The presence of subAB was investigated for 3,453 Escherichia coli strains of various pathogenic categories. The occurrence of other virulence genes in subAB-positive strains was investigated. The subAB operon was detected among some Shiga toxin-producing E. coli (STEC) serotypes devoid of eae and carrying ehxA. Most subAB-positive strains also harbored stx 2 , iha, saa, and lpfA O113 .Subtilase cytotoxin, a new member of the AB 5 toxin family, was identified for the first time in 2004 in a virulent O113:H21 Shiga toxin-producing Escherichia coli (STEC) strain that caused an outbreak of hemolytic-uremic syndrome in South Australia (16,18). The presence of subAB genes was further detected in other STEC strains belonging to different serotypes (19). Subsequently, subAB genes were identified among STEC strains isolated in other countries (3,8,9,14,25).To evaluate how widely distributed the subAB operon is, we studied a large collection of STEC serotypes from nonhuman sources and E. coli strains of different pathogenic categories associated with human infections. The subAB-positive strains were further characterized regarding the presence of other virulence genes.A total of 2,255 E. coli strains isolated from humans and belonging to enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), extraintestinal pathogenic E. coli (ExPEC), and E. coli strains not belonging to the diarrheagenic categories described so far were randomly selected. STEC strains isolated in Brazil from humans have previously been tested for the presence of subAB by our group (3). The 1,198 STEC strains from nonhuman sources were isolated from dairy cattle, beef cattle, buffaloes, and goats. Overall, 109 different STEC serotypes were tested. An STEC strain of serotype O113:H21 (3) was used as a reference strain for subAB, cdt-V, and lpfA O113 , and E. coli strain DH5␣ was used as a negative control.The strains were screened for the presence of the subAB operon (encoding subtilase cytotoxin) using colony hybridization assays (21). The 1,823-bp subAB-specific DNA probe was derived from the STEC serotype O113:H21 (3) strain by PCR as previously described (19). Hybridization assays were performed under stringent conditions, and the probe was labeled with [␣-32 P]dCTP (Amersham), using the Ready-To-Go DNA labeling kit (Amersham). All strains which yielded a positive or weak signal in hybridization assays with the subAB probe were retested by PCR (18,19), and only those confirmed by PCR were considered to be carrying this sequence.The genetic profiles of the subAB-positive strains were determined using our previously reported data for the same strains (6,7,12,13,20,24) regarding the presence of the ehxA, eae, stx 1 , stx 2 , and adhesin-encoding genes (1,10,11,15,17,22,23).A total of 130 STEC strains carrying the subAB operon, representative of each serotype and isolated from different animals, were analyzed by PCR for the presence of genes encoding Lpf O113 and cytolethal distendi...

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BackgroundShiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. Stx is essential in the pathogenesis of HUS, which has been mostly related to Stx2-producing isolates. Very limited data exist on the immune response to STEC in the Brazilian population. In this study, the prevalence of immunoglobulin G (IgG) antibodies to Stx2 was investigated in sera of children diagnosed with HUS and of healthy children in the city of São Paulo, Brazil.MethodsIgG-antibody reactivity to Stx2 was determined by immunoblotting (WB) and enzyme-linked immunosorbent assay (ELISA) in sera from 13 children with HUS aged 8 months to 6 years and 54 healthy urban children aged 5 months to 7 years.ResultsA positive immune response to the A and B subunits of Stx2 was observed in 46.1% HUS patients and in 16.6% healthy individuals by WB. All HUS patients and 62.9% healthy children showed IgG antibodies to the Stx2 A subunit. The frequency of antibodies to both subunits or only to the A subunit of Stx2 was significantly higher in HUS patients than controls (p < 0.05). Also, the mean OD value obtained by ELISA was higher in that group. Considering children’s age, the frequency of reactivity to either the A subunit or both subunits of Stx2 was considerably higher in HUS children up to three years old compared to controls in the same age range. Moreover, in almost 37% of healthy children, no immune response to Stx2 was detected independently of the child’s age.ConclusionsThe seroepidemiolgy of anti-Stx2 antibodies was described for the first time in healthy children and children with HUS in Brazil. The percentage of individuals showing antibodies against Stx2 was higher among HUS patients than controls, and in spite of the low number of notified HUS cases, STEC strains are circulating in our settings. In addition, the results obtained also corroborated previous data on the increased sensitivity and specificity of WB compared to toxin-based enzyme immunoassays.

Little is known on the epitopes derived from metacyclic promastigotes of Leishmania that are important on the regulation or destruction of the parasite, as targets of immune attack in the vertebrate host. In this study we investigated an alternative method to obtain metacyclic promasigotes of Leishmania major, as evaluated by the course of infection and delayed-type hipersensitivity (DTH) in resistant versus susceptible inbred mice. Non-infective (procyclic) promastigotes of L. major recently transformed from tissue amastigotes were attached to a negatively charged glass-wool column, whereas metacyclic promastigotes were not bound to columns and could be easily recovered. Optimal chromatography conditions were validated through statistical analyses. Parasite average yield from glass wool columns and promastigote viability were estimated by light microscopy. Metacyclic promastigotes yielded 43.5% to 57.5%. Different patterns of cutaneous lesions were obtained in BALB/c (susceptible) and C57BL/6 (resistant) mice, the former with highly infective lesions induced by metacyclic promastigotes. DTH responses proved to be higher in groups of C57BL/6 mice which were infected with metacyclic promastigotes. These results indicate that the new method could be integrated with the investigation of metacyclogenesis of Leishmania in vivo.