Lyme Disease Borreliae Research Articles

Erp and Rev Adhesins of the Lyme Disease Spirochete's Ubiquitous cp32 Prophages Assist the Bacterium during Vertebrate Infection.

Almost all spirochetes in the genus Borrelia (sensu lato) naturally contain multiple variants of closely related prophages. In the Lyme disease borreliae, these prophages are maintained as circular episomes that are called circular plasmid 32 kb (cp32s). The cp32s of Lyme agents are particularly unique in that they encode two distinct families of lipoproteins, namely, Erp and Rev, that are expressed on the bacterial outer surface during infection of vertebrate hosts. All identified functions of those outer surface proteins involve interactions between the spirochetes and host molecules, as follows: Erp proteins bind plasmin(ogen), laminin, glycosaminoglycans, and/or components of complement and Rev proteins bind fibronectin. Thus, cp32 prophages provide their bacterial hosts with surface proteins that can enhance infection processes, thereby facilitating their own survival. Horizontal transfer via bacteriophage particles increases the spread of beneficial alleles and creates diversity among Erp and Rev proteins.

Recombinant protein immunoblots for differential diagnosis of tick-borne relapsing fever and Lyme disease.

Lyme disease (LD) is caused by a group of tick-borne bacteria of the genus Borrelia termed Lyme disease Borreliae (LDB). The detection of serum antibodies to specific LDB antigens is widely used to support diagnosis of LD. Recent findings highlight a need for serological tests that can differentiate LD from tick-borne relapsing fever (TBRF) caused by a separate group of Borrelia species termed relapsing fever Borreliae. This is because LD and TBRF share some clinical symptoms and can occur in overlapping locations. The development of serological tests for TBRF is at an early stage compared with LD. This article reviews the application of line immunoblots (IBs), where recombinant proteins applied as lines on nitrocellulose membrane strips are used to detect antibodies in patient sera, for the diagnosis and differentiation of LD and TBRF.

Surveillance of Borrelia miyamotoi-carrying ticks and genomic analysis of isolates in Inner Mongolia, China

BackgroundBorrelia miyamotoi is a newly described relapsing fever spirochete transmitted by ixodid tick species. Little is known about the prevalence of B. miyamotoi infections in humans and ticks in Inner Mongolia, China. Therefore, we investigated the prevalence of B. miyamotoi in Ixodes persulcatus ticks, and we aimed to isolateB. miyamotoi from I. persulcatus from four regions of Greater Khingan, Inner Mongolia, China.MethodsFrom May to June each year during the period 2016–2019, host-seeking adult I. persulcatus ticks were collected from vegetation. Genomic DNA was prepared from half of each tick body for PCR template, and the remaining half was used to cultivate B. miyamotoi in BSK-M medium. We employed quantitative real-time PCR (qPCR) to detect Borrelia DNA in the ticks and to calculate the prevalence of B. miyamotoi and infections with other borreliae. For characterization of the isolated B. miyamotoi, we performed draft genome sequencing and multilocus sequencing analysis (MLSA).ResultsA total of 2656 adult I. persulcatus ticks were collected. The overall prevalence of relapsing fever (RF) borreliae in ticks was 5.0% (134/2656) and that of Lyme disease (LD) borreliae was 43.8% (1164/2656). Co-infection with RF and LD borreliae was observed in 63 ticks (2.4%). Ticks that were positive for RF borreliae by qPCR were subjected to glycerophosphodiester diester phosphodiesterase gene (glpQ) PCR amplification and sequencing, through which we identified the RF borrelia specimens as B. miyamotoi. Furthermore, the B. miyamotoi strain Hetao-1 was isolated from I. persulcatus, and a draft genome sequence was obtained from the isolate. Sequencing determined the strain Hetao-1 genome to be approximately 906.1 kbp in length (28.9% average GC content), and MLSA identified the strain as ST633, which has previously been reported in Japan and Mongolia.ConclusionWe detected B. miyamotoi from I. persulcatus ticks collected in Inner Mongolia, and successfully isolated a B. miyamotoi strain. To our knowledge, this is the first study to culture a B. miyamotoi isolate from China. The data on the prevalence of B. miyamotoi and other borreliae in I. persulcatus ticks will be fundamental for future epidemiological studies of B. miyamotoi disease in Inner Mongolia.

