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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.

Background Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has recently emerged in the field of entomology as a promising method for the identification of arthropods and the detection of associated pathogens. Methodology/Principal findings An experimental model of Ctenocephalides felis (cat fleas) infected with Bartonella quintana and Bartonella henselae was developed to evaluate the efficacy of MALDI-TOF MS in distinguishing infected from uninfected fleas, and its ability to distinguish fleas infected with Bartonella quintana from fleas infected with Bartonella henselae. For B. quintana, two groups of fleas received three successive blood meals, infected or not. A total of 140 fleas (100 exposed fleas and 40 control fleas) were engorged on human blood, infected or uninfected with B. quintana. Regarding the second pathogen, two groups of fleas (200 exposed fleas and 40 control fleas) were fed in the same manner with human blood, infected or not with Bartonella henselae. Fleas were dissected longitudinally; one-half was used for assessment of B. quintana and B. henselae infectious status by real-time PCR, and the second half was subjected to MALDI-TOF MS analysis. Comparison of MS spectra from infected fleas and uninfected fleas revealed distinct MS profiles. Blind queries against our MALDI-TOF MS arthropod database, upgraded with reference spectra from B. quintana and B. henselae infected fleas but also non-infected fleas, provided the correct classification for 100% of the different categories of specimens tested on the first model of flea infection with Bartonella quintana. As for Bartonella henselae, 81% of exposed qPCR-positive fleas, 96% of exposed qPCR-negative fleas and 100% of control fleas were correctly identified on the second model of flea infection. MALDI-TOF MS successfully differentiated Bartonella spp.-infected and uninfected fleas and was also able to correctly differentiate fleas infected with Bartonella quintana and fleas infected with Bartonella henselae. MALDI-TOF MS correctly identified flea species as well as their infectious status, consistent with the results of real-time PCR. Conclusions/Significance MALDI-TOF is a promising tool for identification of the infection status of fleas infected with Bartonella spp., which allows new possibilities for fast and accurate diagnosis in medical entomology and vector surveillance., Author summary Fleas are known vectors of human infectious diseases. Identification of fleas and their associated pathogens is essential for the prevention of flea-borne diseases. Currently, the morphological identification of arthropods based on dichotomous keys, as well as molecular techniques, are the most common approaches for arthropod identification and entomological surveillance. In recent years, MALDI-TOF MS has revolutionized clinical microbiology in enabling the rapid identification of bacteria and fungi by comparing the protein profiles obtained to a database. This proteomic approach has recently been used for arthropod identification and pathogen detection. Here, we developed an experimental model to test MALDI-TOF's ability to differentiate fleas infected with human pathogens, Bartonella quintana and Bartonella henselae, from uninfected fleas.

Tick-Borne Relapsing Fever (TBRF) is caused by spirochetes in the genus Borrelia. Very limited information exists on the incidence of this disease in humans and domestic dogs in the United States. The main objective of this study is to evaluate exposure of dogs to Borrelia turicatae, a causative agent of TBRF, in Texas. To this end, 878 canine serum samples were submitted to Texas A&M Veterinary Medical Diagnostic Laboratory from October 2011 to September 2012 for suspected tick-borne illnesses. The recombinant Borrelial antigen glycerophosphodiester phosphodiesterase (GlpQ) was expressed, purified, and used as a diagnostic antigen in both ELISA assays and Immunoblot analysis. Unfortunately, due to significant background reaction, the use of GlpQ as a diagnostic marker in the ELISA assay was not effective in discriminating dogs exposed to B. turicatae. Nevertheless, immunoblot assays showed that 17 out of 853 samples tested were considered to be seropositive, which constitutes 1.99% of all Texas samples tested in this study. The majority of positive samples were from central and southern Texas. Exposure to TBRF spirochetes may be seasonal, with 70.59% (12 out of 17) of the cases detected between June and December. In addition, 2 out of the 17 sero-reactive cases (11.76%) showed reactivity to both B. burgdorferi (causative agent of Lyme disease) and B. turicatae (a causative agent of TBRF). This is the first report of TBRF sero-prevalence in companion animals in an endemic area. Our findings further indicate that B. turicatae is maintained in domestic canids in Texas in regions where human disease also occurs, suggesting that domestic dogs could serve as sentinels for this disease.

California, with 13 chipmunk (Tamias) species, has more than any other state or country, occupying habitats ranging from chaparral to the high peaks of the Sierra Nevada. Chipmunks host zoonotic pathogens including Yersinia pestis, Anaplasma phagocytophilum, relapsing fever (RF) Borrelia spp., Borrelia burgdorferi, and spotted fever group (SFG) Rickettsia species. Chipmunk species are often not differentiated by public health workers, yet different species utilize different ecological niches and may have intrinsically different capacities for maintaining vector-borne pathogens and infecting vectors. We surveyed over 700 individuals from nine species of chipmunks throughout California for exposure to and infection by Y. pestis, A. phagocytophilum, RF Borrelia spp., Borrelia burgdorferi, and SFG Rickettsia species. DNA of all five pathogens was found and all chipmunks except Merriam's chipmunk (T. merriami) were PCR-positive for at least one of the pathogens. Anaplasma phagocytophilum was most common (40.0%, 2/5) in Sonoma chipmunks (T. sonomae) from Marin county and B. burgdorferi most common (37.5%, 27/72) in redwood chipmunks (T. ochrogenys) from Mendocino county. RF Borrelia spp. was detected in 2% (6/297) of redwood chipmunks in Mendocino county and 10% (1/10) of both least (T. minimus) and lodgepole (T. speciosus) chipmunks in the western Sierra. Exposure to SFG Rickettsia spp. was found in the Northern Coastal region (Del Norte, Humboldt and Mendocino counties) and in the northern and western Sierra in several species of chipmunks. Y. pestis infection was found only in the western Sierra-in a yellow-pine (T. amoenus) and a long-eared (T. quadrimaculatus) chipmunk. Though more data are needed to thoroughly understand the roles that different chipmunk species play in disease transmission, our findings suggest that some chipmunk species may be more important to the maintenance of vector-borne diseases than others within each geographic area.

Previous studies have found that Met52®, which contains the entomopathogenic fungus Metarhizium brunneum, is effective in reducing the abundance of Ixodes scapularis, the tick vector for the bacterium causing Lyme disease and for other tick-borne pathogens. Given widespread interest in effective, safe methods for controlling ticks, Met52 has the potential to be used at increasing scales. The non-target impacts of Met52, as applied for tick control, have not yet been assessed. A Before-After-Control-Impact experiment was conducted to assess the effects of Met52 on non-target arthropods in lawn and forest habitats typical of residential yards. Ground-dwelling arthropods were collected using bulk sampling of soil and litter, and pitfall sampling. Arthropods were sampled once before and twice after treatment of plots with either Met52 or water (control). Multivariate general linear models were used to jointly model the abundance of arthropod orders. For each sampling method and post-spray sampling occasion, Akaike Information Criterion values were used to compare the fits of two alternative models: one that included effects of period (before vs. after spray), habitat (lawn vs. forest), and treatment (Met52 vs. control), versus a nested null model that included effects of period, and habitat, but no treatment effect. The null model was consistently better supported by the data. Significant effects were found of period and habitat but not treatment. Retrospective power analysis indicated the study had 80% power to detect a 50% reduction in arthropod abundance, as measured by bulk samples taken before versus one week after treatment. The deployment of Met52 in suburban settings is unlikely to cause meaningful reductions in the abundance of non-target arthropods.

Lyme disease (LD) diagnosis using the current two-tier algorithm is constrained by low sensitivity for early-stage infection and ambiguity in determining treatment response. We recently developed a protein microarray biochip that measures diagnostic serum antibody targets using grating-coupled fluorescent plasmonics (GC-FP) technology. This strategy requires microliters of blood serum to enable multiplexed biomarker screening on a compact surface and generates quantitative results that can be further processed for diagnostic scoring. The GC-FP biochip was used to detect serum antibodies in patients with active and convalescent LD, as well as various negative controls. We hypothesized that the quantitative, high-sensitivity attributes of the GC-FP approach permit: 1) screening of antibody targets predictive for LD status, and 2) development a diagnostic algorithm that is more sensitive, specific, and informative than the standard ELISA and Western blot assays. Notably, our findings led to a diagnostic algorithm that may be more sensitive than the current standard for detecting early LD, while maintaining 100% specificity. We further show that analysis of relative antibody levels to predict disease status, such as in acute and convalescent stages of infection, is possible with a highly sensitive and quantitative platform like GC-FP. The results from this study add to the urgent conversation regarding better diagnostic strategies and more effective treatment for patients affected by tick-borne disease.

Peromyscus leucopus (the white-footed mouse) is a known reservoir of the Lyme disease spirochete Borrelia burgdorferi. Sampling of white-footed mice allows for year-round B. burgdorferi surveillance as well as opportunities to establish the diversity of the different variants in a geographic region. This study explores the prevalence of B. burgdorferi infections in the tissues of white-footed mice, investigates the correlations between B. burgdorferi infected tissues, and determines the optimum field methods for surveillance of B. burgdorferi in P. leucopus. A total of 90 mice and 573 tissues (spleen, liver, ear, tongue, tail, heart, and kidney) were screened via nested PCR for B. burgdorferi infections. A large number of infections were found in the 90 mice as well as multiple infections within individual mice. Infections in a single mouse tissue (spleen, liver, ear, tongue and tail) were predictive of concurrent infection in other tissues of the same mouse at a statistically significant level. Ear tissue accounted for 68.4% of detected infections, which increased to 78.9% of the infected mice with the inclusion of tail samples. The use of ear punch or tail snip samples (used individually or in tandem) have multiple advantages over current Lyme disease ecological studies and surveillance methodologies, including lower associated costs, minimization of delays, year-round B. burgdorferi testing opportunities, as well as longitudinal monitoring of B. burgdorferi in defined geographic regions. In the absence of an effective vaccine, personal prevention measures are currently the most effective way to reduce Lyme disease transmission to humans. Thus, the identification and monitoring of environmental reservoirs to inform at-risk populations remains a priority. The sampling methods proposed in this study provide a reasonable estimate of B. burgdorferi in white-footed mice in a timely and cost-effective manner.

