outbreaks) and S. hadar (one outbreak). In the S. enteritidis outbreaks, phage type 4 was most frequently isolated (64·8%), followed by phage type 1 (14·8%). The virulence plasmid of 38 megadaltons was found in many different phage types of S. enteritidis.

Ribotyping performed with six restriction endonucleases was used to study the molecular epidemiology of shigellosis in Asturias, Spain. The series included Shigella sonnei from 34 sporadic cases, 3 outbreaks and 3 reference strains, and S. flexneri from 13 sporadic cases and 1 reference strain. The S. sonnei strains were grouped into 5 ribotypes with Sal I, 4 with Hind III and Pvu II, 3 with Bgl II and EcoR I and 2 with Hinc II (Discriminatory Index (DI) between 0·54 and 0·14); the S. flexneri into 5 ribotypes with Sal I, Hinc II and Hind III, and 4 with the other enzymes (DI = 0·71–0·63). The combination of results for 2 or more enzymes facilitated an additional discrimination, the highest values in S. sonnei were for the 6 enzymes (16 types, DI = 0·91) and in S. flexneri for some combinations of 3 or more enzymes (7 types, DI = 0·81). Ribotypes with the 6 enzymes defined 16 clonal lines in S. sonnei and 7 in S. flexneri, which showed a different degree of genetic heterogeneity, and all the lines of each species falling into a different cluster. No line appeared as clearly endemic in the bowels of Asturian people.

An assay was developed for the specific detection of Salmonella enterica serotype Enteritidis, using a novel application of the polymerase chain reaction (PCR). This PCR assay is based on the mismatch amplification mutation assay, an allele-specific reaction, and can discriminate Enteritidis from all other salmonella. PCR primers were selected to amplify a 351-base pair (bp) DNA fragment from the salmonella plasmid virulence A (spvA) gene of Enteritidis. A single base difference at position 272 is present between the nucleotide sequence of the spvA gene of Enteritidis and other salmonellae. The downstream PCR primer, that encompasses position 272 of the Enteritidis spvA gene, was designed to contain a single base mismatch at the penultimate position, resulting in a l-base mismatch with Enteritidis and a 2-base mismatch with other salmonellae that harbour the virulence plasmid. The upstream primer was completely homologous with the region immediately 5′ to the spvA gene. When these primers were used and the annealing and extension reactions were performed at the same temperature, the PCR assay was specific for Enteritidis; no PCR product was detected for 40 other serotypes and 28 different genera examined. In pure culture, 120 colony forming units (c.f.u.) could be detected; a PCR product was observed from template derived from a 5 h enrichment broth culture of chicken seeded with 1 c.f.u. per gram of Enteritidis. This PCR assay is specific, reproducible, and less time consuming than the standard bacteriological methods used to detect Enteritidis.

Traditionally the enteric pathogen Yersinia enterocolitica has been differentiated into biogroups. Despite being considered as non-pathogenic, biogroup 1A isolates have constituted a sizeable fraction of strains from patients with gastroenteritis in many reports. To establish a potential clinical significance for biogroup 1A isolates of Y. enterocolitica, clinical disease in patients with gastroenteritis excreting such isolates was compared with symptoms among patients found infected with pathogenic biogroups. Clinical data and isolates of 66 patients from whom Y. enterocolitica had been isolated by direct plating were available for study. There was an association between patient age below 3 years and infection with ‘pathogenic’ Y. enterocolitica. The severity of gastroenteritis and other symptoms, however, did not depend on the biogroup, or the presence of the virulence plasmid in the yersinia strain isolated from the patients. Strains belonging to biogroup 1A of Y. enterocolitica showed two clusters of ribotypes, one of which encompassed most isolates recovered from humans, the other being associated with environmental isolates. This might indicate the existence of human-adapted and potentially pathogenic strains among biogroup 1A of Y. enterocolitica.

Strains of Salmonella enterica serotype Berta, collected over a period of 6 years from a well documented natural outbreak in Denmark, have been characterized in order to assess the stability of chromosomal typing systems and virulence properties. Outbreak strains were identical in Pvu II and Pst I IS200 profiles, all but two strains showed the same Sma I ribotype, and all but one strain showed the same Not I pulsed field gel electrophoretic pattern, indicating that these molecular markers remained almost constant during the outbreak. In general, strains of S. Berta were found to be of moderate to low virulence; log VC10 values were found to vary between 3·0 and 4·4 after i.p. challenge of mice, and maximum CFU in internal organs of day-old chicks varied between 2 and 4 log10 units following oral challenge. The minor differences observed between strains in vivo did not correlate with differences in in vitro invasion into cultured MDCK cells, nor with in vitro growth characteristics. A succession of different plasmid profile types was observed during the outbreak but a hierarchical selection of clones based on differences in virulence was unlikely to have caused the succession of types of S. Berta during this outbreak.

