Scapularis Ticks Research Articles

Genomic insights into the Ixodes scapularis tick vector of Lyme disease.

Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing ∼57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick–host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host ‘questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.

The putative role of Rhipicephalus microplus salivary serpins in the tick-host relationship

Inflammation and hemostasis are part of the host's first line of defense to tick feeding. These systems are in part serine protease mediated and are tightly controlled by their endogenous inhibitors, in the serpin superfamily (serine protease inhibitors). From this perspective ticks are thought to use serpins to evade host defenses during feeding. The cattle tick Rhipicephalus microplus encodes at least 24 serpins, of which RmS-3, RmS-6, and RmS-17 were previously identified in saliva of this tick. In this study, we screened inhibitor functions of these three saliva serpins against a panel of 16 proteases across the mammalian defense pathway. Our data confirm that Pichia pastoris-expressed rRmS-3, rRmS-6, and rRmS-17 are likely inhibitors of pro-inflammatory and pro-coagulant proteases. We show that rRmS-3 inhibited chymotrypsin and cathepsin G with stoichiometry of inhibition (SI) indices of 1.8 and 2.0, and pancreatic elastase with SI higher than 10. Likewise, rRmS-6 inhibited trypsin with SI of 2.6, chymotrypsin, factor Xa, factor XIa, and plasmin with SI higher than 10, while rRmS-17 inhibited trypsin, cathepsin G, chymotrypsin, plasmin, and factor XIa with SI of 1.6, 2.6, 2.7, 3.4, and 9.0, respectively. Additionally, we observed the formation of irreversible complexes between rRmS-3 and chymotrypsin, rRmS-6/rRmS-17 and trypsin, and rRmS-3/rRmS-17 and cathepsin G, which is consistent with typical mechanism of inhibitory serpins. In blood clotting assays, rRmS-17 delayed plasma clotting by 60 s in recalcification time assay, while rRmS-3 and rRmS-6 did not have any effect. Consistent with inhibitor function profiling data, 2.0 μM rRmS-3 and rRmS-17 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner by up to 96% and 95% respectively. Of significant interest, polyclonal antibodies blocked inhibitory functions of the three serpins. Also notable, antibodies to Amblyomma americanum, Ixodes scapularis, and Rhipicephalus sanguineus tick saliva proteins cross-reacted with the three R. microplus saliva serpins, suggesting the potential of these proteins as candidates for universal anti-tick vaccines.

Growth of Ehrlichia canis, the causative agent of canine monocytic ehrlichiosis, in vector and non-vector ixodid tick cell lines

Canine monocytic ehrlichiosis is caused by Ehrlichia canis, a small gram-negative coccoid bacterium that infects circulating monocytes. The disease is transmitted by the brown dog tick Rhipicephalus sanguineus s.l. and is acknowledged as an important infectious disease of dogs and other members of the family Canidae worldwide. E. canis is routinely cultured in vitro in the canine monocyte-macrophage cell line DH82 and in non-vector Ixodes scapularis tick cell lines, but not in cells derived from its natural vector. Here we report infection and limited propagation of E. canis in the tick cell line RSE8 derived from the vector R. sanguineus s.l., and successful propagation through six passages in a cell line derived from the experimental vector Dermacentor variabilis. In addition, using bacteria semi-purified from I. scapularis cells we attempted to infect a panel of cell lines derived from non-vector species of the tick genera Amblyomma, Dermacentor, Hyalomma, Ixodes and Rhipicephalus with E. canis and, for comparison, the closely-related Ehrlichia ruminantium, causative agent of heartwater in ruminants. Amblyomma and non-vector Dermacentor spp. cell lines appeared refractory to infection with E. canis but supported growth of E. ruminantium, while some, but not all, cell lines derived from Hyalomma, Ixodes and Rhipicephalus spp. ticks supported growth of both pathogens. We also illustrated and compared the ultrastructural morphology of E. canis in DH82, RSE8 and I. scapularis IDE8 cells. This study confirms that E. canis, like E. ruminantium, is able to grow not only in cell lines derived from natural and experimental tick vectors but also in a wide range of other cell lines derived from tick species not known to transmit this pathogen.

