Browsing by Subject "Coxiella burnetii"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Publication
    Entwicklung von spezifischen PCR-ELISA Nachweissystemen für Coxiella burnetii, Francisella tularensis und Orthopockenviren
    (2001) Kohlhaußen, Simone; Böhm, Reinhard
    In the present work specific systems on the basis of the PCR were developed for the detection of Coxiella burnetii, Francisella tularensis and Orthopoxviruses. The detection of the pathogens is possible without additional cultivation. In order to exclude false-negative PCR results the amplification reaction was established as a competitive PCR in which an internal control DNA is included as an amplification check. With regard to a possible automation the detection-systems were constituted in form of a 'PCR ELISA'. The colorimetric detection of the amplicons takes place thereby in streptavidin coated microtiter plates. For each system biotinylated capture probes for both the internal control DNA (ST) and the pathogen specific DNA (WT) have been developed. For the use of these proof systems in the clinical laboratory, the routine application of the tested decontamination procedure by means of UNG during the execution of the PCR ELISA is recommended. For all three systems a detection of the pathogens is possible within one working day. For the species specific detection of F. tularensis and the genus specific detection of Orthopoxviruses the PCR ELISA systems established here represent the first developments of this type with integrated amplification control and potential for quantification.
  • Thumbnail Image
    Publication
    Epidemiologie von Coxiella burnetii, Rickettsia spp., FSME- und Hantaviren in Süddeutschland unter Berücksichtigung klimatischer Veränderungen
    (2011) Pluta, Silvia; Mackenstedt, Ute
    Zoonoses, which are defined as human infections caused by animal pathogens, are of considerable medical importance. Therefore, it is important to evaluate the distribution of tick- and rodent borne pathogens and the risk of infections for humans. Hence, the epidemiology of specific human pathogens (Coxiella burnetii, Rickettsia spp., TBE-virus and Hantavirus) was studied. As climatic conditions are known to have a significant influence on ticks and rodents, the role of climate change in the epidemiology of the examined pathogens was considered. The role of natural foci in the epidemiology of Coxiella burnetii, the causative agent of Q fever, is widely unknown in Germany. Therefore, ticks of the genus Dermacentor and rodents from Q fever endemic areas in Southern Germany were examined for infections with C. burnetii. Altogether, 1120 ticks and 119 rodents were tested by PCR. However, no infections were detected. Rodent blood samples were additionally tested for antibodies against coxiellae, but likewise, all samples were found to be negative. Hence, no natural foci of Q fever were identified in the examined areas. A role of natural foci in the epidemiology of Q fever in Southern Germany is therefore unlikely. Both Dermacentor marginatus and D. reticulatus act as vectors for Rickettsia spp. To elucidate if human pathogenic rickettsiae are distributed in Southern Germany, Dermacentor ticks were investigated for infections by PCR. Moreover, rodents were screened by molecular and serological methods to identify potential reservoir hosts. The overall prevalence of Rickettsia spp. in 1120 ticks was 33.6%. By sequencing of the rOmpA-gene, R. raoultii was identified in 32.8% and R. slovaca in 0.8% of all examined ticks. Both species are pathogenic for humans; thus, there might be a considerable risk of infection. This was confirmed by the detection of an autochthonous case of R. slovaca-infection in Rhineland-Palatinate. Rickettsia spp. was not detected in any rodents by PCR. However, 48 animals (20.2%), muridae as well as arvicolidae, showed antibodies against rickettsiae. It was, therefore, shown that rodents can actually act as reservoir hosts for Rickettsia spp. To determine the current prevalence of TBE virus in ticks, 7842 Ixodes ricinus from Baden-Württemberg were examined by molecular methods. TBE virus was found in four out of 11 examined areas. Prevalences ranged from 0.13% to 0.66%. No infected ticks were found in other areas. The determined prevalences were lower compared to studies from the 1990ies in the same areas. However, reliable comparisons are difficult due to the low numbers of infected ticks. The viruses of ticks from Gengenbach, Hagnau am Bodensee and Hödinger Tobel were cultured and subsequently characterized by sequencing of the E and NS2a genes. Phylogenetic studies were conducted to identify the TBE virus strains present in Baden-Württemberg. The isolate from Hagnau am Bodensee was identified as strain Salem, whereas the isolates of the other two areas differed substantially from all strains found in the database. Therefore, no identification was possible for these isolates. In 2007, the number of notified hantavirus infections exceeded by far the average case numbers of the preceding years. To determine if the steep rise in human infections is associated with an unusual high virus prevalence in reservoir hosts, red bank voles from the endemic area of the Swabian Albs were examined for infections by molecular and serological methods. The overall prevalence of Puumala hantavirus was 57.9%. In a study in 2001 in the same area, the prevalence was only 10%. Therefore, there was a sixfold rise in virus prevalence which reflects the high number of human cases in the study year. Sequence comparisons showed a close relationship of the hantaviruses from the Swabian Alb to hantaviruses detected in other areas of Baden-Württemberg. The results presented in this study provide the basis for further continuous studies. These are necessary to estimate the actual influence of changes in climate conditions on the examined zoonoses and, thereby, the prospective infection risk for humans.
