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The aim of the study was to develop a sensitive and specific ELISA for the detection of LT enterotoxin in E. coli strains. The test was based on the antitoxic antibodies obtained from rabbits immunized with В subunit of cholera toxin (CT). The toxin was structurally closely related with LTB of E. coli. CTB was expressed in a V. cholerae strain harboring a plasmid with a ctxB gene. The toxin was isolated and purified from a growth medium by means of precipitation. A goat antirabbit IgG labeled with HRP antibodies was used as a conjugate. The developed test turned out to be highly specific and sensitive: it was possible to detect from 1.5 to 4.0 ng of LTB. One hundred strains of E. coli isolated from piglets were tested by the LT ELISA. It was found that 67 strains (85.9%) isolated from piglets with diarrhea were LT+ whereas only 2 strains (9.1%) from healthy piglets proved to be enterotoxigenic.
E. coli strains producing Shiga toxins (STEC) are the cause of hemorrhagic colitis, hemolytic uremia syndrome (HUS) and thrombotic thrombocytopenic purpura in humans. The source of these bacteria is usually cattle that are asymptomatic carriers of STEC. Infection of humans is usually by the oral route after consuming food of animal origin, especially beef, contaminated with these microorganisms. Monitoring the presence of STEC in animals and food is very important in determining the source of infection as well as in epidemiological analyses. In the present study a method of STEC identification was applied which utilized the enrichment step of the meat samples and then determined the conserved fragment of the stx gene encoding the production of Shiga toxins. For this purpose a multiplex PCR test was established, that allowed amplification of the stx marker as well as the 16S rRNA E. coli gene, which was used as an internal amplification control. As a result of the reaction, two amplicons were obtained of sizes 230 bp (stx) and 401 bp (16S rRNA), respectively when STEC bacteria were present in the tested meat. The study used 221 beef samples obtained from animals directly in slaughter houses, and 13 (5.9%) positive results were obtained. These results may suggest that beef may be a source of STEC, which, in turn, are a potential threat for human health.
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