Established Population of Blacklegged Ticks with High Infection Prevalence for the Lyme Disease Bacterium, Borrelia burgdorferi Sensu Lato, on Corkscrew Island, Kenora District, Ontario.

We document an established population of blacklegged ticks, Ixodes scapularis, on Corkscrew Island, Kenora District, Ontario, Canada. Primers of the outer surface protein A (OspA) gene, the flagellin (fla) gene, and the flagellin B (flaB) gene were used in the PCR assays to detect Borrelia burgdorferi sensu lato (s.l.), the Lyme disease bacterium. In all, 60 (73%) of 82 adult I. scapularis, were infected with B. burgdorferi s.l. As well, 6 (43%) of 14 unfed I. scapularis nymphs were positive for B. burgdorferi s.l. An I. scapularis larva was also collected from a deer mouse, and several unfed larvae were gathered by flagging leaf litter. Based on DNA sequencing of randomly selected Borrelia amplicons from six nymphal and adult I. scapularis ticks, primers for the flagellin (fla) and flagellin B (flaB) genes reveal the presence of B. burgdorferi sensu stricto (s.s.), a genospecies pathogenic to humans and certain domestic animals. We collected all 3 host-feeding life stages of I. scapularis in a single year, and report the northernmost established population of I. scapularis in Ontario. Corkscrew Island is hyperendemic for Lyme disease and has the highest prevalence of B. burgdorferi s.l. for any established population in Canada. Because of this very high infection prevalence, this population of I. scapularis has likely been established for decades. Of epidemiological significance, cottage owners, island visitors, outdoors enthusiasts, and medical professionals must be vigilant that B. burgdorferi s.l.-infected I. scapularis on Corkscrew Island pose a serious public health risk.

Unexplained Recurrent Fevers and the Importance of Inquiring About Occupation: A Case Report

Many different tick-borne diseases are identified in various geographical areas throughout the United States. Ixodes scapularis tick-borne diseases can present unique diagnostic challenges because of their constellation of vague symptoms. The Ixodes scapularis tick, more commonly known as the blacklegged tick, is responsible for three specific tick-borne infections: Lyme disease (Borellia burgdorferii), Babesiosis (Babesia microti), and Anaplasmosis (Anaplasma phagocytophilum) also known as human granulocytic anaplasmosis. Although these three tick-borne diseases are characteristically found in the New England area, the geographical distribution of the Ixodes tick has expanded to encompass much of the northeastern United States and even states in the upper mid-west.1 Although Lyme disease is the most prevalent of the tick borne illnesses, the incidence of Babesiosis has increased exponentially in the last 50 years and cases have recently been reported in 22 states.2 Here we present a case of Babesiosis which was diagnosed only after an occupational history detailing tick exposure was obtained.

Ixodes scapularis and Ixodes ricinus tick cell lines respond to infection with tick-borne encephalitis virus: transcriptomic and proteomic analysis.

