Branda J, Body B, Boyle J, et al.

AbstractThe cause of Lyme disease, Borrelia burgdorferi, was discovered in 1983. A 2-tiered testing protocol was established for serodiagnosis in 1994, involving an enzyme immunoassay (EIA) or indirect fluorescence antibody, followed (if reactive) by immunoglobulin M and immunoglobulin G Western immunoblots. These assays were prepared from whole-cell cultured B. burgdorferi, lacking key in vivo expressed antigens and expressing antigens that can bind non-Borrelia antibodies. Additional drawbacks, particular to the Western immunoblot component, include low sensitivity in early infection, technical complexity, and subjective interpretation when scored by visual examination. Nevertheless, 2-tiered testing with immunoblotting remains the benchmark for evaluation of new methods or approaches. Next-generation serologic assays, prepared with recombinant proteins or synthetic peptides, and alternative testing protocols, can now overcome or circumvent many of these past drawbacks. This article describes next-generation serodiagnostic testing for Lyme disease, focusing on methods that are currently available or near-at-hand.

Schlachter, S., Seshu, J., Lin, T., Norris, S., Parveen, N.

Lyme disease causing Borrelia burgdorferi is transmitted into the mammalian host by an infected tick bite. Successful infection relies on the ability of this extracellular pathogen to persist and colonize different tissues. B. burgdorferi encodes a large number of adhesins that are able to interact with host ligands to facilitate adherence, and tissue colonization. Multiple glycosaminoglycan binding proteins presence in B. burgdorferi offer a degree of redundancy of function during infection, and highlights the importance for glycosaminoglycans as host cell receptors for spirochete adherence. Of particular interest in this study is Borrelia glycosaminoglycan binding protein (Bgp), which binds to heparin-related glycosaminoglycans. The properties of a bgp transposon mutant and a trans-complemented derivative were compared to the wild type B. burgdorferi in the in vitro binding assays and in infection studies using a C3H/HeJ mouse infection model. We determined that the loss of Bgp impairs spirochete adherence, infectivity, and tissue colonization resulting in a reduction of inflammatory manifestations of Lyme disease. Although Bgp is not essential for infectivity, it is an important virulence factor of B. burgdorferi that allows adherence and tissue colonization, and contributes to disease severity.

Smith, T. C., Helm, S. M., Chen, Y., Lin, Y.-H., Karna, S. L. R., Seshu, J.

Borrelia burgdorferi (Bb), the agent of Lyme disease (LD), uses host-derived signals to modulate gene expression during the vector and mammalian phases of infection. Microarray analysis of mutants lacking Borrelia host-adaptation Regulator (BadR) revealed down-regulation of genes encoding enzymes whose role in the patho-physiology of Bb is unknown. Immunoblot analysis of the badR mutants confirmed reduced levels of these enzymes and one of these enzymes encoded by bb0086, shares homology to prokaryotic magnesium chelatase and Lon-type proteases. BB0086 levels were higher in Bb under conditions mimicking fed ticks. Mutants lacking bb0086 had no apparent in vitro growth defect but were incapable of colonizing immunocompetent C3H/HeN and immunodeficient SCID mice. Immunoblot analysis revealed reduced levels of proteins critical for adaptation of Bb to the mammalian host such as OspC, DbpA and BBK32. Both RpoS and BosR - key regulators of gene expression in Bb - were downregulated in the bb0086 mutants. Therefore, we designated BB0086 as Borrelia host-adaptation Protein (BadP). Unlike badP mutants, the control strains established infection in C3H/HeN mice at 4 days post infection, indicating an early colonization defect in mutants due to reduced levels of lipoproteins/regulators critical for initial stages of infection. However, badP mutants survived within dialysis membrane chambers (DMCs) implanted within rat peritoneal cavity but unlike the control strains did not display complete switching of OspA to OspC suggesting incomplete adaptation to mammalian phase of infection. These findings have opened a novel regulatory mechanism, which impacts the virulence potential of Bb.

Y. Yang et al.

Curtis, M. W., Hahn, B. L., Zhang, K., Li, C., Robinson, R. T., Coburn, J.

