The Lancet

On June 22, 2018, the European Commission issued an updated list of communicable diseases to be covered by epidemiological surveillance in the EU. This list now includes Lyme neuroborreliosis, the neurological manifestation of Lyme borreliosis, which is caused by tick-borne Borrelia burgdorferi sensu lato infection. The decision authorises the European Centre for Disease Prevention and Control (ECDC) to begin monitoring the EU-wide distribution of Lyme neuroborreliosis cases.

Ali Divan, Timothy Casselli, S. Anand Narayanan, Sanjib Mukherjee, David C. Zawieja, John A. Watt, Catherine A. Brissette, M. Karen Newell-Rogers

by Ali Divan, Timothy Casselli, S. Anand Narayanan, Sanjib Mukherjee, David C. Zawieja, John A. Watt, Catherine A. Brissette, M. Karen Newell-Rogers Borrelia burgdorferi, the causative agent of Lyme disease, is a vector-borne bacterial infection that is transmitted through the bite of an infected tick. If not treated with antibiotics during the early stages of infection, disseminated infection can spread to the central nervous system (CNS). In non-human primates (NHPs) it has been demonstrated that the leptomeninges are among the tissues colonized by B. burgdorferi spirochetes. Although the NHP model parallels aspects of human borreliosis, a small rodent model would be ideal to study the trafficking of spirochetes and immune cells into the CNS. Here we show that during early and late disseminated infection, B. burgdorferi infects the meninges of intradermally infected mice, and is associated with concurrent increases in meningeal T cells. We found that the dura mater was consistently culture positive for spirochetes in transcardially perfused mice, independent of the strain of B. burgdorferi used. Within the dura mater, spirochetes were preferentially located in vascular regions, but were also present in perivascular, and extravascular regions, as late as 75 days post-infection. At the same end-point, we observed significant increases in the number of CD3+ T cells within the pia and dura mater, as compared to controls. Flow cytometric analysis of leukocytes isolated from the dura mater revealed that CD3+ cell populations were comprised of both CD4 and CD8 T cells. Overall, our data demonstrate that similarly to infection in peripheral tissues, spirochetes adhere to the dura mater during disseminated infection, and are associated with increases in the number of meningeal T cells. Collectively, our results demonstrate that there are aspects of B. burgdorferi meningeal infection that can be modelled in laboratory mice, suggesting that mice may be useful for elucidating mechanisms of meningeal pathogenesis by B. burgdorferi.

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.

Tammi L. Johnson, Karen A. Boegler, Rebecca J. Clark, Mark J. Delorey, Jenna K. H. Bjork, Frances M. Dorr, Elizabeth K. Schiffman, David F. Neitzel, Andrew J. Monaghan and Rebecca J. Eisen

Abstract. Ixodes scapularis is the vector of at least seven human pathogens in Minnesota, two of which are known to cause Lyme disease (Borrelia burgdorferi sensu stricto and Borrelia mayonii). In Minnesota, the statewide incidence of Lyme disease and other I. scapularis–borne diseases and the geographic extent over which cases have been reported have both increased substantially over the last two decades. These changes correspond with an expanding distribution of I. scapularis over a similar time frame. Because the risk of exposure to I. scapularis–borne pathogens is likely related to the number of ticks encountered, we developed an acarological risk model predicting the density of host-seeking I. scapularis nymphs (DON) in Minnesota. The model was informed by sampling 81 sites located in 42 counties in Minnesota. Two main foci were predicted by the model to support elevated densities of host-seeking I. scapularis nymphs, which included the seven-county Minneapolis-St. Paul metropolitan area and counties in northern Minnesota, including Lake of the Woods and Koochiching counties. There was substantial heterogeneity observed in predicted DON across the state at the county scale; however, counties classified as high risk for I. scapularis–borne diseases and counties with known established populations of I. scapularis had the highest proportion of the county predicted as suitable for host-seeking nymphs (≥ 0.13 nymphs/100 m2). The model provides insight into areas of potential I. scapularis population expansion and identifies focal areas of predicted suitable habitat within counties where the incidence of I. scapularis–borne diseases has been historically low.

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.