In this paper, a simple yet interpretable, probabilistic model is proposed for the prediction of reported case counts of infectious diseases. A spatio-temporal kernel is derived from training data to capture the typical interaction effects of reported infections across time and space, which provides insight into the dynamics of the spread of infectious diseases. Testing the model on a one-week-ahead prediction task for campylobacteriosis and rotavirus infections across Germany, as well as Lyme borreliosis across the federal state of Bavaria, shows that the proposed model performs on-par with the state-of-the-arthhh4model. However, it provides a full posterior distribution over parameters in addition to model predictions, which aides in the assessment of the model. The employed Bayesian Monte Carlo regression framework is easily extensible and allows for incorporating prior domain knowledge, which makes it suitable for use on limited, yet complex datasets as often encountered in epidemiology.Author summaryWhy was this study done?Statistical modeling is invaluable to public-health policy as it helps understand and anticipate the dynamics of the spread of infectious diseases. The available training data is often limited and reported with a low spatial and temporal resolution. This poses a challenge and makes it particularly important to incorporate domain knowledge and prior assumptions to guide the modeling process.In order to evaluate the trustworthiness and reliability of a model’s predictions, it is crucial to be able to interpret the model and quantify the model uncertainty.To address this, we develop an interpretable model that uses Bayesian inference (rather than commonly used maximum likelihood estimation) and provides a probability distribution over inferred parameters.What did the researchers do and find?We develop and test a single probabilistic model that learns to predict the number of weekly case counts for three different diseases (campylobacteriosis, rotaviral enteritis and Lyme borreliosis) at the county level one week ahead of time.We employ a Bayesian Monte Carlo regression approach that provides an estimate of the full probability distribution over inferred parameters as well as model predictions.The model learns an interpretable spatio-temporal kernel that captures typical interactions between infection cases of the tested diseases.The predictive performance of our model compares favorably with a contemporary reference model for all diseases tested.What do these findings mean?Interpretable predictive models can be applied to surveillance data to gain insights into the dynamics of infectious diseases.Probabilistic modeling approaches provide a suitable framework for many challenges of working with epidemiological data.

Objective: Chronic Lyme disease has been a poorly defined term and often dismissed as a fictitious entity. In this paper, the International Lyme and Associated Diseases Society (ILADS) provides its evidence-based definition of chronic Lyme disease. Definition: ILADS defines chronic Lyme disease (CLD) as a multisystem illness with a wide range of symptoms and/or signs that are either continuously or intermittently present for a minimum of six months. The illness is the result of an active and ongoing infection by any of several pathogenic members of the Borrelia burgdorferi sensu lato complex (Bbsl). The infection has variable latency periods and signs and symptoms may wax, wane and migrate. CLD has two subcategories, CLD, untreated (CLD-U) and CLD, previously treated (CLD-PT). The latter requires that CLD manifestations persist or recur following treatment and are present continuously or in a relapsing/remitting pattern for a duration of six months or more. Methods: Systematic review of over 250 peer reviewed papers in the international literature to characterize the clinical spectrum of CLD-U and CLD-PT. Conclusion: This evidence-based definition of chronic Lyme disease clarifies the term’s meaning and the literature review validates that chronic and ongoing Bbsl infections can result in chronic disease. Use of this CLD definition will promote a better understanding of the infection and facilitate future research of this infection.

Some patients have residual non-specific symptoms after therapy for Lyme disease, referred to as post-treatment Lyme disease symptoms or syndrome, depending on whether there is functional impairment. A standardized test battery was used to characterize a diverse group of Lyme disease patients with and without residual symptoms. There was a strong correlation between sleep disturbance and certain other symptoms such as fatigue, pain, anxiety, and cognitive complaints. Results were subjected to a Logistic Regression model using the Neuro-QoL Fatigue t-score together with Short Form-36 Physical Functioning scale and Mental Health component scores; and to a Decision Tree model using only the QoL Fatigue t-score. The Logistic Regression model had an accuracy of 97% and Decision Tree model had an accuracy of 93%, when compared with clinical categorization. The Logistic Regression and Decision Tree models were then applied to a separate cohort. Both models performed with high sensitivity (90%), but moderate specificity (62%). The overall accuracy was 74%. Agreement between 2 time points, separated by a mean of 4 months, was 89% using the Decision Tree model and 87% with the Logistic Regression model. These models are simple and can help to quantitate the level of symptom severity in post-treatment Lyme disease symptoms. More research is needed to increase the specificity of the models, exploring additional approaches that could potentially strengthen an operational definition for post-treatment Lyme disease symptoms. Evaluation of how sleep disturbance, fatigue, pain and cognitive complains interrelate can potentially lead to new interventions that will improve the overall health of these patients.

Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-γ as a biomarker, we developed a new enzyme-linked immunospot method (iSpot LymeTM) to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.

We have previously shown that the Outer surface protein A (OspA) based Lyme borreliosis vaccine VLA15 induces protective immunity in mice. Herein, we report the induction of protective immunity by VLA15 with mouse models using ticks infected with B. burgdorferi (OspA serotype 1), B. afzelii (OspA serotype 2) and B. bavariensis (OspA serotype 4) or with in vitro grown B. garinii (OspA serotype 5 and 6) for challenge. For B. garinii (OspA serotype 3), we have developed a growth inhibition assay using chicken complement and functional antibodies targeting B. garinii (OspA serotype 3) could be demonstrated after immunization with VLA15. Furthermore, following three priming immunizations, a booster dose was administered five months later and the induction of immunological memory could be confirmed. Thus, the antibody titers after the booster dose were increased considerably compared to those after primary immunization. In addition, the half-lives of anti-OspA serotype specific antibodies after administration of the booster immunization were longer than after primary immunization. Taken together, we could show that VLA15 induced protection in mice against challenge with four different clinically relevant Borrelia species (B. burgdorferi, B. afzelii, B. garinii and B. bavariensis) expressing five of the six OspA serotypes included in the vaccine. The protection data is supported by functional assays showing efficacy against spirochetes expressing any of the six OspA serotypes (1 to 6). To our knowledge, this is the first time a Lyme borreliosis vaccine has been able to demonstrate such broad protection in preclinical studies. These new data provide further promise for the clinical development of VLA15 and supports our efforts to provide a new Lyme borreliosis vaccine available for global use.

Background Understanding and quantification of the risk of Lyme borreliosis after a tick bite can aid development of prevention strategies against Lyme borreliosis. Methods We used 3,525 single tick bite reports from three large prospective studies on the transmission risk of tick-borne pathogens to humans, with 50 reports of Lyme borreliosis during the follow-up period, among 1,973 reports with known outcome. A structural equation model was applied to estimate the risk of Lyme borreliosis after a tick bite, and quantify the influence of: developmental stage of the tick, detection of Borrelia burgdorferi s.l. DNA in the tick by PCR, tick engorgement, patient-estimated duration of tick attachment, and patient age. Results The overall risk of developing Lyme borreliosis after a tick bite was 2.6% (95%CI 1.4–5.1). The risk increased with: - Tick engorgement: 1.4% (95%CI 0.7%-2.3%) for low engorgement to 5.5% (95%CI 2.8%-9.2%) for substantially engorged ticks; - Rising patient-estimated tick attachment duration: 2.0% (95%CI 1.3%-2.8%) after

Recent reports suggest that host-seeking nymphs in southern populations of Ixodes scapularis remain below the leaf litter surface, while northern nymphs seek hosts on leaves and twigs above the litter surface. This behavioral difference potentially results in decreased tick contact with humans in the south, and fewer cases of Lyme disease. We studied whether north-south differences in tick survival patterns might contribute to this phenomenon. Four month old larvae resulting from a cross between Wisconsin males and South Carolina females died faster under southern than under northern conditions in the lab, as has previously been reported for ticks from both northern and southern populations. However, newly-emerged larvae from Rhode Island parents did not differ consistently in mortality under northern and southern conditions, possibly because of their younger age. Survival is lower, and so the north-south survival difference might be greater in older ticks. Larval survival was positively related to larval size (as measured by scutal area), while survival was positively related to larval fat content in some, but not all, trials. The difference in larval survival under northern vs. southern conditions might simply result from faster metabolism under warmer southern conditions leading to shorter life spans. However, ticks consistently died faster under southern than under northern conditions in the laboratory when relative humidity was low (75%), but not under moderate (85%) or high (95%) RH. Therefore, mortality due to desiccation stress is greater under southern than under northern conditions. We hypothesize that mortality resulting from the greater desiccation stress under southern conditions acts as a selective pressure resulting in the evolution of host-seeking behavior in which immatures remain below the leaf litter surface in southern I. scapularis populations, so as to avoid the desiccating conditions at the surface. If this hypothesis is correct, it has implications for the effect of climate change on the future distribution of Lyme disease.