Borrelia miyamotoi strain LB-2001 retains plasmids and infectious phenotype throughout continuous culture passages as evaluated by multiplex PCR

Borrelia miyamotoi is a tick-borne spirochete of the relapsing fever borrelia group and an emerging pathogen of public health significance. The genomes of relapsing fever borreliae and Lyme disease borreliae consist of multiple linear and circular plasmids in addition to the chromosome. Previous work with B. burgdorferi sensu lato found diminished infectivity upon continuous in vitro culture passage that was attributable to plasmid loss. The effect of long-term culture passage on B. miyamotoi is not known. We generated a series of plasmid-specific primer sets and developed a multiplex PCR assay to detect the 14 known plasmids of B. miyamotoi North American strains LB-2001 and CT13−2396. We assessed the plasmid content of B. miyamotoi LB-2001 over 64 culture passages spanning 15 months and determined that strain LB-2001 retained all plasmids upon prolonged in vitro cultivation and remained infectious in mice. We also found that strain LB-2001 lacks plasmid lp20−1 which is present in strain CT13−2396. These results suggest that B. miyamotoi remains genetically stable when cultured and passaged in vitro.

Detection of Tick-Borne Pathogens in Ticks from Dogs and Cats in the Yamagata Prefecture of Japan in 2018.

Companion animals can become infected with tick-borne diseases (TBDs) becoming a reservoir for human transfer, thereby damaging human health. To evaluate whether companion animals are infested with ticks harboring human TBD pathogens, we detected TBD pathogens in ticks collected from dogs and cats brought to animal hospitals in the Yamagata prefecture of Japan. An investigation of 164 adult ticks collected from 88 dogs and 41 cats between March and July 2018 revealed that this region was dominated by three tick species, Ixodes ovatus (n = 95, 57.9%), Ixodes nipponensis (n = 37, 22.6%) and Haemaphysalis flava (n = 10, 6.1%). To evaluate their pathogenic potential, we went on to test each tick for spotted fever group rickettsiae, Lyme disease borreliae, relapsing fever borreliae, tick-borne encephalitis virus, and Huaiyangshan banyangvirus (formerly SFTS virus). Our results identified two I. ovatus ticks infected with Borrelia miyamotoi, which causes emerging relapsing fever; several I. nipponensis ticks infected with Rickettsia monacensis, which cause rickettsiosis; and several Ixodes persulcatus ticks infected with Rickettsia helvetica, which can also cause rickettsiosis. These results suggest that dogs and cats, and veterinary professionals and pet owners, in the Yamagata prefecture have some risk of exposure to several TBDs. This means that there should be continuous monitoring and reporting of TBDs, even those known to be uncommon in Japan, in both companion animals and humans to ensure the health and safety of both humans and animals in Japan.

Immunoserological Diagnosis of Human Borrelioses: Current Knowledge and Perspectives

Spirochetes of the genus Borrelia are divided into relapsing fever borreliae and Lyme disease borreliae. Immunoserological assays have been poorly developed for relapsing fever borreliae, where direct detection methods are more adapted to the pathophysiology of these infections presenting with massive bacteraemia. However, emergence of the novel agent of relapsing fever B. miyamotoi has renewed interest in serology in this context. In Lyme disease, because direct detection methods show low sensitivity, serology plays a central role in the diagnostic strategy. This diagnostic strategy is based on a two-tier methodology involving a first test (ELISA) with high sensitivity and acceptable specificity and a second, more specific test (western blot) for diagnostic confirmation. The most frequent limitations and pitfalls of serology are cross reactions, false IgM positivity, a seronegative window period at the early time of the infection, and serologic scars with a suspicion of reinfection. International guidelines have thus been proposed to avoid these difficulties with interpretation. Finally, unconventional diagnostic tests have been developed recently in the context of a highly publicized disease, with widely varying results, some of which have no available evidence-based data. New two-tier testing strategies using two ELISA tests (C6 and WCS for example) to replace immunoblot are currently proposed by some authors and guidelines, and promising new tests such as CXCL-13 in CSF are promising tools for the improvement of the diagnosis of Lyme borreliosis.

Line Immunoblot Assay for Tick-Borne Relapsing Fever and Findings in Patient Sera from Australia, Ukraine and the USA.