Borrelia burgdorferi is a causative agent of Lyme disease, the most common arthropod-borne disease in the United States. B. burgdorferi evades host immune defenses to establish a persistent, disseminated infection. Previous work showed that P66-deficient B. burgdorferi (p66) is cleared quickly after inoculation in mice. We demonstrate that p66 is rapidly cleared from the skin inoculation site prior to dissemination. The rapid clearance of p66 is not due to inherent defects in multiple properties that might affect infectivity: bacterial outer membrane integrity, motility, chemotactic response, or nutrient acquisition. This led us to the hypothesis that P66 may have a role in mCRAMP (major skin antimicrobial peptide) and/or neutrophil evasion. Neither WT nor p66 B. burgdorferi was susceptible to mCRAMP. To examine the role of neutrophil evasion, we administered neutrophil-depleting antibody anti-Ly6G (1A8) to C3H/HeN mice and subsequently followed the course of B. burgdorferi infection. p66 mutants were unable to establish infection in neutrophil-depleted mice suggesting that the important role of P66 during early infection is through another mechanism. Neutrophil depletion did not affect WT B. burgdorferi bacterial burdens in the skin (inoculation site), ear, heart or tibiotarsal joint at early time points post-inoculation. This was unexpected given that prior in vitro studies demonstrated neutrophils phagocytose and kill B. burgdorferi. These data, together with our previous work, suggest that despite the in vitro ability of host innate defenses to kill B. burgdorferi, individual innate immune mechanisms have limited contributions to controlling early B. burgdorferi infection in the laboratory model used.

L. Karan et al.

Koetsveld, J., Manger, A., Hoornstra, D., Draga, R. O., Oei, A., kolyasnikova, N. M., Toporkova, M. G., Sarksyan, D. S., Wagemakers, A., Platonov, A. E., Hovius, J. W.

Objectives: Borrelia miyamotoi is an emerging relapsing fever (RF) Borrelia species that is described to cause human disease in regions where Lyme borreliosis is endemic. We have recently shown that B. miyamotoi tick isolates are resistant to amoxicillin in vitro, however, clinical isolates have not been studied. Therefore, our aim was to show antimicrobial susceptibility of recently obtained clinical isolates of B. miyamotoi.Methods: A dilution series of various antibiotics was made in MKP-F media. Susceptibility of different clinical B. miyamotoi, B. miyamotoi tick, RF Borrelia and B. burgdorferi sensu lato isolates were tested by measuring MICs through colorimetric change and by counting motile spirochetes by darkfield microscopy after 72 hours of incubation.Results: The MIC ranges of the six tested clinical B. miyamotoi isolates to ceftriaxone and azithromycin were 0,03-0,06 mg/L and 0,0016-0,0032 mg/L, respectively. This is similar to MICs from RF Borrelia- and B. miyamotoi tick isolates. All tested RF Borrelia were susceptible to doxycycline (microscopic MIC range 0,0625-0,25 mg/L). In contrast to the MIC of the tested B. burgdorferi sl strains and in line with our previous findings, the MICs for amoxicillin (range 8-32mg/L) of all RF Borrelia -- including B. miyamotoi clinical isolates -- were above the clinical breakpoint for resistance (≤4 mg/L).Conclusions: Clinical isolates of B. miyamotoi are highly susceptible to doxycycline, azithromycin and ceftriaxone in vitro. Interestingly, as earlier described for tick isolates, amoxicillin shows poor in vitro activity against clinical B. miyamotoi isolates.

Sahay, B., Bashant, K., Nelson, N. J., Patsey, R. L., Gadila, S. K., Boohaker, R., Verma, A., Strle, K., Sellati, T. J.

Host genotype influences the severity of murine Lyme borreliosis, caused by the spirochetal bacterium Borrelia burgdorferi. C57BL/6 (B6) mice develop mild, whereas C3H/HeN (C3H) mice develop severe Lyme arthritis. Differential expression of interleukin 10 (IL-10) has long been associated with mouse strain differences in Lyme pathogenesis; however, the underlying mechanism(s) of this genotype-specific IL-10 regulation remained elusive. Herein we reveal a cAMP-mediated mechanism of IL-10 regulation in B6 macrophages that is substantially diminished in C3H macrophages. Under cAMP and CD14-p38 MAPK signaling, B6-macrophage stimulated with B. burgdorferi produce increased amounts of IL-10 and decreased levels of arthritogenic cytokines, including TNF. cAMP relaxes chromatin while p38 increases binding of the transcription factors STAT3 and SP1 to the IL-10 promoter leading to increased IL-10 production in B6 BMDMs. Conversely, macrophages derived from arthritis-susceptible C3H mice possess significantly less endogenous cAMP, produce less IL-10, and thus are ill-equipped to mitigate the damaging consequences of B. burgdorferi-induced TNF. Intriguingly, an altered balance between anti- and pro-inflammatory cytokines and CD14-dependent regulatory mechanisms also is operative in primary human peripheral blood-derived monocytes; providing potential insight into the clinical spectrum of human Lyme disease. In line with this notion, we have demonstrated that cAMP-enhancing drugs increase IL-10 production in myeloid cells, thus curtailing inflammation associated with murine Lyme Borreliosis. Discovery of novel or repurposing of FDA-approved cAMP-modulating medications may be a promising avenue for treatment of patients with adverse clinical outcomes, including certain Post-Lyme complications, in whom dysregulated immune responses may play a role.