D. Hoornstra et al.

Y. Yang et al.

K. Stete et al.

Abstract Background Infection with Borrelia burgdorferi sensu lato complex (B. b. sl) spirochetes can cause Lyme borreliosis, manifesting as localized infection (e.g. erythema migrans) or disseminated disease (e.g. Lyme neuroborreliosis). Generally, patients with disseminated Lyme borreliosis will produce an antibody response several weeks post-infection. So far, no case of neuroborreliosis has been described with persistently negative serology one month after infection. Case presentation We present a patient with a history of Mantle cell lymphoma and treatment with R-CHOP (rituximab, doxorubicine, vincristine, cyclofosfamide, prednisone), with a meningo-encephalitis, who was treated for a suspected lymphoma relapse. However, no malignant cells or other signs of malignancy were found, and microbial tests did not reveal any clues, including Borrelia serology. He did not recall being bitten by ticks, and a Borrelia PCR on CSF was negative. After spontaneous improvement of symptoms, he was discharged without definite diagnosis. Several weeks later, he was readmitted with a relapse of symptoms of meningo-encephalitis. This time however, a Borrelia PCR on CSF was positive, confirmed by two independent laboratories, and the patient received ceftriaxone upon which he partially recovered. Interestingly, during the diagnostic process of this exceptionally difficult case, a variety of different serological assays for Borrelia antibodies remained negative. Only P41 (flagellin) IgG was detected by blot and the Liaison IgG became equivocal 2 months after initial testing. Conclusions To the best of our knowledge this is the first case of neuroborreliosis that is seronegative on repeated sera and multiple test modalities. This unique case demonstrates the difficulty to diagnose neuroborreliosis in severely immunocompromised patients. In this case, a delay in diagnosis was caused by broad differential diagnosis, an absent known history of tick bites, negative serology and the low sensitivity of PCR on CSF. Therefore, awareness of the diagnostic limitations to detect Borrelia infection in this specific patient category is warranted.

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.

Karen O. Johnson, Mark P. Nelder, Curtis Russell, Ye Li, Tina Badiani, Beate Sander, Douglas Sider, Samir N. Patel

by Karen O. Johnson, Mark P. Nelder, Curtis Russell, Ye Li, Tina Badiani, Beate Sander, Douglas Sider, Samir N. Patel Lyme disease (LD) is the most common vector-borne disease in Ontario, Canada. We describe the epidemiology and clinical manifestations of LD in Ontario and examine trends in the incidence of non-disseminated and disseminated LD. LD surveillance data from the integrated Public Health Information System (iPHIS) from 2005–2014 were mapped to symptoms according to syndrome groups (erythema migrans (EM), flu-like, cardiac, neurologic or arthritic) and disease stages (early localized, early disseminated or late disseminated). During the study period, 1,230 cases due to Borrelia burgdoferi were reported in Ontario with annual incidence rates ranging from 0.32 (2006) to 2.16 (2013) cases per 100,000 population. Seventy percent of cases had EM and the proportion of cases with EM increased over time. Other clinical manifestations included flu-like (75%), arthritic (42%), neurologic (41%) and cardiac (6%) symptoms. Early localized disease (n = 415) manifested with EM (87%) and flu-like (57%) symptoms; early disseminated disease (n = 216) manifested with neurologic (94%), cardiac (10%) and EM (63%) symptoms; and late disseminated disease (n = 475) manifested with EM (62%), neurologic (55%), cardiac (9%), and arthritic symptoms (i.e., arthralgia (93%) and arthritis (7%)). Early localized and early disseminated cases (88% each) occurred primarily from May through September, compared to late disseminated cases (81%). The proportion of cases reported to public health within 30 days of illness onset increased during the study period, while the proportion of cases reported within 1–3 months and >3 months decreased. Geographical variations characterized by higher incidence of early localized disease and earlier public health notification (within 30 days of illness onset) occurred in regions with established or recently established LD risk areas, while later public health notification (>3 months after illness onset) was reported more frequently in regions with recently established or no identified risk areas. This is the first study to describe the clinical manifestations of LD in Ontario, Canada. The observed geographical variations in the epidemiology of LD in Ontario reinforce the need for regionally focused public health strategies aimed at increasing awareness, promoting earlier recognition and reporting, and encouraging greater uptake of preventive measures.