BackgroundIxodid ticks are important vectors of a wide variety of viral, bacterial and protozoan pathogens of medical and veterinary importance. Although several studies have elucidated tick responses to bacteria, little is known about the tick response to viruses. To gain insight into the response of tick cells to flavivirus infection, the transcriptomes and proteomes of two Ixodes spp cell lines infected with the flavivirus tick-borne encephalitis virus (TBEV) were analysed.MethodsRNA and proteins were isolated from the Ixodes scapularis-derived cell line IDE8 and the Ixodes ricinus-derived cell line IRE/CTVM19, mock-infected or infected with TBEV, on day 2 post-infection (p.i.) when virus production was increasing, and on day 6 p.i. when virus production was decreasing. RNA-Seq and mass spectrometric technologies were used to identify changes in abundance of, respectively, transcripts and proteins. Functional analyses were conducted on selected transcripts using RNA interference (RNAi) for gene knockdown in tick cells infected with the closely-related but less pathogenic flavivirus Langat virus (LGTV).ResultsDifferential expression analysis using DESeq resulted in totals of 43 and 83 statistically significantly differentially-expressed transcripts in IDE8 and IRE/CTVM19 cells, respectively. Mass spectrometry detected 76 and 129 statistically significantly differentially-represented proteins in IDE8 and IRE/CTVM19 cells, respectively. Differentially-expressed transcripts and differentially-represented proteins included some that may be involved in innate immune and cell stress responses. Knockdown of the heat-shock proteins HSP90, HSP70 and gp96, the complement-associated protein Factor H and the protease trypsin resulted in increased LGTV replication and production in at least one tick cell line, indicating a possible antiviral role for these proteins. Knockdown of RNAi-associated proteins Argonaute and Dicer, which were included as positive controls, also resulted in increased LGTV replication and production in both cell lines, confirming their role in the antiviral RNAi pathway.ConclusionsThis systems biology approach identified several molecules that may be involved in the tick cell innate immune response against flaviviruses and highlighted that ticks, in common with other invertebrate species, have other antiviral responses in addition to RNAi.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-1210-x) contains supplementary material, which is available to authorized users.

Epidemiology of Lyme Disease, Nova Scotia, Canada, 2002-2013.

Ixodes scapularis ticks, which transmit Borrelia burgdorferi, the causative agent of Lyme disease (LD), are endemic to at least 6 regions of Nova Scotia, Canada. To assess the epidemiology and prevalence of LD in Nova Scotia, we analyzed data from 329 persons with LD reported in Nova Scotia during 2002-2013. Most patients reported symptoms of early localized infection with rash (89.7%), influenza-like illness (69.6%), or both; clinician-diagnosed erythema migrans was documented for 53.2%. In a separate serosurvey, of 1,855 serum samples screened for antibodies to B. burgdorferi, 2 were borderline positive (both with an indeterminate IgG on Western blot), resulting in an estimated seroprevalence of 0.14% (95% CI 0.02%-0.51%). Although LD incidence in Nova Scotia has risen sharply since 2002 and is the highest in Canada (16/100,000 population in 2013), the estimated number of residents with evidence of infection is low, and risk is localized to currently identified LD-endemic regions.

Efficacy of a proprietary formulation of fipronil/(S)-methoprene/cyphenothrin against Ixodes scapularis tick infestations on dogs.

BackgroundEfficacy of FRONTLINE® TRITAK® For Dogs (fipronil/(S)-methoprene/cyphenothrin, Merial, Inc., Duluth, GA) against Ixodes scapularis was evaluated in two separate, but concurrent laboratory studies.MethodsOne day after topical treatment with placebo or active, dogs (n = 24) were infested with 50 unfed adult Ixodes scapularis ticks, with repeat infestations on Days 7, 14, 21 and 28. The number of live ticks was counted at 6 hours post-infestation in the first study (n = 12) and at 24 hours post-infestation in the second study (n = 12).ResultsObserved efficacies in study 1 were 93-99 % at 6 hour assessments on Day 1 through Day 28 and in the second study, 98-100 % at 24 hour assessments, occurring on Day 2 through Day 29.ConclusionsA single dose of FRONTLINE® TRITAK® For Dogs (fipronil/(S)-methoprene/cyphenothrin) (0.67 ml or 1.34 ml) prevented the establishment of a new infestation following treatment, as well as the repeated weekly re-infestations with Ixodes scapularis ticks, for 4 weeks.

Different Ecological Niches for Ticks of Public Health Significance in Canada.