This paper models the prevalence of antibodies to Borrelia burgdorferi in domestic dogs in the United States using climate, geographic, and societal factors. We then use this model to forecast the prevalence of antibodies to B. burgdorferi in dogs for 2016. The data available for this study consists of 11,937,925 B. burgdorferi serologic test results collected at the county level within the 48 contiguous United States from 2011-2015. Using the serologic data, a baseline B. burgdorferi antibody prevalence map was constructed through the use of spatial smoothing techniques after temporal aggregation; i.e., head-banging and Kriging. In addition, several covariates purported to be associated with B. burgdorferi prevalence were collected on the same spatio-temporal granularity, and include forestation, elevation, water coverage, temperature, relative humidity, precipitation, population density, and median household income. A Bayesian spatio-temporal conditional autoregressive (CAR) model was used to analyze these data, for the purposes of identifying significant risk factors and for constructing disease forecasts. The fidelity of the forecasting technique was assessed using historical data, and a Lyme disease forecast for dogs in 2016 was constructed. The correlation between the county level model and baseline B. burgdorferi antibody prevalence estimates from 2011 to 2015 is 0.894, illustrating that the Bayesian spatio-temporal CAR model provides a good fit to these data. The fidelity of the forecasting technique was assessed in the usual fashion; i.e., the 2011-2014 data was used to forecast the 2015 county level prevalence, with comparisons between observed and predicted being made. The weighted (to acknowledge sample size) correlation between 2015 county level observed prevalence and 2015 forecasted prevalence is 0.978. A forecast for the prevalence of B. burgdorferi antibodies in domestic dogs in 2016 is also provided. The forecast presented from this model can be used to alert veterinarians in areas likely to see above average B. burgdorferi antibody prevalence in dogs in the upcoming year. In addition, because dogs and humans can be exposed to ticks in similar habitats, these data may ultimately prove useful in predicting areas where human Lyme disease risk may emerge.

The genus Borrelia comprises arthropod-borne bacteria, which are infectious agents in vertebrates. They are mainly transmitted by ixodid or argasid ticks. In Hokkaido, Japan, Borrelia spp. were found in deer and Haemaphysalis ticks between 2011 and 2013; however, the study was limited to a particular area. Therefore, in the present study, we conducted large-scale surveillance of ticks and wild animals in the western part of the main island of Japan. We collected 6,407 host-seeking ticks from two regions and 1,598 larvae obtained from 32 engorged female ticks and examined them to elucidate transovarial transmission. In addition, we examined whole blood samples from 190 wild boars and 276 sika deer, as well as sera from 120 wild raccoons. We detected Borrelia spp. in Haemaphysalis flava, Haemaphysalis megaspinosa, Haemaphysalis kitaokai, Haemaphysalis longicornis, and Haemaphysalis formosensis. In addition, we isolated a strain from H. megaspinosa using Barbour-Stoenner-Kelly medium. The minimum infection rate of ticks was less than 5%. Transovarial transmission was observed in H. kitaokai. Phylogenetic analysis of the isolated strain and DNA fragments amplified from ticks identified at least four bacterial genotypes, which corresponded to the tick species detected. Bacteria were detected in 8.4%, 15%, and 0.8% of wild boars, sika deer, and raccoons, respectively. In this study, we found seasonal differences in the prevalence of bacterial genotypes in sika deer during the winter and summer. The tick activity season corresponds to the season with a high prevalence of animals. The present study suggests that a particular bacterial genotype detected in this study are defined by a particular tick species in which they are present.

Infectious diseases still represent a major challenge for humanity. In this context, their surveillance is critical. From 2010 to 2016, two Point-Of-Care (POC) laboratories have been successfully implemented in the rural Saloum region of Senegal. In parallel, a homemade syndromic surveillance system called EPIMIC was implemented to monitor infectious diseases using data produced by the POC laboratory of the Timone hospital in Marseille, France. The aim of this study is to describe the steps necessary for implementing EPIMIC using data routinely produced by two POC laboratories (POC-L) established in rural Senegal villages. After improving EPIMIC, we started to monitor the 15 pathogens routinely diagnosed in the two POC-L using the same methodology we used in France. In 5 years, 2,577 deduplicated patients-samples couples from 775 different patients have been tested in the Dielmo and Ndiop POC-L. 739 deduplicated patients-samples couples were found to be positive to at least one of the tested pathogens. The retrospective analysis of the Dielmo and Ndiop POC data with EPIMIC allowed to generate 443 alarms. Since January 2016, 316 deduplicated patients-samples couples collected from 298 different patients were processed in the Niakhar POC laboratory. 56 deduplicated patients-samples couples were found to be positive to at least one of the tested pathogens. The retrospective analysis of the data of the Niakhar POC laboratory with EPIMIC allowed to generate 14 alarms. Although some improvements are still needed, EPIMIC has been successfully spread using data routinely produced by two rural POC-L in Senegal, West Africa., Author Summary Infectious diseases are still a major public health problem for humanity, especially because of their unpredictability. In this context, developing surveillance systems is one of the most effective solutions to fight them, including in Sub-Saharan African countries where the situation is particularly worrying. This study describes how automated surveillance systems have been implemented in two rural areas in Senegal for the weekly surveillance of 15 pathogens responsible for fever in Africa and on the basis of laboratory data from two rural laboratories. In addition, the first results of this surveillance are presented. The two surveillance systems prospectively and retrospectively detected several abnormal events, and allowed us to observe that the distribution of the pathogens was not the same depending on the region under surveillance. This study provides evidence on the possibility of developing surveillance systems in rural areas in Africa. Such initiatives are needed, especially to improve our global knowledge on infectious diseases in this continent.

Borrelia mayonii, a Borrelia burgdorferi sensu lato (Bbsl) genospecies, was recently identified as a cause of Lyme borreliosis (LB) among patients from the upper midwestern United States. By microscopy and PCR, spirochete/genome loads in infected patients were estimated at 105 to 106 per milliliter of blood. Here, we present the full chromosome and plasmid sequences of two B. mayonii isolates, MN14-1420 and MN14-1539, cultured from blood of two of these patients. Whole genome sequencing and assembly was conducted using PacBio long read sequencing (Pacific Biosciences RSII instrument) followed by hierarchical genome-assembly process (HGAP). The B. mayonii genome is ~1.31 Mbp in size (26.9% average GC content) and is comprised of a linear chromosome, 8 linear and 7 circular plasmids. Consistent with its taxonomic designation as a new Bbsl genospecies, the B. mayonii linear chromosome shares only 93.83% average nucleotide identity with other genospecies. Both B. mayonii genomes contain plasmids similar to B. burgdorferi sensu stricto lp54, lp36, lp28-3, lp28-4, lp25, lp17, lp5, 5 cp32s, cp26, and cp9. The vls locus present on lp28-10 of B. mayonii MN14-1420 is remarkably long, being comprised of 24 silent vls cassettes. Genetic differences between the two B. mayonii genomes are limited and include 15 single nucleotide variations as well as 7 fewer silent vls cassettes and a lack of the lp5 plasmid in MN14-1539. Notably, 68 homologs to proteins present in B. burgdorferi sensu stricto appear to be lacking from the B. mayonii genomes. These include the complement inhibitor, CspZ (BB_H06), the fibronectin binding protein, BB_K32, as well as multiple lipoproteins and proteins of unknown function. This study shows the utility of long read sequencing for full genome assembly of Bbsl genomes, identifies putative genome regions of B. mayonii that may be linked to clinical manifestation or tissue tropism, and provides a valuable resource for pathogenicity, diagnostic and vaccine studies.

A new wave of portable biosensors allows frequent measurement of health-related physiology. We investigated the use of these devices to monitor human physiological changes during various activities and their role in managing health and diagnosing and analyzing disease. By recording over 250,000 daily measurements for up to 43 individuals, we found personalized circadian differences in physiological parameters, replicating previous physiological findings. Interestingly, we found striking changes in particular environments, such as airline flights (decreased peripheral capillary oxygen saturation [SpO2] and increased radiation exposure). These events are associated with physiological macro-phenotypes such as fatigue, providing a strong association between reduced pressure/oxygen and fatigue on high-altitude flights. Importantly, we combined biosensor information with frequent medical measurements and made two important observations: First, wearable devices were useful in identification of early signs of Lyme disease and inflammatory responses; we used this information to develop a personalized, activity-based normalization framework to identify abnormal physiological signals from longitudinal data for facile disease detection. Second, wearables distinguish physiological differences between insulin-sensitive and -resistant individuals. Overall, these results indicate that portable biosensors provide useful information for monitoring personal activities and physiology and are likely to play an important role in managing health and enabling affordable health care access to groups traditionally limited by socioeconomic class or remote geography., Author Summary A new wave of wearable sensors allows frequent and continuous measurements of body functions (physiology), including heart rate, skin temperature, blood oxygen levels, and physical activity. We investigated the ability of wearable sensors to follow physiological changes that occur over the course of a day, during illness and other activities. Data from these sensors revealed personalized differences in daily patterns of activities. Interestingly, we discovered striking changes in particular environments such as airline flights. Blood oxygen levels decreased during high-altitude flights, and this decrease was associated with fatigue. By combining sensor information with frequent medical measurements, we made two important health-related observations. First, wearable sensors were useful in identifying the onset of Lyme disease and inflammation. From this observation, we then developed a computational algorithm for personalized disease detection using such sensors. Second, we found that wearable sensors can reveal physiological differences between insulin-sensitive and insulin-resistant individuals, raising the possibility that these sensors could help detect risk for type 2 diabetes. Overall, these results indicate that the information provided by wearable sensors is physiologically meaningful and actionable. Wearable sensors are likely to play an important role in managing health.

