Disease Risk & Landscape Attributes of Tick-Borne Borrelia Pathogens in the San Francisco Bay Area,California

Habitat heterogeneity influences pathogen ecology by affecting vector abundance and the reservoir host communities. We investigated spatial patterns of disease risk for two human pathogens in the Borrelia genus–B. burgdorferi and B. miyamotoi–that are transmitted by the western black-legged tick, Ixodes pacificus. We collected ticks (349 nymphs, 273 adults) at 20 sites in the San Francisco Bay Area, California, USA. Tick abundance, pathogen prevalence and density of infected nymphs varied widely across sites and habitat type, though nymphal western black-legged ticks were more frequently found, and were more abundant in coast live oak forest and desert/semi-desert scrub (dominated by California sagebrush) habitats. We observed Borrelia infections in ticks at all sites where we able to collect >10 ticks. The recently recognized human pathogen, B. miyamotoi, was observed at a higher prevalence (13/349 nymphs = 3.7%, 95% CI = 2.0–6.3; 5/273 adults = 1.8%, 95% CI = 0.6–4.2) than recent studies from nearby locations (Alameda County, east of the San Francisco Bay), demonstrating that tick-borne disease risk and ecology can vary substantially at small geographic scales, with consequences for public health and disease diagnosis.     

Tick Surveillance for Relapsing Fever Spirochete Borrelia miyamotoi in Hokkaido,Japan

During 2012–2013, a total of 4325 host-seeking adult ticks belonging to the genus Ixodes were collected from various localities of Hokkaido, the northernmost island of Japan. Tick lysates were subjected to real-time PCR assay to detect borrelial infection. The assay was designed for specific detection of the Relapsing fever spirochete Borrelia miyamotoi and for unspecific detection of Lyme disease-related spirochetes. Overall prevalence of B. miyamotoi was 2% (71/3532) in Ixodes persulcatus, 4.3% (5/117) in Ixodes pavlovskyi and 0.1% (1/676) in Ixodes ovatus. The prevalence in I. persulcatus and I. pavlovskyi ticks were significantly higher than in I. ovatus. Co-infections with Lyme disease-related spirochetes were found in all of the tick species. During this investigation, we obtained 6 isolates of B. miyamotoi from I. persulcatus and I. pavlovskyi by culture in BSK-M medium. Phylogenetic trees of B. miyamotoi inferred from each of 3 housekeeping genes (glpQ, 16S rDNA, and flaB) demonstrated that the Hokkaido isolates were clustered with Russian B. miyamotoi, but were distinguishable from North American and European B. miyamotoi. A multilocus sequence analysis using 8 genes (clpA, clpX, nifS, pepX, pyrG, recG, rplB, and uvrA) suggested that all Japanese B. miyamotoi isolates, including past isolates, were genetically clonal, although these were isolated from different tick and vertebrate sources. From these results, B. miyamotoi-infected ticks are widely distributed throughout Hokkaido. Female I. persulcatus are responsible for most human tick-bites, thereby I. persulcatus is likely the most important vector of indigenous relapsing fever from tick bites in Hokkaido.     

Seasonal dynamics of tick burden and associated Borrelia burgdorferi s.l. and Borrelia miyamotoi infections in rodents in a Dutch forest ecosystem

