TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi naturally persists in a cycle that primarily involves ticks and mammals. We have now identified a tick receptor (TROSPA) that is required for spirochetal colonization of Ixodes scapularis. B. burgdorferi outer surface protein A, which is abundantly expressed on spirochetes within the arthropod and essential for pathogen adherence to the vector, specifically bound to TROSPA. TROSPA mRNA levels in ticks increased following spirochete infestation and decreased in response to engorgement, events that are temporally linked to B. burgdorferi entry into and egress from the vector. The blockade of TROSPA by TROSPA antisera or by the repression of TROSPA expression via RNA interference reduced B. burgdorferi adherence to the I. scapularis gut in vivo, thereby preventing efficient colonization of the vector and subsequently reducing pathogen transmission to the mammalian host. Identification of an I. scapularis receptor for B. burgdorferi is the first step toward elucidating arthropod ligands that are required for survival of spirochetes in nature.     

Species Co-Occurrence Patterns among Lyme Borreliosis Pathogens in the Tick Vector Ixodes ricinus

Mixed infections have important consequences for the ecology and evolution of host-parasite interactions. In vector-borne diseases, interactions between pathogens occur in both the vertebrate host and the arthropod vector. Spirochete bacteria belonging to the Borrelia burgdorferi sensu lato genospecies complex are transmitted by Ixodes ticks and cause Lyme borreliosis in humans. In Europe, there is a high diversity of Borrelia pathogens, and the main tick vector, Ixodes ricinus, is often infected with multiple Borrelia genospecies. In the present study, we characterized the pairwise interactions between five B. burgdorferi sensu lato genospecies in a large data set of I. ricinus ticks collected from the same field site in Switzerland. We measured two types of pairwise interactions: (i) co-occurrence, whether double infections occurred more or less often than expected, and (ii) spirochete load additivity, whether the total spirochete load in double infections was greater or less than the sum of the single infections. Mixed infections of Borrelia genospecies specialized on different vertebrate reservoir hosts occurred less frequently than expected (negative co-occurrence) and had joint spirochete loads that were lower than the additive expectation (inhibition). In contrast, mixed infections of genospecies that share the same reservoir hosts were more common than expected (positive co-occurrence) and had joint spirochete loads that were similar to or greater than the additive expectation (facilitation). Our study suggests that the vertebrate host plays an important role in structuring the community of B. burgdorferi sensu lato genospecies inside the tick vector.     

Inhibition of Borrelia burgdorferi-tick interactions in vivo by outer surface protein A antibody

Borrelia burgdorferi outer surface protein (Osp) A is preferentially expressed by spirochetes in the Ixodes scapularis gut and facilitates pathogen-vector adherence in vitro. Here we examined B. burgdorferi-tick interactions in vivo by using Abs directed against OspA from each of the three major B. burgdorferi sensu lato genospecies: B. burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. Abs directed against B. burgdorferi sensu stricto (isolate N40) destroy the spirochete and can protect mice from infection. In contrast, antisera raised against OspA from B. afzelii (isolate ACA-1) and B. garinii (isolate ZQ-1) bind to B. burgdorferi N40 but are not borreliacidal against the N40 isolate. Our present studies assess whether these selected OspA Abs interfere with B. burgdorferi-tick attachment in a murine model of Lyme disease with I. scapularis. We examined engorged ticks that had fed on B. burgdorferi N40-infected scid mice previously treated with OspA (N40, ACA-1, ZQ-1, or mAb C3.78) or control Abs. OspA-N40 antisera or mAb C3.78 destroyed B. burgdorferi N40 within the engorged ticks. In contrast, treatment of mice with OspA-ACA-1 and OspA-ZQ-1 antisera did not kill B. burgdorferi N40 within the ticks but did effectively interfere with B. burgdorferi-I. scapularis adherence, thereby preventing efficient colonization of the vector. These studies show that nonborreliacidal OspA Abs can inhibit B. burgdorferi attachment to the tick gut, highlighting the importance of OspA in spirochete-arthropod interactions in vivo.     

