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.

Lyme borreliosis, more commonly referred to as Lyme disease, is the fastest growing zoonotic disease in North America with approximately 30,000 confirmed cases and 300,000 estimated infections per year. In nature, the causative agent of Lyme disease, the bacterium Borrelia burgdorferi, cycles between Ixodes sp. ticks and small mammals. Humans become infected with Lyme disease after being bitten by an infected tick. The primary indicator of a Borrelia burgdorferi infection is a bull’s eye rash typically followed by flu-like symptoms with treatment consisting of a 2-4 week course of antibiotics. If not treated, later stages of the disease can result in arthritis, cardiovascular and neurological symptoms. Diagnosis of Lyme disease is challenging and currently requires a complex laboratory diagnostic using indirect detection of host-generated antibodies by a two-tiered approach consisting of an enzyme linked immunosorbent assay (ELISA) followed by IgM and IgG immunoblots. Although two-tier testing has provided an adequate approach for Lyme disease diagnosis, it has weaknesses including subjective analysis, complex protocols and lack of reagent standardization. Immuno-PCR (iPCR) is a method that combines ELISA-based detection specificity with the sensitivity of PCR signal amplification and has demonstrated increased sensitivity for many applications such as detection of disease biomarkers but has yet to be applied for diagnosis of Lyme disease. Herein, using iPCR and recombinant B. burgdorferi antigens, an assay for both the direct and the indirect detection of Lyme disease was developed iv and demonstrated improved sensitivity for detection of B. burgdorferi antibodies using a murine model. Moreover, we present evidence using human Lyme disease patient serum samples that iPCR using both multiple antigens and a unique single hybrid antigen is capable of achieving increased sensitivity and specificity compared to existing methodology. These data represent the first demonstration of iPCR for Lyme disease diagnosis and support the replacement of two-tier testing with a more simplified and objective approach.

No description available.

Emerging infectious diseases continue to threaten human health and healthcare resources across the world, and with the sustained emergence of some like Lyme disease, this trend is only expected to get worse. Even though some research has examined the spread of Lyme disease in different parts of the United States, focusing on its origin, spread, surveillance, and reporting, there has been minimal research on the recent spread of Lyme disease into and within Central and Southern Appalachia. This is an important gap, considering that this region is at the edge of the disease's expanding incidence and range. Considering this factor and the consequences that this disease may have on the residents of Appalachia who already face an array of underlying problems like chronic conditions, decreased access to health care, and exposure through outdoor work like resource extraction, addressing this gap will be important in understanding the current and future spatial range and impacts of the continued emergence of Lyme disease in Appalachia. This study thus sought to examine the extent of the emergence of Lyme disease in Appalachia between 2000 and 2019 using spatial scan statistic. This type of analysis uses spatiotemporal information to quantify disease emergence and diffusion using retrospective case and location data within a GIS environment; hence it helped quantify the spatial distribution and diffusion of Lyme disease into and within Appalachia and identify additional areas that can be targeted with public health prevention measures. We found five statistically significant clusters of Lyme disease across the contiguous US, indicating that the disease expanded spatially over the study period. Lyme disease has more than tripled across the US, with the number of counties with RR>1 increasing from 130 in 2000 to 453 by 2019. Lyme disease also increased more than eightfold in Appalachia, with spread southwards into and within Appalachia. The number of Appalachian counties with RR>1 increased from 16 in 2000 to 127 in 2019. These findings are important in understanding the current and future spatial range and the impacts of the continued emergence of Lyme disease in Appalachia. With this understanding, we can minimize the misdiagnosis of Lyme disease and inform public health action to reduce public vulnerability. / Master of Science / Emerging infectious diseases are diseases that have not been present, the persistence of diseases that were previously unknown, or those diseases that are rapidly increasing in a given location. These diseases continue to threaten human health and healthcare resources across the world, and with the sustained emergence of some like Lyme disease, this trend is only expected to get worse. Even though some research has investigated the spread of Lyme disease in different parts of the United States, focusing on its origin, spread, monitoring, and reporting, there has been minimal research on the recent spread of Lyme disease in Central and Southern Appalachia. This is an important gap, considering that this region is next to areas where the disease has recently spread, thereby making it vulnerable to continued spread. Considering this factor and the consequences that this disease may have on the residents of Appalachia who already face many underlying problems like long-term conditions, decreased access to health care, and exposure through outdoor work like resource extraction, addressing this gap will be important in understanding the current and future geographic occurrence and impacts of the continued spread of Lyme disease in Appalachia. This study thus sought to investigate the extent of the spread of Lyme disease in Appalachia between 2000 and 2019 using statistics and identification of places with a higher than expected number of cases. This type of analysis uses location information to quantify disease occurrence and spread using past case and location data using GIS software; hence it helped identify the distribution and spread of Lyme disease into and within Appalachia and identify additional areas that can be targeted with public health prevention measures. We found five areas with higher than expected number of Lyme disease cases across the contiguous US, indicating that the disease increased over the study period. Lyme disease has more than tripled across the US, with the number of counties with higher cases than expected increasing from 130 in 2000 to 453 by 2019. Lyme disease also increased more than eightfold in Appalachia, with spread southwards into and within Appalachia. The number of Appalachian counties with higher than expected Lyme disease cases increased from 16 in 2000 to 127 in 2019. These findings are important in understanding the current and future spread and the impacts of this continued spread of Lyme disease in Appalachia. With this understanding, we can properly detect Lyme disease and inform public health action to reduce public vulnerability.