Ixodes ricinus ticks transmit Borrelia burgdorferi sensu lato (s.l.) as well as Borrelia miyamotoi. Larvae become infected when feeding on infected rodents, with horizontal transmission of B. burgdorferi and horizontal and vertical transmission of B. miyamotoi. We studied seasonal dynamics of infection rates of I. ricinus and their rodent hosts, and hence transmission risk of these two distinctly different Borrelia species. Rodents were live-trapped and inspected for ticks from May to November in 2013 and 2014 in a forest in The Netherlands. Trapped rodents were temporarily housed in the laboratory and detached ticks were collected. Borrelia infections were determined from the trapped rodents and collected ticks. Borrelia burgdorferi s.l. and B. miyamotoi were found in ticks as well as in rodents. Rodent density was higher in 2014, whereas tick burden as well as the Borrelia infection rates in rodents were higher in 2013. The density of B. miyamotoi-infected nymphs did not differ between the years. Tick burdens were higher on Apodemus sylvaticus than on Myodes glareolus, and higher on males than on females. Borrelia-infection rate of rodents varied strongly seasonally, peaking in summer. As the larval tick burden also peaked in summer, the generation of infected nymphs was highest in summer. We conclude that the heterogeneity of environmental and host-specific factors affects the seasonal transmission of Borrelia spp., and that these effects act more strongly on horizontally transmitted B. burgdorferi spp. than on the vertically transmitted B. miyamotoi.

     

Survival rates of immature Ixodes pacificus (Acari: Ixodidae) ticks estimated using field‐placed enclosures

Granulocytic anaplasmosis (GA) and Lyme borreliosis are emerging tick‐borne diseases caused by infection with Anaplasma phagocytophilum and Borrelia burgdorferi, respectively, and maintained in rodent‐Ixodes spp. tick cycles, including I. pacificus in the western U.S. Ixodes pacificus has a multiple‐year life cycle and B. burgdorferi and A. phagocytophilum are transstadially, but not transovarially, transmitted within ticks, thus ticks function importantly in maintaining infection in nature. In this study, the survival of larval and nymphal I. pacificus was determined using ticks placed in tubes in leaf litter from June 2005 to September 2006 at two field sites in the California northern coast range mountains and a laboratory control. In all three sites, nymphal and larval survival ranged from 90–400 d, with differences in mean survival among sites. Fewer ticks died in the autumn in the moister field sites compared with the drier incubator control treatment. The first large die‐off event in late autumn occurred at all sites shortly before relative humidity increased from 80–100% and temperature declined from approximately 22–15° C. The concurrent die‐off in the incubator population, subject to relative humidity and temperature regimes that were invariant, suggests that survival time was dependent on other factors in addition to environmental conditions. These results suggested that many ticks exhausted resources or tolerance for relatively low humidity within six months of questing, and that higher humidity prolonged survival. Based on observed longevity, humans and other animals could acquire A. phagocytophilum infection from adult I. pacificus that were infected up to three years earlier.     

Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.     

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.     

Climate and Tick Seasonality Are Predictors of Borrelia burgdorferi Genotype Distribution