Production of outer surface protein A by Borrelia burgdorferi during transmission from infected mammals to feeding ticks is insufficient to trigger OspA seroconversion

The Lyme disease spirochete, Borrelia burgdorferi, produces two outer surface lipoproteins, OspA and OspB, that are essential for colonization of tick vectors. Both proteins are highly expressed during transmission from infected mammals to feeding ticks and during colonization of tick midguts, but are repressed when bacteria are transmitted from ticks to mammals. Humans and other infected mammals generally do not produce antibodies against either protein, although some Lyme disease patients do seroconvert and produce antibodies against OspA for unknown reasons. We hypothesized that, if such patients had been fed upon by additional ticks, bacteria moving from the patients' bodies to the feeding ticks would have produced OspA and OspB proteins, which then led to immune system recognition and antibody production. This hypothesis was tested by analyzing immune responses of infected mice following feedings by additional Ixodes scapularis ticks. However, results of the present studies demonstrate that expression of OspA and OspB by B. burgdorferi during transmission from infected mammals to feeding ticks does not trigger seroconversion.     

Borrelia burgdorferi sensu lato in ticks (Acari: Ixodidae) from two different foci in Spain

The prevalence of Borrelia burgdorferi sensu lato in several tick species was studied over a 2 year period in two ecologically different areas in Spain. One area was an endemic area for Lyme disease, with a number of autochthonous human cases and supported large populations of Ixodes ricinus on cattle and birds; the second area was characterized by the absence of I. ricinus together with the presence of foxes and their associated tick species. While I. ricinus was the main vector of B. burgdoreri in the endemic area (with a mean prevalence of 14% in adults and 51% in nymphs), adults of both Ixodes canisuga and Ixodes hexagonus had high rates of B. burgdorferi prevalence (30 and 28%, respectively) in the zone where I. ricinus was absent. Immatures of Ixodes frontalis were found to be carriers of the spirochete only in those zones where I. ricinus is present, suggesting evidence for reservoir competence in a tick-bird cycle.     

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.     

Tick-host-pathogen interactions in Lyme borreliosis

Borrelia burgdorferi, the spirochetal agent of Lyme borreliosis, is predominantly transmitted by Ixodes ticks. Spirochetes have developed many strategies to adapt to the different environments that are present in the arthropod vector and the vertebrate host. This review focuses on B. burgdorferi genes that are preferentially expressed in the tick and the vertebrate host, and describes how selected gene products facilitate spirochete survival throughout the enzootic life cycle. Interestingly, B. burgdorferi also enhances expression of specific Ixodes scapularis genes, such as TROSPA and salp15. The importance of these genes and their products for B. burgdorferi survival within the tick, and during the transmission process, will also be reviewed. Moreover, we discuss how such vector molecules could be used to develop vector-antigen-based vaccines to prevent the transmission of B. burgdorferi and, potentially, other arthropod-borne microbes.     

Host and pathogen DNA identification in blood meals of nymphal Ixodes ricinus ticks from forest parks and rural forests of Poland

DNA analysis of blood meals from unfed nymphal Ixodes ricinus allows for the identification of tick host and tick-borne pathogens in the host species. The recognition of host species for tick larvae and the reservoirs of Borrelia, Rickettsia and Anaplasma species were simultaneously carried out by analysis of the blood meals of 880 questing nymphal I. ricinus ticks collected in forest parks of Szczecin city and rural forests in northwestern Poland that are endemic areas for Lyme borreliosis. The results obtained from the study indicate that I. ricinus larvae feed not only on small or medium animals but also on large animals and they (i.e. roe deer, red deer and wild boars) were the most prevalent in all study areas as the essential hosts for larvae of I. ricinus. The composition of medium and small vertebrates (carnivores, rodents, birds and lizards) provided a more diverse picture depending on study site. The reservoir species that contain the most pathogens are the European roe deer Capreolus capreolus, in which two species of Rickettsia and two species of Borrelia were identified, and Sus scrofa, in which one Rickettsia and three Borrelia species were identified. Rickettsia helvetica was the most common pathogen detected, and other included species were the B. burgdorferi s.l. group and B. miyamotoi related to relapsing fever group. Our results confirmed a general association of B. garinii with birds but also suggested that such associations may be less common in the transmission cycle in natural habitats than what was thought previously.     

