Mitotic catastrophe as a prestage to necrosis in mouse liver cells treated with Helicobacter pullorum sonicates.

Helicobacter pullorum infections have been associated with several enterohepatic diseases, but the mechanism of action is currently undefined. The present study was therefore set up to investigate possible cytotoxic effects of this pathogen on liver cells. A mouse hepatic cell line was exposed to H. pullorum sonicate and cytotoxicity was observed for all isolates after incubation for 72 h. Features characteristic for mitotic catastrophe characterized by chromatin condensation, formation of multinuclear distended cells and micronucleation, were recorded. In addition, intranuclear pseudoinclusions were seen in sonicate-treated cells. Finally, cells exposed to sonicate eventually underwent cell death with the morphological features of necrosis, which occurred without activation of caspase-3. The toxic factor involved in the cytotoxic activity proved to be soluble, trypsin-sensitive and stable at 56 degrees C and at -70 degrees C with a molecular weight to be over 50 kDa. The current study shows for the first time that H. pullorum causes mitotic catastrophe resulting in primary necrosis in mouse hepatocytes.

Pathogenesis of Helicobacter pullorum infections in broilers.

Four groups of 23 one-day-old broiler chickens were each inoculated by gavage with a different Helicobacter pullorum strain isolated from humans or poultry. As a control, a fifth group of eight animals was inoculated with phosphate-buffered saline. Faecal samples were collected weekly and tested for the presence of H. pullorum DNA using PCR. At 1, 8, 15, 22 and 42 days postinoculation, birds were euthanized and samples from the liver and intestinal tract were histologically, immunohistochemically and bacteriologically examined. The samples were also tested for the presence of H. pullorum DNA by PCR. All animals remained clinically healthy throughout the experiment although mild lesions in the caeca were present in animals inoculated with H. pullorum. In all H. pullorum-inoculated groups, DNA of this bacterium was detected in faecal samples until 42 days postinoculation. The main site of colonization was the caecum. Immunohistochemical examination revealed that the bacterium was closely associated with the caecal epithelial cells. It was concluded that H. pullorum may colonize the caecum of broilers and is excreted in their faeces until slaughter age. This implies that chicken meat might constitute a source of infection for human beings.

Helicobacter pullorum in chickens, Belgium.

A total of 110 broilers from 11 flocks were tested for Helicobacter pullorum by polymerase chain reaction; positive samples were reexamined with a conventional isolation method. H. pullorum isolates were examined by amplified fragment length polymorphism (AFLP) fingerprinting for interstrain genetic diversity and relatedness. Sixteen isolates from cecal samples from 2 different flocks were obtained. AFLP analysis showed that these isolates and 4 additional isolates from a different flock clustered according to their origin, which indicates that H. pullorum colonization may occur with a single strain that disseminates throughout the flock. Strains isolated from different hosts or geographic sources displayed a distinctive pattern. H. pullorum is present in approximately one third of live chickens in Belgium and may represent a risk to human health.

Cytolethal distending toxin generates cell death by inducing a bottleneck in the cell cycle.

Cytolethal distending toxin (CDT) is a bacterial protein that is widely distributed among gram-negative bacteria including Escherichia coli, Campylobacter spp., enterohepatic Helicobacter spp., Actinobacillus actinomycetemcomitans and Haemophilus ducreyi. In vitro studies demonstrated that it is able to stop proliferation of various cell lines. The toxin is composed of three subunits designated CDTs A, B and C. The B subunit targets the eukaryotic DNA and triggers a signalling pathway involving different protein kinases which results in a cell block before entering into mitosis. To date, the individual role of the A and C subunits has not been totally elucidated. There are indications that the CDT is also produced in vivo. Its exact role in pathogenesis is not yet clear, but possible actions include inhibition of epithelial cell proliferation, apoptosis of immune cells and inhibition of a fibrotic response.

The cytolethal distending toxin among Helicobacter pullorum strains from human and poultry origin.

Helicobacter pullorum has been associated with diarrhoea, gastroenteritis and liver disease in humans and with hepatitis and enteritis in poultry. The purpose of the present study was to examine whether cytolethal distending toxin was present among 10 poultry and three human H. pullorum isolates and whether a different level of cytolethal distending toxin activity was noted. A PCR assay was performed to detect the cdtB gene. In addition, epithelial Hep-2 cells inoculated with sonicate from all strains were observed microscopically and DNA analysis of these cells was done by flow cytometry. All H. pullorum isolates harboured the cdtB gene, but functional cytolethal distending toxin activity was only demonstrated in the human H. pullorum strain CCUG 33839. A significant number of cells treated with sonicate from this strain were enlarged. The nuclei were distended proportionally. Giant cells and multinucleated cells were observed as well. In addition, stress fibers accumulated. DNA analysis by flow cytometry revealed 31.0% of these cells at the S/G2 stage of the cell cycle. The tested poultry and human H. pullorum isolates all possess the cdtB gene, but under the circumstances adopted in this study only the human strain CCUG 33839 seems to show biological activity typically for CDT in vitro.