The blacklegged tick, Ixodes scapularis, is of significant public health importance as a vector of Borrelia burgdorferi, the agent of Lyme borreliosis. The timing of seasonal activity of each immature I. scapularis life stage relative to the next is critical for the maintenance of B. burgdorferi because larvae must feed after an infected nymph to efficiently acquire the infection from reservoir hosts. Recent studies have shown that some strains of B. burgdorferi do not persist in the primary reservoir host for more than a few weeks, thereby shortening the window of opportunity between nymphal and larval feeding that sustains their enzootic maintenance. We tested the hypothesis that climate is predictive of geographic variation in the seasonal activity of I. scapularis, which in turn differentially influences the distribution of B. burgdorferi genotypes within the geographic range of I. scapularis. We analyzed the relationships between climate, seasonal activity of I. scapularis, and B. burgdorferi genotype frequency in 30 geographically diverse sites in the northeastern and midwestern United States. We found that the magnitude of the difference between summer and winter daily temperature maximums was positively correlated with the degree of seasonal synchrony of the two immature stages of I. scapularis. Genotyping revealed an enrichment of 16S-23S rRNA intergenic spacer restriction fragment length polymorphism sequence type 1 strains relative to others at sites with lower seasonal synchrony. We conclude that climate-associated variability in the timing of I. scapularis host seeking contributes to geographic heterogeneities in the frequencies of B. burgdorferi genotypes, with potential consequences for Lyme borreliosis morbidity.An increasingly important area of research in infectious disease epidemiology is the influence of pathogen strain diversity on patterns of disease risk and clinical outcome. Strain-specific pathogenicity or transmissibility can be important clinical and epidemiological parameters; for example, only a subset of Neisseria meningitidis strains are responsible for invasive infections leading to meningitis (1). Geography and environmental features influence the genetic structure of certain pathogens by regulating their distribution, dispersal, or population size (8, 31, 49). Accordingly, a heterogeneous environment will result in spatial structuring of genotype frequencies, with possible epidemiological implications.Lyme borreliosis is a tick-borne zoonosis caused by Borrelia burgdorferi, a spirochetal bacterium that exhibits genetic diversity throughout its range in eastern North America (12, 60), where it is maintained in a horizontal transmission cycle between its vector, the blacklegged tick Ixodes scapularis, and vertebrate reservoir hosts. I. scapularis has a two-year life cycle in which it takes three blood meals, one per life stage, with the two subadult stages responsible for the enzootic maintenance of B. burgdorferi (2, 3, 51). Larval ticks hatch uninfected from eggs (41) and acquire the spirochetes from infected reservoir hosts. Infected larvae maintain the spirochetes transstadially, allowing them to transmit B. burgdorferi to uninfected reservoirs during their nymphal blood meal the following summer. The seasonal timing of activity, or phenology, of each tick life stage relative to the next is a critical factor in the maintenance of B. burgdorferi because larvae typically must feed after an infected nymph in order to acquire the bacteria (32).Previous studies in Europe of tick-borne encephalitis virus have shown that seasonal synchrony of immature ticks is necessary for the maintenance of the virus in natural enzootic cycles because nonsystemic infections are transmitted from nymphs to larvae feeding in close proximity on the same individual reservoir rodent (48). Furthermore, seasonal synchrony of immature tick activity, a prerequisite of cofeeding, was found to be correlated with climate (47). Although it is possible for an I. scapularis larva to become infected with B. burgdorferi by simultaneously feeding in close proximity to an infected nymph, a role for cofeeding transmission in the enzootic maintenance of B. burgdorferi in North America has not been established (43). Rather, until recently, the existing evidence indicated that B. burgdorferi causes life-long systemic infections in reservoirs that allow for its maintenance in the absence of seasonal synchrony of I. scapularis immatures (18). However, recent studies suggest that this may not always be the case (34) and that there are differences in the duration of infectiousness that are strain specific (16, 28).We hypothesized that large-scale, climate-driven geographic variability in the host seeking phenology of immature I. scapularis ticks is associated with heterogeneity in the frequencies of strains acquired by larval ticks. Using regression models and accounting for spatial autocorrelation, we examined the relationships between climate, the temporal synchrony of larval and nymphal seasonal host seeking activity, and B. burgdorferi genotype frequency in ticks collected from 30 geographically diverse sites systematically selected for their locations throughout the northeastern and midwestern United States.Here we present empirical evidence that climate patterns, specifically, regional variation in summer and winter temperature cycle extremes, are associated with variation in the seasonal synchrony of I. scapularis larval and nymphal host seeking activity. Furthermore, both climate and the differences in the seasonal synchrony of the two immature tick stages are related to geographic variation in B. burgdorferi genotype frequency. Our results point to the impact of climate upon the natural dynamics of enzootic transmission and population genetic structure of an important vector-borne human pathogen, with possible implications for the distribution of human disease risk and epidemiology.     

