Escherichia coli O157:H7 is now recognized as an important human pathogen. Illnesses caused by E. coli O157:H7 infection can range from self-limited, watery diarrhea to life-threatening manifestations such as hemolytic uremic syndrome or thrombotic thrombocytopenic purpura. The mode of transmission is primarily through food; however, person-to-person transmission also has been identified in some day-care center and nursing home out-breaks. Studies to date indicate that cattle are an important reservoir of the organism. Although adhesion to intestinal epithelial cells and verotoxins are considered important virulence factors in the pathogenesis of the organism, more research is are necessary to determine the exact mechanism of pathogenicity. There is need for a rapid diagnostic test for the detection of E. coli O157:H7 in food and in clinical samples. Several useful research reagents have been developed for detecting E. coli O157:H7; however, they must be applied to a procedure that is specific, sensitive, rapid, easy to use, and commercially available so that microbiological laboratories can readily use them.
A sensitive, specific procedure was developed for detecting Escherichia coil 0157:H7 in food in less than 20 h. The procedure involves enrichment of 25 g of food in 225 ml of a selective enrichment medium for 16 to 18 h at 37°C with agitation (150 rpm). The enrichment culture is applied to a sandwich enzyme-linked immunosorbent assay (ELISA) with a polyclonal antibody specific for E. coli 0157 antigen as the capture antibody and a monoclonal antibody specific for enterohemorrhagic E. coli of serotypes 0157:H7 and 026:H11 as the detection antibody. The ELISA can be completed within 3 h. The sensitivity of the procedure, determined by using E. coli 0157:H7-inoculated ground beef and dairy products, including different varieties of cheese, was 0.2 to 0.9 cell per g of food. A survey of retail fresh ground beef and farm raw milk samples with this procedure revealed that 3 (2.8%) of 107 ground beef samples and 11 (10%) of 115 raw milk samples were positive for E. coli 0157:H7. Most-probable-number determinations revealed E. coli 0157:H7 populations of 0.4 to 1.5 cells per g in the three ground beef samples. In addition to being highly specific, sensitive, and rapid, this procedure is easy to perform and is amenable to use by laboratories performing routine microbiological testing.
A monoclonal antibody (MAb 4E8C12) specific for Escherichia coli 0157:H7 and 026:H11 was produced by immunizing BALB/c mice with a rough strain of E. coli 0157:H7. The antibody reacted strongly by a direct enzyme-linked immunosorbent assay with each of 36 strains of E. coli 0157:H7. No cross-reactivity was observed with strains of Salmonella spp., Yersinia enterocolitica, Shige11a dysenteriae, Proteus spp., Escherichia hermanii, Klebsiella pneumoniae, Campylobacter jejuni, Serratia marcescens, Citrobacter spp., Enterobacter cloacae, Hafnia alvei, Aeromonas hydrophila, and all except five strains of E. coli other than serotype 0157:H7 (including strains of serotype 0157 but not H7). The E. coli strains (all of serotype 026:H11) that reacted with the antibody were enterohemorrhagic E. coli (EHEC) that were isolated from patients with hemolytic uremic syndrome or hemorrhagic colitis and produced verotoxin similar to that of E. coli 0157:H7. MAb 4E8C12 belongs to the subclass immunoglobulin G2a and has a kappa light chain. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis of outer membrane proteins of E. coli of different serotypes followed by Western immunoblot analysis revealed that MAb 4E8C12 reacted specifically with two proteins of EHEC strains of serotypes 0157:H7 and 026:H11 with apparent molecular weights of 5,000 to 6,000. These proteins appeared to be markers specific for EHEC strains of serotypes 0157:H7 and 026:H11. This MAb, because of its specificity, may be a useful reagent of an immunoassay for the rapid detection of these types of EHEC isolates in clinical and food specimens.
A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or I-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and ,-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and-negative sera. The data
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