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Since C. pseudotuberculosis is a facultative intracellular pathogen the aim of this study was focused on evaluating mechanisms that allowed these bacteria to survive in macrophages and determining their influence on induction of cell death. The influence of Corynebacteria on the programmed cell death of macrophages was determined on the basis of induction the autophagy and apoptosis in the cultures of murine macrophage cell lines J774 infected with bacteria. Corynebacterium pseudotuberculosis strains could survive within macrophages more than 48 hours. During that time bacteria were released as a result of the process that lead to death of phagocytes. This property varied among studied strains. There was no increase of micro- tubule-associated protein I light chain 3 (MAP I LC3) activity in macrophages infected with examined strains comparing with uninfected cultures and cultures treated with autophagy inducer (rapamycin) that served as negative and positive controls, respectively. The study with confocal microscopy did not show the increasing of caspase-3 activity in the infected macrophages and their nucleus did not reveal the fragmentation.
Epidemiological studies on caseous lymphadenitis were carried out in Poland in 1996 and 2002 among goat herds covered by a milk recording program. Between-herd seroprevalence was 13.2% in 1996 and increased to 62.5% in 2002. The average size of seropositive herds was statistically significantly higher than that of seronegative ones, however there was no statistically significant difference in the age between the herds. A statistically significant prevalence ratio (PR) was identified and relevant attributable risk for exposed animals (ARexp) was calculated for the following risk factors: presence of seropositive males in a herd (PR=8.350; ARexp=0.651), presence of superficial abscesses in animals (PR=6.142; ARexp=0.620), presence of respiratory signs (PR=2.900; ARexp=0.393), presence of animals in poor condition in a herd (PR=2.774; ARexp=0.390) and occurrence of reproductive failures in a herd (PR=1.798; ARexp=0.230). Purchase of animals from abroad, mastitis and husbandry conditions (housing system, grazing system, hygienic conditions) were not shown to be statistically significant risk factors.
The aim of the present study was the phenotypic and genotypic characterization of Corynebacterium pseudotuberculosis strains isolated from goats with symptoms of caseous lymphadenitis, derived from herds in Poland. Sixty one isolates were studied and their morphology of colonies and cells, biochemical properties, production of phospholipase D were determined. All strains were genotyped by ADSRRS-fingerprinting. The results of the study showed the significant variability in biochemical and genetic properties of C. pseudotuberculosis strains. Among all isolates, 11 distinct biochemical groups were determined. All strains produced phospholipase D, the main virulence factor of C. pseudotuberculosis, and all but one isolate had no ability to reduce nitrate to nitrite. We also found a variability in the activity of alkaline phosphatase, alfa-glucosidase, pyrazinamidase and fermentation of maltose and sucrose. Genotyping of strains showed 10 distinct genome patterns consisting of 11 to 20 bands between approximately 1400-100 bp. The obtained results indicate that ADSRRS-fingerprinting technique could be a useful tool for the screening of genome differentiation and establishing relationships between clinical isolates of C. pseudotuberculosis.
Phenotypic approaches based on metabolic and biological characteristics of Corynebacterium pseudotuberculosis have been limited due to insufficient discrimination between closely related isolates. In this paper we present performance and convenience of three molecular typing methods: BOX-PCR, random amplification of polymorphic DNA (RAPD) and amplification of DNA fragments surrounding rare restriction site (ADSRRS-fingerprinting) in genome analysis of these bacteria. Among examined 61 strains there were distinguished four, eight and 10 different genotypes by BOX-PCR, RAPD and ADSRRS-fingerprinting, respectively. The value of discrimination index was the lowest for BOX-PCR (D = 0.265), much bigger for RAPD (D = 0.539) and the highest for ADSRRS-fingerprinting (D = 0.604). The good discriminatory ability and reproducibility of RAPD and ADSRRS-fingerprinting indicates that those techniques may be particularly applied for epidemiological studies of C. pseudotuberculosis isolates. We found that ADSRRS-fingerprinting is a rapid method offering good discrimination power, excellent reproducibility and may be applied for epidemiological studies of intraspecific genetic relatedness of C. pseudotuberculosis strains.
Coryneform bacteria occur in different environments: soil, water, plants, organisms, humans and animals. Many of them are a part of the normal flora of human and animals while others are opportunistic bacteria. Coryneform bacteria are not new microbes, but their role in infections of humans and animals is still underestimated. The genus of Corynebacterium comprises more than 70 species, including 43 of clinical significance. Among the known Corynebacterium species are distinguished: human and animal pathogens, commensal colonizing of the skin and mucous membranes of the respiratory system and genital tract, as well as plant pathogens and saprophytes living in an abiotic environment (plant debris, soil, water). Predisposing factors for Corynebacterium spp infection include: immunosuppression, long-lasting and broad-band spectrum antibiotics, steroids, an age of over 65-years-of-age, ischemic heart disease, kidney failure, respiratory failure, diabetes, cancer, multi-organ injuries, infections by HIV and CMV viruses, prematurity, tears of the skin and mucous membranes, and invasive medical procedures. The paper presents the occurrence and virulence factors of Corynebacterium spp. Infections caused by Corynebacterium spp. Their resistance to antibiotics are also described.
In the study, phenotypic and genotypie properties of 42 Corynebacterium pseudotuberculosis strains isolated from 16 goat flocks were determined by random amplified polymorphic DNA and amplified fragment length polymorphism techniques. Several sets of RAPD primers and AFLP protocols were experimentally evaluated for potential of generating amplification profiles of the best discriminatory power. The overall similarity level of profiles by cluster analysis generated with RAPD3 (64%) set was higher than obtained for both RAPD 2 (70%) and AFLP (88%). The results confirmed usefulness of both AFLP and RAPD methods to discriminate strains of C. pseudotuberculosis, even at the flocks' level, and confirmed a single genotype as the most prevalent in goats coming from flocks in Poland.
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