In Lyme borreliosis, the skin is the key site for bacterial inoculation by the infected tick and for cutaneous manifestations. We previously showed that different strains of Borrelia burg-dorferi sensu stricto isolated from tick and from different clinical stages of the Lyme borrelio-sis (erythema migrans, and acrodermatitis chronica atrophicans) elicited a very similar transcriptional response in normal human dermal fibroblasts. In this study, using whole transcriptome microarray chips, we aimed to compare the transcriptional response of normal human dermal fibroblasts stimulated by 3 Borrelia burgdorferi sensu lato strains belonging to 3 main pathogenic species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) in order to determine whether " species-related " inflammatory pathways could be identified. The three Borrelia strains tested exhibited similar transcriptional profiles, and no species-specific fingerprint of transcriptional changes in fibroblasts was observed. Conversely , a common core of chemokines/cytokines (CCL2, CXCL1, CXCL2, CXCL6, CXCL10, IL-6, IL-8) and interferon-related genes was stimulated by all the 3 strains. Dermal fibroblasts appear to play a key role in the cutaneous infection with Borrelia, inducing a homogeneous inflammatory response, whichever Borrelia species was involved.

International audience; IntroductionLouse-borne relapsing fever (LBRF) is an acute febrile infection that is typically characterized by one to three fairly regular waves of bacteremia [1,2]. It is caused by Borrelia recurrentis, a motile spirochete that measures 5 to 40 μm in length. The microorganism is transmitted from person to person by the human body louse (Pediculus humanus humanus). Disruptions in sanitation during wartime and mass migrations of people provide conditions that favor the propagation of body lice and thus the occurrence of outbreaks of the disease [1,3]. LBRF is endemic in East Africa (e.g., Ethiopia, Eritrea, Somalia, and Sudan) with the highest number of cases observed in Ethiopia, where it is the seventh most common cause of hospital admission and the fifth most common cause of death [4,5]. We report here the first case of imported LBRF observed in Lombardy (northern Italy) in a Somali refugee.

Conclusion: Results from a database for sudden sensorineural hearing loss (SSNHL) demonstrate no correlation between laboratory findings, treatment, and outcome in 400 patients. The patients with pathological test results were not treated differently from those with normal test results. The value of laboratory findings and MRI might increase if the results are categorized to more specific diagnoses. Objectives: To investigate diagnostic test batteries for SSNHL and evaluate their value in the management of idiopathic SSNHL. Methods: A total of 400 patients submitted to the Swedish national database for SSNHL were analyzed. Information was collected about the patients past medical history, potential precipitating events, trauma, medical history, hearing loss, current disease, diagnostic protocol, and treatment, using questionnaires as well as two audiograms, one at the first ENT clinic visit and another 3 months later. Results: In all, 65% of these 400 patients underwent hematological tests and 40% had an MRI/CT scan. Twenty-two of 160 MRI investigated had pathological findings including 5 acoustic neuromas. Also, 300 of these 400 patients were evaluated as having idiopathic sudden sensorineural hearing loss (ISSNHL); 24% of them had one or more pathological test results. No significant correlation was found between either the MRI findings or the laboratory findings with regard to treatment or hearing recovery in patients with ISSNHL. This is an electronic version of an article published in: Ramesh Zarenoe, Magnus Hansson and Elisabeth Hultcrantz, Assessment of diagnostic approaches to idiopathic sudden sensorineural hearing loss and their influence on treatment and outcome, 2010, ACTA OTO-LARYNGOLOGICA, (130), 3, 384-391. ACTA OTO-LARYNGOLOGICA is available online at informaworldTM: http://dx.doi.org/10.3109/00016480903161541 Copyright: Taylor & Francis http://www.tandf.co.uk/journals/default.asp

Borrelia persica, a bacterium transmitted by the soft tick Ornithodoros tholozani, causes tick-borne relapsing fever in humans in the Middle East, Central Asia and the Indian peninsula. Immunocompetent C3H/HeOuJ mice were infected intradermally with B. persica at varying doses: 1 x 106, 1 x 104, 1 x 102 and 4 x 100 spirochetes/mouse. Subsequently, blood samples were collected and screened for the presence of B. persica DNA. Spirochetes were detected in all mice infected with 1 x 106, 1 x 104 and 1 x 102 borrelia by real-time PCR targeting the flaB gene of the bacterium. Spirochetemia developed with a one- to two-day delay when 1 x 104 and 1 x 102 borrelia were inoculated. Mice injected with only four organisms were negative in all tests. No clinical signs were observed when infected mice were compared to negative control animals. Organs (heart, spleen, urinary bladder, tarsal joint, skin and brain) were tested for B. persica-specific DNA and cultured for the detection of viable spirochetes. Compiled data show that the target organs of B. persica infections are the brain and the skin. A newly developed serological two-tiered test system (ELISA and western blot) for the detection of murine IgM, IgG and IgA antibody titers against B. persica showed a vigorous antibody response of the mice during infection. In conclusion, the infection model described here for B. persica is a platform for in vivo studies to decipher the so far unexplored survival strategies of this Borrelia species., Author Summary The spirochete Borrelia persica is a tick-borne bacterium that is transmitted by the vector Ornithodoros tholozani to its vertebrate host in the Middle East, Central Asia and the Indian peninsula. Current migration of vast numbers of individuals from this area increases the likelihood that B. persica infections will be introduced into new geographic regions. After infection and distribution by the bloodstream, relapsing fever episodes occur in humans. Since no reliable in vivo tools have existed so far to study this organism, a murine model was established in this study to characterize the infection kinetics in immunocompetent mice. Aspects of the potential infectivity of the laboratory strain and of potential clinical signs, spirochetemia and antibody response as well as organ tropism and histopathological reactions were studied. With the successful infection model presented here, further studies are conceivable in order to gain advanced insights into the pathogenesis of B. persica infection and to characterize in detail the host immune response mounted against the bacterium. We propose that this model could also be used for the development of new rapid diagnostic approaches to initiate or monitor treatment regimes in order to clear or prevent the infection with B. persica.

Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag) OspC (rOspC) could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a) with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b) with free rOspC and Montanide PET GEL A; (c) with free rOspC and alum; or (d) with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants.

Based on chromosome sequences, the human pathogen Borrelia miyamotoi phylogenetically clusters with species that cause relapsing fever. But atypically for relapsing fever agents, B. miyamotoi is transmitted not by soft ticks but by hard ticks, which also are vectors of Lyme disease Borrelia species. To further assess the relationships of B. miyamotoi to species that cause relapsing fever, I investigated extrachromosomal sequences of a North American strain with specific attention on plasmid-borne vsp and vlp genes, which are the underpinnings of antigenic variation during relapsing fever. For a hybrid approach to achieve assemblies that spanned more than one of the paralogous vsp and vlp genes, a database of short-reads from next-generation sequencing was supplemented with long-reads obtained with real-time DNA sequencing from single polymerase molecules. This yielded three contigs of 31, 16, and 11 kb, which each contained multiple and diverse sequences that were homologous to vsp and vlp genes of the relapsing fever agent B. hermsii. Two plasmid fragments had coding sequences for plasmid partition proteins that differed from each other from paralogous proteins for the megaplasmid and a small plasmid of B. miyamotoi. One of 4 vsp genes, vsp1, was present at two loci, one of which was downstream of a candiate prokaryotic promoter. A limited RNA-seq analysis of a population growing in the blood of mice indicated that of the 4 different vsp genes vsp1 was the one that was expressed. The findings indicate that B. miyamotoi has at least four types of plasmids, two or more of which bear vsp and vlp gene sequences that are as numerous and diverse as those of relapsing fever Borrelia. The database and insights from these findings provide a foundation for further investigations of the immune responses to this pathogen and of the capability of B. miyamotoi for antigenic variation.

Background Our study aimed to assess the presence of different pathogens in ticks collected in two regions in Côte d’Ivoire. Methodology/Principal Findings Real-time PCR and standard PCR assays coupled to sequencing were used. Three hundred and seventy eight (378) ticks (170 Amblyomma variegatum, 161 Rhipicepalus microplus, 3 Rhipicephalus senegalensis, 27 Hyalomma truncatum, 16 Hyalomma marginatum rufipes, and 1 Hyalomma impressum) were identified and analyzed. We identified as pathogenic bacteria, Rickettsia africae in Am. variegatum (90%), Rh. microplus (10%) and Hyalomma spp. (9%), Rickettsia aeschlimannii in Hyalomma spp. (23%), Rickettsia massiliae in Rh. senegalensis (33%) as well as Coxiella burnetii in 0.2%, Borrelia sp. in 0.2%, Anaplasma centrale in 0.2%, Anaplasma marginale in 0.5%, and Ehrlichia ruminantium in 0.5% of all ticks. Potential new species of Borrelia, Anaplasma, and Wolbachia were detected. Candidatus Borrelia africana and Candidatus Borrelia ivorensis (detected in three ticks) are phylogenetically distant from both the relapsing fever group and Lyme disease group borreliae; both were detected in Am. variegatum. Four new genotypes of bacteria from the Anaplasmataceae family were identified, namely Candidatus Anaplasma ivorensis (detected in three ticks), Candidatus Ehrlichia urmitei (in nine ticks), Candidatus Ehrlichia rustica (in four ticks), and Candidatus Wolbachia ivorensis (in one tick). Conclusions/Significance For the first time, we demonstrate the presence of different pathogens such as R. aeschlimannii, C. burnetii, Borrelia sp., A. centrale, A. marginale, and E. ruminantium in ticks in Côte d’Ivoire as well as potential new species of unknown pathogenicity., Author Summary The management of febrile illnesses represents a veritable challenge in sub Saharan-Africa. Until recently most of them were considered as malaria. However, it was showed that a large part of non-malarial febrile diseases in African rural regions (for instance, in Senegal) may be caused by tick-borne infections. Unfortunately, no data exist about the prevalence and incidence of tick-borne diseases in Côte d'Ivoire and their role in public health. We aimed to search for different pathogenic bacteria in ticks in order to understand if there is the background for tick-borne diseases. We detected pathogenic bacteria responsible for many infectious diseases like Rickettsia (spotted fevers), Borrelia (relapsing fevers), Anaplasma, Ehrlichia (ehrlichiosis and anaplasmosis) and Coxiella burnetii (Q fever). These finding suggested that, as in others sub-Saharan African countries, tick-borne disease may be considered as a health care problem in Cote d'Ivoire.