L. Karan et al.

Abstract Background With a prevalence of 4.7–13% in Danish Ixodes ricinus ticks, Rickettsia helvetica is one of the most frequently detected tick-borne organisms in Denmark. Most reports of human exposure have described asymptomatic seroconversion or a mild, self-limiting flu-like illness but it has also been implicated as a cause of subacute lymphocytic meningitis. Because Borrelia burgdorferi sensu lato (Bbsl) and R. helvetica are both found in the same tick species, potential co-transmission is a possibility. We examined 1) the seroprevalence of anti-rickettsia antibodies in patients investigated for Lyme neuroborreliosis (LNB), and 2) the cerebrospinal fluid (CSF) and sera of same patients for the presence of Rickettsia DNA. Methods Ninety-nine sera and 87 CSF samples from patients with intrathecal synthesis of anti-Borrelia antibodies and 101 sera and 103 CSF samples from patients with no detectable intrathecal synthesis were retrospectively examined for this study. Sera were analyzed for antibodies against spotted fever group (SFG) rickettsiae and both the CSF and sera were tested for Rickettsia DNA using a genus-specific real-time PCR. Results Of the patients tested for LNB, 32% (64/200) had IgG antibodies against SFG rickettsiae. Among patients with confirmed intrathecal synthesis of Borrelia-specific antibodies, 38% (38/99) exhibited IgG antibodies. None of these values were statistically significant when compared with sera from healthy blood donors (p = 0.7 and 0.19). Rickettsia DNA was found in the CSF of 4% (8/190) of patients. Conclusion No statistically significant difference was found in the seroprevalence of anti-rickettsia antibodies in patients tested for LNB and healthy blood donors, indicative of a low rate of exposure in this group of patients. Eight patients showed evidence of Rickettsia DNA in the CSF, five of whom had LNB. However, cycle threshold (Ct) values were high, indicating low concentrations of DNA, and no apparent alteration in the clinical manifestations of LNB were noted in the medical records of these patients.

Sébastien Bontemps-Gallo, Kevin A. Lawrence, Crystal L. Richards, Frank C. Gherardini

by Sébastien Bontemps-Gallo, Kevin A. Lawrence, Crystal L. Richards, Frank C. Gherardini In recent years, the number of Lyme disease or borreliosis cases in Eurasia has been dramatically increasing. This tick-borne disease is caused by Borrelia burgdorferi sensu lato, which includes B. burgdorferi sensu stricto, the main species found in North America, and B. afzelii and B. garinii, which are primarily responsible for the disease in Eurasia. Currently, research on Lyme disease has focused mainly on B. burgdorferi while B. afzelii and B. garinii, which cause disease with distinctly different symptoms, are less studied. The purpose of this study is to evaluate B. afzelii BO23 and B. garinii CIP 103362 as model organisms to study Eurasian Lyme disease. To begin our analyses, we sequenced, annotated the chromosomes of both species and compared them to B. burgdorferi strain B31. We also assayed shuttle vector, pBSV2, for transformation efficacy and demonstrated that these strains can be cultured on solid media. In addition, we characterized how physicochemical parameters (e.g., oxygen, osmolarity, oxidative stress) affect both growth and motility of the bacteria. Finally, we describe each strain’s antibiotic susceptibility and accessed their ability to infect mice. In conclusion, B. afzelii BO23 was more practical for in vitro and in vivo studies than B. garinii CIP 103362.

Shanna K. Williams, Zachary P. Weiner, Robert D. Gilmore

by Shanna K. Williams, Zachary P. Weiner, Robert D. Gilmore Borrelia burgdorferi, the agent of Lyme borreliosis, can elude hosts’ innate and adaptive immunity as part of the course of infection. The ability of B. burgdorferi to invade or be internalized by host cells in vitro has been proposed as a mechanism for the pathogen to evade immune responses or antimicrobials. We have previously shown that B. burgdorferi can be internalized by human neuroglial cells. In this study we demonstrate that these cells take up B. burgdorferi via coiling phagocytosis mediated by the formin, Daam1, a process similarly described for human macrophages. Following coincubation with glial cells, B. burgdorferi was enwrapped by Daam1-enriched coiling pseudopods. Coiling of B. burgdorferi was significantly reduced when neuroglial cells were pretreated with anti-Daam1 antibody indicating the requirement for Daam1 for borrelial phagocytosis. Confocal microscopy showed Daam1 colocalizing to the B. burgdorferi surface suggesting interaction with borrelial membrane protein(s). Using the yeast 2-hybrid system for identifying protein-protein binding, we found that the B. burgdorferi surface lipoprotein, BBA66, bound the FH2 subunit domain of Daam1. Recombinant proteins were used to validate binding by ELISA, pull-down, and co-immunoprecipitation. Evidence for native Daam1 and BBA66 interaction was suggested by colocalization of the proteins in the course of borrelial capture by the Daam1-enriched pseudopodia. Additionally, we found a striking reduction in coiling for a BBA66-deficient mutant strain compared to BBA66-expressing strains. These results show that coiling phagocytosis is a mechanism for borrelial internalization by neuroglial cells mediated by Daam1.

