A qPCR assay to detect and quantify Shiga toxin-producing E. coli (STEC) in cattle and on farms: a potential predictive tool for STEC culture-positive farms.

Shiga toxin-producing E. coli (STEC), of various serogroups harboring the intimin gene, form a serious threat to human health. They are asymptomatically carried by cattle. In this study, a quantitative real-time PCR (qPCR) method was developed as a molecular method to detect and quantify Shiga toxin genes stx1 and stx2 and the intimin gene eae. Subsequently, 59 fecal samples from six farms were tested using qPCR and a culture method as a reference. Three farms had contaminated animals as demonstrated by the culture method. Culture-positive farms showed moderate significantly higher stx prevalences than culture-negative farms (p = 0.05). This is the first study which showed preliminary results that qPCR can predict STEC farm contamination, with a specificity of 77% and a sensitivity of 83%, as compared with the culture method. Furthermore, the presence or quantity of stx genes in feces was not correlated to the isolation of STEC from the individual animal. Quantitative data thus did not add value to the results. Finally, the detection of both stx and eae genes within the same fecal sample or farm using qPCR was not correlated with the isolation of an eae-harboring STEC strain from the respective sample or farm using the culture method.

Use of antibody responses against locus of enterocyte effacement (LEE)-encoded antigens to monitor enterohemorrhagic Escherichia coli infections on cattle farms.

Enterohemorrhagic Escherichia coli (EHEC) is a significant zoonotic pathogen causing severe disease associated with watery and bloody diarrhea, hemorrhagic colitis, and the hemolytic-uremic syndrome (HUS) in humans. Infections are frequently associated with contact with EHEC-contaminated ruminant feces. Both natural and experimental infection of cattle induces serum antibodies against the LEE-encoded proteins intimin, EspA, EspB, and Tir and the Shiga toxins Stx1 and Stx2, although the latter are poorly immunogenic in cattle. We determined whether antibodies and/or the kinetics of antibody responses against intimin, Tir, EspA, and/or EspB can be used for monitoring EHEC infections in beef cattle herds in order to reduce carcass contamination at slaughter. We examined the presence of serum antibodies against recombinant O157:H7 E. coli intimin EspA, EspB, and Tir during a cross-sectional study on 12 cattle farms and during a longitudinal time course study on two EHEC-positive cattle farms. We searched for a possible correlation between intimin, Tir, EspA, and/or EspB antibodies and fecal excretion of EHEC O157, O145, O111, O103, or O26 seropathotypes. The results indicated that serum antibody responses to EspB and EspA might be useful for first-line screening at the herd level for EHEC O157, O26, and most likely also for EHEC O103 infections. However, antibody responses against EspB are of less use for monitoring individual animals, since some EHEC-shedding animals did not show antibody responses and since serum antibody responses against EspB could persist for several months even when shedding had ceased.

Longitudinal follow-up of the persistence and dissemination of EHEC on cattle farms in Belgium.

A longitudinal survey was performed on three cattle herds known to be positive for, respectively, Enterohemorrhagic Eschericia coli (EHEC) O157, O26/O103, and O26 in a slaughterhouse study. This study aimed to investigate the persistence and dissemination of EHEC in beef cattle and beef cattle farms. At each farm, a cohort of 10 animals was sampled, seven times on farm B and eight times on farms A and C, at intervals of approximately 4-6 weeks. In addition, incoming cattle and environmental samples were also examined for the presence of EHEC at each sampling occasion. In 65 (18.8%) out of 345 samples, EHEC was detected, of which 41 were from cohort animals, four from incoming cattle and 20 from environmental samples (cats 3/23; dogs 2/7; feed 4/23, water 2/23, and dust 9/23). On two farms, non-EHEC strains harboring either vtx or eae genes were detected in 21 samples. EHEC was detected at least once in 23 of the cohort animals, with a maximum of four positive sampling occasions. Genetic typing by pulsed-field gel electrophoresis (PFGE) demonstrated that a same strain occurred for several months (up to 11 months) in two of three cattle farms. Among the environmental samples, dust harbored EHEC most frequently. In conclusion, transmission and dissemination of EHEC might have occurred not only in the bovine reservoir but also in the farm environment and in other farm animals.

Loss of vtx genes after the first subcultivation step of verocytotoxigenic Escherichia coli O157 and Non-O157 during isolation from naturally contaminated fecal samples.

Verocytotoxins VT1 and VT2,produced by Verocytotoxigenic Escherichia coli (VTEC), are encoded on temperate bacteriophages. Several studies reported the loss of the vtx genes after multiple subcultivation steps or long preservation. The objective of this study was to determine if the loss of the verocytotoxin genes can already occur during the first subcultivation step. Consequently, the stability of the vtx genes were tested in 40 isolates originating from 40 vtx-positive fecal samples after the first subcultivation step following the isolation procedure. The loss occurred in 12 out of 40 strains tested and was rather rare among the O157 strains compared to the non-O157 strains. This is the first study demonstrating that the loss of the verocytotoxin genes can already occur after the first subcultivation step. This may lead to an underestimation of VTEC positive samples.