Tick-borne diseases are a growing public health concern as their incidence and range have increased in recent decades. Lyme disease is an emerging infectious disease in Canada due to northward expansion of the geographic range of Ixodes scapularis, the principal tick vector for the Lyme disease agent Borrelia burgdorferi, into central and eastern Canada. In this study the geographical distributions of Ixodid ticks, including I. scapularis, and environmental factors associated with their occurrence were investigated in New Brunswick, Canada, where few I. scapularis populations have been found to date. Density of host-seeking ticks was evaluated by drag sampling of woodland habitats in a total of 159 sites. Ixodes scapularis ticks (n = 5) were found on four sites, Ixodes muris (n = 1) on one site and Haemaphysalis leporispalustris (n = 243) on 41 sites. One of four adult I. scapularis ticks collected was PCR-positive for B. burgdorferi. No environmental variables were significantly associated with the presence of I. scapularis although comparisons with surveillance data in neighbouring provinces (Québec and Nova Scotia) suggested that temperature conditions may be too cold for I. scapularis (< 2800 annual degree days above 0°C [DD > 0°C]) across much of New Brunswick. In contrast, the presence of H. leporispalustris, which is a competent vector of tularaemia, was significantly (P < 0.05) associated with specific ranges of mean DD > 0°C, mean annual precipitation, percentage of clay in site soil, elevation and season in a multivariable logistic regression model. With the exception of some localized areas, temperature conditions and deer density may be too low for the establishment of I. scapularis and Lyme disease risk areas in New Brunswick, while environmental conditions were suitable for H. leporispalustris at many sites. These findings indicate differing ecological niches for two tick species of public health significance.

Atraumatic splenic rupture from Babesia: A disease of the otherwise healthy patient

Babesiosis, an infection caused by the protozoan Babesia microti and transmitted by the Ixodes scapularis tick, is commonly described in the literature with an approximate incidence of 1000 cases per year (Herwaldt et al., 2012). Infections in North America occur most frequently during the spring and summer months in the Northeastern and Midwestern United States. Babesia can cause a wide spectrum of clinical manifestations ranging from a self-limited febrile illness or mild anemia to severe illness causing acute respiratory distress syndrome (ARDS), disseminated intravascular coagulopathy (DIC) and multisystem organ failure. Severe illness most commonly occurs in elderly, immunocompromised, or asplenic patients (Vannier and Krause, 2012). Splenic rupture has been generally described as a complication of severe illness secondary to babesiosis. We describe a case of spontaneous splenic rupture in an otherwise healthy woman that required emergent splenectomy. Recent case reports suggest that splenic rupture occurs in people without known risk factors for severe babesiosis. Physicians should be aware of this acute presentation in otherwise healthy individuals.

Association between body size and reservoir competence of mammals bearing Borrelia burgdorferi at an endemic site in the northeastern United States.

BackgroundThe reservoirs for the Lyme disease agent, Borrelia burgdorferi, are dominated by several different small to medium sized mammals in eastern North America.FindingsTo experimentally assess the competence of different mammalian species to transmit this pathogen to ticks, we carried out quantitative species-specific PCR of individual nymphal Ixodes scapularis ticks, which had been collected as replete larvae from animals captured at a field site in eastern Connecticut and then allowed to molt in the laboratory. The mammals, in order of increasing body mass, were the white-footed mouse, pine vole, eastern chipmunk, gray squirrel, Virginia opossum, striped skunk, and common raccoon. The prevalence of infection in the nymphs and the counts of spirochetes in infected ticks allometrically scaled with body mass with exponents of −0.28 and −0.29, respectively. By species, the captured animals from the site differed significantly in the mean counts of spirochetes in the ticks recovered from them, but these associations could not be distinguished from an effect of body size per se.ConclusionsThese empirical findings as well as inferences from modeling suggest that small mammals on the basis of their sizes are more competent as reservoirs of B. burgdorferi in this environment than medium-to large-sized mammals.

