Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 284-323.

Universiẗat, Diss., 2004--München.

Universiẗat, Diss., 2003--Jena.

During a period of 3 years, from March to July 2014, 2015 and 2016, ticks were collected from dogs and cats in shelter facilities for abandon animals in Česke Budejovice, South Bohemia. In total, 343 ticks were found on 106 pets: 67 domestic dogs and 39 cats. All collected ticks, that were identified as Ixodes ricinus and Ixodes hexagonus, were tested for the presence of spirochetes from Borrelia burgdorferi sensu lato (s.l.) complex, Rickettsia spp., Anaplasma spp, and Babesia spp using conventional PCR and nested PCR. Identification of pathogens was done by following sequencing of amplicons. Out of all tested ticks, 49,56% were proved to be infected at least with one pathogen. Co-infection of at least two different pathogens was determined in 18 ticks (5,2%). The aim of the present study was to estimate the role of accompanying animals (cats and dogs) in the circulation of ticks and tick-borne pathogens, to determine the frequency of pathogenic infections in dog and cat associated ticks, to evaluate the current risk of infection for dogs and cats, with respect torisk forhumans living in the area of České Budějovice.

I describe procedures for the introduction of Borrelia burgdorferi, the spirochetal agent of Lyme disease, into larval and nymphal stage of the tick vector, Ixodes ricinus. The goal of this Mgr. Thesis is to find an optima system, that would reliably and reproducibly allow the infection of large numbers of ticks with the Lyme disease spirochete.

The aim of present master’s thesis was to compare the results of serological methods for diagnosing borreliosis that are commonly used in Spadia laboratories (ELISA, immunoblots) in terms of recommendation on how and when to indicate and interpret them. The theoretical part is focusing on the characteristics and history of borreliosis, microbiological description of Borrelia, immune system and pathogenesis of the disease as well as the therapy and prevention. The experimental part is focusing on the analysis of results obtained from common examinations of antibodies to Borrelia made in Spadia Lab laboratories from January 1st 2014 to December 31st 2015. Screening of antibodies to Borrelia made by ELISA in IgM and IgG was done for all samples according to recommendation of CDC. In 2014 the ELISA screening was done using ELISA kits from Euroimmun and Evolis sample processors whereas in 2015 it was done using DiaSorin’s CLIA kits on Liaison analyser. Positive results were then confirmed by Westernblot or lineblot alternatively if the physician did not ask otherwise. It must be remembered that ELISA and Westernblot belong among serological methods that are using antibodies, i.e. substances produced by the immune system. The immune system plays the key role in protecting the body against infection and the antibodies are its important tool. Serological methods belong among immunoassay methods, which is still not standardized. Diagnosis of infections cann‘t be based only on antibody testing. It is necessary to assess the results in the context of the entire clinical picture, history and in the case of antibodies it is recommended retesting with an interval.

