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The purpose of the study was to estimate the influence of temperature increase on Hsp70 induction in M. haemolytica serovar 1 strains. Three wildtype M. haemolytica strains, obtained from calves respiratory tracts and incubated at a temp of 41.5°C for 2 hours were used as the research material. Analyses of particular fractions were carried out by SDS-PAGE electrophoresis and identification of obtained proteins by immunoblotting (Western blotting) using polyclonal rabbit anti Hsp70 antibodies. The first step was to separate the capillaries in gradient pH 5/7 and 3/10 which was carried out in two-dimensional electrophoresis. The second step was carried out in SDS-PAGE electrophoresis using 4% stocking and 12% resolving gels. An analysis of SDS-PAGE electrophoresis revealed additional protein fractions, displaying positive reactions with anti-Hsp70 antibodies. The presence of these proteins was observed both in membrane and cytoplasmatic bacterial cell fractions. The molecular weight of the obtained proteins ranged between 77.5-79 kDa. The additional protein fractions were present in membrane fractions between molecules, weighed 22-26 kDa, as well as displaying a positive reaction with anti-Hsp70 antibodies. The electrophoregrams obtained in 2D electrophoresis revealed the presence of additional spots in membrane, cyto- and periplasmatic fractions. The obtained results suggest the potential for M. haemolytica strains to produce Hsp70 during stress induced by temperature increase.
Transport oraz adaptacja do nowych warunków środowiskowych należą do podstawowych czynników stresogennych, usposabiających do zachorowań i upadków wszystkich gatunków zwierząt hodowlanych. Analiza poziomu kortyzolu pozwala na ocenę intensywności przebiegu reakcji stresowej jako skutku negatywnego oddziaływania środowiska zewnętrznego na organizm. Stężenie kortyzolu we krwi oraz 11.17 DOA w kale uzyskanym od bydła i owiec po transporcie było istotnie wyższe (p≤0,05), w porównaniu do wartości uzyskanych przed transportem zwierząt. W okresie adaptacji zwierząt do nowych warunków środowiska hodowlanego istotnie wyższe wartości (p≤0,05) poziomu kortyzolu oraz 11.17 DOA obserwowano także w 9 oraz 14 dniu po transporcie. Uzyskane wyniki sugerują istotny wpływ stresu związanego z transportem oraz adaptacją przeżuwaczy na homeostatyczne mechanizmy organizmu, co wyraża się wzrostem sterydowych hormonów nadnerczowych. Obserwowany wzrost poziomu 11.17 DOA w kale, istotnie skorelowany z poziomem kortyzolu we krwi, stwarza nieograniczone możliwości analizy dobrostanu zwierząt w określonych środowiskach hodowlanych, bez dodatkowych zabiegów manipulacyjnych, mogących wpływać na zwiększenie intensywności reakcji stresowej u zwierząt.
The investigations were carried out in 10 sows that developed post-partum MMA syndrome (experimental group) and in 10 healthy sows (control) from one closed production cycle farm. The levels of TNFα and IL-6 were measured 12-24 and 48-72 h before, as well as 12-24 and 48-72 h after parturition using the ELISA. The findings revealed significantly increased postpartum levels of TNFα in both groups. Compared to the control group, the levels of TNFα in the experimental group were significantly higher 12-24 h before and 48-72 h after the parturition. The IL-6 levels significantly increased in the experimental group 48-72 h after the parturition. At both postpartum measurement points, the levels of IL-6 were significantly higher in the experimental group than those in controls. The results indicate that TNFα and IL-6 are involved in the pathogenesis of the MMA syndrome and that their determinations are useful for early diagnosis and monitoring of the disease.
Iron-regulated outer membrane proteins (IROMPs) in Mannheimia haemolytica A1, which function as a receptor for complexes containing iron ions, are induced by iron deficiency in the growth environment of the bacteria. Densitometric analysis of SDS-PAGE separation showed expression of IROMPs of 71, 77, and 100 kDa in the case of bacteria grown in a medium with 2,2-dipyridyl. The electrophoregrams obtained in 2-DE separations confirmed the presence of protein fractions with these molecular weights and isoelectric points ranging from 5.4 to 6.4. The results of the study also confirmed the ability of M. haemolytica A1 proteins involved in iron uptake to induce a protective immune response. In Western blot with serum from convalescent calves naturally infected with M. haemolytica A1, distinct reactions were obtained for IROMPs of 71, 77, and 100 kDa.
The purpose of the study was estimating the viability and susceptibility effect of leukocytes isolated from cattle before and after the transportation in vitro on M. haemolytica leukotoxin (Lkt) cytotoxicity. 40 Simentaler heifers that were transported by truck a distance of 1700 km for 72 hours were used in the experiment. The material for the study was the blood (40 samples) collected on heparin directly before and after transportation. In relation to leukocytes the examination of susceptibility on cytotoxic effect of Lkt has been carried out with the use of MTT (microtitration assay) and the viability of leukocytes after 1, 2, 3 and 6 hour of incubation. The results obtained in the cell viability test did not show statistically significant differences (P≥0.05) in 1st and 2nd hour of incubation in leukocytes obtained from heifers before and after transportation. After the 1st hour of incubation the percentage of leukocyte viability was on a very high level and showed 87% in both groups of animals. The significantly lower cell viability values in comparison to leukocytes isolated from animals before the transportation was observed in the transported heifers from 3rd to 6th hour of incubation. The analysis of the results obtained by MTT test indicated statistically significant differences in the susceptibility of leukocytes for cytotoxic activity of Lkt. The average values of toxic activity of Lkt in relation to leukocytes isolated before and after transportation was 79% and 92% respectively. The lytic activity of Lkt for 50% of the cell population referred as 1 unit (1U) was observed in Lkt concentration 15 mg/ml (leukocytes before transportation) and 7.5 mg/ml (after the transportation). The increase of susceptibility of leukocytes isolated after transportation on the cytotoxic effect of leukotoxin suggest the significant influence of transporting stress on the increase of respiratory diseases caused by M. haemolytica strains.
The aim of this study was to compare the immunostimulatory properties of Lkt of M. haemolytica inactivated by formaldehyde and glutaraldehyde and to evaluate the neutralizing properties of anti-Lkt antibodies. The experiment was conducted on 20 Black-and-White Lowland calves of 100 kg body weight, assigned to 4 experimental groups. The animals were given subcutaneous vaccine injections with native Lkt, Lkt inactivated by formaldehyde or Lkt inactivated by glutaraldehyde. The anti-Lkt antibody titres were measured using an enzyme-linked immunosorbent assay (ELISA), based on absorb- ance of the sera obtained from the animals immunized with the different forms of Lkt. The protective effects of the antibodies present in the sera isolated from the vaccinated animals were estimated using an MIT assay. Analysis of the ELISA absorbance values in the sera from calves in the vaccinated groups did not show any significant differences between the groups. The highest increase in absorbance of sera was observed in calves from the group that received formaldehyde-inactivated Lkt. In the case of calves immunized with native Lkt, the absorbance values were lower than in the group immunized with Lkt inactivated by formaldehyde. The lowest absorbance values were observed in sera obtained from calves vaccinated with Lkt inactivated by glutaraldehyde. Analysis of the MTT assay results revealed the greatest Lkt-neutralizing properties of antibodies in the sera of calves immunized with two doses of a vaccine containing native Lkt and Lkt inactivated with formaldehyde.
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