The Anemic and Thrombocytopenic Febrile Neonate

BackgroundBabesiosis is a tick-borne, protozoal disease seen primarily in the northeastern and northern Midwest portions of the United States. It is primarily transmitted through the bite of the Ixodes scapularis tick and may cause hemolysis. Case ReportIn the following case report, we present a 4-week-old male neonate with a temperature of 38.8°C (102.0°F) who presented to our pediatric emergency department with anemia, thrombocytopenia, and splenomegaly. Blood smear analysis demonstrated U-shaped parasitic rings within red blood cells consistent with babesiosis, confirmed by a positive Babesia microti antibody screen. Because the patient was never exposed to a vector for babesiosis, and never lived in an endemic area, the most likely exposure was in-utero transmission of the disease through transplacental migration of the parasite prior to birth. The patient was treated with atovaquone and azithromycin and discharged from the hospital after 6 days. The patient's mother also tested positive and was treated with the same medications. The evaluation of a febrile neonate includes a broad differential diagnosis. Although babesiosis is still uncommon in many areas of the United States, a specific constellation of laboratory findings and symptoms should prompt its consideration, even in patients who have not been exposed to an endemic area or a typical vector. Vertical transmission of babesiosis has been previously reported, but the occurrence is quite rare. Why Should an Emergency Physician Be Aware of This?Practitioners should include babesiosis in their differential diagnosis where appropriate and be aware that it can be transmitted not only via tick bite, but also from mother to neonate during pregnancy.

Identification of 24 h Ixodes scapularis immunogenic tick saliva proteins

Ixodes scapularis is arguably the most medically important tick species in the United States. This tick transmits 5 of the 14 human tick-borne disease (TBD) agents in the USA: Borrelia burgdorferi, Anaplasma phagocytophilum, B. miyamotoi, Babesia microti, and Powassan virus disease. Except for the Powassan virus disease, I. scapularis-vectored TBD agents require more than 24h post attachment to be transmitted. This study describes identification of 24h immunogenic I. scapularis tick saliva proteins, which could provide opportunities to develop strategies to stop tick feeding before transmission of the majority of pathogens. A 24h fed female I. scapularis phage display cDNA expression library was biopanned using rabbit antibodies to 24h fed I. scapularis female tick saliva proteins, subjected to next generation sequencing, de novo assembly, and bioinformatic analyses. A total of 182 contigs were assembled, of which ∼19% (35/182) are novel and did not show identity to any known proteins in GenBank. The remaining ∼81% (147/182) of contigs were provisionally identified based on matches in GenBank including ∼18% (27/147) that matched protein sequences previously annotated as hypothetical and putative tick saliva proteins. Others include proteases and protease inhibitors (∼3%, 5/147), transporters and/or ligand binding proteins (∼6%, 9/147), immunogenic tick saliva housekeeping enzyme-like (17%, 25/147), ribosomal protein-like (∼31%, 46/147), and those classified as miscellaneous (∼24%, 35/147). Notable among the miscellaneous class include antimicrobial peptides (microplusin and ricinusin), myosin-like proteins that have been previously found in tick saliva, and heat shock tick saliva protein. Data in this study provides the foundation for in-depth analysis of I. scapularis feeding during the first 24h, before the majority of TBD agents can be transmitted.

Motor rotation is essential for the formation of the periplasmic flagellar ribbon, cellular morphology, and Borrelia burgdorferi persistence within Ixodes scapularis tick and murine hosts.

Borrelia burgdorferi must migrate within and between its arthropod and mammalian hosts in order to complete its natural enzootic cycle. During tick feeding, the spirochete transmits from the tick to the host dermis, eventually colonizing and persisting within multiple, distant tissues. This dissemination modality suggests that flagellar motor rotation and, by extension, motility are crucial for infection. We recently reported that a nonmotile flaB mutant that lacks periplasmic flagella is rod shaped and unable to infect mice by needle or tick bite. However, those studies could not differentiate whether motor rotation or merely the possession of the periplasmic flagella was crucial for cellular morphology and host persistence. Here, we constructed and characterized a motB mutant that is nonmotile but retains its periplasmic flagella. Even though ΔmotB bacteria assembled flagella, part of the mutant cell is rod shaped. Cryoelectron tomography revealed that the flagellar ribbons are distorted in the mutant cells, indicating that motor rotation is essential for spirochetal flat-wave morphology. The ΔmotB cells are unable to infect mice, survive in the vector, or migrate out of the tick. Coinfection studies determined that the presence of these nonmotile ΔmotB cells has no effect on the clearance of wild-type spirochetes during murine infection and vice versa. Together, our data demonstrate that while flagellar motor rotation is necessary for spirochetal morphology and motility, the periplasmic flagella display no additional properties related to immune clearance and persistence within relevant hosts.