Differential Transmission of the Genospecies of Borrelia burgdorferi Sensu Lato by Game Birds and Small Rodents in England

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in a focus of Lyme borreliosis in southern Britain dominated by game birds. Ticks, rodents, and pheasants were analyzed for spirochete infections by PCR targeting the 23S-5S rRNA genes, followed by genotyping by the reverse line blot method. In questing Ixodes ricinus ticks, three genospecies of B. burgdorferi sensu lato were detected, with the highest prevalences found for Borrelia garinii and Borrelia valaisiana. B. burgdorferi sensu stricto was rare (<1%) in all tick stages. Borrelia afzelii was not detected in any of the samples. More than 50% of engorged nymphs collected from pheasants were infected with borreliae, mainly B. garinii and/or B. valaisiana. Although 19% of the rodents harbored B. burgdorferi sensu stricto and/or B. garinii in internal organs, only B. burgdorferi sensu stricto was transmitted to xenodiagnostic tick larvae (it was transmitted to 1% of the larvae). The data indicate that different genospecies of B. burgdorferi sensu lato can be maintained in nature by distinct transmission cycles involving the same vector tick species but different vertebrate host species. Wildlife management may have an influence on the relative risk of different clinical forms of Lyme borreliosis.     

Survey of Ixodes pacificus Ticks in California Reveals a Diversity of Microorganisms and a Novel and Widespread Anaplasmataceae Species

Ixodes pacificus ticks can harbor a wide range of human and animal pathogens. To survey the prevalence of tick-borne known and putative pathogens, we tested 982 individual adult and nymphal I. pacificus ticks collected throughout California between 2007 and 2009 using a broad-range PCR and electrospray ionization mass spectrometry (PCR/ESI-MS) assay designed to detect a wide range of tick-borne microorganisms. Overall, 1.4% of the ticks were found to be infected with Borrelia burgdorferi, 2.0% were infected with Borrelia miyamotoi and 0.3% were infected with Anaplasma phagocytophilum. In addition, 3.0% were infected with Babesia odocoilei. About 1.2% of the ticks were co-infected with more than one pathogen or putative pathogen. In addition, we identified a novel Anaplasmataceae species that we characterized by sequencing of its 16S rRNA, groEL, gltA, and rpoB genes. Sequence analysis indicated that this organism is phylogenetically distinct from known Anaplasma species with its closest genetic near neighbors coming from Asia. The prevalence of this novel Anaplasmataceae species was as high as 21% at one site, and it was detected in 4.9% of ticks tested statewide. Based upon this genetic characterization we propose that this organism be called ‘Candidatus Cryptoplasma californiense’. Knowledge of this novel microbe will provide awareness for the community about the breadth of the I. pacificus microbiome, the concept that this bacterium could be more widely spread; and an opportunity to explore whether this bacterium also contributes to human or animal disease burden.     

Relative importance of lizards and mammals as hosts for ixodid ticks in northern California

Lizards and mammals were trapped and examined for ticks from August 1992 to June 1993 in two habitat types, chaparral and woodland-grass, in northern California. Five tick species were collected from mammals (Dermacentor occidentalis, Haemaphysalis leporispalustris, Ixodes pacificus, I. spinipalpis, I. woodi), but only I. pacificus was found on lizards. Dermacentor occidentalis, I. pacificus, and I. woodi occurred in both habitats, whereas H. leporispalustris and I. spinipalpis were found only on animals trapped in chaparral. The tick species most commonly encountered on mammals was D. occidentalis in chaparral and I. pacificus in woodland-grass. Peak infestation of mammals occurred in spring for I. pacificus immatures and H. leporispalustris, summer for D. occidentalis immatures, fall through spring for I. woodi immatures, and fall through winter for I. spinipalpis. The primary aim of the study was to quantify the relative importance of the western fence lizard (Sceloporus occidentalis), which is reservoir-incompetent for Borrelia burgdorferi sensu lato (s.l.), and mammalian B.burgdorferi s.l.-reservoirs as hosts for the immature stages of I. pacificus in spring. The estimated relative utilization by I. pacificus of the western fence lizard versus mammals was 88% for larvae and 99% for nymphs in chaparral in May. When tick infestation data were corrected for a two-fold lower efficiency of field examinations for rodents than for lizards, the western fence lizard still accounted for 78% of larval and 98% of nymphal feedings. In woodland-grass, 46% of 100 I. pacificus larvae and 100% of 52 nymphs recovered from mammals or western fence lizards during May-June were collected from the lizards. However, this may represent an underestimate of the importance of the western fence lizard as a larval host in this habitat because inclement weather during the late May sampling period doubtless resulted in significantly decreased lizard activity. In conclusion, the western fence lizard was more heavily utilized by I. pacificus immatures, especially nymphs, than were rodents. This revised version was published online in July 2006 with corrections to the Cover Date.     

