Variation in the Microbiota of Ixodes Ticks with Regard to Geography,Species, and Sex

Ixodes scapularis is the principal vector of Lyme disease on the East Coast and in the upper Midwest regions of the United States, yet the tick is also present in the Southeast, where Lyme disease is absent or rare. A closely related species, I. affinis, also carries the pathogen in the South but does not seem to transmit it to humans. In order to better understand the geographic diversity of the tick, we analyzed the microbiota of 104 adult I. scapularis and 13 adult I. affinis ticks captured in 19 locations in South Carolina, North Carolina, Virginia, Connecticut, and New York. Initially, ticks from 4 sites were analyzed by 454 pyrosequencing. Subsequently, ticks from these sites plus 15 others were analyzed by sequencing with an Illumina MiSeq machine. By both analyses, the microbiomes of female ticks were significantly less diverse than those of male ticks. The dissimilarity between tick microbiomes increased with distance between sites, and the state in which a tick was collected could be inferred from its microbiota. The genus Rickettsia was prominent in all locations. Borrelia was also present in most locations and was present at especially high levels in one site in western Virginia. In contrast, members of the family Enterobacteriaceae were very common in North Carolina I. scapularis ticks but uncommon in I. scapularis ticks from other sites and in North Carolina I. affinis ticks. These data suggest substantial variations in the Ixodes microbiota in association with geography, species, and sex.     

A Chitin Deacetylase-Like Protein Is a Predominant Constituent of Tick Peritrophic Membrane That Influences the Persistence of Lyme Disease Pathogens within the Vector

Ixodes scapularis is the specific arthropod vector for a number of globally prevalent infections, including Lyme disease caused by the bacterium Borrelia burgdorferi. A feeding-induced and acellular epithelial barrier, known as the peritrophic membrane (PM) is detectable in I. scapularis. However, whether or how the PM influences the persistence of major tick-borne pathogens, such as B. burgdorferi, remains largely unknown. Mass spectrometry-based proteome analyses of isolated PM from fed ticks revealed that the membrane contains a few detectable proteins, including a predominant and immunogenic 60 kDa protein with homology to arthropod chitin deacetylase (CDA), herein termed I. scapularis CDA-like protein or IsCDA. Although IsCDA is primarily expressed in the gut and induced early during tick feeding, its silencing via RNA interference failed to influence either the occurrence of the PM or spirochete persistence, suggesting a redundant role of IsCDA in tick biology and host-pathogen interaction. However, treatment of ticks with antibodies against IsCDA, one of the most predominant protein components of PM, affected B. burgdorferi survival, significantly augmenting pathogen levels within ticks but without influencing the levels of total gut bacteria. These studies suggested a preferential role of tick PM in limiting persistence of B. burgdorferi within the vector. Further understanding of the mechanisms by which vector components contribute to pathogen survival may help the development of new strategies to interfere with the infection.     

Population-Based Passive Tick Surveillance and Detection of Expanding Foci of Blacklegged Ticks Ixodes scapularis and the Lyme Disease Agent Borrelia burgdorferi in Ontario,Canada

We identified ticks submitted by the public from 2008 through 2012 in Ontario, Canada, and tested blacklegged ticks Ixodes scapularis for Borrelia burgdorferi and Anaplasma phagocytophilum. Among the 18 species of ticks identified, I. scapularis, Dermacentor variabilis, Ixodes cookei and Amblyomma americanum represented 98.1% of the 14,369 ticks submitted. Rates of blacklegged tick submission per 100,000 population were highest in Ontario''s Eastern region; D. variabilis in Central West and Eastern regions; I. cookei in Eastern and South West regions; and A. americanum had a scattered distribution. Rates of blacklegged tick submission per 100,000 population were highest from children (0–9 years old) and older adults (55–74 years old). In two health units in the Eastern region (i.e., Leeds, Grenville & Lanark District and Kingston-Frontenac and Lennox & Addington), the rate of submission for engorged and B. burgdorferi-positive blacklegged ticks was 47× higher than the rest of Ontario. Rate of spread for blacklegged ticks was relatively faster and across a larger geographic area along the northern shore of Lake Ontario/St. Lawrence River, compared with slower spread from isolated populations along the northern shore of Lake Erie. The infection prevalence of B. burgdorferi in blacklegged ticks increased in Ontario over the study period from 8.4% in 2008 to 19.1% in 2012. The prevalence of B. burgdorferi-positive blacklegged ticks increased yearly during the surveillance period and, while increases were not uniform across all regions, increases were greatest in the Central West region, followed by Eastern and South West regions. The overall infection prevalence of A. phagocytophilum in blacklegged ticks was 0.3%. This study provides essential information on ticks of medical importance in Ontario, and identifies demographic and geographic areas for focused public education on the prevention of tick bites and tick-borne diseases.     