Bacteria associated with Amblyomma cajennense tick eggs.

Ticks represent a large group of pathogen vectors that blood feed on a diversity of hosts. In the Americas, the Ixodidae ticks Amblyomma cajennense are responsible for severe impact on livestock and public health. In the present work, we present the isolation and molecular identification of a group of culturable bacteria associated with A. cajennense eggs from females sampled in distinct geographical sites in southeastern Brazil. Additional comparative analysis of the culturable bacteria from Anocentor nitens, Rhipicephalus sanguineus and Ixodes scapularis tick eggs were also performed. 16S rRNA gene sequence analyses identified 17 different bacterial types identified as Serratia marcescens, Stenotrophomonas maltophilia, Pseudomonas fluorescens, Enterobacter spp., Micrococcus luteus, Ochrobactrum anthropi, Bacillus cereus and Staphylococcus spp., distributed in 12 phylogroups. Staphylococcus spp., especially S. sciuri, was the most prevalent bacteria associated with A. cajennense eggs, occurring in 65% of the samples and also frequently observed infecting A. nitens eggs. S. maltophilia, S. marcescens and B. cereus occurred infecting eggs derived from specific sampling sites, but in all cases rising almost as pure cultures from infected A. cajennense eggs. The potential role of these bacterial associations is discussed and they possibly represent new targets for biological control strategies of ticks and tick borne diseases.

Defensins from the tick Ixodes scapularis are effective against phytopathogenic fungi and the human bacterial pathogen Listeria grayi.

BackgroundIxodes scapularis is the most common tick species in North America and a vector of important pathogens that cause diseases in humans and animals including Lyme disease, anaplasmosis and babesiosis. Tick defensins have been identified as a new source of antimicrobial agents with putative medical applications due to their wide-ranging antimicrobial activities. Two multigene families of defensins were previously reported in I. scapularis. The objective of the present study was to characterise the potential antimicrobial activity of two defensins from I. scapularis with emphasis on human pathogenic bacterial strains and important phytopathogenic fungi.MethodsScapularisin-3 and Scapularisin-6 mature peptides were chemically synthesised. In vitro antimicrobial assays were performed to test the activity of these two defensins against species of different bacterial genera including Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Listeria spp. as well as Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa along with two plant-pathogenic fungi from the genus Fusarium. In addition, the tissue-specific expression patterns of Scapularisin-3 and Scapularisin-6 in I. scapularis midgut, salivary glands and embryo-derived cell lines were determined using PCR. Finally, tertiary structures of the two defensins were predicted and structural analyses were conducted.ResultsScapularisin-6 efficiently killed L. grayi, and both Scapularisin-3 and Scapularisin-6 caused strong inhibition (IC50 value: ~1 μM) of the germination of plant-pathogenic fungi Fusarium culmorum and Fusarium graminearum. Scapularisin-6 gene expression was observed in I. scapularis salivary glands and midgut. However, Scapularisin-3 gene expression was only detected in the salivary glands. Transcripts from the two defensins were not found in the I. scapularis tick cell lines ISE6 and ISE18.ConclusionOur results have two main implications. Firstly, the anti-Listeria and antifungal activities of Scapularisin-3 and Scapularisin-6 suggest that these peptides may be useful for (i) treatment of antibiotic-resistant L. grayi in humans and (ii) plant protection. Secondly, the antimicrobial properties of the two defensins described in this study may pave the way for further studies regarding pathogen invasion and innate immunity in I. scapularis.

