Molecular identification of a novel deltaproteobacterium as the etiologic agent of epizootic bovine abortion (foothill abortion)

Epizootic bovine abortion (EBA) is endemic in California's coastal range and the foothill regions of the Sierra Nevada, where it has been the primary diagnosed cause of abortion in beef cattle for >50 years. Investigation of these losses has defined a specific fetal syndrome characterized by late-term abortion or birth of weak or dead calves. Although the unusual clinical presentation and unique fetal pathology associated with EBA have been recognized since the 1950s, the identity of the etiologic agent is unknown. In this study, suppression-hybridization PCR was used to identify a fragment of the 16S rRNA gene of a previously undescribed bacterium in thymus tissue derived from affected fetuses. Phylogenetic analysis revealed that this pathogen was a deltaproteobacterium closely related to members of the order Myxococcales. A specific PCR was subsequently developed to detect the presence of this bacterium in DNA extracted from fetal thymuses. Using histopathology as the definitive diagnosis for EBA, this PCR demonstrated 100% specificity and 88% sensitivity. The bacterium was also detected in the argasid tick Ornithodoros coriaceus, which is the recognized vector of EBA. These data imply a close association between this novel agent and the etiology of EBA.     

Association of Ixodes pacificus (Acari: ixodidae) with the spatial and temporal distribution of equine granulocytic ehrlichiosis in California.

This study was conducted to determine if the biology of certain ticks associated with horses regulates the spatial and temporal distribution of equine granulocytic ehrlichiosis (EGE) in California north of Monterey County. We compared the spatial and temporal distribution of EGE cases with the seasons of activity and life histories of ticks that infest horses. Spatially, cases collected from equine veterinarians clustered around each other in a manner different from the way in which control cities of practice were distributed, with foci limited to the Sierra Nevada and coastal foothills. Cases also clustered seasonally: most were diagnosed between November and April. The spatial and temporal pattern of EGE cases closely parallels the well-characterized life history and distribution of Ixodes pacificus Cooley & Kohls, but not other ticks commonly associated with horses. Building on previous studies, there is compelling evidence that this tick has the vectorial capacity to transmit Ehrlichia equi to horses. Based on the life history and distribution of I. pacificus in relation to EGE cases, we reason that this tick is the only biologically plausible vector of E. equi in California, and provide evidence for a tightly linked association between I. pacificus and the epidemiology of EGE.     

Genetic characteristics of Borrelia coriaceae isolates from the soft tick Ornithodoros coriaceus (Acari: Argasidae)

Two Borrelia isolates (CA434 and CA435) cultured from the soft tick Ornithodoros coriaceus were analyzed by contour-clamped homogeneous electric field gel electrophoresis of unrestricted and ApaI-restricted DNA, standard electrophoresis of BamHI- and HindIII-restricted DNA, Southern hybridization, restriction fragment length polymorphism and sequencing of the 16S rRNA gene, and amplification of the 5S-23S intergenic spacer region. These isolates were compared with Borrelia coriaceae type strain Co53, B. burgdorferi sensu stricto strain CA4, and the relapsing-fever spirochete B. parkeri (undesignated). The 16S rRNA region of CA434 and CA435 differed from that of B. coriaceae type strain Co53 by the presence of 1 base (C) at position 367 (GenBank accession no. U42286). The linear plasmid profile of CA434 was similar to that of Co53, and the ApaI, BamHI, and HindIII restriction fingerprints of the total cellular DNA of CA434 and Co53 were similar. In contrast, CA435 differed somewhat from CA434 and Co53, which demonstrates that B. coriaceae is genetically diverse. Southern hybridization showed that the DNAs of CA434 and CA435 hybridized strongly with the digoxigenin-labeled DNA of Co53. Low homology was found between the DNA of Co53 and that of B. parkeri. The 16S rRNA sequence of B. parkeri was identical to previously published results for B. parkeri strain M3001 (GenBank accession number U42296). CA434 and CA435 represent only the second and third isolates of B. coriaceae obtained from any source since its initial isolation from an O. coriaceus tick in 1985. All three B. coriaceae isolates were derived from adult ticks collected from the same locality in northwestern California. Difficulties encountered in detecting B. coriaceae in, and isolating this spirochete from, the tissues of O. coriaceus are discussed. The lack of concordance between different detection or isolation methods suggests that reliance upon a single technique may grossly underestimate the true prevalence of spirochetal infection in wild-caught O. coriaceus ticks.     