A Tick Gut Protein with Fibronectin III Domains Aids Borrelia burgdorferi Congregation to the Gut during Transmission

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.     

Cross-Immunity and Community Structure of a Multiple-Strain Pathogen in the Tick Vector

Many vector-borne pathogens consist of multiple strains that circulate in both the vertebrate host and the arthropod vector. Characterization of the community of pathogen strains in the arthropod vector is therefore important for understanding the epidemiology of mixed vector-borne infections. Borrelia afzelii and B. garinii are two species of tick-borne bacteria that cause Lyme disease in humans. These two sympatric pathogens use the same tick, Ixodes ricinus, but are adapted to different classes of vertebrate hosts. Both Borrelia species consist of multiple strains that are classified using the highly polymorphic ospC gene. Vertebrate cross-immunity against the OspC antigen is predicted to structure the community of multiple-strain Borrelia pathogens. Borrelia isolates were cultured from field-collected I. ricinus ticks over a period spanning 11 years. The Borrelia species of each isolate was identified using a reverse line blot (RLB) assay. Deep sequencing was used to characterize the ospC communities of 190 B. afzelii isolates and 193 B. garinii isolates. Infections with multiple ospC strains were common in ticks, but vertebrate cross-immunity did not influence the strain structure in the tick vector. The pattern of genetic variation at the ospC locus suggested that vertebrate cross-immunity exerts strong selection against intermediately divergent ospC alleles. Deep sequencing found that more than 50% of our isolates contained exotic ospC alleles derived from other Borrelia species. Two alternative explanations for these exotic ospC alleles are cryptic coinfections that were not detected by the RLB assay or horizontal transfer of the ospC gene between Borrelia species.     

Transmission Dynamics of Borrelia turicatae from the Arthropod Vector

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.     

Prevalence of Borrelia burgdorferi Sensu Lato Genospecies in Ixodes ricinus Ticks in Europe: a Metaanalysis

In Europe, Borrelia burgdorferi genospecies causing Lyme borreliosis are mainly transmitted by the tick Ixodes ricinus. Since its discovery, B. burgdorferi has been the subject of many epidemiological studies to determine its prevalence and the distribution of the different genospecies in ticks. In the current study we systematically reviewed the literature on epidemiological studies of I. ricinus ticks infected with B. burgdorferi sensu lato. A total of 1,186 abstracts in English published from 1984 to 2003 were identified by a PubMed keyword search and from the compiled article references. A multistep filter process was used to select relevant articles; 110 articles from 24 countries contained data on the rates of infection of I. ricinus with Borrelia in Europe (112,579 ticks), and 44 articles from 21 countries included species-specific analyses (3,273 positive ticks). These data were used to evaluate the overall rate of infection of I. ricinus with Borrelia genospecies, regional distributions within Europe, and changes over time, as well as the influence of different detection methods on the infection rate. While the infection rate was significantly higher in adults (18.6%) than in nymphs (10.1%), no effect of detection method, tick gender, or collection period (1986 to 1993 versus 1994 to 2002) was found. The highest rates of infection of I. ricinus were found in countries in central Europe. B. afzelii and B. garinii are the most common Borrelia species, but the distribution of genospecies seems to vary in different regions in Europe. The most frequent coinfection by Borrelia species was found for B. garinii and B. valaisiana.     

Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.     

Prevalence,Diversity, and Load of Borrelia species in Ticks That Have Fed on Humans in Regions of Sweden and ?land Islands,Finland with Different Lyme Borreliosis Incidences

The incidence of Lyme borreliosis (LB) in a region may reflect the prevalence of Borrelia in the tick population. Our aim was to investigate if regions with different LB incidences can be distinguished by studying the prevalence and diversity of Borrelia species in their respective tick populations. The Borrelia load in a feeding tick increases with the duration of feeding, which may facilitate a transmission of Borrelia Spirochetes from tick to host. Therefore, we also wanted to investigate how the Borrelia load in ticks that have fed on humans varies with the duration of tick feeding. During 2008 and 2009, ticks that had bitten humans were collected from four regions of Sweden and Finland, regions with expected differences in LB incidence. The duration of tick feeding was estimated and Borrelia were detected and quantified by a quantitative PCR assay followed by species determination. Out of the 2,154 Ixodes ricinus ticks analyzed, 26% were infected with Borrelia and seven species were identified. B. spielmanii was detected for the first time in the regions. The tick populations collected from the four regions exhibited only minor differences in both prevalence and diversity of Borrelia species, indicating that these variables alone cannot explain the regions’ different LB incidences. The number of Borrelia cells in the infected ticks ranged from fewer than ten to more than a million. We also found a lower number of Borrelia cells in adult female ticks that had fed for more than 36 hours, compared to the number of Borrelia cells found in adult female ticks that had fed for less than 36 hours.     