Detection and Genetic Characterization of Relapsing Fever Spirochete Borrelia miyamotoi in Estonian Ticks

During the years 2008–2010 I. ricinus and I. persulcatus ticks were collected from 64 sites in mainland Estonia and on the island Saaremaa. Presence of B. miyamotoi was found in 0.9% (23/2622) of ticks. The prevalence in I. persulcatus and I. ricinus ticks differed significantly, 2.7% (15/561) and 0.4% (8/2061), respectively. The highest prevalence rates were in found South-Eastern Estonia in an area of I. persulcatus and I. ricinus sympatry and varied from 1.4% (1/73) to 2.8% (5/178). Co-infections with B. burgdorferi s.l. group spirochetes and tick-borne encephalitis virus were also revealed. Genetic characterization of partial 16S rRNA, p66 and glpQ genes demonstrated that Estonian sequences belong to two types of B. miyamotoi and cluster with sequences from Europe and the European part of Russia, as well as with sequences from Siberia, Asia and Japan, here designated as European and Asian types, respectively. Estonian sequences of the European type were obtained from I. ricinus ticks only, whereas the Asian type of B. miyamotoi was shown for both tick species in the sympatric regions.     

The spatial distribution of Borrelia burgdorferi-infected Ixodes ricinus in the Connemara region of County Galway,Ireland

Studies were carried out in the Connemara area of County Galway in the west of Ireland in order to determine the abundance and distribution of the tick, Ixodes ricinus and the prevalence of its infection with Borrelia burgdorferi. The tick was very abundant locally, in particular when associated with cattle, sheep and enclosed red deer. Large numbers of ticks not only occurred on the pastures, but also on adjacent roadside verges. No infections with B. burgdorferi could be demonstrated when nymphal ticks were sampled from central areas of the pastures, suggesting that livestock and red deer are probably not significant reservoirs of the spirochaete. Small numbers of infected nymphal and adult ticks were associated with hedges, dry stone walls, the margins of woodland adjoining infested pastures and in woodland from which livestock were excluded. Woodmice (Apodemus sylvaticus) were most numerous in such habitats and the majority were infected with B. burgdorferi.     

Geographic Risk for Lyme Disease and Human Granulocytic Ehrlichiosis in Southern New York State

Ixodes scapularis, the tick vector of Lyme disease and human granulocytic ehrlichiosis (HGE), is prevalent in much of southern New York state. The distribution of this species has increased, as have reported cases of both Lyme disease and HGE. The unreliability of case reports, however, demonstrates the need for tick and pathogen surveillance in order to accurately define areas of high risk. In this study, a total of 89,550 m² at 34 study sites was drag sampled in 1995 and a total of 51,540 m² at 40 sites was sampled in 1996 to determine tick and pathogen distribution in southern New York state. I. scapularis was collected from 90% of the sites sampled, and regionally, a 2.5-fold increase in nymphal abundance occurred from 1995 to 1996. I. scapularis individuals from all sites were infected with Borrelia burgdorferi in 1995, while an examination of ticks for both B. burgdorferi and the agent of HGE in 1996 confirmed that these organisms were present in all counties; the average coinfection rate was 1.9%. No correlation was found between estimated risk and reported cases of Lyme disease. The geographic disparity of risk observed among sites in this study underscores the need for vector and pathogen surveillance on a regional level. An entomologic risk index can help identify sites for targeted tick control efforts.     