Detection of Borrelia burgdorferi, Ehrlichia chaffeensis, and Anaplasma phagocytophilum in ticks (Acari: Ixodidae) from a coastal region of California

A study was conducted in Santa Cruz County to estimate the prevalence and distribution of the agents of Lyme disease, human granulocytic (HGE), and human monocytic (HME) ehrlichiosis in 1,187 adult ixodid ticks collected from eight public-use recreation areas over a 2-yr period. Borrelia burgdorferi, the causative agent of Lyme disease, was detected by a polymerase chain reaction (PCR) in 44 of 776 (5.67%) Ixodes pacificus ticks and in 3 of 58 (5.17%) Dermacentor variabilis ticks. Anaplasma phagocytophilum, the causative agent of HGE, was detected by PCR in 48 (6.19%) I. pacificus ticks and 5 (8.62%) D. variabilis ticks. Ehrlichia chaffeensis, the causative agent of HME, was detected by nested PCR in just five (0.64%) I. pacificus ticks and four (6.9%) D. variabilis ticks. Interestingly, eight (1.03%) I. pacificus ticks were co-infected with B. burgdorferi and A. phagocytophilum, and just one (0.12%) tick was co-infected with B. burgdorferi and E. chaffeensis. Less than 1% of 353 Dermacentor occidentalis ticks showed evidence of infection with any of the agents tested. To our knowledge, this is the first reported identification of A. phagocytophilum and E. chaffeensis in D. occidentalis ticks from California This study represents the first extensive survey of Lyme and the ehrlichial diseases across multiple areas of Santa Cruz County, and suggests that prevalence of B. burgdorferi in Santa Cruz County may be higher than other areas of the state.     

Infection rate ofIxodes ricinus ticks withBorrelia afzelii,Borrelia garinii,andBorrelia burgdorferi sensu stricto in Slovenia

In spring 1993,Ixodes ricinus ticks were collected from six regions of Slovenia to determine their overall rate of infection withBorrelia burgdorferi sensu lato and to assess the frequency of individual species in these tick populations. Ticks were dissected and midgut tissue inoculated into modified Barbour-Stoenner-Kelly (BSK II) medium.Borrelia isolates were differentiated into separate species using species-specific polymerase chain reaction (PCR) primers and by large restriction fragment pattern (LRFP) analysis. Infected ticks were found in all six regions surveyed. Spirochaetes were isolated from 69 of 363 ticks (19 %): the isolation rate from adult female ticks was 35 % (23/66 ticks cultured), from adult male ticks 22 % (20/91), and from nymphal ticks 13 % (26/206). Determination of the species of 60 isolates revealed that 32 wereBorrelia afzelii (53 %), 20 wereBorrelia garinii (33 %), and 8 wereBorrelia burgdorferi sensu stricto (13 %). In the Ljubljana regionBorrelia afzelii andBorrelia garinii predominated (43 % and 40 %, respectively), whereasBorrelia burgdorferi sensu stricto constituted only 17 % of isolates. In three other regions of the countryBorrelia afzeliiwas isolated exclusively, although the number of isolates investigated was small. This study demonstrates the presence of all three European species ofBorrelia burgdorferi sensu lato within the Slovenian tick population and also within a geographic area of less than 100 m².     

Host-seeking behavior of adult Ixodes pacificus (Acari: Ixodidae) as determined by flagging vegetation

Diurnal host-seeking by adults of the western black-legged tick, Ixodes pacificus Cooley & Kohls in relation to vegetative habitat type and meterological factors was investigated at a coastal and an inland site in northern California. Host-seeking behavior and relative tick abundance were determined with a tick drag method from late fall to midwinter 1987-1988. At the inland site, tick abundance usually was significantly greater in chaparral-grassland ecotones than in adjoining dense chaparral on the south-facing slope of a mountaintop, whereas both of these vegetative types produced significantly fewer ticks on a north slope compared with a contiguous south-facing slope. There was no evidence for an association between tick abundance and plant species within ecotonal chaparral. Multiple regression analyses revealed that tick abundance in ecotonal chaparral at the inland site and in grassland at the coastal site was not associated consistently with either ambient temperature or relative humidity. Compared with the inland site, ticks were considerably more abundant at the coastal site where sampling occasionally yielded more than 100 ticks per 100 drag samples. The incidence of infection with Borrelia burgdorferi Johnson, Schmid, Hyde, Steigerwalt & Brenner, the etiologic agent of Lyme borreliosis, in adult ticks collected in the morning (1.5%) versus afternoon (2.2%), and in male (1.3%) versus female ticks (2.5%), at the coastal site in winter was similar.     