Jun-Gu Kang, Jeong-Byoung Chae, Yoon-Kyoung Cho, Young-Sun Jo, Nam-Shik Shin, Hang Lee, Kyoung-Seong Choi, Do-Hyeon Yu, Jinho Park, Bae-Keun Park and Joon-Seok Chae

Abstract. Anaplasmosis, cat-scratch disease, and Lyme disease are emerging vector-borne infectious diseases in Korea. Although the prevalence of vector-borne pathogens (VBPs) in domestic animals and vector arthropods has been documented, there is limited information on the presence of VBPs in wild animals. The raccoon dog (Nyctereutes procyonoides), a wild canid found in East Asia and Europe, represents a potential wildlife reservoir for zoonotic diseases. To investigate the prevalence of VBPs in raccoon dogs, 142 carcasses and 51 blood samples from captured raccoon dogs were collected from 2003 to 2010 and from 2008 to 2009, respectively, in Korea. In addition, 105 Haemaphysalis flava (14 larvae, 43 nymphs, 32 males, and 16 females) and nine Haemaphysalis longicornis (all female) were collected from three raccoon dogs. Samples of the spleen and blood were tested for the presence of VBPs by using nested polymerase chain reaction. Among the samples collected from 193 raccoon dogs and 114 ticks, two samples were positive for Anaplasma phagocytophilum, four for Anaplasma bovis, two for Borrelia theileri, and two for Bartonella henselae. To the best of our knowledge, this study is the largest survey of raccoon dogs aimed at the analysis of VBPs in this species. Moreover, the present study represents the first identification of A. phagocytophilum, B. henselae, and B. theileri in raccoon dogs in their native habitat (East Asia).

The American Journal of Tropical Medicine and Hygiene

Authors: Kang JG, Chae JB, Cho YK, Jo YS, Shin NS, Lee H, Choi KS, Yu DH, Park J, Park BK, Chae JS Abstract Anaplasmosis, cat-scratch disease, and Lyme disease are emerging vector-borne infectious diseases in Korea. Although the prevalence of vector-borne pathogens (VBPs) in domestic animals and vector arthropods has been documented, there is limited information on the presence of VBPs in wild animals. The raccoon dog (Nyctereutes procyonoides), a wild canid found in East Asia and Europe, represents a potential wildlife reservoir for zoonotic diseases. To investigate the prevalence of VBPs in raccoon dogs, 142 carcasses and 51 blood samples from captured raccoon dogs were collected from 2003 to 2010 and from 2008 to 2009, respectively, in Korea. In addition, 105 Haemaphysalis flava...

Manning J, Morens D, Kamhawi S, et al.

AbstractArthropod-borne viruses (arboviruses) are taxonomically diverse causes of significant morbidity and mortality. In recent decades, important mosquito-borne viruses such as West Nile, chikungunya, dengue, and Zika have re-emerged and spread widely, in some cases pandemically, to cause serious public health emergencies. There are no licensed vaccines against most of these viruses, and vaccine development and use has been complicated by the number of different viruses to protect against, by subtype and strain variation, and by the inability to predict when and where outbreaks will occur. A new approach to preventing arboviral diseases is suggested by the observation that arthropod saliva facilitates transmission of pathogens, including leishmania parasites, Borrelia burgdorferi, and some arboviruses. Viruses carried within mosquito saliva may more easily initiate host infection by taking advantage of the host’s innate and adaptive immune responses to saliva. This provides a rationale for creating vaccines against mosquito salivary proteins, rather than against only the virus proteins contained within the saliva. As proof of principle, immunization with sand fly salivary antigens to prevent leishmania infection has shown promising results in animal models. A similar approach using salivary proteins of important vector mosquitoes, such as Aedes aegypti, might protect against multiple mosquito-borne viral infections.

Eckman E, Pacheco-Quinto J, Herdt A, et al.