A Dityrosine Network Mediated by Dual Oxidase and Peroxidase Influences the Persistence of Lyme Disease Pathogens within the Vector

Ixodes scapularis ticks transmit a wide array of human and animal pathogens including Borrelia burgdorferi; however, how tick immune components influence the persistence of invading pathogens remains unknown. As originally demonstrated in Caenorhabditis elegans and later in Anopheles gambiae, we show here that an acellular gut barrier, resulting from the tyrosine cross-linking of the extracellular matrix, also exists in I. scapularis ticks. This dityrosine network (DTN) is dependent upon a dual oxidase (Duox), which is a member of the NADPH oxidase family. The Ixodes genome encodes for a single Duox and at least 16 potential peroxidase proteins, one of which, annotated as ISCW017368, together with Duox has been found to be indispensible for DTN formation. This barrier influences pathogen survival in the gut, as an impaired DTN in Doux knockdown or in specific peroxidase knockdown ticks, results in reduced levels of B. burgdorferi persistence within ticks. Absence of a complete DTN formation in knockdown ticks leads to the activation of specific tick innate immune pathway genes that potentially resulted in the reduction of spirochete levels. Together, these results highlighted the evolution of the DTN in a diverse set of arthropod vectors, including ticks, and its role in protecting invading pathogens like B. burgdorferi. Further understanding of the molecular basis of tick innate immune responses, vector-pathogen interaction, and their contributions in microbial persistence may help the development of new targets for disrupting the pathogen life cycle.     

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.     

Ixodid tick salivary gland products target host wound healing growth factors

For successful blood-feeding, ticks must confront the host immune system comprising many cells and signaling molecules, mainly cytokines and growth factors. These factors bind to specific receptors on the cell membranes, thereby initiating a signaling cascade that leads to distinct cellular activities. Ticks are able to manipulate host immune responses via molecules secreted from their salivary glands. Saliva of ixodid ticks contains factors binding important cytokines and their subgroup, chemokines. Here we demonstrate that constituents of tick salivary gland extract (SGE) also appear to bind growth factors: transforming growth factor beta (TGF-β1), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF-2), and hepatocyte growth factor (HGF), depending on tick species. SGE derived from Amblyommavariegatum reacted with TGF-β1, PDGF, FGF-2 and HGF; Dermacentorreticulatus and Rhipicephalusappendiculatus with TGF-β1, FGF-2 and HGF; and Ixodes ricinus and Ixodesscapularis with PDGF. SGE from the species targeting PDGF (A. variegatum and I. ricinus) also inhibited cell proliferation in vitro and induced a change in morphology of different cell lines. These effects correlated with disruption of the actin cytoskeleton. Such effects were not observed with SGE of the two species that did not target PDGF. Targeting of wound healing growth factors appears to be yet another strategy ixodid ticks adopt for suppression of inflammation and successful haematophagy.     

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

Background

The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype.

Results

We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying.

Conclusion

We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.     

Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector,Ixodes scapularis

Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10–15% and 65–71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.     

Active surveillance to update county scale distribution of four tick species of medical and veterinary importance in Oklahoma

The incidence of tick‐borne disease continues to increase in humans and companion animals in the United States, yet distribution maps for several tick vectors in Oklahoma, including Dermacentor variabilis, Dermacentor albipictus, Ixodes scapularis, and Amblyomma maculatum, are not available or are outdated. To address this issue, county‐scale tick records from peer‐reviewed literature and passive collections were reviewed for Oklahoma. Additionally, dry ice traps, tick drags, and harvested deer were utilized to actively collect adult ticks throughout the state. Through these methods, D. variabilis, D. albipictus, I. scapularis, and A. maculatum were identified in 88% (68/77), 45.4% (35/77), 66.2% (51/77), and 64.9% (50/77) of the counties in Oklahoma, respectively. Baseline maps were developed for the distribution of D. variabilis and D. albipictus and distribution maps were updated for I. scapularis and A. maculatum. This data confirms that these four species of ticks continue to be widespread within Oklahoma with a western expansion of the range of I. scapularis within the state. These results assist efforts to better understand the epidemiology of the different diseases caused by pathogens transmitted by these tick species within the Great Plains region.     

