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The present study investigated the effectiveness of three different disinfectants: preparation H1 (two-component preparation based on hydrogen peroxide); Pedox (multi-component preparation based on peroxyacetic acid) and Savo hypochlorite preparation) against Malassezia pachydermatis. The antifungal activity of disinfectants was tested by quantitative suspension method according to STN EN 1650. The results confirmed 100% effectiveness of these disinfectants at all concentrations and exposure times tested.
Considering the fact that certain phenotypic properties found in M. pachydermatis strains can indirectly indicate their pathogenicity, we have evaluated the morphological traits of the fungus (cell size and shape as well as colony type), expression of extracellular hydrolases (API-ZYM) and the activity of catalase, urease, casein, beta-glucosidase, phospholipase and the Tween hydrolases: 20, 40, 60 and 80. Considering the source of the analyzed strains’ origin as a differentiation criterium (animals clinically healthy and with otitis externa symptoms), it was found that the isolates obtained from the pathological material were generally (80%) characterized with a smooth growth type of light cream, large or medium-sized colonies, as well as high a activity of lipolytic enzymes, such as phospholipase and hydrolase Tween 80 and valine arylamidase, cystine arylamidase and basic phosphatase. Determining a profile of M. pachydermatis phenotypic characteristics may substantially facilitate defining the pathogenicity of the strains isolated directly from the clinical material.
Yeast like fungi of Malassezia pachydermatis species are numbered among the opportunistic agents accounting for acute dermatoses or even systemic infections in people and animals. They exhibit the clear heterogeneity pertaining to the phenotypic traits and growth requirements. The objective of the present study was to determine the degree of variation as well as the biochemical profiles of M. pachydermatis strains. The studies were conducted on 40 strains isolated from the auditory canal of healthy dogs and those with otitis externa symptoms recognized. An assessment was conducted of the traits of fungus morphology (size and shape of cells and colony type), expression of extracellular hydrolases (API-ZYM) and the activity of catalase, urease, caseinase, beta-glucosidase, phospholipase and Tween hydrolases: 20, 40, 60 and 80. The obtained results made it possible to determine the general metabolism pattern for this species: no fermentability or capacity for assimilation of most carbohydrates, poor proteolytic properties, high activity of enzymes from the phosphatase and lipolytic enzymes groups. On the grounds of the statistical analysis, the examined strains were classified into 7 separate groups of congenial morphology and a determined level of enzymatic activity. Biotype I includes large, smooth and creamy-white colonies of high metabolic activity; moreover, they exhibit high sensitivity to the inhibitory operation of Tween 20 and Cremophor EL; biotype II exhibits a rough type of colony growth, the lowest metabolic activity and a lack of expression of lipase C14 and enzymes from the arylamidase group; biotype III comprises the strains of large, smooth colonies, poor total enzymatic activity and with no activity of cystynic arylamidase; biotype IV includes the strains of small and smooth colonies, average metabolic activity but with optimal usability of Tween 40 and 60 hydrolysis products for growth; biotype V groups the strains of the average metabolic activity and without phospholipase activity recorded; biotype VI comprises the strains of smooth, large or medium-sized colonies and a relatively high enzymatic activity as well as the highest level for alkaline phosphatase and valine arylimidase, phospholipase, catalase, caseine, Tween 80 hydrolase; biotype VII is characterized by the highest total enzymatic activity, high capacity for eskulin break-down, Tween 40, 60 and 80 hydrolysis. Further studies are needed to demonstrate a correlation between the strain classification into a defined biotype and its ecologic or clinical status.
The aim of the study was to make an attempt at showing the intraspecies heterogenecity of Malassezia pachydermatis strains with regards to their origin (strains isolated from healthy dogs and with otitis externa symptoms). The study included 41 strains of Malassezia pachydermatis species isolated in a pure culture from dogs with clinical otitis externa symptoms (n = 20), clinically healthy dogs (n = 20) and a reference strain, M. pachydermatis (CBS7925). In order to isolate the genetic material from the fungal cells, the following four procedures were selected: mechanical, enzymatic, thermal and chemical. Considering the yield and repeatability of a method for the genomic DNA extraction, a mechanical method was applied. The genetic material research of each strain was performed according to PCR-REA technique with the amplification of three genome regions: ITS, LSU rRNA and a gene encoding beta-tubuline. The ITS and LSU rRNA regions were amplified employing the standard PCR reagents, whereas the region coding beta-tubuline with the so called touch down. The obtained amplification products were subjected to restrictive analysis by means of the following enzymes: EcoRI, Ncol, Hinfl, Alul, and Eco881 (Aval). The performed investigations made it possible to reveal the genotypic differentiation within M.pachydermatis species as well as some correlation between a genotypic profile and the origin of a strain (from healthy animals or with otitis externa symptoms), which may imply the existence of genetic conditioning of the Malassezia strains’ pathogenicity.
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The aim of the work was analyzing of genomic DNA of Malassezia pachydermatis isolates from clinical cases otitis externa from dogs using RAPD method with arbitrary primers Eric1R, Eric2, BG2 and FM1. Materials and methods. 47 strains of M. pachydermatis isolates from clinical cases otitis externa from dogs were tested. Isolation of genomic DNA was provided according with MasterPureTM Yeast DNA Purification Kit EPICENTRE procedure. The quality of isolated genomic DNA was determined electrophoreticaly. For differentiation the following primers were used: Eric1R, Eric2, BG2 and FM1. Primers Eric 1R and Eric 2 were used together in one reaction or amplificated separately. Obtained products were analyzed electrophoreticaly in 1.5% agarose gel. For determination of phylogenic tree Quantity one VersaDoc (BioRad) and Statgraphics plus 4.1 programs were used. Results. High degree of heterogeneity of DNA among investigated isolates of M. pachydermatis was shown using FM1 primer. Dendrograms were prepared by calculation euclid's distance of different parameters (size and count of RAPD products) by nearest neighbor method. Basing on phylogenic tree four main types (phylogenic groups) of M. pachydermatis isolates were shown. The other five groups non-count was shown also.
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