Lyme disease has been a growing problem in the United States over the last few decades, and is currently the most common vector-borne disease in the country. This research evaluates the land cover within specified counties of northern Virginia to determine if a correlation exists between forest fragmentation, suburbanization, and cases of human Lyme disease as has been demonstrated in other Lyme endemic regions in the United States. Few studies have focused specifically on northern Virginia when considering the impacts of land cover change on Lyme disease. Discovered through the use of geospatial and statistical analysis, the cluster of Lyme disease cases in northern Virginia are associated with forest fragmentation within the study region, which creates an ideal habitat for black-legged ticks and the white-footed mouse, allowing for an increase in Lyme disease transfer from vector to humans. The goal is for the research findings to be applicable to other regions with similar land cover types. Regions with similar characteristics would then be able to recognize the potential risk of human Lyme disease and implement ways to reduce the Lyme disease risk associated with suburban development. The purpose of this study is to answer the following research questions: 1) How has the spatial distribution of Lyme disease in Northern Virginia changed since 2005 with respect to land cover? 2) Which suburban communities are more at risk for Lyme disease when considering their land cover types and the increasing spatial distribution of Lyme disease? / Master of Science / Lyme disease has been a growing problem in the United States over the last few decades, and is currently the most common vector-borne disease in the country. This research evaluates the land cover within specified counties of northern Virginia to determine if a correlation exists between forest fragmentation, suburbanization, and cases of human Lyme disease as has been demonstrated in other Lyme endemic regions in the United States. Few studies have focused specifically on northern Virginia when considering the impacts of land cover change on Lyme disease. Discovered through the use of geospatial and statistical analysis, the cluster of Lyme disease cases in northern Virginia are associated with forest fragmentation within the study region, which creates an ideal habitat for black-legged ticks and the white-footed mouse, allowing for an increase in Lyme disease transfer from vector to humans. The goal is for the research findings to be applicable to other regions with similar land cover types. Regions with similar characteristics would then be able to recognize the potential risk of human Lyme disease and implement ways to reduce the Lyme disease risk associated with suburban development. The purpose of this study is to answer the following research questions: 1) How has the spatial distribution of Lyme disease in Northern Virginia changed since 2005 with respect to land cover? 2) Which suburban communities are more at risk for Lyme disease when considering their land cover types and the increasing spatial distribution of Lyme disease?