AbstractBackgroundLyme encephalopathy, characterized by non-specific neurobehavioral symptoms including mild cognitive difficulties, may occur in patients with systemic Lyme disease and is often mistakenly attributed to CNS infection. Identical symptoms occur in innumerable other inflammatory states and may reflect the effect of systemic immune mediators on the CNS.MethodsMultiplex immunoassays were used to characterize the inflammatory profile in serum and CSF from Lyme and non-Lyme patients with a range of symptoms to determine if there are specific markers of active CNS infection (neuroborreliosis), or systemic inflammatory mediators associated with neurobehavioral syndromes.ResultsCSF CXCL13 was elevated dramatically in confirmed neuroborreliosis (n=8) and to a lesser extent in possible neuroborreliosis (n=11) and other neuroinflammatory conditions (n=44). Patients with Lyme (n=63) or non-Lyme (n=8) encephalopathy had normal CSF findings, but had elevated serum levels of IL-7, TSLP, IL-17A, IL-17F, and MIP-1α/CCL3.ConclusionsCSF CXCL13 is a sensitive and specific marker of neuroborreliosis in individuals with Borrelia-specific intrathecal antibody (ITAb) production. However, CXCL13 does not distinguish individuals strongly suspected of having neuroborreliosis, but lacking confirmatory ITAb, from those with other neuroinflammatory conditions. Patients with mild cognitive symptoms occurring during acute Lyme disease, and/or following appropriate treatment, have normal CSF but elevated serum levels of T-helper 17 markers and T-cell growth factors. These markers are also elevated in non-Lyme disease patients experiencing similar symptoms. Our results support that in the absence of CSF abnormalities, neurobehavioral symptoms are associated with systemic inflammation, not CNS infection or inflammation, and are not specific to Lyme disease.

J. Koetsveld, N.M. Kolyasnikova, A. Wagemakers, O.A. Stukolova, D. Hoornstra, D.S. Sarksyan, M.G. Toporkova, A.J. Henningsson, D. Hvidsten, W. Ang, R. Dessau, A.E. Platonov, J.W. Hovius

Borrelia miyamotoi disease (BMD) is an emerging tick-borne disease in the Northern hemisphere. Serodiagnosis by measuring antibodies against glycerophosphodiester-phosphodiesterase (GlpQ) has been performed experimentally but has not been extensively clinically validated. Because we had previously shown the differential expression of antigenic variable major proteins (Vmps) in B. miyamotoi, our aim was to study antibody responses against GlpQ and Vmps in PCR-proven BMD patients and controls.

Cristina Fevola, Suvi Kuivanen, Teemu Smura, Antti Vaheri, Hannimari Kallio-Kokko, Heidi C. Hauffe, Olli Vapalahti, Anne J. J??skel?inen

Background: Directly-transmitted rodent-borne zoonotic viruses, such as lymphocytic choriomeningitis virus (LCMV) can cause nervous system infections. Rodent-borne Ljungan virus (LV) is considered potentially zoonotic possibly causing neurological symptoms. Our objective was to understand the role of these two viruses compared to other pathogens in causing neurological infections in Finnish patients. Methods: Routine screening data were available for 400 patients aged 5-50 years, collected from December 2013-December 2014 with suspected neurological infection. Depending on symptoms, patients were variously tested for herpesviruses, enteroviruses, varicella zoster virus and Mycoplasma pneumoniae, while those suspected of tick bite were further tested for Borrelia spp. and tick-borne encephalitis virus using antibody and/or nucleic acid tests. For 380 patients, we also screened the RNA and antibody prevalence of LCMV and LV in order to test if either of these viruses were the causative agent. Results: Data collected indicated that the causative microbial agent was confirmed in only 15.5% of all Finnish patients with neurological symptoms, with M. pneumoniae (26 cases) being the most common causative agent found in sera, whereas Borrelia spp. (15), herpes simplex viruses (7) and enteroviruses (5) were the most common agents confirmed in the CSF. The seroprevalences for LV and LCMV were 33.8% and 5.0%, respectively, but no samples were PCR-positive. Conclusions: In this study, M. pneumoniae and Borrelia spp. were the most common causative agents of neurological infections in Finland. No LCMV or LV infections were detected. We conclude there was no association of LV with neurological diseases in this patient cohort. This article is protected by copyright. All rights reserved …

Molloy P, Weeks K, Todd B, et al.