Adaptation is key for survival as vector-borne pathogens transmit between the arthropod and vertebrate, and temperature change is an environmental signal inducing alterations in gene expression of tick-borne spirochetes. While plasmids are often associated with adaptation, complex genomes of relapsing fever spirochetes have hindered progress in understanding the mechanisms of vector colonization and transmission. We utilized recent advances in genome sequencing to generate the most complete version of the Borrelia turicatae 150 kb linear megaplasmid (lp150). Additionally, a transcriptional analysis of open reading frames (ORFs) in lp150 was conducted and identified regions that were up-regulated during in vitro cultivation at tick-like growth temperatures (22°C), relative to bacteria grown at 35°C and infected murine blood. Evaluation of the 3’ end of lp150 identified a cluster of ORFs that code for putative surface lipoproteins. With a microbe’s surface proteome serving important roles in pathogenesis, we confirmed the ORFs expression in vitro and in the tick compared to spirochetes infecting murine blood. Transcriptional evaluation of lp150 indicates the plasmid likely has essential roles in vector colonization and/or initiating mammalian infection. These results also provide a much needed transcriptional framework to delineate the molecular mechanisms utilized by relapsing fever spirochetes during their enzootic cycle.

Borrelia burgdorferi is a spirochetal bacterium transmitted by the Ixodes tick that causes Lyme disease in humans due to its ability to evade the host immune response and disseminate to multiple immunoprotective tissues. The pathogen undergoes dynamic genetic alterations important for adaptation from the tick vector to the mammalian host, but little is known regarding the changes at the transcriptional level within the distal tissues they colonize. In this study, B. burgdorferi infection and gene expression of the essential virulence determinant ospC was quantitatively monitored in a spatial and temporal manner utilizing reporter bioluminescent borrelial strains with in vivo and ex vivo imaging. Although expressed from a shuttle vector, the PospC-luc construct exhibited a similar expression pattern relative to native ospC. Bacterial burden in skin, inguinal lymph node, heart, bladder and tibiotarsal joint varied between tissues and fluctuated over the course of infection possibly in response to unique cues of each microenvironment. Expression of ospC, when normalized for changes in bacterial load, presented unique profiles in murine tissues at different time points. The inguinal lymph node was infected with a significant B. burgdorferi burden, but showed minimal ospC expression. B. burgdorferi infected skin and heart induced expression of ospC early during infection while the bladder and tibiotarsal joint continued to display PospC driven luminescence throughout the 21 day time course. Localized skin borrelial burden increased dramatically in the first 96 hours following inoculation, which was not paralleled with an increase in ospC expression, despite the requirement of ospC for dermal colonization. Quantitation of bioluminescence representing ospC expression in individual tissues was validated by qRT-PCR of the native ospC transcript. Taken together, the temporal regulation of ospC expression in distal tissues suggests a role for this virulence determinant beyond early infection.

Tick-transmitted diseases are an emerging health problem, and the hard tick Ixodes ricinus is the main vector for Borrelia spp., tick-borne encephalitis virus and most of the spotted fever Rickettsiae in Europe. The aim of the present study was to examine the incidence of rickettsial infection in the southernmost and south central parts of Sweden and the Aland Islands in Finland the risk of infection in humans and its correlation with a bite of a Rickettsia-infected tick, the self-reported symptoms of rickettsial disease, and the prevalence of co-infection between Rickettsia spp. and Borrelia spp. Persons with a recent tick bite were enrolled through public media and asked to answer a questionnaire, provide a blood sample and bring detached ticks at enlistment and at follow-up three months later. Blood samples were previously analysed for Borrelia spp. antibodies and, for this report, analysed for antibodies to Rickettsia spp. by immunofluorescence and in 16 cases also using Western Blot. Ninety-six (44.0%) of the 218 participants were seropositive for IgG antibodies to Rickettsia spp. Forty (18.3%) of the seropositive participants had increased titres at the follow-up, indicating recent/current infection, while four (1.8%) had titres indicating probable recent/current infection (amp;gt;= 1: 256). Of 472 ticks, 39 (8.3%) were Rickettsia sp. positive. Five (31.3%) of 16 participants bitten by a Rickettsia-infected tick seroconverted. Experience of the selfreported symptoms nausea (p = 0.006) and radiating pain (p = 0.041) was more common among those with recent, current or probable infection compared to those who did not seroconvert. Participants who showed seroreactivity or seroconversion to Rickettsia spp. had more symptoms than those who were seronegative. Seven (3.2%) participants showed seroconversion to Borrelia spp., and three (1.4%) of these showed seroconversion to both Rickettsia spp. and Borrelia spp., in accordance with previous studies in Sweden. Symptoms of rickettsial disease were in most of the cases vague and general that were difficult to differentiate from other tick-borne diseases. Funding Agencies|Swedish Research Council Branch of Medicine [K2008-58X-14631-06-3]; EU Interreg IV A project ScandTick [167226]; County Council of Ostergotland [L10-56191]; Stiftelsen Olle Engqvist Byggmastare [11877]; Uppsala-Orebro-Regional Research 324 Council [25021]; Center for Clinical Research Dalarna [9028]

Lyme borreliosis is caused by the spirochete Borrelia burgdorferi sensu lato, a fastidious bacterium that replicates slowly and requires special conditions to grow in the laboratory. Borrelia isolation from clinical material is a golden standard for microbiological diagnosis of borrelial infection. Important factors that affect in vitro borrelia growth are temperature of incubation and number of borrelia cells in the sample. The aim of the study was to assess the influence of temperature on borrelia growth and survival by evaluation and comparison of growth of 31 different borrelia strains at five different temperatures and to determine the influence of different inoculums on borrelia growth at different temperatures. Borreliae were cultured in the MKP medium; the initial and final number of spirochetes was determined by dark field microscopy using Neubauer counting chamber. The growth of borrelia was defined as final number of cells/mL after three days of incubation. For all three Borrelia species, the best growth was found at 33°C, followed by 37, 28, and 23°C, while no growth was detected at 4°C (P0.05), respectively. Inoculum had statistically significant influence on growth of all three Borrelia species at all tested temperatures except at 4°C.

Introduction Ticks are the most common arthropod vectors of both human and animal diseases in Europe, and the Ixodes ricinus tick species is able to transmit a large number of bacteria, viruses and parasites. Ticks may also be co-infected with several pathogens, with a subsequent high likelihood of co-transmission to humans or animals. However few data exist regarding co-infection prevalences, and these studies only focus on certain well-known pathogens. In addition to pathogens, ticks also carry symbionts that may play important roles in tick biology, and could interfere with pathogen maintenance and transmission. In this study we evaluated the prevalence of 38 pathogens and four symbionts and their co-infection levels as well as possible interactions between pathogens, or between pathogens and symbionts. Methodology/principal findings A total of 267 Ixodes ricinus female specimens were collected in the French Ardennes and analyzed by high-throughput real-time PCR for the presence of 37 pathogens (bacteria and parasites), by rRT-PCR to detect the presence of Tick-Borne encephalitis virus (TBEV) and by nested PCR to detect four symbionts. Possible multipartite interactions between pathogens, or between pathogens and symbionts were statistically evaluated. Among the infected ticks, 45% were co-infected, and carried up to five different pathogens. When adding symbiont prevalences, all ticks were infected by at least one microorganism, and up to eight microorganisms were identified in the same tick. When considering possible interactions between pathogens, the results suggested a strong association between Borrelia garinii and B. afzelii, whereas there were no significant interactions between symbionts and pathogens. Conclusion/significance Our study reveals high pathogen co-infection rates in ticks, raising questions about possible co-transmission of these agents to humans or animals, and their consequences to human and animal health. We also demonstrated high prevalence rates of symbionts co-existing with pathogens, opening new avenues of enquiry regarding their effects on pathogen transmission and vector competence., Author Summary Ticks transmit more pathogens than any other arthropod, and one single species can transmit a large variety of bacteria and parasites. Because co-infection might be much more common than previously thought, we evaluated the prevalence of 38 known or neglected tick-borne pathogens in Ixodes ricinus ticks. Our results demonstrated that co-infection occurred in almost half of the infected ticks, and that ticks could be infected with up to five pathogens. Moreover, as it is well established that symbionts can affect pathogen transmission in arthropods, we also evaluated the prevalence of four symbiont species and demonstrated that all ticks were infected by at least one microorganism. This work highlights the co-infection phenomenon in ticks, which may have important implications for human and animal health, emphasizing the need for new diagnostic tests better adapted to tick-borne diseases. Finally, the high co-occurrence of symbionts and pathogens in ticks, reveals the necessity to also account for these interactions in the development of new alternative strategies to control ticks and tick-borne disease.