Infection and co-infection rates of Anaplasma phagocytophilum variants, Babesia spp., Borrelia burgdorferi, and the rickettsial endosymbiont in Ixodes scapularis (Acari: Ixodidae) from sites in Indiana, Maine, Pennsylvania, and Wisconsin

In total, 394 questing adult blacklegged ticks, Ixodes scapularis Say (Acari: Ixodidae), collected at four sites were analyzed by polymerase chain reaction (PCR) for five microbial species: Anaplasma phagocytophilum, Babesia microti, Babesia odocoilei, Borrelia burgdorferi, and the rickettsial I. scapularis endosymbiont. Identities of genetic variants of A. phagocytophilum were determined by sequencing a portion of the 16S DNA. In 55% of infected ticks (193/351), a single agent was detected. In 45% (158/351), two or more agents were detected; 37% harbored two agents and 8% harbored three agents. One male tick, collected from Ft. McCoy, WI, harbored all four microbial genera The highest rates of co-infection were by the Ixodes endosymbiont and B. burgdorferi (95/351). Two species of Babesia co-occurred within a single tick population in Wells National Estuarine Research Reserve, Wells, ME, whereas only B. odocoilei was found in other tick populations. Only A. phagocytophilum human anaplasmosis variant was detected in questing ticks from Tippecanoe River State Park, IN; from Wells; and Ft. McCoy, whereas a single infected tick from Presque Isle, PA, was infected by AP-Variant 1. Partially engorged ticks from deer in Tippecanoe River State Park were all infected with AP-Variant 1. Frequency of infections with each agent varied among populations. Rates and types of co-infections were not significantly different from random except for the Ixodes endosymbiont and B. burgdorferi in male ticks, which co-occurred less frequently than expected. Thus, I. scapularis hosts an array of pathogenic and symbiotic agents and potential evidence of interactions among microbial species was observed.     

Sexual transmission of spotted fever group rickettsiae by infected male ticks: detection of rickettsiae in immature spermatozoa of Ixodes ricinus.

Electron microscopic evidence is provided showing that the newly described "Swiss agent," a spotted fever group rickettsia, is incorporated into the reproductive cells of its male tick vector, Ixodes ricinus. Rickettsiae were found in spermatogonia, spermatocytes, and maturing spermatids. The potential significance of these observations is briefly discussed in relation to published data on sexual transmission of rickettsiae by ticks.     

Detection of the agent of heartwater, Cowdria ruminantium, in Amblyomma ticks by PCR: validation and application of the assay to field ticks

We have previously reported that the pCS20 PCR detection assay for Cowdria ruminantium, the causative agent of heartwater disease of ruminants, is more sensitive than xenodiagnosis and the pCS20 DNA probe for the detection of infection in the vector Amblyomma ticks. Here, we further assessed the reliability of the PCR assay and applied it to field ticks. The assay detected DNA of 37 isolates of C. ruminantium originating from sites throughout the distribution of heartwater and had a specificity of 98% when infected ticks were processed concurrently with uninfected ticks. The assay did not detect DNA of Ehrlichia chaffeensis, which is closely related to C. ruminantium. PCR sensitivity varied with tick infection intensity and was high (97 to 88%) with ticks bearing 10(7) to 10(4) organisms but dropped to 61 and 28%, respectively, with ticks bearing 10(3) and 10(2) organisms. The assay also detected C. ruminantium in collections of Amblyomma hebraeum and Amblyomma variegatum field ticks from 17 heartwater-endemic sites in four southern African countries. Attempts at tick transmission of infection to small ruminants failed with four of these collections. The pCS20 PCR assay is presently the most characterized and reliable test for C. ruminantium in ticks and thus is highly useful for field and laboratory epidemiological investigations of heartwater.     

