Verotoxin-producing Escherichia coli isolates from feces of healthy cattle were identified by DNA hybridization with verotoxin 1-and verotoxin 2-specific gene probes. Among 259 animals investigated, 28 (10.8%) were found to carry verotoxin-producing E. coli strains. Characterization of the verotoxin-producing isolates revealed a heterogeneous population in terms of serotype and toxin type. Nearly 40% of the strains belonged to serogroups known to be pathogenic for humans, i.e.,

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A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an Escherichia coli strain, ONT:H19, isolated from bovine feces. The complete stx 1 gene of this strain was amplified and sequenced. Nucleotide sequence homology with stx 1 from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein. Cell culture supernatant of this strain, which was negative for stx 2 by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx. PCR primers were constructed for the specific detection of the new variant. The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods. Shiga toxins (Stx) are highly potent cytotoxins and essential virulence factors of enterohemorrhagic Escherichia coli (EHEC).Based on antigenic, cytotoxic and genotypic differences, two major types of these toxins, Stx1 and Stx2, are known, both of which are phage encoded. Stx2 includes several subtypes, Stx2c (26), Stx2d (22), Stx2e (30), and Stx2f (25) being the most important ones. In contrast, Stx1 is rather homogenous and basically identical with Shiga toxin of Shigella dysenteriae, only one amino acid is replaced and three nucleotide changes were detected (28). However, during the last decade, several minor variants of Stx1 have been described as well. Paton et al. (20,21) identified three variants which differed by two amino acids in the A subunit. Nucleotide sequence homology of these variants with stx 1 of phage 933J was more than 99%. A more substantial deviation was observed only in strain

In the present study, a robust TaqMan real-time PCR amplifying the F57 and the ISMav2 sequences of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples was developed and validated. The validation was based on the recommendations of International Organization for Standardization protocols for PCR and real-time PCR methods. For specificity testing, 205 bacterial strains were selected, including 105 M. avium subsp. paratuberculosis strains of bovine, ovine, and human origin and 100 non-M. avium subsp. paratuberculosis strains. Diagnostic quality assurance was obtained by use of an internal amplification control. By investigating six TaqMan reagents from different suppliers, the 100% detection probability was assessed to be 0.1 picogram M. avium subsp. paratuberculosis DNA per PCR. The amplification efficiency was 98.2% for the single-copy gene F57 and 97.8% for the three-copy insertion sequence ISMav2. The analytical method was not limited due to instrument specificity. The triplex real-time PCR allowed the reliable detection of M. avium subsp. paratuberculosis DNA using the ABI Prism 7000 sequence detection system, and the LightCycler 1.0. TaqMan mgb and locked nucleic acid fluorogenic probes were suitable for fluorescent signal detection. To improve the detection of M. avium subsp. paratuberculosis from bovine fecal samples, a more efficient DNA extraction method was developed, which offers the potential for automated sample processing. The 70% limit of detection was assessed to be 10 2 CFU per gram of spiked bovine feces. Comparative analysis of 108 naturally contaminated samples of unknown M. avium subsp. paratuberculosis status resulted in a relative accuracy of 98.9% and a sensitivity of 94.4% for fecal samples containing <10 CFU/g feces compared to the traditional culture method.Mycobacterium avium subsp. paratuberculosis is the causative agent of ruminant paratuberculosis (Johne's disease), which has become a worldwide problem. There is controversy regarding its zoonotic capacity and potential role in the human Crohn's disease (14). Because of these reasons, a rapid, costeffective, and automated diagnosis of this pathogen is a high priority task not only for animal breeders but also for the food production industry and for public health institutions. Culturebased detection of M. avium subsp. paratuberculosis is timeconsuming, labor-intensive, and therefore not suitable. The PCR has been shown to be a powerful tool in microbiological diagnostics (12,43). Guidelines for diagnostic quality assurance have been set by the International Organization for Standardization (7,8). Standardized PCR and real-time PCR methods should fulfill numerous criteria, including a high detection probability with regard to the investigated matrix, the sample preparation, and DNA extraction as well as high specificity, robustness, and user-friendly protocols. In this context the real-time PCR technology offers the possibility for a onestep and closed-tube reaction (13).As a molecular reference marker for the confirm...

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