A Doença de Lyme-símile brasileira ou Síndrome Baggio-Yoshinari (SBY) é uma zoonose emergente, transmitida por carrapatos e até o momento, de descrição restrita ao território brasileiro. O agente etiológico da SBY era desconhecido até o presente trabalho. O objetivo principal do estudo foi identificar a etiologia da SBY. Foi selecionado 2 grupos de pacientes: grupo A (n=68) composto por pacientes com suspeita diagnóstica de SBY, a maioria na fase latente da doença; grupo B (n=10), composto por pacientes com diagnóstico de SBY, que apresentaram obrigatoriamente eritema migratório e que encontravam-se sintomáticos no momento da coleta. Foi utilizado também um grupo controle composto por indivíduos saudáveis e com epidemiologia negativa (n=50). Amostras de sangue foram coletadas para a realização de sorologias, culturas, análises microscópicas (óptica e eletrônica) e reação de cadeia da polimerase (PCR) para diferentes micro-organismos (Mycoplasma spp, Chlamydia spp e Borrelia spp). Além disso, foi realizado um estudo preliminar, através da PCR para Borrelia spp em 47 amostras de carrapatos oriundos de áreas de risco do Espírito Santo (sendo 17 Rhipicephalus microplus e 30 Rhipicephalus sanguineus), e amostras de sangue total de 27 bovinos e 26 equinos, animais estes oriundos da Universidade Federal Rural do Rio de Janeiro. Os resultados mostraram que a SBY não se trata de uma zoonose causada por um conjunto de micro-organismos como pensado inicialmente e sim pela Borrelia burgdorferi sensu lato. Descoberta essa que foi possível empregando-se novos primers amplificadores do principal gene envolvido na síntese do gancho flagelar da Borrelia, chamado flgE. A positividade para flgE foi confirmada em 6 pacientes do grupo B, 2 carrapatos, 1 bovino e 1 equino, os quais apresentaram homologia de 99% com o gene da proteína do gancho flagelar da Borrelia burgdorferi (flgE) depositado no GenBank (L43849). Esta importante descoberta, associada às pesquisas anteriores, permitiu definir a SBY como zoonose emergente e própria do país, causada pela bactéria B. burgdorferi na apresentação morfológica atípica, transmitida por carrapatos não pertencentes ao complexo Ixodes ricinus, responsável por manifestações clínicas semelhantes à Doença de Lyme, exceto pela grande frequência de sintomas recorrentes / Brazilian Lyme disease-like illness (BLDL) or Baggio-Yoshinari Syndrome (BYS) is an emerging zoonosis, transmitted by ticks and so far, restricted to the description of the Brazilian territory. The causative agent of BYS was unknown until now. The main objective of this study was to identify the etiology of BYS. We have selected two groups of patients: group A (n = 68) consisting of patients suspected of BYS, mostly in the latent stage of disease; group B (n = 10), composed of patients diagnosed with BYS, who had compulsorily erythema migrans and that were symptomatic at the time of blood collection. We also used a control group composed of healthy individuals with negative epidemiology (n = 50). Blood samples were collected, in which we performed serology, cultures, microscopic analysis (optical and electron) and polymerase chain reaction (PCR) for different microorganisms (Mycoplasma spp, Chlamydia spp and Borrelia spp). In addition, a preliminary study was conducted by PCR for Borrelia spp in 47 samples of ticks from risk areas at Espirito Santo State (being 17 Rhipicephalus microplus and 30 Rhipicephalus sanguineus), 27 cattle and 26 horses, being these animals from the Universidade Federal Rural do Rio de Janeiro. The results showed that BYS is not a zoonosis caused by a set of microorganisms as initially thought, but by Borrelia burgdorferi sensu lato. These findings were possible after employing new primers that are able to amplify portions of the main genes involved in the synthesis of the Borrelia flagellar hook protein, called flgE. We confirmed positivity for the flgE in 6 patients from group B, 2 ticks, a cow, and a horse, which showed 99% homology with the gene of Borrelia burgdorferi flagellar hook protein (flgE) deposited in GenBank (L43849). This important discovery, coupled with previous research, helped to define BYS as an emerging zoonosis particular from Brazil, caused by B. burgdorferi of atypical morphologic presentation, transmitted by ticks outside the Ixodes ricinus complex, responsible for clinical signs similar to Lyme disease, except for the high frequency of relapsing symptoms