World-wide concern over emerging vector-borne diseases has increased in recent years for both animal and human health. In the United Sates, concern about vector-borne diseases in canines has focused on Lyme disease, anaplasmosis, ehrlichiosis, and heartworm which infect domestic and wild canids. Of these diseases, Lyme and anaplasmosis are also frequently diagnosed in humans. Gray wolves (Canis lupus) recolonized Wisconsin in the 1970s, and we evaluated their temporal and geographic patterns of exposure to these four vector-borne diseases in Wisconsin as the population expanded between 1985 and 2011. A high proportion of the Wisconsin wolves were exposed to the agents that cause Lyme (65.6%) and anaplasma (47.7%), and a smaller proportion to ehrlichiosis (5.7%) and infected with heartworm (9.2%). Wolf exposure to tick borne diseases was consistently higher in older animals. Wolf exposure was markedly higher than domestic dog (Canis familiaris) exposure for all 4 disease agents during 2001–2013. We found a cluster of wolf exposure to Borrelia burgdorferi in northwestern Wisconsin, which overlaps human and domestic dog clusters for the same pathogen. In addition, wolf exposure to Lyme disease in Wisconsin has increased, corresponding with the increasing human incidence of Lyme disease in a similar time period. Despite generally high prevalence of exposure none of these diseases appear to have slowed the growth of the Wisconsin wolf population.

To determine whether human Borrelia miyamotoi infection occurs in the far-western United States, we tested archived sera from northwestern California residents for antibodies to this emerging relapsing fever spirochete. These residents frequently were exposed to I. pacificus ticks in a region where B. miyamotoi tick infection has been reported. We used a two-step B. miyamotoi rGlpQ assay and a B. miyamotoi whole-cell lysate (WCL) assay to detect B. miyamotoi antibody. We also employed Borrelia hermsii and Borrelia burgdorferi WCL assays to examine if these Borrelia induce cross reacting antibody to B. miyamotoi. Sera were collected from 101 residents in each of two consecutive years. The sera of 12 and 14 residents in years one and two, respectively, were B. miyamotoi rGlpQ seroreactive. Sufficient sera were available to test 15 of the 26 seropositive samples using B. miyamotoi and B. hermsii WCL assays. Two residents in year one and seven residents in year two were seroreactive to both Borrelia antigens. Although discernible differences in seroreactivity were evident between the B. miyamotoi and B. hermsii WCL assays, infection with one or the other could not be determined with certainty. Sera from two Borrelia burgdorferi /B. miyamotoi seropositive subjects reacted strongly against B. miyamotoi and B. hermsii WCL antigens. Ecological, epidemiological, and clinical data implicated B. miyamotoi as the probable cause of infection among those whose sera reacted against both antigens. Our findings suggest that human B. miyamotoi infection occurs in northern California and that B. hermsii and B. burgdorferi infections produce antibodies that cross-react with B. miyamotoi antigens. Health care professionals in the far-western United States should be aware that B. miyamotoi disease may occur throughout the geographic distribution of I. pacificus and that improved relapsing fever group spirochete antibody assays are urgently needed.

Background Argasid ticks (soft ticks) are blood-feeding arthropods that can parasitize rodents, birds, humans, livestock and companion animals. Ticks of the Ornithodoros genus are known to be vectors of relapsing fever borreliosis in humans. In Algeria, little is known about relapsing fever borreliosis and other bacterial pathogens transmitted by argasid ticks. Methodology/Principal findings Between May 2013 and October 2015, we investigated the presence of soft ticks in 20 rodent burrows, 10 yellow-legged gull (Larus michahellis) nests and animal shelters in six locations in two different bioclimatic zones in Algeria. Six species of argasid ticks were identified morphologically and through 16S rRNA gene sequencing. The presence and prevalence of Borrelia spp., Bartonella spp., Rickettsia spp. and Anaplasmataceae was assessed by qPCR template assays in each specimen. All qPCR-positive samples were confirmed by standard PCR, followed by sequencing the amplified fragments. Two Borrelia species were identified: Borrelia hispanica in Ornithodoros occidentalis in Mostaganem, and Borrelia cf. turicatae in Carios capensis in Algiers. One new Bartonella genotype and one new Anaplasmataceae genotype were also identified in Argas persicus. Conclusions The present study highlights the presence of relapsing fever borreliosis agents, although this disease is rarely diagnosed in Algeria. Other bacteria of unknown pathogenicity detected in argasid ticks which may bite humans deserve further investigation., Author summary Argasid ticks (soft ticks) are obligate blood-feeding arthropods that may bite humans. To increase understanding of the bacteria that are associated with these arthropods, we collected soft ticks from different sites in Algeria and used molecular tools to detect and identify bacteria in these ticks. Molecular tools revealed that some argasid ticks tested positive for agents of relapsing fever in humans, and other bacteria of unknown pathogenicity.

International audience; Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDITOF MS) has been demonstrated to be useful for tick identification at the species level. More recently, this tool has been successfully applied for the detection of bacterial pathogens directly in tick vectors. The present work has assessed the detection of Borrelia burgdorferi sensu lato in Ixodes ricinus tick vector by MALDI-TOF MS. To this aim, experimental infection model of I. ricinus ticks by B. afzelii was carried out and specimens collected in the field were also included in the study. Borrelia infectious status of I. ricinus ticks was molecularly controlled using half-idiosome to classify specimens. Among the 39 ticks engorged on infected mice, 14 were confirmed to be infected by B. afzelii. For field collection, 14.8% (n = 12/81) I. ricinus ticks were validated molecularly as infected by B. burgdorferi sl. To determine the body part allowing the detection of MS protein profile changes between noninfected and B. afzelii infected specimens, ticks were dissected in three compartments (i.e. 4 legs, capitulum and half-idiosome) prior to MS analysis. Highly reproducible MS spectra were obtained for I. ricinus ticks according to the compartment tested and their infectious status. However, no MS profile change was found when paired body part comparison between non-infected and B. afzelii infected specimens was made. Statistical analyses did not succeed to discover, per body part, specific MS peaks distinguishing Borrelia-infected from non-infected ticks whatever their origins, laboratory reared or field collected. Despite the unsuccessful of MALDI-TOF MS to classify tick specimens according to their B. afzelii infectious status, this proteomic tool remains a promising method for rapid, economic and accurate identification of tick species. Moreover, the singularity of MS spectra between legs and half-idiosome of I. ricinus could be used to reinforce this proteomic identification by submission of both these compartments to MS.

Borrelia burgdorferi transmission to the vertebrate host commences with growth of the spirochete in the tick gut and migration from the gut to the salivary glands. This complex process, involving intimate interactions of the spirochete with the gut epithelium, is pivotal to transmission. We utilized a yeast surface display library of tick gut proteins to perform a global screen for tick gut proteins that might interact with Borrelia membrane proteins. A putative fibronectin type III domain-containing tick gut protein (Ixofin3D) was most frequently identified from this screen and prioritized for further analysis. Immunization against Ixofin3D and RNA interference-mediated reduction in expression of Ixofin3D resulted in decreased spirochete burden in tick salivary glands and in the murine host. Microscopic examination showed decreased aggregation of spirochetes on the gut epithelium concomitant with reduced expression of Ixofin3D. Our observations suggest that the interaction between Borrelia and Ixofin3D facilitates spirochete congregation to the gut during transmission, and provides a “molecular exit” direction for spirochete egress from the gut., Author Summary Lyme borreliosis, the most common vector-borne illness in Northeastern parts of USA, is caused by Borrelia burgdorferi sensu lato spirochetes, and transmitted by the Ixodes scapularis ticks. Currently there is no vaccine available to prevent Lyme borreliosis. A better understanding of tick proteins that interact with Borrelia to facilitate spirochete transmission could identify new targets for the development of a tick-based vaccine to prevent Lyme borreliosis. Spirochete growth and exit from the gut is central to transmission, and might involve intimate interactions between the spirochete and the tick gut. We therefore performed a global screen to identify Borrelia-interacting tick gut proteins. One of the four Borrelia-interacting tick proteins, referred to as Ixofin3D, was further characterized. RNA-interference-mediated down-regulation of Ixofin3D resulted in decreased spirochete numbers in the salivary glands and consequently decreased transmission to the host during tick feeding. We demonstrate that Ixofin3D aids spirochete congregation to the gut epithelium, a critical first step that might direct spirochete exit from the gut.