Bivalve molluscs, (cockles, mussels, scallops and oysters) were examined according to EC shellfish bed classification regulations for faecal coliforms, Escherichia coli and salmonella, and for coliforms and campylobacter which are not specified by these regulations. Salmonella serotypes were detected in 8% of 433 molluscs. Seven salmonella isolations (2%) were made from category A beds, nominally suitable for immediate consumption according to E. coli counts. A higher percentage of salmonella isolates (6%) was detected in shellfish which require relaying or depuration prior to eating. In another survey, thermophilic Campylobacter spp. were found in 42% of 380 shellfish. These findings show that shellfish bed classification on the basis of indicator organisms alone is not sufficient to assure the absence of bacterial, and no doubt viral, pathogens. Depuration and end product specifications which require the absence of salmonellae are an essential part of these regulations. Microbiologists may wish to consider whether tests for pathogens such as salmonella and campylobacter should be included when determining the suitability of shellfish for human consumption.

Digital image processing (DIP) of bacterial smears is a new method of analysing the composition of the gut microbial flora. This method provides the opportunity to compare and evaluate differences in the complex highly concentrated anaerobic fraction of gut microbial flora, based on micromorphological differences. There is ample evidence that this fraction can be characterized as related or unrelated to the host organism by its immunogenicity. In this study germfree ND2 mice were associated with either related (rodent) SPF microflora (SPF-MF) or unrelated human MF (HUM-MF). DIP analysis was performed on original SPF-MF and HUM-MF and on the faeces of ex-germfree mice 4 weeks after association. The micromorphological pattern of highly concentrated anaerobic bacteria in faeces of HUM-MF associated ex-germfree mice was significantly different from SPF-MF associated counterparts with regard to the scores for elongation (P < 0·01) and morphological variety (P < 0·05). Moreover, gross morphological variability was present between individual HUM-MF associated mice but not between individual SPF-MF associated animals. No differences were found between original SPF and HUM-MF. The data are discussed with regard to differences in the presence of (non-)immunogenic bacteria and the ability for related and unrelated flora to colonize the murine gut. This study provides evidence that murine host specificity of microbial flora may not only be reflected in the number of non-immunogenic bacteria but also in the micromorphological pattern of highly concentrated anaerobic bacteria in faeces measured by DIP analysis.

At the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR, B) Dhaka we studied the trends in cholera for the period January 1992 to May 1995. Vibrio cholerae O139 Bengal emerged as a second aetiologic agent of cholera in Dhaka in January 1993. In 1993, the majority of cholera cases was due to V. cholerae O139, with V. cholerae O1 accounting for a small proportion of cases. During the latter part of the study period (Jan 1994–May 1995), V. cholerae O1 re-emerged as the predominant cholera strain. The predominant age group affected in endemic cholera due to V. cholerae O1 was children 2–9 years old, and the organism was isolated from more females than from males at all ages. In contrast, cholera due to V. cholerae O139 caused disease mostly in adults 15 years and older, which indicated that this organism was new in this population. As with V. cholerae O1, V. cholerae O139 was isolated from more females than males. The initial rapid emergence and predominance of V. cholerae O139 was considered possibly to herald the start of the eighth pandemic of cholera. However, just after a year, the prevalence of V. cholerae O139 decreased dramatically with V. cholerae O1 resuming the role of the dominant cholera strain. The factor(s) contributing to the dramatic decline in prevalence of V. cholerae O139 is not well understood.

Genetic relationships amongst 115 mainly Australian isolates of Mycobacterium avium were assessed using multilocus enzyme electrophoresis (MEE). The isolates were divided into 58 electrophoretic types (ETs), with a mean genetic diversity of 0·29. Isolates from humans were closely related to but distinct from those cultured from birds, whilst some porcine isolates belonged to the same ETs as certain human isolates. Pulsed field gel electrophoresis (PFGE) was used to differentiate related isolates, and those from birds and some from other animals, including pigs, were distinguished from the human isolates. The results of MEE and PFGE suggested that certain strains of M. avium may be transmitted between birds and pigs, but there was no clear evidence of transmission to humans. The serovar of the M. avium isolates was not obviously related to their ET assignment or their PFGE type.

In an outbreak of plasmid-free Salmonella enteritidis phage type 4 (PT4) food poisoning at a hospital for mentally handicapped people in July 1990, 101 residents and 8 staff were affected and a cohort study implicated beef rissoles cooked by deep-fat frying as the vehicle of infection (relative risk 2·92, 95% confidence interval 1·73–4·93, P ≪ 0·001). Replication of the cooking process demonstrated that the rissoles achieved core temperatures of only 48–60 °C despite external temperatures of 91–95 °C and an oil temperature of 142–154 °C. No residual food was available for microbiological testing but plasmid-containing S. enteritidis PT 4 was isolated in shell eggs from the hospital kitchen.