A Doença de Lyme-símile brasileira ou Síndrome Baggio-Yoshinari (SBY) é uma zoonose emergente, transmitida por carrapatos e até o momento, de descrição restrita ao território brasileiro. O agente etiológico da SBY era desconhecido até o presente trabalho. O objetivo principal do estudo foi identificar a etiologia da SBY. Foi selecionado 2 grupos de pacientes: grupo A (n=68) composto por pacientes com suspeita diagnóstica de SBY, a maioria na fase latente da doença; grupo B (n=10), composto por pacientes com diagnóstico de SBY, que apresentaram obrigatoriamente eritema migratório e que encontravam-se sintomáticos no momento da coleta. Foi utilizado também um grupo controle composto por indivíduos saudáveis e com epidemiologia negativa (n=50). Amostras de sangue foram coletadas para a realização de sorologias, culturas, análises microscópicas (óptica e eletrônica) e reação de cadeia da polimerase (PCR) para diferentes micro-organismos (Mycoplasma spp, Chlamydia spp e Borrelia spp). Além disso, foi realizado um estudo preliminar, através da PCR para Borrelia spp em 47 amostras de carrapatos oriundos de áreas de risco do Espírito Santo (sendo 17 Rhipicephalus microplus e 30 Rhipicephalus sanguineus), e amostras de sangue total de 27 bovinos e 26 equinos, animais estes oriundos da Universidade Federal Rural do Rio de Janeiro. Os resultados mostraram que a SBY não se trata de uma zoonose causada por um conjunto de micro-organismos como pensado inicialmente e sim pela Borrelia burgdorferi sensu lato. Descoberta essa que foi possível empregando-se novos primers amplificadores do principal gene envolvido na síntese do gancho flagelar da Borrelia, chamado flgE. A positividade para flgE foi confirmada em 6 pacientes do grupo B, 2 carrapatos, 1 bovino e 1 equino, os quais apresentaram homologia de 99% com o gene da proteína do gancho flagelar da Borrelia burgdorferi (flgE) depositado no GenBank (L43849). Esta importante descoberta, associada às pesquisas anteriores, permitiu definir a SBY como zoonose emergente e própria do país, causada pela bactéria B. burgdorferi na apresentação morfológica atípica, transmitida por carrapatos não pertencentes ao complexo Ixodes ricinus, responsável por manifestações clínicas semelhantes à Doença de Lyme, exceto pela grande frequência de sintomas recorrentes / Brazilian Lyme disease-like illness (BLDL) or Baggio-Yoshinari Syndrome (BYS) is an emerging zoonosis, transmitted by ticks and so far, restricted to the description of the Brazilian territory. The causative agent of BYS was unknown until now. The main objective of this study was to identify the etiology of BYS. We have selected two groups of patients: group A (n = 68) consisting of patients suspected of BYS, mostly in the latent stage of disease; group B (n = 10), composed of patients diagnosed with BYS, who had compulsorily erythema migrans and that were symptomatic at the time of blood collection. We also used a control group composed of healthy individuals with negative epidemiology (n = 50). Blood samples were collected, in which we performed serology, cultures, microscopic analysis (optical and electron) and polymerase chain reaction (PCR) for different microorganisms (Mycoplasma spp, Chlamydia spp and Borrelia spp). In addition, a preliminary study was conducted by PCR for Borrelia spp in 47 samples of ticks from risk areas at Espirito Santo State (being 17 Rhipicephalus microplus and 30 Rhipicephalus sanguineus), 27 cattle and 26 horses, being these animals from the Universidade Federal Rural do Rio de Janeiro. The results showed that BYS is not a zoonosis caused by a set of microorganisms as initially thought, but by Borrelia burgdorferi sensu lato. These findings were possible after employing new primers that are able to amplify portions of the main genes involved in the synthesis of the Borrelia flagellar hook protein, called flgE. We confirmed positivity for the flgE in 6 patients from group B, 2 ticks, a cow, and a horse, which showed 99% homology with the gene of Borrelia burgdorferi flagellar hook protein (flgE) deposited in GenBank (L43849). This important discovery, coupled with previous research, helped to define BYS as an emerging zoonosis particular from Brazil, caused by B. burgdorferi of atypical morphologic presentation, transmitted by ticks outside the Ixodes ricinus complex, responsible for clinical signs similar to Lyme disease, except for the high frequency of relapsing symptoms

Indiana University-Purdue University Indianapolis (IUPUI) / Borrelia burgdorferi, a bacterium which causes Lyme disease, is maintained in nature through a cycle involving two distinct hosts: a tick vector and a mammalian host. To adapt to these two diverse environments, B. burgdorferi undergoes dramatic alterations in its surface lipoprotein. Two essential lipoproteins, outer surface protein A (OspA) and outer surface protein C (OspC), are reciprocally regulated throughout the B. burgdorferi lifecycle. Very little is known about the regulation of OspA. These studies elucidate the regulatory mechanisms controlling the expression of OspA. Various truncations of the ospA promoter were created and then studied in our novel in vitro model of ospA repression or grown within the host-adapted model. A T-Rich region of the ospA promoter was determined to be a cis-element essential for both the full expression and full repression of ospA.

The main research problem concerns the characteristics of tick-borne disease and determine the quantity and quality of information that students get in health education and biology of these diseases. The theoretical part deals with the history, etiology, clinical features, diagnosis, treatment and prevention of tick-borne encephalitis and Lyme disease. It also discusses the general problems of tick and other diseases that ticks carry and cause. Also is mentioned the inclusion of tick-borne diseases in the educational program. The practical part is devoted to the awareness of upper primary school students and changes in their knowledge about the issue during their elementary education. The aim of this thesis is to determine how the students are informed about diseases transmitted by ticks, especially about Lyme disease and tick-borne encephalitis. This method is used questionnaire, which includes questions regarding this issue. Although the results of the research show a certain degree of knowledge of students at upper primary school, especially in some classes is unsatisfactory. Based on research, there is provided a recommendation for pedagogical practices. KEYWORDS: tick, tick-borne encephalitis, Lyme disease, knowledge, disease, upper primary school