Tick-borne relapsing fever (TBRF) is caused by spirochete bacteria of the genus Borrelia termed relapsing fever Borreliae (RFB). TBRF shares symptoms with Lyme disease (LD) caused by related Lyme disease Borreliae (LDB). TBRF and LD are transmitted by ticks and occur in overlapping localities worldwide. Serological detection of antibodies used for laboratory confirmation of LD is not established for TBRF. A line immunoblot assay using recombinant proteins from different RFB species, termed TBRF IB, was developed and its diagnostic utility investigated. The TBRF IBs were able to differentiate between antibodies to RFB and LDB and had estimated sensitivity, specificity, and positive and negative predictive values of 70.5%, 99.5%, 97.3%, and 93.4%, respectively, based on results with reference sera from patients known to be positive and negative for TBRF. The use of TBRF IBs and analogous immunoblots for LD to test sera of patients from Australia, Ukraine, and the USA with LD symptoms revealed infection with TBRF alone, LD alone, and both TBRF and LD. Diagnosis by clinical criteria alone can, therefore, underestimate the incidence of TBRF. TBRF IBs will be useful for laboratory confirmation of TBRF and understanding its epidemiology worldwide.

Evaluation of the clinical relevance of vancomycin for the treatment of Lyme disease.

Vancomycin is active in vitro and in vivo in mouse systems against Lyme disease borrelia; however, there are no published data on the efficacy of vancomycin in patients with Lyme disease and no convincing theoretical advantages of vancomycin over the currently used and highly effective orally administered antimicrobial agents, including doxycycline, amoxicillin and cefuroxime axetil. In addition, vancomycin may cause awide variety of potentially serious adverse effects and requires the placement of an intravenous catheter. It is concluded that vancomycin is amuch less attractive option for the treatment of patients with early Lyme disease (or any other manifestation of Lyme disease), compared with the antimicrobials currently being used. Based on available evidence, clinical studies to evaluate the safety and efficacy of vancomycin for Lyme disease cannot be recommended.

Case control study: Serological evidence that Borrelia miyamotoi disease occurs nationwide in Japan.

Since 2011, Borrelia miyamotoi disease (BMD) has been reported in five countries in the northern hemisphere. The causative agent of BMD is transmitted by Ixodes ticks, which are also vectors of Lyme disease borreliae. In this study, we examined 459 cases of clinically suspected Lyme disease (LD group), and found twelve cases that were seropositive for the glycerophosphodiester phosphodiesterase (GlpQ) antigen derived from B. miyamotoi. The retrospective surveillance revealed that the seroprevalence of anti-GlpQ in the LD group was significantly higher than in a healthy cohort. Seropositive cases were observed from spring through autumn when ticks are active, and the cases were geographically widespread, being found in Hokkaido-Tohoku, Kanto, Chubu, Kinki, and Kyushu-Okinawa regions. Seropositive cases for GlpQ were most frequent in the Chubu region (6.3%) where B. miyamotoi has been found in Ixodes ticks. Out of the twelve cases that were found in the LD group, three cases exhibited concomitant seropositivity to Lyme disease borreliae by western blot assay. This is the first report of serological surveillance for BMD in Japan, and we conclude that BMD occurs nationwide.

Characterization of Borrelia burgdorferi Binding to Mammalian Cells and Extracellular Matrix.

Lyme disease Borreliae produces outer surface adhesins to confer bacterial attachment to the extracellular matrix (ECM) components on the surface of mammalian cells. Here, we describe protocols to characterize the activity and specificity of these adhesins by flow cytometry or measurement of the binding of radiolabeled spirochetes to immobilized ECM or mammalian cells.

Sleeper cells: the stringent response and persistence in the Borreliella (Borrelia) burgdorferi enzootic cycle.

Infections with tick-transmitted Borreliella (Borrelia) burgdorferi, the cause of Lyme disease, represent an increasingly large public health problem in North America and Europe. The ability of these spirochetes to maintain themselves for extended periods of time in their tick vectors and vertebrate reservoirs is crucial for continuance of the enzootic cycle as well as for the increasing exposure of humans to them. The stringent response mediated by the alarmone (p)ppGpp has been determined to be a master regulator in B. burgdorferi. It modulates the expression of identified and unidentified open reading frames needed to deal with and overcome the many nutritional stresses and other challenges faced by the spirochete in ticks and animal reservoirs. The metabolic and morphologic changes resulting from activation of the stringent response in B. burgdorferi may also be involved in the recently described non-genetic phenotypic phenomenon of tolerance to otherwise lethal doses of antimicrobials and to other antimicrobial activities. It may thus constitute a linchpin in multiple aspects of infections with Lyme disease borrelia, providing a link between the micro-ecological challenges of its enzootic life-cycle and long-term residence in the tissues of its animal reservoirs, with the evolutionary side effect of potential persistence in incidental human hosts.