Detection of Lyme disease spirochete,Borrelia burgdorferi sensu lato,including three novel genotypes in ticks (Acari: Ixodidae) collected from songbirds (Passeriformes) across Canada

Lyme disease is reported across Canada, but pinpointing the source of infection has been problematic. In this three‐year, bird‐tick‐pathogen study (2004–2006), 366 ticks representing 12 species were collected from 151 songbirds (31 passerine species/subspecies) at 16 locations Canada‐wide. Of the 167 ticks/pools tested, 19 (11.4%) were infected with Borrelia burgdorferi sensu lato (s.l.). Sequencing of the rrf‐rrl intergenic spacer gene revealed four Borrelia genotypes: B. burgdorferi sensu stricto (s.s.) and three novel genotypes (BC genotype 1, BC genotype 2, BC genotype 3). All four genotypes were detected in spirochete‐infected Ixodes auritulus (females, nymphs, larvae) suggesting this tick species is a vector for B. burgdorferi s.l. We provide first‐time records for: ticks in the Yukon (north of 60° latitude), northernmost collection of Amblyomma americanum in North America, and Amblyomma imitator in Canada. First reports of bird‐derived ticks infected with B. burgdorferi s.l. include: live culture of spirochetes from Ixodes pacificus (nymph) plus detection in I. auritulus nymphs, Ixodes scapularis in New Brunswick, and an I. scapularis larva in Canada. We provide the first account of B. burgdorferi s. l. in an Ixodes muris tick collected from a songbird anywhere. Congruent with previous data for the American Robin, we suggest that the Common Yellowthroat, Golden‐crowned Sparrow, Song Sparrow, and Swainson's Thrush are reservoir‐competent hosts. Song Sparrows, the predominant hosts, were parasitized by I. auritulus harboring all four Borrelia genotypes. Our results show that songbirds import B. burgdorferi s.l.‐infected ticks into Canada. Bird‐feeding I. scapularis subadults were infected with Lyme spirochetes during both spring and fall migration in eastern Canada. Because songbirds disperse millions of infected ticks across Canada, people and domestic animals contract Lyme disease outside of the known and expected range.     

Molecular Detection of Tick-Borne Pathogens in Humans with Tick Bites and Erythema Migrans,in the Netherlands

BackgroundTick-borne diseases are the most prevalent vector-borne diseases in Europe. Knowledge on the incidence and clinical presentation of other tick-borne diseases than Lyme borreliosis and tick-borne encephalitis is minimal, despite the high human exposure to these pathogens through tick bites. Using molecular detection techniques, the frequency of tick-borne infections after exposure through tick bites was estimated.MethodsTicks, blood samples and questionnaires on health status were collected from patients that visited their general practitioner with a tick bite or erythema migrans in 2007 and 2008. The presence of several tick-borne pathogens in 314 ticks and 626 blood samples of this cohort were analyzed using PCR-based methods. Using multivariate logistic regression, associations were explored between pathogens detected in blood and self-reported symptoms at enrolment and during a three-month follow-up period.ResultsHalf of the ticks removed from humans tested positive for Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Candidatus Neoehrlichia mikurensis, Rickettsia helvetica, Rickettsia monacensis, Borrelia miyamotoi and several Babesia species. Among 92 Borrelia burgdorferi s. l. positive ticks, 33% carried another pathogen from a different genus. In blood of sixteen out of 626 persons with tick bites or erythema migrans, DNA was detected from Candidatus Neoehrlichia mikurensis (n = 7), Anaplasma phagocytophilum (n = 5), Babesia divergens (n = 3), Borrelia miyamotoi (n = 1) and Borrelia burgdorferi s. l. (n = 1). None of these sixteen individuals reported any overt symptoms that would indicate a corresponding illness during the three-month follow-up period. No associations were found between the presence of pathogen DNA in blood and; self-reported symptoms, with pathogen DNA in the corresponding ticks (n = 8), reported tick attachment duration, tick engorgement, or antibiotic treatment at enrolment.ConclusionsBased on molecular detection techniques, the probability of infection with a tick-borne pathogen other than Lyme spirochetes after a tick bite is roughly 2.4%, in the Netherlands. Similarly, among patients with erythema migrans, the probability of a co-infection with another tick-borne pathogen is approximately 2.7%. How often these infections cause disease symptoms or to what extend co-infections affect the course of Lyme borreliosis needs further investigations.     