Lack of spatial autocorrelation in fine-scale distributions of Ixodes scapularis (Acari: Ixodidae)

Spatial patterns of Ixodes scapularis Say, the vector of the Lyme disease agent, have been examined at various geographic scales, demonstrating that distributions of these ticks are spatially autocorrelated at both national and state scales. We tested the hypothesis that distributions of nymphal I scapularis ticks at the fine scale of an endemic community also are spatially autocorrelated. Nymphal tick densities were determined by collecting ticks from 51 and 47 wooded residential properties in a southern Rhode Island town in 2002 and 2003, respectively. The average tick density at residences during 2002 was 51.17 ( +/- 46.04) nymphs per hour, with a range of 3-297 and median of 40.82. In 2003, the average tick density was 44.48 (+/- 38.31) nymphs per hour, with a range of 3-153 and median of 36. Semivariance analysis revealed no spatial autocorrelation in tick densities between residences, likely due to the high variability of tick distributions at this scale. Further analysis of drag-sampling data at individual residences by using Lloyd's patchiness index (m*/m) demonstrated a patchy distribution of nymphs. High variability of nymphal I. scapularis densities may greatly affect predicting spatial patterns of ticks at a fine scale.     

The present knowledge of tick-borne encephalitis vectors

The most important aspects of the relationships of tick-borne encephalitis virus and its vectors--Ixodes ticks. These include the establishment of a rule of viral exchange between the co-fed ticks, which makes its reverse transfer from nymphs to larvae possible; enhancement of tick aggressiveness under the influence of the incremental concentrations of the virus increasing their epidemiological danger; the association of the spread of viral genospecies with the genetic structure of tick populations; the emergence of phenotypically different vector subpopulations with new vector properties (under the influence of anthropogenic factors); and the elucidation of a possible role of the virus as an enhancer in mixed viral and bacterial infections of tick vectors.     

Geographical distribution patterns and habitat suitability models for presence of host-seeking ixodid ticks in dense woodlands of Mendocino County, California

We used drag sampling to examine the geographical distribution patterns of ixodid ticks engaging in open (non-nidicolous) host-seeking behavior in dense woodland habitats of the climatically and ecologically diverse Mendocino County in north coastal California. The findings based on this sampling methodology reflect risk of human exposure to host-seeking ticks rather than the true distribution of the ticks. Drag sampling in 78 sites yielded 7,860 nymphal or adult Ixodes pacificus Cooley & Kohls, 220 Dermacentor occidentalis Marx, 150 Ixodes spinipalpis Hadwen & Nuttall, 15 Hemaphysalis leporispalustris (Packard), 12 Ixodes angustus Neumann, 12 Ixodes auritulus Neumann, and a single Dermacentor variabilis (Say). I. pacificus, which is the primary vector of the Lyme disease spirochete Borrelia burgdorferi to humans in California, occurred in all 78 sites examined. D. occidentalis, another tick species commonly biting humans in California, and H. leporispalustris typically were encountered in oak-associated woodlands in the central or eastern parts of the county. In contrast, three species of Ixodes ticks (I. angustus, I. auritulus, and I. spinipalpis) most commonly were found questing openly in woodlands with redwood present in the western part of the county. I. angustus and I. spinipalpis are occasional human biters and known experimental vectors of B. burgdorferi. Our study represents the first collection of large numbers of openly host-seeking I. spinipalpis ticks. Univariate tests of associations between presence of ticks (D. occidentalis, H. leporispalustris, I. angustus, I. auritulus, or I. spinipalpis) and environmental geographical information systems-remote sensing (GIS/ RS)-based data indicated that elevation, number of growing degree-days, and tasseled cap brightness, greenness, and wetness are especially useful predictors of presence of openly hostseeking ticks. Combinations of the above-mentioned GIS/RS-based data yielded significant logistic regression models for habitat suitability of host-seeking ticks for all five above-mentioned species. The model equations were used to create spatial surfaces of predicted presence of suitable habitat for openly host-seeking ticks in Mendocino County dense woodlands.     

Computer simulation of management strategies for American dog ticks (Acari: Ixodidae) and Rocky Mountain spotted fever