  • Thumbnail Image
    Publication
    Untersuchungen zum Potential biotechnologischer Methoden zur Inaktivierung von tier- und humanmedizinischen Krankheitserregern der Schutzstufe 3
    (2017) Hartmann, Nadja; Hölzle, Ludwig
    With the increasing use of slurry in biogas plants there remains the question of the extent of the potential hazard for human and animal beings due to infectious pathogens which enter biogas plants through contaminated substrates. The aim of this doctoral thesis was to establish a suitable approach for the inactivation of infectious and zoonotic agents. Therefore, the fermentation process itself as well as potential pre- and post-procedures which can contribute to the inactivation of pathogens were analyzed. First of all, a laboratory process was established concerning an increase of temperature and dwelling time to analyze the consequences for the inactivation of the pathogens. Additionally, the possible impact of pasteurization and diverse substrates on the inactivation of different pathogens were investigated. The influence of storage to the contaminated substrates after the biogas process was also considered. Due to the distinctive tenacity of the bovine tuberculosis pathogens M. bovis and M. caprae as well as the paratuberculosis pathogen M. avium ssp. paratuberculosis (MAP). Those pathogens are used within this study. As another major cause of zoonotic, the obligate intracellular bacterium C. burnetii was used. This bacterium occurs also in high concentrations in slurry from animal populations which are tested positively of coxelliosis. Beside bacterial infection and zoonotic pathogens, viral agents are playing a major role, such as the highly contagious foot-and-mouth disease (FMD) and the virus classical swine fever (CSF). FMD was substituted by the equine rhinitis A virus (ERAV) and CSF by the bovine virus diarrhea (BVD). The results can be transferred to FMD and CSF because of the close phylogenetic relation of the surrogate viruses. The inactivation studies of M. bovis through storage over 21 days showed that there are besides the substrate and temperature specific differences also intraspecific differences. This fact should be included in future selection of the inactivation methods. At temperatures of 4 and 20 °C it was possible to detect mycobacteria throughout the entire experimental duration time. At 37 °C already after seven days no pathogens could be detected. Mycobacteria which were suspended in PBS were detectable during the whole experimental time. The storage studies with ERAV and BVDB were performed over 15 days with the different substrates. At low temperatures of 4 and 20 °C there was no significant virus reduction detectable. At higher temperatures (40 – 42 °C) after three days, a significant virus reduction for ERAV was detectable and a complete inactivation for BVD, respectively. Additionally, it could be proven, that the substrate has no impact on the reduction of ERAV. Experiments using ERAV absorbed membranes showed a substrate dependent inactivation at 40 °C. As a consequence, those adsorbed membranes were used for a biogas plant in a laboratory dimension for 24 hours in the style of the Hohenheim biogas yield test. The reduction of the titer could be seen at least after 120 minutes. Furthermore, it could be shown, that there is a significant reduction of the ERAV titer between 120 minutes and 24 hours. In regard to the success of pasteurization dependent on the substrate, no substrate dependency was found for mycobacteria. In experiments with M. bovis and M. caprae no pathogens could be found after 30 minutes incubation time at 60 °C. Investigations with MAP showed after 360 minutes at 70 °C no culturable pathogens. Pasteurization studies with C. burnetii showed no reliable data. The pasteurization studies of ERAV in dependence of three different substrates showed, that the reduction was only caused by temperature. Therefore, BVD was only combined with one substrate which had the most heterogenic composition. Concerning ERAV, after 15 minutes at 55 °C a significant reduction of the titer was detectable. For BVD at 40 °C no significant virus reduction was achieved, but a dependency of the substrate was proven. A significant reduction of the titer was achieved after 60 minutes at 45 °C in the substrate suspended viruses, 30 minutes at 50 °C, and for in cell culture suspended viruses after 60 – 120 minutes at 50 °C. The results of this thesis show the massive effect of the properties of individual pathogens on the duration of inactivation. This subordinate role should be considered within the choice of the suitable biotechnological process. To reduce the infectious risk the procedure should always orientate at the worst case: the pathogen with the highest tenacity.