Background The importance of tick-borne diseases is increasing all over the world, including Turkey. The tick-borne disease outbreaks reported in recent years and the abundance of tick species and the existence of suitable habitats increase the importance of studies related to the epidemiology of ticks and tick-borne pathogens in Turkey. The aim of this study was to investigate the presence of and to determine the infection rates of some tick-borne pathogens, including Babesia spp., Borrelia burgdorferi sensu lato and spotted fever group rickettsiae in the ticks removed from humans in different parts of Ankara. Methodology/Principal Findings A total of 169 ticks belonging to the genus Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus were collected by removing from humans in different parts of Ankara. Ticks were molecularly screened for Babesia spp., Borrelia burgdorferi sensu lato and spotted fever group rickettsiae by PCR and sequencing analysis. We detected 4 Babesia spp.; B. crassa, B. major, B. occultans and B. rossi, one Borrelia spp.; B. burgdorferi sensu stricto and 3 spotted fever group rickettsiae; R. aeschlimannii, R. slovaca and R. hoogstraalii in the tick specimens analyzed. This is the report showing the presence of B. rossi in a region that is out of Africa and in the host species Ha. parva. In addition, B. crassa, for which limited information is available on its distribution and vector species, and B. occultans, for which no conclusive information is available on its presence in Turkey, were identified in Ha. parva and H. marginatum, respectively. Two human pathogenic rickettsia species (R. aeschlimannii and R. slovaca) were detected with a high prevalence in ticks. Additionally, B. burgdorferi sensu stricto was detected in unusual tick species (H. marginatum, H. excavatum, Hyalomma spp. (nymph) and Ha. parva). Conclusions/Significance This study investigates both the distribution of several tick-borne pathogens affecting humans and animals, and the presence of new tick-borne pathogens in Turkey. More epidemiological studies are warranted for B. rossi, which is very pathogenic for dogs, because the presented results suggest that B. rossi might have a wide distribution in Turkey. Furthermore, we recommend that tick-borne pathogens, especially R. aeschlimannii, R. slovaca, and B. burgdorferi sensu stricto, should be taken into consideration in patients who had a tick bite in Turkey., Author Summary Ticks are widespread in over all Turkey. Primary tick-borne diseases (TBDs), such as theileriosis, babesiosis, and anaplasmosis, affecting animals have been known for a long time in Turkey. However, TBDs have become a major concern in humans in recent years due to the recent Crimean-Congo hemorrhagic fever outbreak in Turkey. We know that some TBDs like CCHF, Lyme borreliosis, spotted fever group rickettsiosis, babesiosis and anaplasmosis exist in this geography. However, the other diseases except for CCHF are neglected in patients. In this study, we aimed to investigate Babesia spp., Borrelia burgdorferi sensu lato and spotted fever group rickettsiae in ticks removed from humans in different parts of Ankara by using PCR and sequencing. The result of this study showed that 4 Babesia species, one B. burgdorferi sensu lato, and 3 spotted fever group rickettsia are detected in ticks. The most striking result of this study is that B. rossi, which is very pathogenic for dogs, was reported for the first time from a region that is out of Africa and in Ha. parva. Therefore, B. rossi should be considered in dogs in Turkey. Furthermore, we propose that R. aeschlimannii, R. slovaca, and B. burgdorferi sensu stricto should be taken into consideration in patients who had a tick bite in Ankara.

Background With the global distribution, morbidity, and mortality associated with tick and louse-borne relapsing fever spirochetes, it is important to understand the dynamics of vector colonization by the bacteria and transmission to the host. Tick-borne relapsing fever spirochetes are blood-borne pathogens transmitted through the saliva of soft ticks, yet little is known about the transmission capability of these pathogens during the relatively short bloodmeal. This study was therefore initiated to understand the transmission dynamics of the relapsing fever spirochete Borrelia turicatae from the vector Ornithodoros turicata, and the subsequent dissemination of the bacteria upon entry into murine blood. Methodology/Principal Findings To determine the minimum number of ticks required to transmit spirochetes, one to three infected O. turicata were allowed to feed to repletion on individual mice. Murine infection and dissemination of the spirochetes was evaluated by dark field microscopy of blood, quantitative PCR, and immunoblotting against B. turicatae protein lysates and a recombinant antigen, the Borrelia immunogenic protein A. Transmission frequencies were also determined by interrupting the bloodmeal 15 seconds after tick attachment. Scanning electron microscopy (SEM) was performed on infected salivary glands to detect spirochetes within acini lumen and excretory ducts. Furthermore, spirochete colonization and dissemination from the bite site was investigated by feeding infected O. turicata on the ears of mice, removing the attachment site after engorment, and evaluating murine infection. Conclusion/Significance Our findings demonstrated that three ticks provided a sufficient infectious dose to infect nearly all animals, and B. turicatae was transmitted within seconds of tick attachment. Spirochetes were also detected in acini lumen of salivary glands by SEM. Upon host entry, B. turicatae did not require colonization of the bite site to establish murine infection. These results suggest that once B. turicatae colonizes the salivary glands the spirochetes are preadapted for rapid entry into the mammal., Author Summary Relapsing fever spirochetes cause recurrent febrile episodes, rigors, nausea, vomiting, malaise, and pregnancy complications, and are a leading cause of hospital admissions in regions of Africa. Routes of pathogen transmission include crushed human body lice and feces, or through bites by Ornithodoros spp. of ticks. The life cycle of Ornithodoros turicatae, the vector of Borrelia turicatae, includes over six nymphal stages, upwards of a ten year life span, and a bloodmeal that is completed within an hour. We investigated B. turicatae transmission from the tick vector and assessed the rapidity of spirochete entry into the mammal and dissemination in the blood. Salivary glands from infected ticks were also evaluated to visualize B. turicatae within the tissues to determine spirochete localization. We conclude that given the transmission dynamics of B. turicatae, it may be important to target conserved surface proteins that relapsing fever spirochetes produce in the salivary glands in order to develop preventative measures against the pathogens.

Parry-Romberg syndrome (PRS) or progressive hemifacial atrophy is a rare entity characterized by unilateral atrophy of the skin, subcutaneous tissue and sometimes bone and cartilage. Although this syndrome has overlapping features of scleroderma 'en coup de sabre', it shows little or no sclerosis and may affect the entire distribution of the trigeminal nerve including the eye and tongue. As the pathogenesis is unknown, no effective therapy exists. We present here the third case of PRS associated with borreliosis and more interestingly two cases whose progressive course have been stabilized with gel PUVA therapy.

In the Plasmodium infected host, a balance between pro- and anti-inflammatory responses is required to clear the parasites without inducing major host pathology. Clinical reports suggest that bacterial infection in conjunction with malaria aggravates disease and raises both mortality and morbidity in these patients. In this study, we investigated the immune responses in BALB/c mice, co-infected with Plasmodium berghei NK65 parasites and the relapsing fever bacterium Borrelia duttonii. In contrast to single infections, we identified in the co-infected mice a reduction of L-Arginine levels in the serum. It indicated diminished bioavailability of NO, which argued for a dysfunctional endothelium. Consistent with this, we observed increased sequestration of CD8+ cells in the brain as well over expression of ICAM-1 and VCAM by brain endothelial cells. Co-infected mice further showed an increased inflammatory response through IL-1 beta and TNF-alpha, as well as inability to down regulate the same through IL-10. In addition we found loss of synchronicity of pro- and anti-inflammatory signals seen in dendritic cells and macrophages, as well as increased numbers of regulatory T-cells. Our study shows that a situation mimicking experimental cerebral malaria (ECM) is induced in co-infected mice due to loss of timing and control over regulatory mechanisms in antigen presenting cells.

There is currently no Lyme borreliosis vaccine available for humans, although it has been shown that the disease can be prevented by immunization with an OspA-based vaccine (LYMErix). Outer surface protein A (OspA) is one of the dominant antigens expressed by the spirochetes when present in a tick. The Borrelia species causing Lyme borreliosis in Europe express different OspA serotypes on their surface, B. burgdorferi (serotype 1), B. afzelii (serotype 2), B. garinii (serotypes, 3, 5 and 6) and B. bavariensis (serotype 4), while only B. burgdorferi is present in the US. In order to target all these pathogenic Borrelia species, we have designed a multivalent OspA-based vaccine. The vaccine includes three proteins, each containing the C-terminal half of two OspA serotypes linked to form a heterodimer. In order to stabilize the C-terminal fragment and thus preserve important structural epitopes at physiological temperature, disulfide bonds were introduced. The immunogenicity was increased by introduction of a lipidation signal which ensures the addition of an N-terminal lipid moiety. Three immunizations with 3.0 µg adjuvanted vaccine protected mice from a challenge with spirochetes expressing either OspA serotype 1, 2 or 5. Mice were protected against both challenge with infected ticks and in vitro grown spirochetes. Immunological analyses (ELISA, surface binding and growth inhibition) indicated that the vaccine can provide protection against the majority of Borrelia species pathogenic for humans. This article presents the approach which allows for the generation of a hexavalent vaccine that can potentially protect against a broad range of globally distributed Borrelia species causing Lyme borreliosis.

Humans in the northeastern and midwestern United States are at increasing risk of acquiring tickborne diseases – not only Lyme disease, but also two emerging diseases, human granulocytic anaplasmosis and human babesiosis. Co-infection with two or more of these pathogens can increase the severity of health impacts. The risk of co-infection is intensified by the ecology of these three diseases because all three pathogens (Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti) are transmitted by the same vector, blacklegged ticks (Ixodes scapularis), and are carried by many of the same reservoir hosts. The risk of exposure to multiple pathogens from a single tick bite and the sources of co-infected ticks are not well understood. In this study, we quantify the risk of co-infection by measuring infection prevalence in 4,368 questing nymphs throughout an endemic region for all three diseases (Dutchess County, NY) to determine if co-infections occur at frequencies other than predicted by independent assortment of pathogens. Further, we identify sources of co-infection by quantifying rates of co-infection on 3,275 larval ticks fed on known hosts. We find significant deviations of levels of co-infection in questing nymphs, most notably 83% more co-infection with Babesia microti and Borrelia burgdorferi than predicted by chance alone. Further, this pattern of increased co-infection was observed in larval ticks that fed on small mammal hosts, but not on meso-mammal, sciurid, or avian hosts. Co-infections involving A. phagocytophilum were less common, and fewer co-infections of A. phagocytophilum and B. microti than predicted by chance were observed in both questing nymphs and larvae fed on small mammals. Medical practitioners should be aware of the elevated risk of B. microti/B. burgdorferi co-infection.