Computer models were developed to simulate the effects of management technologies on populations of the American dog tick, Dermacentor variabilis (Say), principal vector of Rocky Mountain spotted fever (RMSF) in eastern North America. The technologies modeled were area-wide acaricide application, acaricide-food-baited tubes for self-treatment by small mammals, dipping of dogs in acaricides, acaricide-impregnated plastic dog collars, reduction of small mammal host populations (host management), and removal of vegetation that protects free-living tick stages (vegetative management). Submodels for each of these technologies were incorporated into a model (ADTSIM) for the population dynamics of the tick and RMSF transmission. Comparisons of simulated and observed data were used to verify reasonable accuracy of the submodels. Repetitive simulations were made to identify levels and timing of each control method (alone or combined) required to reduce tick populations below a RMSF transmission threshold of 252 unfed adults/ha. Eight to 30 acaricide applications, depending on acaricide and percentage of population treated, were needed during a 10-yr period to reduce densities of ticks below the threshold. The baited-tube method, host management, and vegetative management (depending on level and frequency of treatment) also were capable of reducing tick density below the threshold. However, acaricide-impregnated plastic dog collars did not reduce tick density below the threshold unless at least 50% of the hosts of adult ticks were domestic dogs. Integrated strategies were developed for management of ticks and RMSF in six selected states. These strategies reduced numbers of human cases of RMSF 90% or more by year 20 by maintaining tick densities between 100 and 252 unfed adults/ha.     

Ehrlichia chaffeensis (Rickettsiales: Ehrlichieae) infection in Amblyomma americanum (Acari: Ixodidae) at Aberdeen Proving Ground, Maryland

Human monocytic ehrlichiosis (HME) is a sometimes fatal, emerging tick-borne disease caused by the bacterium Ehrlichia chaffeensis. It is frequently misdiagnosed because its symptoms mimic those of the flu. Current evidence indicates that Amblyomma americanum (L.), the lone star tick, is the major vector of HME. To determine if E. chaffeensis is present in ticks at Aberdeen Proving Ground, MD, questing A. americanum ticks were collected from 33 sites. Nucleic acid was extracted from 34 adult and 81 nymphal pools. Sequences diagnostic for E. chaffeensis from three different loci (16S rRNA, 120-kDa protein, and a variable-length polymerase chain reaction [PCR] target, or VLPT) were targeted for amplification by the PCR. Fifty-two percent of the collection sites yielded pools infected with E. chaffeensis, confirming the presence and widespread distribution of E. chaffeensis at Aberdeen Proving Ground. Analysis with the both the 120-kDa protein primers and the VLPT primers showed that genetic variance exists. A novel combination of variance for the two loci was detected in two tick pools. The pathogenic implications of genetic variation in E. chaffeensis are as yet unknown.     

Lyme disease: a selective medium for isolation of the suspected etiological agent, a spirochete.

A simple procedure with a new selective culture medium for the isolation of the suspected etiological agent of Lyme disease from ticks is described. Live ticks (Ixodes dammini) were ground with a mortar and pestle, and the suspensions were inoculated into a selective and nonselective medium. The selective medium, which contained kanamycin and 5-fluorouracil, yielded positive spirochete cultures from 100% of the pooled ticks and from 79% of the single tick specimens. The isolation rate for the nonselective medium was 0% from the tick pools and 58% from the single tick specimens.     

Detection of a Borrelia miyamotoi sensu lato relapsing-fever group spirochete from Ixodes pacificus in California

We investigated whether host-seeking nymphs and adults of the western blacklegged tick, Ixodes pacificus Cooley & Kohls, the primary vector of Lyme disease spirochetes in far-western North America, are infected naturally with relapsing-fever group spirochetes in Mendocino County, California. Relapsing-fever group borreliae were detected in four (1.7%) of 234 nymphal and two (0.7%) of 282 adult host-seeking I. pacificus ticks by polymerase chain reaction and sequence analysis of the 16S rRNA and flagellin genes, respectively, exhibiting 99 and 98.5% sequence homology to Borrelia miyamotoi Fukunaga. Phylogenetic analysis based on these two genes revealed that the borreliae detected in these ticks belong to the relapsing-fever group and that these are closely related to, if not identical with, B. miyamotoi.     

Molecular typing of isolates of Rickettsia rickettsii by use of DNA sequencing of variable intergenic regions

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is found throughout the Americas, where it is associated with different animal reservoirs and tick vectors. No molecular typing system currently exists to allow for the robust differentiation of isolates of R. rickettsii. Analysis of eight completed genome sequences of rickettsial species revealed a high degree of sequence conservation within the coding regions of chromosomes in the genus. Intergenic regions between coding sequences should be under less selective pressure to maintain this conservation and thus should exhibit greater nucleotide polymorphisms. Utilizing these polymorphisms, we developed a molecular typing system that allows for the genetic differentiation of isolates of R. rickettsii. This typing system was applied to a collection of 38 different isolates collected from humans, animals, and tick vectors from different geographic locations. Serotypes 364D, from Dermacentor occidentalis ticks, and Hlp, from Haemaphysalis leporispalustris ticks, appear to be distinct genotypes that may not belong to the species R. rickettsii. We were also able to differentiate 36 historical isolates of R. rickettsii into three different phylogenetic clades containing seven different genotypes. This differentiation correlated well, but not perfectly, with the geographic origin and likely tick vectors associated with the isolates. The few apparent typing discrepancies found suggest that the molecular ecology of R. rickettsii needs more investigation.     

