Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.Although Escherichia coli belongs to the normal microflora present in the gastrointestinal tracts of most mammals and birds, certain E. coli strains have been associated with intestinal or extraintestinal infections. Among these pathogenic E. coli strains, enteropathogenic E. coli (EPEC) is a major cause of infant diarrhea in developing countries (for a recent review, see reference 51) and is a significant category of diarrheagenic E. coli in different animal species. In addition, EPEC is an important cause of morbidity and mortality in weaned rabbits (5,54,56). EPEC is also pathogenic in neonatal calves (20,53) and seems to be isolated more frequently in farms with recurrent diarrhea (7). In swine, EPEC is involved in cases of postweaning diarrhea (67). There is also increasing evidence for a diarrheagenic role of EPEC in dogs (16,64). Finally, EPEC has been isolated ...
Flavobacterium columnare (Flexibacter columnaris) is an important cause of gill and skin disease in freshwater fish species, often causing high mortality. In previous studies, virulence of F. columnare was correlated with the ability to adhere to the gill tissue. To gain insight into the factors responsible for adherence, a gill perfusion model was used. The bacterial cells of the high virulence strain AJS 1 were exposed to various treatments, after which they were added to the organ bath of an isolated gill arch and adherence to the gill tissue assessed. Adherence capabilities were significantly reduced following treatment of the bacteria with sodium metaperiodate or incubating them with d‐glucose, N‐acetyl‐d‐glucosamine, d‐galactose and d‐sucrose. Incubation of the bacteria with trypsin and pronase did not significantly inhibit adherence. The binding sites for F. columnare on the gill tissue were also partially characterised. Treatment of the gill with sodium metaperiodate reduced adhesion, but treatment with pronase or trypsin did not cause any significant reduction, indicating that the major component of the receptor is of carbohydrate nature. Adherence ability of the bacteria correlated well with their haemagglutination capacity using chicken and guinea pig erythrocytes. Higher haemagglutination titres were obtained with the highly virulent strain AJS 1 than with strain AJS 4, a strain with low virulence and adherence capacity. Haemagglutination was partially inhibited after incubation of the bacteria with d‐glucose and N‐acetyl‐d‐glucosamine and after treatment of the bacteria at 41_°C for 10_min (minor heat treatment). It was completely abolished following incubation of the bacterial cells with sodium metaperiodate and intensive heat treatment (65_°C, 25_min). Haemagglutination was also in‐sensitive to pronase and trypsin treatment. Transmission electron microscopy (TEM) revealed that the high virulence strain had a thick capsule (120–130_nm) with a regular, dense appearance, whereas the capsule of the low virulence strain was much thinner (80–90_nm) and less dense. TEM also demonstrated the loss of the capsule of the high virulence strain after treatment of the bacterial cells with minor heat and sodium metaperiodate. These results indicate that a lectin‐like carbohydrate‐binding substance incorporated in the capsule is responsible for the attachment of F. columnare to the gill tissue.
Structural aspects of the bovine zona pellucida (ZP) of in vitro-matured (IVM) oocytes and in vitro-produced (IVP) embryos were studied in two experiments to find a tentative explanation for the zona's barrier function against viral infection. In Experiment 1, the ultrastructure of the outer ZP surface was studied. The diameter (nm) and the number of the outer pores within an area of 5000 microm(2) of 10 IVM oocytes, 10 zygotes, 10 8-cell-stage embryos, and 10 morulae were evaluated by scanning electron microscopy. In oocytes and morulae, the ZP surface showed a rough and spongy appearance with numerous pores. In zygotes, the ZP surface was found to have a smooth, melted appearance with only a few pores. In 8-cell-stage embryos, both surface patterns were found. The mean number (per 5000 microm(2)) and the mean diameter of the outer pores were different between the four stages of development (P < 0.001): 1511 pores in oocytes, 1187 in zygotes, 1658 in 8-cell-stage embryos, and 3259 in morulae, with mean diameters of 182, 223, 203, and 155 nm, respectively. In Experiment 2, the continuity of the meshes (network of pores) towards the embryonic cells was examined by confocal laser scanning microscopy. Therefore, the passage through and the location in the ZP of fluorescent microspheres, with similar dimensions as bovine viral diarrhea virus (BVDV, 40-50 nm) and bovine herpesvirus-1 (BHV-1; 180-200 nm), were evaluated. For all stages, the smallest beads were detected halfway through the thickness of the ZP, whereas the beads with a size of 200 nm were found only within the outer-fourth part of the ZP. It can be concluded that the intact ZP of bovine IVM oocytes and IVP embryos are constructed in such a way that BVDV and BHV-1 should not be able to traverse the ZP and reach the embryonic cells. However, the risk exists that viral particles can be trapped in the outer layers of the ZP.
The ability of Flavobacterium columnare (Flexibacter columnaris) to attach to the gills of common carp, Cyprinus carpio L., was evaluated using a gill perfusion model. A comparison between a high and a low virulence strain of F. columnare was made and evaluated in comparison to results obtained previously with an in vivo model. The ion composition of the water of the organ bath in which the gills were suspended was varied and the influence on adhesion processes assessed. Experiments were carried out to examine the influence of water quality (i.e. nitrite and organic matter) and temperature on the capacity of the bacteria to adhere. It was found that the high virulence strain adhered more readily than the low virulence strain, as was found during the in vivo experiments. Moreover, it was observed that adhesion of the high virulence strain was enhanced by a number of factors. These were immersion of the gill in bivalent, ion‐rich water, the presence of nitrite or organic matter, and high temperatures.
Enterotoxigenic Escherichia coli strains express fimbriae which mediate binding to intestinal mucosal cells. The F17 fimbriae mediate binding to N-acetylglucosamine-containing receptors present on calf intestinal mucosal cells. These fimbriae consist of F17-A subunit peptides. Analysis of the F17 gene cluster indicated that at least the F17-A, F17-C, F17-D, and F17-G genes are indispensable to obtain adhesive F17 fimbriae (unpublished data). Genetic evidence is presented that the F17-G protein, a minor fimbrial component, is required for the binding of the F17 fimbriae to the intestinal villi. The F17-G gene was cloned and sequenced. An open reading frame of 1,032 bp encoding a polypeptide of 344 amino acids, starting with a signal sequence of 22 residues, was localized. The F17-G mutant strain produced F17 fimbriae which were morphologically identical to the fimbriae purified from strains which contained the intact F17 gene cluster. However, this F17-G mutant could no longer adhere to calf villi. The F17-G locus was shown to act in trans: transformation of the F17-G mutant strain, still expressing the genes F17-A, F17-C, and F17-D, with a vector expressing the F17-G gene restored the binding activity of this mutant strain.
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