In the Thousand Islands region of eastern Ontario, Canada, Lyme disease is emerging as a serious health risk. The factors that influence Lyme disease risk, as measured by the number of blacklegged tick (Ixodes scapularis) vectors infected with Borrelia burgdorferi, are complex and vary across eastern North America. Despite study sites in the Thousand Islands being in close geographic proximity, host communities differed and both the abundance of ticks and the prevalence of B. burgdorferi infection in them varied among sites. Using this archipelago in a natural experiment, we examined the relative importance of various biotic and abiotic factors, including air temperature, vegetation, and host communities on Lyme disease risk in this zone of recent invasion. Deer abundance and temperature at ground level were positively associated with tick abundance, whereas the number of ticks in the environment, the prevalence of B. burgdorferi infection, and the number of infected nymphs all decreased with increasing distance from the United States, the presumed source of this new endemic population of ticks. Higher species richness was associated with a lower number of infected nymphs. However, the relative abundance of Peromyscus leucopus was an important factor in modulating the effects of species richness such that high biodiversity did not always reduce the number of nymphs or the prevalence of B. burgdorferi infection. Our study is one of the first to consider the interaction between the relative abundance of small mammal hosts and species richness in the analysis of the effects of biodiversity on disease risk, providing validation for theoretical models showing both dilution and amplification effects. Insights into the B. burgdorferi transmission cycle in this zone of recent invasion will also help in devising management strategies as this important vector-borne disease expands its range in North America.

Borrelia burgdorferi, the agent of Lyme disease, is a vector-borne pathogen that transits between Ixodes ticks and vertebrate hosts. During the natural infectious cycle, spirochetes must globally adjust their transcriptome to survive in these dissimilar environments. One way B. burgdorferi accomplishes this is through the use of alternative sigma factors to direct transcription of specific genes. RpoS, one of only three sigma factors in B. burgdorferi, controls expression of genes required during tick-transmission and infection of the mammalian host. How spirochetes switch between different sigma factors during the infectious cycle has remained elusive. Here we establish a role for a novel protein, BBD18, in the regulation of the virulence-associated sigma factor RpoS. Constitutive expression of BBD18 repressed transcription of RpoS-dependent genes to levels equivalent to those observed in an rpoS mutant. Consistent with the global loss of RpoS-dependent transcripts, we were unable to detect RpoS protein. However, constitutive expression of BBD18 did not diminish the amount of rpoS transcript, indicating post-transcriptional regulation of RpoS by BBD18. Interestingly, BBD18-mediated repression of RpoS is independent of both the rpoS promoter and the 5' untranslated region, suggesting a mechanism of protein destabilization rather than translational control. We propose that BBD18 is a novel regulator of RpoS and its activity likely represents a first step in the transition from an RpoS-ON to an RpoS-OFF state, when spirochetes transition from the host to the tick vector.

Babesia microti and Borrelia burgdorferi, the respective causative agents of human babesiosis and Lyme disease, are maintained in their enzootic cycles by the blacklegged tick (Ixodes scapularis) and use the white-footed mouse (Peromyscus leucopus) as primary reservoir host. The geographic range of both pathogens has expanded in the United States, but the spread of babesiosis has lagged behind that of Lyme disease. Several studies have estimated the basic reproduction number (R 0) for B. microti to be below the threshold for persistence (

Aim: Here we investigated the role of complement activation in phagocytosis and the release of cytokines and chemokines in response to two clinical isolates: Borrelia afzelii K78, which is resistant to complement-mediated lysis, and Borrelia garinii LU59, which is complement-sensitive. Methods: Borrelia spirochetes were incubated in hirudin plasma, or hirudin-anticoagulated whole blood. Complement activation was measured as the generation of C3a and sC5b-9. Binding of the complement components C3, factor H, C4, and C4BP to the bacterial surfaces was analyzed. The importance of complement activation on phagocytosis, and on the release of cytokines and chemokines, was investigated using inhibitors acting at different levels of the complement cascade. Results: 1) Borrelia garinii LU59 induced significantly higher complement activation than did Borrelia afzelii K78. 2) Borrelia afzelii K78 recruited higher amounts of factor H resulting in significantly lower C3 binding. 3) Both Borrelia strains were efficiently phagocytized by granulocytes and monocytes, with substantial inhibition by complement blockade at the levels of C3 and C5. 4) The release of the pro-inflammatory cytokines and chemokines IL-1 beta, IL-6, TNF, CCL20, and CXCL8, together with the anti-inflammatory IL-10, were increased the most (by>10-fold after exposure to Borrelia). 5) Both strains induced a similar release of cytokines and chemokines, which in contrast to the phagocytosis, was almost totally unaffected by complement blockade. Conclusions: Our results show that complement activation plays an important role in the process of phagocytosis but not in the subsequent cytokine release in response to live Borrelia spirochetes.

Background: Relapsing fever is the most frequent bacterial disease in Africa. Four main vector / pathogen complexes are classically recognized, with the louse Pediculus humanus acting as vector for B. recurrentis and the soft ticks Ornithodoros sonrai, O. erraticus and O. moubata acting as vectors for Borrelia crocidurae, B. hispanica and B. duttonii, respectively. Our aim was to investigate the epidemiology of the disease in West, North and Central Africa. Methods And Findings: From 2002 to 2012, we conducted field surveys in 17 African countries and in Spain. We investigated the occurrence of Ornithodoros ticks in rodent burrows in 282 study sites. We collected 1,629 small mammals that may act as reservoir for Borrelia infections. Using molecular methods we studied genetic diversity among Ornithodoros ticks and Borrelia infections in ticks and small mammals. Of 9,870 burrows investigated, 1,196 (12.1%) were inhabited by Ornithodoros ticks. In West Africa, the southern and eastern limits of the vectors and Borrelia infections in ticks and small mammals were 13 degrees N and 01 degrees E, respectively. Molecular studies revealed the occurrence of nine different Ornithodoros species, including five species new for science, with six of them harboring Borrelia infections. Only B. crocidurae was found in West Africa and three Borrelia species were identified in North Africa: B. crocidurae, B. hispanica, and B. merionesi. Conclusions: Borrelia Spirochetes responsible for relapsing fever in humans are highly prevalent both in Ornithodoros ticks and small mammals in North and West Africa but Ornithodoros ticks seem absent south of 13 degrees N and small mammals are not infected in these regions. The number of Ornithodoros species acting as vector of relapsing fever is much higher than previously known.

Blood is the specimen of choice for most laboratory tests for diagnosis and disease monitoring. Sampling exhaled breath is a noninvasive alternative to phlebotomy and has the potential for real-time monitoring at the bedside. Improved instrumentation has advanced breath analysis for several gaseous compounds from humans. However, application to small animal models of diseases and physiology has been limited. To extend breath analysis to mice, we crafted a means for collecting nose-only breath samples from groups and individual animals who were awake. Samples were subjected to gas chromatography and mass spectrometry procedures developed for highly sensitive analysis of trace volatile organic compounds (VOCs) in the atmosphere. We evaluated the system with experimental systemic infections of severe combined immunodeficiency Mus musculus with the bacterium Borrelia hermsii. Infected mice developed bacterial densities of ∼10(7) per ml of blood by day 4 or 5 and in comparison to uninfected controls had hepatosplenomegaly and elevations of both inflammatory and anti-inflammatory cytokines. While 12 samples from individual infected mice on days 4 and 5 and 6 samples from uninfected mice did not significantly differ for 72 different VOCs, carbon monoxide (CO) was elevated in samples from infected mice, with a mean (95% confidence limits) effect size of 4.2 (2.8-5.6), when differences in CO2 in the breath were taken into account. Normalized CO values declined to the uninfected range after one day of treatment with the antibiotic ceftriaxone. Strongly correlated with CO in the breath were levels of heme oxygenase-1 protein in serum and HMOX1 transcripts in whole blood. These results (i) provide further evidence of the informativeness of CO concentration in the exhaled breath during systemic infection and inflammation, and (ii) encourage evaluation of this noninvasive analytic approach in other various other rodent models of infection and for utility in clinical management.

The agent of Lyme borreliosis, Borrelia burgdorferi, evades host immunity and establishes persistent infections in its varied mammalian hosts. This persistent biology may pose challenges to effective antibiotic treatment. Experimental studies in dogs, mice, and non-human primates have found persistence of B. burgdorferi DNA following treatment with a variety of antibiotics, but persisting spirochetes are non-cultivable. Persistence of B. burgdorferi DNA has been documented in humans following treatment, but the significance remains unknown. The present study utilized a ceftriaxone treatment regimen in the C3H mouse model that resulted in persistence of non-cultivable B. burgdorferi in order to determine their long-term fate, and to examine their effects on the host. Results confirmed previous studies, in which B. burgdorferi could not be cultured from tissues, but low copy numbers of B. burgdorferi flaB DNA were detectable in tissues at 2, 4 and 8 months after completion of treatment, and the rate of PCR-positive tissues appeared to progressively decline over time. However, there was resurgence of spirochete flaB DNA in multiple tissues at 12 months, with flaB DNA copy levels nearly equivalent to those found in saline-treated mice. Despite the continued non-cultivable state, RNA transcription of multiple B. burgdorferi genes was detected in host tissues, flaB DNA was acquired by xenodiagnostic ticks, and spirochetal forms could be visualized within ticks and mouse tissues by immunofluorescence and immunohistochemistry, respectively. A number of host cytokines were up- or down-regulated in tissues of both saline- and antibiotic-treated mice in the absence of histopathology, indicating host response to the presence of non-cultivable, despite the lack of inflammation in tissues.