Out of 174 bovine Shiga toxin-producing Escherichia coli (STEC) strains isolated from diarrheic calves in Germany and Belgium, 122 strains (70.1%) were selected because of their reactivity with the eae (E. coli attaching and effacing gene) probe ECW1-ECW2. One hundred seven of these eae-positive strains (87.7%) harbored stx1 genes, 13 strains (10.7%) had stx2 genes, and 2 strains (1.6%) had both stx genes. The strains displayed 17 different O types, the majority (97 strains [79.5%]) belonging to O5 (5 strains), O26 (21 strains), O111 (13 strains) O118 (36 strains), O145 (9 strains), and O157 (13 strains). In the HEp-2 cell adhesion assay, 99 strains (81.1%) showed a localized adhesion, and 80 strains (65.6%) stimulated actin accumulation, as determined in the fluorescence actin staining test. None of the strains harbored genes coding for bundle-forming pili (bfpA), clearly differentiating them from enteropathogenic E. coli. espB gene sequences were only detectable in 23 (18.9%) of the eae-positive bovine STEC strains. Three different PCRs were established, differentiating between eae sequences of enteropathogenic E. coli strain E2348/69 (O127:H6) and STEC strain EDL933 (O157: H7). Primers matching in the more heterologous downstream eae sequences gave amplicons in only 8 of the 17 O types (O84:

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Bovine vaginal cytobrush specimens were analyzed for the presence of Chlamydia spp. by a high-sensitivity, high-specificity quantitative PCR. The 53% prevalence of low-level Chlamydia psittaci and C. pecorum genital infection detected in virgin heifers suggests predominantely extragenital transmission of Chlamydia in cattle and conforms to the high seroprevalence of anti-Chlamydia antibodies.Over the last 40 years, evidence has accumulated to suggest the ubiquitous presence of infections with intracellular bacteria of the genus Chlamydia in cattle and other livestock species. Despite some improvement in diagnostic techniques, our understanding about the prevalence and pathogenetic significance of these infections, succinctly reviewed by Shewen (11) in 1980, has not substantially changed since that time. In cattle, enzyme-linked immunosorbent assay examinations of sera for the antibody against Chlamydia psittaci suggest a high level of exposure to C. psittaci (6,8,10). The application of nested PCR to bovine clinical specimens substantiated such widespread, but mostly clinically inapparent, presumably low-level infections (3, 7), similar to the findings for human C. pneumoniae infections (1). However, due to high technical demands, these PCR methods were rarely transferred from research settings to systematic epidemiological investigations and diagnostic use. A simple, highly specific, fluorescentprobe-based single-tube LightCycler quantitative PCR (qPCR) platform was recently developed for the detection of Chlamydia DNA. This platform optimizes sample nucleic acid preservation, extraction, and recovery, as well as qPCR methodology, for maximum sensitivity in the detection of Chlamydia (2). This has opened the possibility for the sensitive routine diagnosis of chlamydial infection as well as for systematic epidemiological studies. Such investigations would benefit from both the high sensitivity and the ability to ascertain quantitative differences in chlamydial burdens between animals, which may be required to understand disease mechanisms. In this initial epidemiological application of the Chlamydia qPCR platform, we addressed the question of chlamydial infection of the bovine genital tract in animals that had not previously been exposed to the possibility of sexual transmission of chlamydiae. We report here a high prevalence of genital tract infection with C. psittaci and C. pecorum in clinically normal virgin cattle.A herd of 51 virgin Holstein heifers, 14 to 16 months old, was sampled four times at weekly intervals. These animals were clinically normal, but low-grade vaginitis was common.Vaginal cytobrush specimens (Histobrush; Fisher Scientific, Suwanee, Ga.) were obtained by 10-s rotation in the vaginal vestibulum, brushes were immediately transferred to 400 l of RNA-DNA stabilization reagent (Roche Applied Science, Indianapolis, Ind.) in a 1.5-ml screw-cap microcentrifuge tube, samples were stirred, and the brushes were cut off. Brushes were removed after a 5-min centrifugation at 3,000 ϫ g at room tempera...

Shiga toxin-producing Escherichia coli (STEC) is widespread in the cattle population, but the clinical significance of Shiga toxins (Stx’s) for the bovine species remains obscure. Since Stx’s exert immunomodulating effects in other species, we examined the effect of purified Stx1 on a bovine B lymphoma cell line (BL-3) and peripheral blood mononuclear cells (PBMC) isolated from adult bovine blood by viability assays and flow cytometry analysis. Stx1 markedly induced apoptosis in stimulated BL-3 cells. The susceptibility of this B-cell-derived cell line was induced only by either lipopolysaccharide (LPS) or pokeweed mitogen, while cultures stimulated with T-cell mitogens were unaffected by the toxin. In contrast, Stx1 did not induce cellular death—neither apoptosis nor necrosis—in primary cultures of PBMC but hindered the mitogen-induced increase in metabolic activity. The influence of Stx1 on single PBMC subpopulations varied with the type of mitogenic stimulus applied. Stimulation with phytohemagglutinin P particularly induced the proliferation of bovine CD8-expressing (BoCD8+) cells, and this proliferative response was blocked by Stx1. On the other hand, Stx1 reduced the portion of viable B cells in the presence of LPS. Modulation of activation marker expression (BoCD25 and BoCD71) by Stx1 indicated that the toxin hindered the proliferation of cells by blocking their activation. In conclusion, we assume that Stx1 contributes to the pathogenesis of STEC-associated diarrhea in calves by suppressing the mucosa-associated immune response. The usefulness of cattle as a model in which to study Stx-induced immunomodulation is discussed.