Batool, M., Caoili, S. E. C., Dangott, L. J., Gerasimov, E., Ionov, Y., Piontkivska, H., Zelikovsky, A., Waghela, S. D., Rogovskyy, A. S.

The tick-borne pathogen, Borrelia burgdorferi, is responsible for approximately 300,000 Lyme disease (LD) cases per year in the United States. Recent increases in the number of LD cases in addition to the spread of the tick vector and a lack of vaccine highlights an urgent need for designing and developing an efficacious LD vaccine. Identification of protective epitopes that could be used to develop a second-generation vaccine is therefore imperative. Despite the antigenicity of several lipoproteins and integral outer membrane proteins (OMPs) on B. burgdorferi surface, the spirochetes successfully evade antibodies primarily due to the VlsE-mediated antigenic variation. VlsE is thought to sterically block antibody access to protective epitopes of B. burgdorferi. However, it is highly unlikely that VlsE shields the entire surface epitome. Thus, identification of subdominant epitope targets that induce protection, when made dominant, is necessary to generate an efficacious vaccine. Towards the identification, we repeatedly immunized immunocompetent mice with live-attenuated VlsE-deleted B. burgdorferi and then challenged the animals with VlsE-expressing (host-adapted) wild type. Passive immunization and western blot data suggested that the protection of the 50% of repeatedly-immunized animals against the highly immune-evasive B. burgdorferi was antibody-mediated. Comparison of serum antibody repertoires identified in protected and non-protected animals permitted the identification of several putative epitopes significantly associated with the protection. Most linear putative epitopes were conserved between the main pathogenic Borrelia genospecies and found within known subdominant regions of OMPs. Currently, we are performing immunization studies to test whether the identified protection-associated epitopes are protective for mice.

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.

G. J. Hickling et al.

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...

Abstract Background The studies on the occurrence and diversity of tick-borne infections in HIV-infected individuals have been few, and the subject has been relatively neglected when compared with other common infections associated with HIV. In HIV-positive patients in whom a serological diagnostics is complicated due to reduced positive predictive value, a method where the microorganism is detected directly is of great value. Therefore, we performed a molecular study to ascertain the prevalence and incidence of tick-borne infections in HIV-infected persons in Poland, an endemic area for Ixodes ricinus ticks. Methods Genomic DNA was isolated from whole blood of tested patients. Detection of tick-borne pathogens was performed by amplification and sequencing of different loci. Molecular and phylogenetic analyses of obtained nucleotide sequences were performed. Serum samples were analyzed for antibodies against tick-borne pathogens by using commercial tests in all patients. Results Among 148 studied blood samples from HIV-infected patients, two cases (1.4%) of infection with tick-borne pathogen were reported. No symptoms of tick-borne infection were observed in these cases. In one case a patient was infected with Anaplasma phagocytophilum – the agent of human granulocytic anaplasmosis (HGA) and in the other with Borrelia garinii. Conclusions Our study revealed the first case of HIV positive patient infected with A. phagocytophilum. Asymptomatic tick-borne infection can occur in HIV-positive patients. The detailed history of tick bites, especially in endemic tick areas, should be considered as part of anamnesis in routine clinical care of HIV-positive patients.

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.

