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W pracy zastosowano technikę RAPD (Random Amplified Polymorphic DNA) w klasyfikacji gatunkowo swoistej oraz w ocenie zmienności wewnątrzgatun- kowej szczepów 8 gatunków z rodzaju Bordetella. Przeprowadzona optymalizacja reakcji RAPD umożliwiła uzyskanie powtarzalnych i różnicujących profili RAPD. Wykazano przydatność metody RAPD w identyfikacji gatunkowej szczepów B. avium i B. holmesii oraz w różnicowaniu wewnątrzgatunkowym szczepów B. bronchiseptica i B. hinzii.
Technikę AFLP (Amplified Fragment Length Polymorphism) zastosowano do identyfikacji gatunku і do oceny wewnątrzgatunkowej zmienności szczepów B. pertussis, В. parapertussis oraz В. bronchiseptica. Wykazano przydatność metody AFLP do określania gatunkowej przynależności szczepów B. pertussis, В. parapertussis і В. bronchiseptica oraz wewnątrzgatunkowego różnicowania szczepów B. pertussis i B. bronchiseptica.
The aim of the study was the evaluation and optimisation of PCR test for the detection of dermonecrotoxin gene (DNT) of Bordetella bronchiseptica. For the optimisation of the test, vaccine strain B16 was used. The optimisation procedure included: estimation of optimal Mg²⁺ concentration, annealing temperature, numbers of cycles, as well as sensitivity. The specificity of PCR test was checked with DNA of other pathogens existing in pigs' respiratory tract. The elaborated test was specific and sensitive to detect DNT gene of B. bronchiseptica, in both clinical samples, as well as in pure culture of the bacteria.
Dermonecrotoxigenic (DNT) strains of Pasteurella multocida (Pm) and Bordetella bronchiseptica (Bbr), etiological agents of atrophic rhinitis (AR) of swine, are usually detected by conventional microbiological methods. Currently the main tasks in the diagnosis of infectious diseases is to detect the specific genes of pathogens directly from clinical samples within the shortest possible period of time by using sufficiently specific and sensitive methods. In this study two PCR tests were developed for the detection of genes encoding DNT in Pm and Bbr directly in nasal swabs. They were used for examining 481 nasal swabs collected from pigs raised in 56 farms, suspected of AR or displaying clinical symptoms of PAR. Additionally, the usefulness of PCR in the routine diagnosis of AR was compared with standard microbiological and serological methods. DNT+ Pm were directly evidenced in 74 samples (15.4%) from 27 tested herds (48.2%) while DNT+ Bbr were detected in 196 swabs (40.7%) from 41 farms (73.2%). Using the standard microbiological examination, the presence of bacterial cultures of the morphology typical of Pm was observed in 114 tested samples (23.7%) and those typical of Bbr, in 156 samples (32.45%). In the PCR test performed to confirm the presence of the DNT- -encoding gene in Pm isolates, positive results were obtained in 26 samples (22.8%), while among isolated Bbr strains, 95 out of 156 (60.1%) possessed the gene encoding DNT. In the ELISA test, 22 out of 26 isolates (84.6%) were DNT+. In the case of DNT- isolates of PM, the results of ELISA were in full agreement with PCR results. In total, PAR was detected in 27 farms (48.2%), NPAR in 21 farms (37.5%), and 8 farms (14.8%) were diagnosed as free from AR since no positive results were obtained by either method. Summarizing, the results of serological, bacteriological and molecular examinations clearly demonstrated the usefulness of PCR in the routine diagnosis of AR directly from clinical material.
The response of five positive acute phase proteins (APP) was evaluated in pigs after infection with Bordetella bronchiseptica (Bbr). Twelve piglets from a herd with high health status were used. Six of them were infected intranasally with Bbr (3.4 x 10⁸ cfu). The standard bacteriological methods and PCR were used for detection of Bbr in nasal swabs, lungs, and bronchoalveolar fluids. Serum APP concentrations were measured using commercial tests. Various kinetics of response was identified within the APP tested. C-reactive protein and serum amyloid A was characterised as a ven fast, transient responder, while haptoglobin as a fast and very prolonged responder. Pig major acute phase protein was characterised as a fast, protracted responder. Alfa-l-acid-glycoprotein did not respond significantly after inoculation. No correlations were found between levels of APP in serum and changes in the turbinates. On the basis of the investigation we cannot state that evaluation of APP concentrations in serum may provide useful information about severity of atrophic rhinitis. However, the concentration of most investigated APP increased after inoculation. Thus, APP measurement could be the primary screening test prior to specific disease diagnosis. "APP-high" animals may be chosen next for pathogen-specific diagnostics. Moreover, monitoring of serum APP concentrations may be useful for selecting clinically healthy pigs before integration into an uninfected herd.
Pasteurella multocida (Pm) and Bordetella bronchiseptica (Bbr) are important pathogens of humans and many species of animals. At present, several classic and modern techniques are applied for their detection and identification. The review presents an overview of the diagnostic methods that are the most widely used in laboratories, with special emphasis placed on highlighting their advantages and limitations. The choice of the technique depends on the aim of the study. The most often used are classical methods such as microbiological, serological, biochemical, and - in the case of atrophic rhinitis - likewise the morphometric analysis of the cross-section of a turbinated bone. The above-mentioned techniques are not accurate enough because most of the test results must be confirmed by more precise and sensitive techniques. The development of a wide spectrum of molecular techniques has facilitated the undoubted identification of Pm and Bbr. The widest used is PCR basing on the amplification of the gene encoding dermonecrotoxin of Pm and Bbr. Other molecular techniques could be useful for describing the connection among individual components of bacterial cells and their ability to develop the disease. Moreover they could also be used to describe the degree of phylogenetical relationship among field strains. Such information could be used to make a prognosis regarding the appearance of diseases on a particular area. Taking into account the significance of the rapid and precise detection of the pathological agents for immediate and accurate therapy, the continuation of studies aimed at the developing of rapid and sensitive diagnostic techniques is fully recommended and justified.
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