Prevalence of Bartonella henselae and Borrelia burgdorferi Sensu Lato DNA in Ixodes ricinus Ticks in Europe

Bartonella spp. can cause persistent bloodstream infections in humans and animals. To determine whether Bartonella henselae is present in questing Ixodes ricinus ticks, we analyzed the prevalence of B. henselae DNA among tick stages compared to the prevalence of DNA from Borrelia burgdorferi sensu lato, the pathogen most frequently transmitted by ticks. B. henselae DNA was present with a prevalence of up to ∼40% in tick populations sampled in four European sites (Eberdingen, Germany; Klasdorf, Germany; Lembach, France; and Madeira, Portugal). The odds of detecting B. henselae DNA in nymphal ticks was ∼14-fold higher than in adult ticks. No tick was found to be coinfected with B. henselae and B. burgdorferi sensu lato. Taken together, our data indicate that ticks might serve as a vector for the transmission of B. henselae to humans.In immunocompetent patients, Bartonella henselae infections often result in cat scratch disease (CSD), a self-limiting but often prolonged lymphadenitis; immunocompromised patients (e.g., AIDS patients) can suffer from vasculoproliferative disorders (bacillary angiomatosis, peliosis hepatis [1]). Cats are a confirmed reservoir host of B. henselae transmitting the pathogen by cat scratches or bites.Several Bartonella species (e.g., B. henselae, B. quintana, and B. vinsonii) cause a persistent intraerythrocytic bacteremia in their respective mammalian reservoir hosts (7). B. henselae was detected in the peripheral blood of a wide range of mammals including domestic (e.g., cats, dogs, and horses) and wild animals (e.g., porpoise, lions, cheetahs, and wild felids). Obviously, such an asymptomatic, persistent bacteremia with B. henselae represents an important factor for the spread of the pathogens via blood-sucking arthropods. Mechanistic details determining the intraerythrocytic presence of Bartonella spp. have been investigated in detail in a B. tribocorum rat infection model mimicking Trench fever (a human disease caused by B. quintana); here, the pathogen persists several weeks in the circulating blood in an immunoprivileged intraerythrocytic niche (28).Cat fleas are well established vectors for B. henselae (1). However, transmission by other arthropods, in particular ticks, has been suggested: B. henselae DNA was detected in questing Ixodes pacificus and I. persulcatus ticks in North America, Eastern Europe, and Russia, respectively (4, 13, 14, 22, 25) and in I. ricinus ticks feeding on people or domestic animals in Central Europe (24, 26). DNA of various Bartonella spp. has also been detected in keds, biting flies, and mites (reviewed in reference 2). Recently, ticks (I. ricinus) were experimentally infected with B. henselae. Inoculation of cats with salivary glands of infected ticks resulted in a B. henselae bacteremia (5). Nevertheless, controversial data about the prevalence of Bartonella spp. in ticks and their role as vectors for B. henselae exist (29).Here, we present data on the prevalence of B. henselae and Lyme disease spirochetes in 654 questing ticks (I. ricinus) collected at four locations in Europe, suggesting that ticks might serve as potential vectors for the transmission of B. henselae to humans.     