In der vorliegenden Arbeit wurde erstmalig die in unter anderen in Mitteleuropa heimische Pflanze Dipsacus sylvestris HUDS. in einer Arbeit gleichzeitig phytochemisch-analytisch und pharmakologisch untersucht. Zur sicheren Identifizierung der Pflanze wurden dünnschicht- und kapillarchromato-graphische Methoden entwickelt und geeignete Markersubstanzen identifiziert. Dabei konnte eine Stufen-DC etabliert werden, die ein breites Fingerprintchromatogramm über das polare und apolare Inhaltsstoffspektrum auf einer DC-Folie ermöglicht. Mithilfe von CZE und MEKC konnten instru-mentelle Methoden entwickelt werden, die sowohl eine Pflanzen-Identifizierung, als auch im Falle der MEKC eine quantitative Erfassung ausgewählter iridoider und phenolischer Markersubstanzen zulassen. Dabei konnten Kaffee-, Chlorogensäure, Cantleyosid und Loganin quantifiziert werden. Die Methode wurde auch auf Dipsacus asperoides CHENG übertragen. Um analytische Markersubstanzen und Testsubstanzen für sich anschließende mikrobiologische Untersuchungen an Borrelia burgdorferi s.s. zu erhalten, wurden die Iridoide Loganin und Cantleyosid, das Lignan Prinsepiol und das Triterpen Ursolsäure aus D. sylvestris isoliert. Dazu wurden säulen- und planarchromatographische Methoden angewandt. Mittels NMR- und ESI-MS-Messungen wurden die erhaltenen Substanzen identifiziert. Neben den phytochemischen Untersuchungen wurden Extrakte und isolierte Reinstoffe aus D. sylv. in vitro an mediumsadaptierten B. burgdorferi s.s. auf wachstumshemmende Effekte hin untersucht. Vor allem das Lignan Prinsepiol, das erstmals aus D. sylvestris isoliert werden konnte, zeigte signifikante wachstumshemmende Effekte (0,2 mg/ml). Daneben wurden im Modell auch Ladanum und Manoyloxide sowie Carvacrol aus Cistus creticus eingesetzt. Ladanum (2,0 mg/ml), Carvacrol (0,2 und 0,05 mg/ml) und ent-13-epi-Manoyloxid (0,2 mg/ml) waren hier in vitro signifikant aktiv, während stereoisomere Manoyloxide kaum Effekte zeigten. Mit der Arbeit wurden interessante Grundlagen geschaffen, die Ausgangspunkt für weitere Forschungen sein können. So könnten sich z.B. Studien zur Toxizität, Metabolisierung und Bioverfüg-barkeit der aktiven Substanzen anschließen.:EINLEITUNG 1 Einführung in die Aufgabenstellung 2 Wilde Karde 3 Graubehaarte Zistrose 4 Borrelia burgdorferi 5 Ziele der Arbeit MATERIAL UND METHODEN 6 Verwendete Materialien 7 Methoden ERGEBNISSE 8 Ergebnisse DISKUSSION 9 Diskussion ZUSAMMENFASSUNG UND THESEN 10 Zusammenfassung und Thesen 11 Summary and theses ANHANG 12 GC-MS-Chromatogramme und Massenspektren 13 CE-Daten, Spektren und Elektropherogramme 14 NMR-Spektren 15 Zähldaten der Bbss-Bestimmungen 16 Weitere Diagramme der Bbss-Bestimmungen (Wiederholungsexperimente) VERZEICHNISSE 17 Literaturverzeichnis 18 Abbildungsverzeichnis 19 Tabellenverzeichnis 20 Formelverzeichnis 21 Abkürzungsverzeichnis WEITERE INFORMATIONEN 22 Lebenslauf, Publikationen 23 Erklärung über die eigenständige Abfassung der Arbeit / In the present study, the plant Dipsacus sylvestris HUDS., which is native in Central Europe among others, was examined for the first time both phytochemically and pharmacologically. Thin-layer and capillary chromatographic methods have been developed and suitable marker substances have been identified for the reliable identification of the plant. A stepwise developed TLC was established, which enables a broad fingerprint chromatogram over the polar and apolar ingredient spectrum on one single TLC plate. With the help of CZE and MEKC, instrumental methods have been developed that allow both plant identification and, in the case of MEKC, quantitative detection of selected iridoid and phenolic marker substances. Caffeic acid, chlorogenic acid, cantleyoside, and loganin could be quantified. The method was also transferred to Dipsacus asperoides CHENG. In order to obtain analytical marker substances and test substances for subsequent microbiological investigations on Borrelia burgdorferi s.s., the iridoids loganin and cantleyoside, the lignan prinsepiol and the triterpenoid ursolic acid were isolated from D. sylvestris. Column and planar chromatographic methods were used for this purpose. The isolated substances were identified by NMR and ESI-MS measurements. In addition to phytochemical investigations, extracts and isolated pure substances from D. sylv. were tested in vitro for growth-inhibiting effects on medium-adapted B. burgdorferi s.s. Especially the lignan prinsepiol, which could be isolated from D. sylvestris for the first time, showed significant growth-inhibiting effects (0.2 mg/ml). Ladanum and various manoyl oxides as well as carvacrol from Cistus creticus were also used in the model. Ladanum (2.0 mg/ml), carvacrol (0.2 and 0.05 mg/ml) and ent-13-epi manoyl oxide (0.2 mg/ml) were significantly active in vitro, while stereoisomeric manoyl oxides showed hardly any effects. The results obtained in this work set the stage for further research including e.g. studies on toxicity, metabolism, and bioavailability of active substances.:EINLEITUNG 1 Einführung in die Aufgabenstellung 2 Wilde Karde 3 Graubehaarte Zistrose 4 Borrelia burgdorferi 5 Ziele der Arbeit MATERIAL UND METHODEN 6 Verwendete Materialien 7 Methoden ERGEBNISSE 8 Ergebnisse DISKUSSION 9 Diskussion ZUSAMMENFASSUNG UND THESEN 10 Zusammenfassung und Thesen 11 Summary and theses ANHANG 12 GC-MS-Chromatogramme und Massenspektren 13 CE-Daten, Spektren und Elektropherogramme 14 NMR-Spektren 15 Zähldaten der Bbss-Bestimmungen 16 Weitere Diagramme der Bbss-Bestimmungen (Wiederholungsexperimente) VERZEICHNISSE 17 Literaturverzeichnis 18 Abbildungsverzeichnis 19 Tabellenverzeichnis 20 Formelverzeichnis 21 Abkürzungsverzeichnis WEITERE INFORMATIONEN 22 Lebenslauf, Publikationen 23 Erklärung über die eigenständige Abfassung der Arbeit