AbstractBackgroundThere are no Food and Drug Administration (FDA) approved diagnostic tests for Borrelia miyamotoi infection, an emerging tick-borne illness in the United States. The purpose of this study was to evaluate whether the FDA-approved C6 peptide ELISA currently used to diagnose Lyme disease may potentially serve as a diagnostic test for B. miyamotoi infections.MethodsSerum specimens from 30 patients from the Northeastern United States with B. miyamotoi infection established by a polymerase chain reaction assay of a blood specimen were tested using the C6 ELISA. To reduce confounding with B. burgdorferi co-infection, 6 sera were excluded: 3 from patients with a positive Western immunoblot for antibodies to B. burgdorferi and 3 from patients for whom immunoblot testing had not been performed. ResultsTwenty-two (91.7%; (95% C.I. 73.0%-98.8%) of 24 evaluable B. miyamotoi patients were C6 ELISA reactive, principally on a convalescent phase serum specimen. C6 ELISA index values were often well above the positive cut-off value of 1.1, exceeding 4 in 11 (50.0%) of the 22 C6 ELISA reactive patients.ConclusionsAlthough previously regarded as a highly specific test for Lyme disease, the C6 ELISA is also regularly positive on convalescent phase serum samples of patients from the Northeastern United States with B. miyamotoi infection.

Molloy P, Weeks K, Todd B, et al.

AbstractBackgroundThere are no Food and Drug Administration (FDA) approved diagnostic tests for Borrelia miyamotoi infection, an emerging tick-borne illness in the United States. The purpose of this study was to evaluate whether the FDA-approved C6 peptide ELISA currently used to diagnose Lyme disease may potentially serve as a diagnostic test for B. miyamotoi infections.MethodsSerum specimens from 30 patients from the Northeastern United States with B. miyamotoi infection established by a polymerase chain reaction assay of a blood specimen were tested using the C6 ELISA. To reduce confounding with B. burgdorferi co-infection, 6 sera were excluded: 3 from patients with a positive Western immunoblot for antibodies to B. burgdorferi and 3 from patients for whom immunoblot testing had not been performed. ResultsTwenty-two (91.7%; (95% C.I. 73.0%-98.8%) of 24 evaluable B. miyamotoi patients were C6 ELISA reactive, principally on a convalescent phase serum specimen. C6 ELISA index values were often well above the positive cut-off value of 1.1, exceeding 4 in 11 (50.0%) of the 22 C6 ELISA reactive patients.ConclusionsAlthough previously regarded as a highly specific test for Lyme disease, the C6 ELISA is also regularly positive on convalescent phase serum samples of patients from the Northeastern United States with B. miyamotoi infection.

Kingry L, Anacker M, Pritt B, et al.

AbstractBackgroundTick-transmitted Borrelia species fall into two heterogeneous bacterial complexes comprised of multiple species, the relapsing fever (RF) group and the Borrelia burgdorferi sensu lato group, which are the causative agents of Lyme borreliosis (LB), the most common tickborne disease in the northern hemisphere. Geographic expansion of human LB in the United States and discovery of emerging Borrelia pathogens underscores the importance of surveillance for disease causing Borrelia.MethodsDe-identified clinical specimens, submitted by providers throughout the United States, for patients suspected of LB, anaplasmosis, ehrlichiosis, or babesiosis, were screened using a Borrelia genus level TaqMan PCR. Borrelia species and sequence types (STs) were characterized by multi-locus sequence typing (MLST) utilizing next generation sequencing.ResultsAmong the 7,292 tested specimens tested, five different Borrelia species were identified: two causing LB, B. burgdorferi (n=25) and B. mayonii (n=9), and three RF borreliae, B. hermsii (n=1), B. miyamotoi (n=8), and CandidatusB. johnsonii (n=1), a species previously detected only in the bat tick, Carios kelleyi. ST diversity was greatest for B. burgdorferi positive specimens, with new STs identified primarily among synovial fluids.ConclusionThese results demonstrate broad PCR screening followed by MLST is a powerful surveillance tool for uncovering the spectrum of Borrelia species causing human disease, improving understanding of their geographic distribution, and investigating the correlation between B. burgdorferi STs and joint involvement. Detection of CandidatusB. johnsonii in a patient with suspected tickborne disease suggests this species may be a previously undetected cause of illness in humans with exposure to bat ticks.

Rebecca J. Eisen, Lars Eisen

In the United States, the blacklegged tick, Ixodes scapularis, is a vector of seven human pathogens, including those causing Lyme disease, anaplasmosis, babesiosis, Borrelia miyamotoi disease, Powassan virus disease, and ehrlichiosis associated with Ehrlichia muris eauclarensis. In addition to an accelerated rate of discovery of I. scapularis-borne pathogens over the past two decades, the geographic range of the tick, and incidence and range of I. scapularis-borne disease cases, have increased. Despite knowledge of when and where humans are most at risk of exposure to infected ticks, control of I.