The Western Progression of Lyme Disease: Infectious and Nonclonal Borrelia burgdorferi Sensu Lato Populations in Grand Forks County,North Dakota

Scant attention has been paid to Lyme disease, Borrelia burgdorferi, Ixodes scapularis, or reservoirs in eastern North Dakota despite the fact that it borders high-risk counties in Minnesota. Recent reports of B. burgdorferi and I. scapularis in North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, ND, were identified as B. burgdorferi sensu lato through sequence analyses of the 16S rRNA gene, the 16S rRNA gene-ileT intergenic spacer region, flaB, ospA, ospC, and p66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the upper Midwest and Canada. BALB/c mice were infected with B. burgdorferi isolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive, and B. burgdorferi M3 was detected by quantitative PCR (qPCR) in the tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larval I. scapularis ticks were able to acquire B. burgdorferi M3 from infected mice; M3 was maintained in I. scapularis during the molt from larva to nymph; and further, M3 was transmitted from infected I. scapularis nymphs to naive mice, as evidenced by cultures and qPCR analyses. These results demonstrate that isolate M3 is capable of disseminated infection by both artificial and natural routes of infection. This study confirms the presence of unique (nonclonal) and infectious B. burgdorferi populations in eastern North Dakota.     

Geography,Deer, and Host Biodiversity Shape the Pattern of Lyme Disease Emergence in the Thousand Islands Archipelago of Ontario,Canada

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.     

Risk indicators for the tick Ixodes ricinus and Borrelia burgdorferi sensu lato in Sweden

The distributional area of the tick Ixodes ricinus (L.), the primary European vector to humans of Lyme borreliosis spirochaetes (Borrelia burgdorferi sensu lato) and tick‐borne encephalitis virus, appears to be increasing in Sweden. It is therefore important to determine which environmental factors are most useful to assess risk of human exposure to this tick and its associated pathogens. The geographical distribution of I. ricinus in Sweden was analysed with respect to vegetation zones and climate. The northern limit of I. ricinus and B. burgdorferi s.l. in Sweden corresponds roughly to the northern limit of the southern boreal vegetation zone, and is characterized climatically by snow cover for a mean duration of 150 days and a vegetation period averaging 170 days. The zoogeographical distribution of I. ricinus in Sweden can be classified as southerly–central, with the centre of the distribution south of the Limes Norrlandicus. Ixodes ricinus nymphs from 13 localities in different parts of Sweden were examined for the presence of B. burgdorferi s.l. and found to be infected with Borrelia afzelii and Borrelia garinii. Tick sampling localities were characterized on the basis of the density of Borrelia‐infected I. ricinus nymphs, presence of specific mammals, dominant vegetation and climate. Densities of I. ricinus nymphs and Borrelia‐infected nymphs were significantly correlated, and nymphal density can thus serve as a general indicator of risk for exposure to Lyme borreliosis spirochaetes. Analysis of data from this and other studies suggests that high densities of Borrelia‐infected nymphs typically occur in coastal, broadleaf vegetation and in mixed deciduous/spruce vegetation in southern Sweden. Ixodes ricinus populations consistently infected with B. burgdorferi s.l. can occur in: (a) biotopes with shrews, rodents, hares and birds; (b) biotopes with shrews, rodents, hares, deer and birds, and (c) island locations where the varying hare (Lepus timidus) is the only mammalian tick host.     

Characterization of Monoclonal Antibodies for Identification of Borrelia japonica,Isolates from Ixodes ovatus

Monoclonal antibodies for identification of Borrelia japonica isolated from tick, Ixodes ovatus and long-tailed shrew, Sorex unguiculatus in Japan and Borrelia related to Lyme disease (Borrelia burgdorferi sensu lato) were prepared and characterized. All isolates belonging to B. japonica and isolates from I. dentatus and cottontail rabbit in North America reacted with MAb O1441b against flagellin which was prepared from immunized mice with strain HO14, type strain of B. japonica, but isolates from I. persulcatus, patient, and wood mouse, Apodemus speciosus ainu, in Japan, and isolates belonging to B. burgdorferi, B. garinii and B. afzelii from North America and Europe did not. Strains used in this study reacted with MAb P62 against common antigen which was prepared from immunized mice with strain NT24 isolated from I. persulcatus in Japan, but B. japonica did not. These MAbs are useful for identification and differentiation of B. japonica and B. burgdorferi sensu lato in Japan.