Browsing by Subject "M. avium ssp. paratuberculosis"

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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.