Indiana University-Purdue University Indianapolis (IUPUI) / Lyme disease is a multi-system inflammatory disorder that is currently the fastest growing arthropod-borne disease in the United States. The Lyme disease pathogen, Borrelia burgdorferi, exists within an enzootic cycle consisting of Ixodes tick vectors and a variety of vertebrate hosts. Borrelia lies within a distinct clade of microorganisms known as spirochetes which exhibit a unique spiral morphology. The underlying genetic mechanisms controlling for borrelial morphologies are still being discovered. One flagellar protein, FlaB, has been indicated to affect both spiral shape and motility of the organisms and significantly impacts the organism’s ability to establish infection. Due to the potential connection between morphological characteristics and pathogenesis, we sought to screen and identify morphological mutants in an attempt to identify genes associated with morphological phenotypes of Borrelia burgdorferi. Among Borrelia’s unique features is the presence of abundant lipoproteins making up its cellular membrane as opposed to the typical lipopolysaccharides. These proteins confer a wide variety of functions to the microorganism, among which include the abilities to circulate between widely differing hosts and to establish infection. Two important outer surface proteins, OspC and OspA, are found to be inversely expressed throughout the borrelial life cycle. OspC, in particular, becomes highly expressed during tick-feeding and transmission to the mammalian host. It has been found to be essential for establishment of infection. A global regulatory pathway has been shown to control for OspC, however there are missing links in this pathway between the external stimuli (such as temperature, pH, and cell density) and the regulatory pathway. We have performed a screening process to identify OspC expression mutants in order to identify novel genes associated with this pathway.

The use of the periplasmic alkaline phosphatase (PhoA) reporter protein from E. coli has been critical for definition of the topology of transmembrane proteins of multiple bacterial species. This report demonstrates development of a PhoA reporter system in B. burgdorferi. Codon usage of the E. coli phoA in B. burgdorferi was analyzed and an optimized version of the gene was obtained. In order to assess the differential activity of the reporter system, two optimized PhoA-fusion construct using B. burgdorferi proteins were engineered: one using the periplasmic protein OppAIV and one using the cytoplasmic protein PncA. The activity of PhoA requires periplasmic localization. The periplasmic OppAIV-PhoA fusion as well as the cytoplasmic PncA-PhoA fusion produced detectable PhoA protein in E. coli and in B. burgdorferi. The periplasmic fusion construct, but not the cytoplasmic fusion construct, resulted in functional alkaline phosphatase (AP) activity in E. coli, as observed by blue colonies on agar plates containing a chromogenic substrate for AP. In contrast, both of the fusion constructs produced limited detectable levels of functional alkaline phosphatase activity in B. burgdorferi, as observed by yellow color change in liquid protein lysate containing a chromogenic substrate for AP. Development of a PhoA fusion reporter system for use in B. burgdorferi will provide a new molecular genetics tool for analyzing the topology of B. burgdorferi transmembrane proteins. These types of studies are critical for understanding the function of B. burgdorferi transport systems and may identify novel molecular approaches for the treatment of Lyme disease.

The spirochete Borrelia burgdorferi is the etiologic agent of Lyme disease. This pathogen has a complex enzootic life cycle that involves passage between the tick vector (Ixodes scapularis) and various vertebrate hosts with humans being inadvertent hosts. There is a pressing need to study the genetic aspects of the B. burgdorferi infectious cycle and particularly spirochete genes involved in mammalian infectivity so as to develop novel therapeutic and diagnostic strategies to combat Lyme disease. The B. burgdorferi genome is fragmented and comprised of a single 900 kb linear chromosome and multiple linear and circular plasmids. It has been observed that plasmids are lost during serial passage and manipulation in vitro and the loss of some of the plasmids has been shown to be related to the loss of infectivity and persistence in the host. One such plasmid is linear plasmid 36 (lp36). lp36 is approximately 36kb in size and carries 56 putative open reading frames a majority of which have no predicted function. B. burgdorferi lacking lp36 show no deficiency in survival in ticks; however, these mutant spirochetes are highly attenuated for mammalian infectivity. The genetic components of this plasmid that contribute to its function in mammalian infectivity have yet to be clearly defined. Using an in vivo expression technology (IVET) based genetic screen the lp36- encoded gene bbk46 was identified as a candidate B. burgdorferi gene that is expressed during mammalian infection. Herein we present evidence that bbk46 is required for B. burgdorferi persistent infection of immunocompetent mice. Our data iii support a molecular model of immune evasion by which bbk46 functions as an RNA to regulate expression of the antigenic variation protein VlsE. These data represent the first demonstration of a regulatory mechanism critical for controlling vlsE gene expression. Moreover these findings further define the critical role of linear plasmid 36 in Borrelia burgdorferi pathogenesis.