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

Borrelia burgdorferi (Bb) responds to a variety of host-derived factors and appropriately alters its gene expression for adaptation under different host-specific conditions. We previously showed that varying levels of acetate, a Short-Chain Fatty Acid (SCFA), altered the protein profile of Bb. In this study, we determined the effects of other physiologically relevant SCFAs in the regulation of metabolic/virulence-associated proteins using mutant borrelial strains. No apparent increase in the synthesis of Outer surface protein C (OspC) was noted when carbon storage regulator A (csrABb) mutant was propagated within dialysis membrane chambers implanted within rat peritoneal cavity while the parental wild type (B31-A3 strain, wt) and csrABb cis-complemented strain (ct) had increased OspC with a reciprocal reduction in OspA levels. Growth rates of wt, mt, ct, 7D (csrABb mutant lacking 7 amino acids at the C-terminus) and 8S (csrABb with site-specific changes altering its RNA-binding properties) borrelial strains were similar in the presence of acetate. Increased levels of propionate and butyrate reduced the growth rates of all strains tested with mt and 8S exhibiting profound growth deficit at higher concentrations of propionate. Transcriptional levels of rpoS and ospC were elevated on supplementation of SCFAs compared to untreated spirochetes. Immunoblot analysis revealed elevated levels of RpoS, OspC and DbpA with increased levels of SCFAs. Physiological levels of SCFAs prevalent in select human and rodent fluids were synergistic with mammalian host temperature and pH to increase the levels of aforementioned proteins, which could impact the colonization of Bb during the mammalian phase of 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.

Francesco Cerutti, Paola Modesto, Francesca Rizzo, Alessandra Cravero, Irena Jurman, Stefano Costa, Mauro Giammarino, Maria Lucia Mandola, Mariella Goria, Slobodanka Radovic, Federica Cattonaro, Pier Luigi Acutis, Simone Peletto

by Francesco Cerutti, Paola Modesto, Francesca Rizzo, Alessandra Cravero, Irena Jurman, Stefano Costa, Mauro Giammarino, Maria Lucia Mandola, Mariella Goria, Slobodanka Radovic, Federica Cattonaro, Pier Luigi Acutis, Simone Peletto Arthropod vectors are responsible for the transmission of human pathogens worldwide. Several arthropod species are bird ectoparasites, however, no study to date has characterized their microbiota as a whole. We sampled hematophagous ectoparasites that feed on migratory birds and performed 16S rRNA gene metabarcoding to characterize their microbial community. A total of 194 ectoparasites were collected from 115 avian hosts and classified into three groups: a) Hippoboscidae diptera; b) ticks; c) other arthropods. Metabarcoding showed that endosymbionts were the most abundant genera of the microbial community, including Wolbachia for Hippoboscidae diptera, Candidatus Midichloria for ticks, Wolbachia and Arsenophonus for the other arthropod group. Genera including pathogenic species were: Rickettsia, Borrelia, Coxiella, Francisella, Bartonella, Anaplasma. Co-infection with Borrelia-Rickettsia and Anaplasma-Rickettsia was also observed. A global overview of the microbiota of ectoparasites sampled from migratory birds was obtained with the use of 16S rRNA gene metabarcoding. A novel finding is the first identification of Rickettsia in the common swift louse fly, Crataerina pallida. Given their possible interaction with pathogenic viruses and bacteria, the presence of endosymbionts in arthropods merits attention. Finally, molecular characterization of genera, including both pathogenic and symbiont species, plays a pivotal role in the design of targeted molecular diagnostics.

Nathan C. Nieto, W. Tanner Porter, Julie C. Wachara, Thomas J. Lowrey, Luke Martin, Peter J. Motyka, Daniel J. Salkeld

by Nathan C. Nieto, W. Tanner Porter, Julie C. Wachara, Thomas J. Lowrey, Luke Martin, Peter J. Motyka, Daniel J. Salkeld Tick-borne pathogens are increasing their range and incidence in North America as a consequence of numerous factors including improvements in diagnostics and diagnosis, range expansion of primary vectors, changes in human behavior, and an increasing understanding of the diversity of species of pathogens that cause human disease. Public health agencies have access to human incidence data on notifiable diseases e.g., Borrelia burgdorferi, the causative agent of Lyme disease, and often local pathogen prevalence in vector populations. However, data on exposure to vectors and pathogens can be difficult to determine e.g., if disease does not occur. We report on an investigation of exposure to ticks and tick-borne bacteria, conducted at a national scale, using citizen science participation. 16,080 ticks were submitted between January 2016 and August 2017, and screened for B. burgdorferi, B. miyamotoi, Anaplasma phagocytophilum, and Babesia microti. These data corroborate entomologic investigations of tick distributions in North America, but also identify patterns of local disease risk and tick contact with humans throughout the year in numerous species of ticks and associated pathogens.