Oral secretion elicited by effectors of signal transduction pathways in the salivary glands of Amblyomma americanum (Acari: Ixodidae).

The abilities of various effectors of signal pathways in tick salivary glands to stimulate oral secretions in partially fed Amblyomma americanum (L.) female ticks were compared. Pilocarpine stimulated secretion the most rapidly following tick stimulation, but its effectiveness declined with time and subsequent injections. Secretion rates induced by dopamine and theophylline increased with time of collection and additional injections and were as effective as pilocarpine after 60 min. Activators of protein kinase C, phorbol 12-myristate 13-acetate (PMA), and 1-oleoyl-2-acetyl-sn-glycerol inhibited dopamine, and theophylline stimulated secretion. gamma-amino-butyric acid either by itself or in combination with other drugs had no effect on volume of oral secretions. Peptides were visible in the oral secretion stimulated by the various drugs after separation and staining in SDS-polyacrylamide gels. More peptides were present in the oral secretion obtained from the smallest partially fed (slow feeding) ticks. In some ticks, proteins were observed in oral secretions obtained from the same tick at one collection time but not another in response to the same pharmacological agent.     

Host-dependent differences in feeding and reproduction of Ixodes dammini (Acari: Ixodidae)

The frequencies with which adult Ixodes dammini Spielman, Clifford, Piesman and Corwin feed upon white-tailed deer (Odocoileus virginianus), raccoons (Procyon lotor), opossums (Didelphis virginiana), and domestic cats were compared in a wooded site in northeastern United States. By combining estimates of the quantity of ticks feeding on each host species with host densities, we derived the relative contribution made by each kind of host to the feeding of the tick population. To compare engorgement success and reproductive efficacy, we weighed ticks found on these hosts and compared tick weights with the quantity of eggs produced. Deer harbored the most female ticks per individual and, although deer were less abundant than raccoons, they harbored 94.6% of the feeding tick population. Raccoons, cats, and opossums, respectively, provided blood meals to 3.6, 1.6, and 0.2% of feeding adult ticks. Ticks engorged more successfully on cats and raccoons than on deer, and egg production correlated linearly with weight of female ticks collected from both cats and deer. Although these ticks engorged better on other hosts, the vast majority of eggs resulted from ticks that had fed on deer. We conclude that, in this site, the abundance of deer largely determines the abundance of I. dammini, the vector of the agent of Lyme disease.     

"Candidatus Neoehrlichia mikurensis," Anaplasma phagocytophilum, and lyme disease spirochetes in questing european vector ticks and in feeding ticks removed from people

To estimate the likelihood of people coming into contact with the recently described tick-borne agent "Candidatus Neoehrlichia mikurensis," we compared its prevalence to those of Lyme disease spirochetes and Anaplasma phagocytophilum in questing adult Ixodes ricinus ticks collected in various Central European sites and examined ticks, which had been removed from people, for the presence of these pathogens. Whereas spirochetes infected questing adult ticks most frequently (22.3%), fewer than a third as many ticks were infected by "Ca. Neoehrlichia mikurensis" (6.2%), and about a sixth harbored A. phagocytophilum (3.9%). On average, every twelfth encounter of a person with an I. ricinus tick (8.1%) may bear the risk of acquiring "Ca. Neoehrlichia mikurensis." Although a fifth of the people (20%) had removed at least one tick infected by "Ca. Neoehrlichia mikurensis," none displayed symptoms described for this pathogen, suggesting that its transmission may not be immediate and/or that immunocompetent individuals may not be affected. Because immunosuppressed patients may be at a particular risk of developing symptoms, it should be considered that "Ca. Neoehrlichia mikurensis" appears to be the second most common pathogen in I. ricinus ticks. In our survey, only Borrelia afzelii appears to infect Central European vector ticks more frequently.