There Is a Method to the Madness: Strategies to Study Host Complement Evasion by Lyme Disease and Relapsing Fever Spirochetes

Lyme disease and relapsing fever are caused by various Borrelia species. Lyme disease borreliae, the most common vector-borne pathogens in both the U.S. and Europe, are transmitted by Ixodes ticks and disseminate from the site of tick bites to tissues leading to erythema migrans skin rash, arthritis, carditis, and neuroborreliosis. Relapsing fever borreliae, carried by ticks and lice, trigger reoccurring fever episodes. Following transmission, spirochetes survive in the blood to induce bacteremia at the early stages of infection, which is thought to promote evasion of the host complement system. The complement system acts as an important innate immune defense mechanism in humans and vertebrates. Upon activation, the cleaved complement components form complexes on the pathogen surface to eventually promote bacteriolysis. The complement system is negatively modulated by a number of functionally diverse regulators to avoid tissue damage. To evade and inhibit the complement system, spirochetes are capable of binding complement components and regulators. Complement inhibition results in bacterial survival in serum (serum resistance) and is thought to promote bloodstream survival, which facilitates spirochete dissemination and disease manifestations. In this review, we discuss current methodologies to elucidate the mechanisms of Borrelia spp. that promote serum resistance and bloodstream survival, as well as novel methods to study factors responsible for bloodstream survival of Lyme disease borreliae that can be applied to relapsing fever borreliae. Understanding the mechanisms these pathogens utilize to evade the complement system will ultimately aid in the development of novel therapeutic strategies and disease prevention to improve human health.

Borrelia valaisiana Resist Complement-Mediated Killing Independently of the Recruitment of Immune Regulators and Inactivation of Complement Components

Spirochetes belonging to the Borrelia (B.) burgdorferi sensu lato complex differ in their resistance to complement-mediated killing, particularly in regard to human serum. In the present study, we elucidate the serum and complement susceptibility of B. valaisiana, a genospecies with the potential to cause Lyme disease in Europe as well as in Asia. Among the investigated isolates, growth of ZWU3 Ny3 was not affected while growth of VS116 and Bv9 was strongly inhibited in the presence of 50% human serum. Analyzing complement activation, complement components C3, C4 and C6 were deposited on the surface of isolates VS116 and Bv9, and similarly the membrane attack complex was formed on their surface. In contrast, no surface-deposited components and no aberrations in cell morphology were detected for serum-resistant ZWU3 Ny3. While further investigating the protective role of bound complement regulators in mediating complement resistance, we discovered that none of the B. valaisiana isolates analyzed bound complement regulators Factor H, Factor H-like protein 1, C4b binding protein or C1 esterase inhibitor. In addition, B. valaisiana also lacked intrinsic proteolytic activity to degrade complement components C3, C3b, C4, C4b, and C5. Taken together, these findings suggest that certain B. valaisiana isolates differ in their capability to resist complement-mediating killing by human serum. The molecular mechanism utilized by B. valaisiana to inhibit bacteriolysis appears not to involve binding of the key host complement regulators of the alternative, classical, and lectin pathways as already known for serum-resistant Lyme disease or relapsing fever borreliae.

DipA, a Pore-Forming Protein in the Outer Membrane of Lyme Disease Spirochetes Exhibits Specificity for the Permeation of Dicarboxylates

Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a single-channel conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of 0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions. Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may play an important role for the uptake of specific nutrients in different Borrelia species.

Deciphering the role of Toll-like receptors in humoral responses to Borreliae

The bacteria of the genus Borrelia are arthropod-borne spirochetes that cause relapsing fever and Lyme disease in humans. Like most arthropod-borne pathogens, Borreliae must survive in the periphery of their vertebrate hosts to allow for transmission to another arthropod vector. These spatial and temporal restrictions require that Borreliae evade the adaptive immune response. Borreliae have evolved genetic mechanisms that alter their surface protein expression, thereby altering the antigenic target presented to the host. To control Borreliae infection, the host relies on a rapid humoral response. While it is clear that B cell antigen receptor signaling is a critical requirement for the specific antibody responses, growing evidence suggests that additional signaling by innate immune receptors such as Toll-like receptors is necessary for optimal T cell-dependent and T cell-independent antibody responses. This review is focused on the role of Toll-like receptors in B cell responses to relapsing fever and Lyme disease Borreliae.