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.

Partial pathogen protection by tick-bite sensitization and epitope recognition in peptide-immunized HLA DR3 transgenic mice

Ticks are notorious vectors of disease for humans, and many species of ticks transmit multiple pathogens, sometimes in the same tick bite. Accordingly, a broad-spectrum vaccine that targets vector ticks and pathogen transmission at the tick/host interface, rather than multiple vaccines against every possible tickborne pathogen, could become an important tool for resolving an emerging public health crisis. The concept for such a tick protective vaccine comes from observations of an acquired tick resistance (ATR) that can develop in non-natural hosts of ticks following sensitization to tick salivary components. Mice are commonly used as models to study immune responses to human pathogens but normal mice are natural hosts for many species of ticks and fail to develop ATR. We evaluated HLA DR3 transgenic (tg) “humanized” mice as a potential model of ATR and assessed the possibility of using this animal model for tick protective vaccine discovery studies. Serial tick infestations with pathogen-free Ixodes scapularis ticks were used to tick-bite sensitize HLA DR3 tg mice. Sensitization resulted in a cytokine skew favoring a Th2 bias as well as partial (57%) protection to infection with Lyme disease spirochetes (Borrelia burgdorferi) following infected tick challenge when compared to tick naïve counterparts. I. scapularis salivary gland homogenate (SGH) and a group of immunoinformatic-predicted T cell epitopes identified from the I. scapularis salivary transcriptome were used separately to vaccinate HLA DR3 tg mice, and these mice also were assessed for both pathogen protection and epitope recognition. Reduced pathogen transmission along with a Th2 skew resulted from SGH vaccination, while no significant protection and a possible T regulatory bias was seen in epitope-vaccinated mice. This study provides the first proof-of-concept for using HLA DR tg “humanized” mice for studying the potential tick protective effects of immunoinformatic- or otherwise-derived tick salivary components as tickborne disease vaccines.

Antivirulence Properties of an Antifreeze Protein

As microbial drug-resistance increases, there is a critical need for new classes of compounds to combat infectious diseases. The Ixodes scapularis tick antifreeze glycoprotein, IAFGP, functions as an antivirulence agent against diverse bacteria, including methicillin-resistant Staphylococcus aureus. Recombinant IAFGP and a peptide, P1, derived from this protein bind to microbes and alter biofilm formation. Transgenic iafgp-expressing flies and mice challenged with bacteria, as well as wild-type animals administered P1, were resistant to infection, septic shock, or biofilm development on implanted catheter tubing. These data show that an antifreeze protein facilitates host control of bacterial infections and suggest therapeutic strategies for countering pathogens.

Filarial Nematode Infection in Ixodes scapularis Ticks Collected from Southern Connecticut

It was recently demonstrated that the lone star tick Amblyomma americanum could harbor filarial nematodes within the genus Acanthocheilonema. In this study, Ixodes scapularis (deer) ticks collected from Southern Connecticut were evaluated for their potential to harbor filarial nematodes. Non-engorged nymphal and adult stage Ixodes scapularis ticks were collected in Southern Connecticut using the standard drag method. In situ hybridization with filarial nematode specific sequences demonstrated the presence of filarial nematodes in Ixodes ticks. Filarial nematode specific DNA sequences were amplified and confirmed by direct sequencing in Ixodes nymphal and adult ticks using either general filarial nematode or Onchocercidae family specific PCR primers. Phylogenetic analysis of the 12S rDNA gene sequence indicated that the filarial nematode infecting Ixodes scapularis ticks is most closely related to the species found in Amblyoma americanum ticks and belongs to the genus of Acanthocheilonema. Our data also demonstrated that infection rate of these filarial nematode in Ixodes ticks is relatively high (about 22% and 30% in nymphal and adult Ixodes ticks, respectively). In summary, the results from our studies demonstrated that filarial nematode infection was found in Ixodes ticks similar to what has been found in Amblyomma americanum ticks.