Thesis (Ph. D.)--University of Missouri-Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 70-85). Also available on the Internet.

Thesis (Ph.D.) -- University of Rhode Island, 2008. / Typescript. Includes bibliographical references (leaves 136-146).

Sensitivity of spirochetes from Borrelia burgdorferi sensu lato complex to serum complement of humans of different age and sex was analyzed. Complement-mediated Borrelia killing was observed in different combination of serum and selected Borrelia genospecies. The obtained results confirmed that age itself does not influence the sensitivity of human to Borrelia infection. However, the females seem to be more vulnerable to it. Each of ten tested Borrelia species was proved to be potentially infective for human in different ratio. The clear separation of all ten checked Borrelia species into two groups was revealed after the reaction with human sera: species with low sensitivity to human serum complement (mortality below 1 percent) and species with higher sensitivity (mortality over 3-4 percent).

No description available.

Lyme disease is the most common vector-borne infection in the Western world with an annual incidence usually in excess of 100 cases per 100 000 people in temperate areas of the United States and Europe. Same as other infectious diseases, Lyme borreliosis wreaks havoc on the host they have invaded. B. burgdorferi, the causative agent of this disease, circulates among wildlife vertebrate hosts and Ixodes tick vectors but may sometimes infect humans. Its natural enzootic cycle usually occurs as follows: The larval/nymphal stage tick feeds on an infected host. During this engorgement, the spirochetes reach the tick gut and stay confined to it. After the tick molts into the next developmental stage, it finds a second host. The new bloodmeal triggers the spirochetes to multiply within the gut and traverse the gut endothelium in a highly organized manner. They finally disseminate through the hemocoel up to the tick salivary glands and into the new host. We studied whether B. burgdorferi is capable of reaching the tick salivary glands during the first infective feeding period in uninfected ticks.

The samples under investigation were collected in Department of Pediatric Infectious Diseases of the University Hospital (Brno). Group of patients (100) was heterogeneous in terms of symptoms, age and sex. The samples were taken from patients with an LB diagnosis and from those with nonspecific symptoms. Molecular typing of LB spirochetes in clinical samples (104 blood/serum, 89 cerebro-spinal fluid and 1 synovial fluid) became necessary when the general immunological tests gave unclear results. The samples were analyzed using PCR-based and molecular biology techniques that include: nested- and spacer-PCR, specie-specific PCR, sequence, virtual hybridization, in silico RFLP analysis, similarity search. Results of conducted analysis confirmed that 51% of samples (98) were positive on B. bugrdorferi sensu lato. Using above mentioned techniques 6 spirochete species from B. burgdorferi sensu lato complex were identified; two of them weren?t detected in samples of human origin in Europe yet. Comparative analysis of two media for Borrelia cultivation from samples of human origin definitely proved the adventage of using MKP instead of traditionally used BSK-H Complete.

abstract: Borrelia burgdorferi (Bb), the causative agent of Lyme disease, is a unique pathogen, with a complex genome and unique immune evasion tactics. It lacks genes encoding proteins involved in nutrient synthesis and typical metabolic pathways, and therefore relies on the host for nutrients. The Bb genome encodes both an unusually high number of predicted outer surface lipoproteins of unknown function but with multiple complex roles in pathogenesis, and an unusually low number of predicted outer membrane proteins, given the necessity of bringing in the required nutrients for pathogen survival. Cellular processing of bacterial membrane proteins is complex, and structures of proteins from Bb have all been solved without the N-terminal signal sequence that directs the protein to proper folding and placement in the membrane. This dissertation presents the first membrane-directed expression in E. coli of several Bb proteins involved in the pathogenesis of Lyme disease. For the first time, I present evidence that the predicted lipoprotein, BBA57, forms a large alpha-helical homo-multimeric complex in the OM, is soluble in several detergents, and purifiable. The purified BBA57 complex forms homogeneous, 10 nm-diameter particles, visible by negative stain electron microscopy. Two-dimensional class averages from negative stain images reveal the first low-resolution particle views, comprised of a ring of subunits with a plug on top, possibly forming a porin or channel. These results provide the first evidence to support our theories that some of the predicted lipoproteins in Bb form integral-complexes in the outer membrane, and require proper membrane integration to form functional proteins. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2020