Role of Migratory Birds in Introduction and Range Expansion of Ixodes scapularis Ticks and of Borrelia burgdorferi and Anaplasma phagocytophilum in Canada

During the spring in 2005 and 2006, 39,095 northward-migrating land birds were captured at 12 bird observatories in eastern Canada to investigate the role of migratory birds in northward range expansion of Lyme borreliosis, human granulocytic anaplasmosis, and their tick vector, Ixodes scapularis. The prevalence of birds carrying I. scapularis ticks (mostly nymphs) was 0.35% (95% confidence interval [CI] = 0.30 to 0.42), but a nested study by experienced observers suggested a more realistic infestation prevalence of 2.2% (95% CI = 1.18 to 3.73). The mean infestation intensity was 1.66 per bird. Overall, 15.4% of I. scapularis nymphs (95% CI = 10.7 to 20.9) were PCR positive for Borrelia burgdorferi, but only 8% (95% CI = 3.8 to 15.1) were positive when excluding nymphs collected at Long Point, Ontario, where B. burgdorferi is endemic. A wide range of ospC and rrs-rrl intergenic spacer alleles of B. burgdorferi were identified in infected ticks, including those associated with disseminated Lyme disease and alleles that are rare in the northeastern United States. Overall, 0.4% (95% CI = 0.03 to 0.41) of I. scapularis nymphs were PCR positive for Anaplasma phagocytophilum. We estimate that migratory birds disperse 50 million to 175 million I. scapularis ticks across Canada each spring, implicating migratory birds as possibly significant in I. scapularis range expansion in Canada. However, infrequent larvae and the low infection prevalence in ticks carried by the birds raise questions as to how B. burgdorferi and A. phagocytophilum become endemic in any tick populations established by bird-transported ticks.     

A blood meal-induced Ixodes scapularis tick saliva serpin inhibits trypsin and thrombin,and interferes with platelet aggregation and blood clotting

Ixodes scapularis is a medically important tick species that transmits causative agents of important human tick-borne diseases including borreliosis, anaplasmosis and babesiosis. An understanding of how this tick feeds is needed prior to the development of novel methods to protect the human population against tick-borne disease infections. This study characterizes a blood meal-induced I. scapularis (Ixsc) tick saliva serine protease inhibitor (serpin (S)), in-house referred to as IxscS-1E1. The hypothesis that ticks use serpins to evade the host’s defense response to tick feeding is based on the assumption that tick serpins inhibit functions of protease mediators of the host’s anti-tick defense response. Thus, it is significant that consistent with hallmark characteristics of inhibitory serpins, Pichia pastoris-expressed recombinant IxscS-1E1 (rIxscS-1E1) can trap thrombin and trypsin in SDS- and heat-stable complexes, and reduce the activity of the two proteases in a dose-responsive manner. Additionally, rIxscS-1E1 also inhibited, but did not apparently form detectable complexes with, cathepsin G and factor Xa. Our data also show that rIxscS-1E1 may not inhibit chymotrypsin, kallikrein, chymase, plasmin, elastase and papain even at a much higher rIxscS-1E1 concentration. Native IxscS-1E1 potentially plays a role(s) in facilitating I. scapularis tick evasion of the host’s hemostatic defense as revealed by the ability of rIxscS-1E1 to inhibit adenosine diphosphate- and thrombin-activated platelet aggregation, and delay activated partial prothrombin time and thrombin time plasma clotting in a dose-responsive manner. We conclude that native IxscS-1E1 is part of the tick saliva protein complex that mediates its anti-hemostatic, and potentially inflammatory, functions by inhibiting the actions of thrombin, trypsin and other yet unknown trypsin-like proteases at the tick–host interface.     

Intracellular Symbionts and Other Bacteria Associated with Deer Ticks (Ixodes scapularis) from Nantucket and Wellfleet, Cape Cod, Massachusetts

The diversity of bacteria associated with the deer tick (Ixodes scapularis) was assessed using PCR amplification, cloning, and sequencing of 16S rRNA genes originating from seven ticks collected from Nantucket Island and Wellfleet, Cape Cod, Mass. The majority of sequences obtained originated from gram-negative proteobacteria. Four intracellular bacteria were detected including strains of Ehrlichia, Rickettsia, and Wolbachia and an organism related to intracellular insect symbionts from the Cytophaga-Flavobacterium-Bacteroides group. Several strains of members of the Sphingomonadaceae were also detected in all but one tick. The results provide a view of the diversity of bacteria associated with I. scapularis ticks in the field.     

An update on the ecological distribution of Ixodid ticks infesting cattle in Rwanda: countrywide cross-sectional survey in the wet and the dry season

As part of the epidemiological studies aimed at developing an East Coast fever (ECF) immunisation control strategy, which combines an infection and treatment method with strategic tick control, a countrywide tick survey was carried out in both the dry and the wet season to determine the abundance and the dynamics of the tick populations infesting cattle in Rwanda. Six Ixodid tick species where identified from a total of 12,814 tick specimens collected. Rhipicephalus appendiculatus, the main vector of ECF was the most abundant (91.8%) followed by Boophilus decoloratus (6.1%) and Ambyomma variegatum (1.2%). Few ticks from the three other less economically important Ixodid species (Rhipicephalus compositus, R. evertsi evertsi and Ixodes cavipalpus) were recovered. Both adult and immature stages of the most dominant tick species were found to be widespread with a year round presence. The numbers of ticks were high in low land and medium zones and declined markedly in the higher regions of Rwanda. The geographical distribution of various tick species throughout the country and their epidemiological implications are discussed.