Out of the Woods: the Remarkable Genomes of the Genus Borrelia

Out of the Woods: the Remarkable Genomes of the Genus Borrelia

Coexistence of Pathogens in Host-Seeking and Feeding Ticks within a Single Natural Habitat in Central Germany

The importance of established and emerging tick-borne pathogens in Central and Northern Europe is steadily increasing. In 2007, we collected Ixodes ricinus ticks feeding on birds (n = 211) and rodents (n = 273), as well as host-seeking stages (n = 196), in a habitat in central Germany. In order to find out more about their natural transmission cycles, the ticks were tested for the presence of Lyme disease borreliae, Anaplasma phagocytophilum, spotted fever group (SFG) rickettsiae, Francisella tularensis, and babesiae. Altogether, 20.1% of the 680 ticks examined carried at least one pathogen. Bird-feeding ticks were more frequently infected with Borrelia spp. (15.2%) and A. phagocytophilum (3.2%) than rodent-feeding ticks (2.6%; 1.1%) or questing ticks (5.1%; 0%). Babesia spp. showed higher prevalence rates in ticks parasitizing birds (13.2%) and host-seeking ticks (10.7%), whereas ticks from small mammals were less frequently infected (6.6%). SFG rickettsiae and F. tularensis were also found in ticks collected off birds (2.1%; 1.2%), rodents (1.8%; 1.5%), and vegetation (4.1%; 1.6%). Various combinations of coinfections occurred in 10.9% of all positive ticks, indicating interaction of transmission cycles. Our results suggest that birds not only are important reservoirs for several pathogens but also act as vehicles for infected ticks and might therefore play a key role in the dispersal of tick-borne diseases.

Lyme Borreliosis, Po River Valley, Italy

We aimed to determine the presence of Ixodes ricinus ticks in heavily populated areas of the Po River Valley after report of a Lyme disease case. Eighteen percent of ticks examined from 3 locations were positive for Lyme disease borreliae. Lyme disease was diagnosed for 3 workers at risk for tick bite.

Binding of the complement inhibitor C4b-binding protein to Lyme disease borreliae

The Lyme disease spirochetes, Borrelia burgdorferi sensu stricto, Borrelia afzelii and Borrelia garinii, are tick-borne pathogens that can cause chronic disseminated infections. To survive in the human host borreliae need to evade the immune system. It is already well known that B. burgdorferi ss. and B. afzelii bind the complement (C) alternative pathway inhibitor factor H from serum using OspE and CRASP-1/Bba68 proteins to escape C attack. In the presence of natural antibodies and in chronic infections, when specific antibodies develop, borreliae have to protect themselves from antibody-induced classical pathway C attack. In this study we demonstrate binding of the classical pathway inhibitor, C4b-binding protein (C4bp), to three genospecies of B. burgdorferi sensu lato. Binding was strongest to B. garinii, which has been found to be the weakest factor H binder. The bacteria bound both purified 125I-labeled C4bp and C4bp from serum. Unlabeled C4bp competed for binding with 125I-C4bp, whereas BSA had no effect. Binding was salt-sensitive and inhibited by C4b and partially by heparin. C4bp maintained its cofactor activity for factor I in cleaving C4b when bound to the bacterial surface. Ligand blotting analysis of whole cell lysates and fractionated outer cell membranes of the bacteria suggested one major receptor of approximately 43kDa (P43) for C4bp in B. garinii and B. burgdorferi sensu stricto. Binding of C4bp may thus allow Lyme disease borreliae to escape activation of the classical C pathway and allow chronic infections in humans even in the presence of specific antibodies.

Detection of tick and tick-borne pathogen in some ports of Inner Mongolia

To investigate the distribution, composition and situation of natural infection pathogen of tick species in the main ports of Inner Mongolia. All ticks were collected manually with white cloth, from the grassland and searching for the hosts followed by detection of pathogens, with PCR. 1313 ticks identified, belonged to 1 family, 4 geniuses and 7 species in the three surveyed areas, with Dermacentor nuttallia distributed in the Ceke, Mandula and Manzhouli bordering ports. 69.08% of the total species were discovered at Port Ceke, with Rhipicephalus pumilio as the predominant one, which accounted for 74.86%. 5 kinds of tick-borne disease pathogens were detected from ticks in these three bordering ports while only Coxiella burnetii was found at the Port Ceke. In these three ports, the average infection rates of Lyme disease borrelia, Human babesia microti, Spotted fever group Rickettsia, Coxiella burnetii, Ehrlichiosis were 15.08%, 3.35%, 1.98%, 1.07%, 0.99% respectively. The positive rate of tick infected with Borrelia burgdorferi were 13.56%,22.88%,5.00% in the 3 bordering ports, respectively with significant differences. The positive rates of Babesia microti and Spotted fever group Rickettsia infections were also significantly different among these areas. The natural infection rates of the above mentioned five kinds of tick-borne pathogens were different in the Ports Ceke, Mandula and Manzhouli.