Borrelia burgdorferi is the etiological agent of Lyme disease. Not much is known about the susceptibility of this organism to chemical disinfection. Current antimicrobial susceptibility test methods, such as those published by the American Society for Testing and Materials (ASTM), usually require assessment of the number of colony forming units (cfu) of growing organisms on plates following exposure to an agent. For fast-growing organisms, plates are ready for counting 1-2 days post plating, while several weeks may be needed for slower growing organisms. Spirochetes, like B. burgdorferi are difficult to grow on solid media and typically require long incubation periods, sometimes up to several weeks, to generate visible colonies. These issues make B. burgdorferi cfu assessment by plate counting difficult and unreliable. Furthermore, Borrelia have a demonstrated capacity for pleomorphic forms, and can exist in spirochete, round body, or biofilm forms, depending on culture conditions. Plate counts, by nature, do not allow for assessment of morphological form changes. Additionally, the susceptibility of B. burgdorferi pleomorphic forms to chemical disinfectants has not been tested. In this study, we used the SYBR GREEN I/Propidium Iodide (SG I/PI) viability assay to rapidly estimate the percent kill of B. burgdorferi pleomorphic forms to chemical disinfection. Planktonic spirochete populations in 30-second treated samples showed viability percent values of: >95% for Hanks balanced salt solution (HBSS), ~60% for distilled deionized H2O (dd H2O), <5% for ACS 200, and 1% for 1% glutaraldehyde (GTA). Solutions containing 70% ethanol (ETH) and 1% hypochlorite (HC) showed no viable spirochetes following treatment. The percent of live round body cells following different treatments were: >99% for HBSS and <25% for dd H2O. ACS 200, 1% GTA, and 70% ETH treatments resulted in <1% live round body forms, whereas HC showed no live round cell forms. The susceptibility of B. burgdorferi biofilms to various treatments was also assayed using a SG I/PI viability stain after 30-minute contact times. The percent of viable organisms (green) in the treated biofilms was estimated by microscopic observations. HBSS controls showed >98% of bacteria in the biofilm were alive, while treated biofilms showed the following percent viabilities: ACS 200 - ~2%, 1% HC - <1%, 5% HC - <1%, 1% GTA - ~10%, 70% ETH - ~ 2%, and dd H2O ~40%. These techniques merged standardized assessment of antimicrobial activity in liquid culture using an ASTM-type kill-time procedure with viability techniques used in antibiotic susceptibility testing to rapidly evaluate the percent kill of B. burgdorferi pleomorphic forms in vitro following disinfectant exposure. These results showed that B. burgdorferi biofilm forms are orders of magnitude more resistant to chemical disinfection than other morphological forms of this organism.

The Western Fence Lizard (Sceloporus occidentalis) is a major host of juvenile stages of the Western Black-legged Tick (Ixodes pacificus), which is the vector for the Lyme disease causative spirochete bacterium Borrelia burgdorferi in the western United States. Because S. occidentalis is reservoir incompetent and capable of eliminating spirochetes from infected ticks, it has been implicated as a major factor in the ecology of Lyme disease in the West. Although complement proteins in lizard blood have been established as the borreliacidal factor, no studies have examined intraspecific variability in host lizard borreliacidal capacity. In Chapter 1 of this thesis, we introduce the complexity of the Borrelia burgdorferi transmission cycle and it’s implications for transmission risk. In Chapter 2 we tested the hypothesis that host lizard physiological condition impacts their borreliacidal capacity. Blood plasma of lizards in varying physiological conditions was challenged against cultured B. burgdorferi, and the complement-mediated inactivation of spirochetes was quantified. Adult lizards had higher bactericidal activity than first-year juveniles, suggesting that complement-mediated inactivation develops with maturity and/or exposure to spirochete antigens. Also, bactericidal activity was positively associated with lizard tick load and body condition. Adult lizard sex did not significantly affect spirochete mortality. Lizards from an inland site with little exposure to ticks had higher bactericidal activity than lizards from a coastal population that is heavily parasitized by ticks.

The macrophage receptors that mediate phagocytosis of Borrelia burgdorferi, the Lyme disease spirochete, are unknown despite this cell type's importance in promoting pathogen clearance and inflammation-mediated tissue damage. We now demonstrate that the β2 integrin, Complement Receptor 3 (CR3), mediates the phagocytosis of opsonized and non-opsonized spirochetes by murine macrophages and human monocytes. Although, expression of the surface proteins, CspA and OspE, protects B. burgdorferi from complement-mediated phagocytosis, the versatility of CR3 counteracts the ability of B. burgdorferi to interfere with complement activation and complement-derived opsonins, thus minimizing the bacteria's anti-complement strategy. Interaction of the spirochete with the integrin is not sufficient to internalize B. burgdorferi; however, phagocytosis occurs when the GPI-anchored protein, CD14, is coexpressed in CHO-CR3 cells. CR3-mediated phagocytosis occurs independently of MyD88-induced or inside-out signals but requires the translocation of the integrin to cholesterol rich membrane microdomains. Interestingly, the absence of CR3 leads to marked increases in production of TNF in vitro and in vivo, in spite of reduced spirochetal uptake. Overall, our data establish CR3 as a MyD88-independent phagocytic receptor for B. burgdorferi that also participates in the modulation of the proinflammatory output of macrophages. Macrophages are critical cellular components of the immune response to infectious agents. During infection with B. burgdorferi, macrophages infiltrate the cardiac tissue and induce the activation of invariant NKT cells, leading to the production of the protective cytokine IFNγ. The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen activated protein kinases, such as p38 MAP kinase. We now demonstrate that p38 MAP kinase-mediated responses are critical components to the immune response during infection with B. burgdorferi. The inhibition of p38 MAP kinase does not alter the ability of macrophages to phagocytose B. burgdorferi; however, inhibition of p38 during infection with B. burgdorferi results in increased carditis. Through the generation of transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, we demonstrate that this kinase regulates the production of the iNKT attracting chemokine, MCP-1 and the infiltration of these cells to the cardiac tissue during infection. Overall, the inhibition of p38 MAP kinase during infection with B. burgdorferi specifically in macrophages results in the deficient infiltration of iNKT cells and their diminished production of IFNγ, leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.