The ecology of Ixodes trianguliceps ticks and their role in the natural foci of ixodid tick-borne borrelioses in the Middle Urals

From 1994 to 2011, over 7000 individuals of small mammals were captured and examined for ticks in the natural foci of ixodid tick-borne borrelioses (ITBB) in the Middle Urals (Chusovskoy District of Perm Territory). Alongside with the taiga tick (Ixodes persulcatus), which is the main Borrelia vector, approximately 5700 feeding individuals of Ixodes trianguliceps have been identified. The latter species has been found to be about five times less abundant than the former. I. trianguliceps has been collected from small mammals belonging to 19 species. Its main hosts are common shrews (Sorex araneus), bank voles (Clethrionomys glareolus), and northern red-backed voles (C. rutilus). I. trianguliceps shows two seasonal peaks of abundance, spring-summer, and summer-autumn. Plating in BSK II medium yielded 72 isolates of Borrelia from a total of 1142 individuals of I. trianguliceps; 64 isolates have been identified with PCR and RFLP. The mean values of the Borrelia infestation rate in I. trianguliceps larvae, nymphs, and adults are 2.6, 10.2, and 8.1%, respectively, which is 5–10 times lower than in the taiga tick individuals collected from the same mammals. Borreliae obtained from I. trianguliceps (as well as those from I. persulcatus) have been identified as Borrelia garinii and B. afzelii, the former spirochete species being more frequent (about 90% of isolates from I. trianguliceps). Our results indicate that I. trianguliceps ticks participate in circulation of the ITBB causative agents in the forests of the Middle Urals. Rare occurrence of the tick and low rates of its infestation with borreliae suggest that the species is unlikely to play a significant role in the epizootic development in the natural foci of ITBB.     

Borrelia burgdorferi sensu lato and co‐infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg,Germany

To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe‐based quantitative real‐time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co‐infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co‐infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia‐positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.     

Borrelia burgdorferi sensu lato in Ixodes ricinus ticks from Norway: evaluation of a PCR test targeting the chromosomal flaB gene

A consensus TaqMan real-time PCR test targeting the chromosomal flaB gene of Borrelia burgdorferi sensu lato was constructed. The test was compared with a recently published generic Light Upon eXtension (LUX) 16S rRNA real-time PCR test (Wilhelmsson et al. in J Clin Microbiol 48:4169–4176, 2010) on material consisting of 242 Ixodes ricinus ticks collected from dogs and cats in Northern Norway (n?=?139) and Telemark County in Southern Norway (n?=?103). Ticks positive in either test were further tested by nested PCR amplification of the 5S-23S rRNA intergenic-spacer region followed by sequencing for species identification. A tick was defined as Borrelia positive if two of three tests were positive. Thirty-four of the 242 (14?%) ticks satisfied this definition of positivity. Of these ticks 32 were positive both in the rRNA and flaB test, while two were positive only in the rRNA test. One tick was positive only in the rRNA test and was considered false positive since PCR for sequencing failed. The sensitivity of the flaB test was 94?% and the specificity 100?%. It was possible to determine the species present using Tm analysis. Among ticks from Northern Norway the prevalence of Borrelia was 13?%, whereas the prevalence in Telemark was 16?%. Among identified species (n?=?33) B. afzelii was found in 16 (47?%), B. garinii in 15 (44?%) and B. valaisiana in 2 (6?%) ticks, respectively. The flaB test is a rapid, sensitive and specific test for detection and quantification of Borrelia burgdorferi s.l. in I. ricinus ticks. This is the first report on Borrelia prevalence in I. ricinus in Northern Norway.