Evaluation of Doxycycline-Laden Oral Bait and Topical Fipronil Delivered in a Single Bait Box to Control Ixodes scapularis (Acari: Ixodidae) and Reduce Borrelia burgdorferi and Anaplasma phagocytophilum Infection in Small Mammal Reservoirs and Host-Seeking Ticks.

A field trial was conducted on residential properties in a Lyme disease endemic area of New Jersey to determine the efficacy of Maxforce Tick Management System (TMS) bait boxes modified with doxycycline hyclate-laden bait to reduce the acarological risk of Lyme disease and the utility of galvanized steel shrouds to protect the bait boxes from squirrel depredation and ability to routinely service these devices. The strategy began with a 9-wk deployment against larvae followed by a 17-wk deployment against nymphs and larvae the second year. Passive application of fipronil reduced nymphal and larval tick burdens on small mammals by 76 and 77%, respectively, and nymphal tick abundance by 81% on treated properties. In addition, the percentage of infected small mammals recovered from intervention areas following treatment was reduced by 96% for Borrelia burgdorferi and 93% for Anaplasma phagocytophilum. Infection prevalence in host-seeking nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 93 and 61%, respectively. Results indicate that Maxforce TMS bait boxes fitted with doxycycline-impregnated bait is an effective means of reducing ticks and infection prevalence for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing Ixodes scapularis Say ticks. The protective shroud allows the device to be routinely serviced and protect against squirrel depredation.

Coxiella symbionts are widespread into hard ticks.

Ticks are blood-feeding arthropods and can harbor several bacteria, including the worldwide zoonotic disease Q-fever agent Coxiella burnetii. Recent studies have reported a distinct group of Coxiella mostly associated with Ixodidae ticks, including the primary endosymbionts of Amblyomma americanum. In the present work, a screening for Coxiella infection was performed by 16S ribosomal DNA (rDNA) gene analyses in 293 tick samples of 15 different species sampled worldwide, including Brazil, Colombia, Kenya, and China. Different Coxiella phylotypes were identified, and these putative symbiotic bacteria were detected in ten different Amblyomma tick species. Approximately 61 % of Rhipicephalus sanguineus and ∼37 % of Rhipicephalus microplus DNA samples were positive for Coxiella. Sequence analysis and phylogenetic reconstruction grouped all the detected Coxiella with Coxiella-like symbionts from different Ixodidae ticks. This well-defined clade clearly excludes known phylotypes of C. burnetii pathogens and other Coxiella spp. detected in different environmental samples and other invertebrate hosts.

Borrelia bissettiae sp. nov. and Borrelia californiensis sp. nov. prevail in diverse enzootic transmission cycles.

Two species of the genus Borrelia, Borrelia bissettiae sp. nov. and Borrelia californiensis sp. nov., were first described by Postic and co-workers on the basis of genetic analyses of several loci. Multilocus sequence analysis of eight housekeeping loci confirmed that these two Borrelia genomospecies are distinct members of the Borrelia burgdorferi sensu lato complex. B. bissettiae sp. nov. was initially described in transmission cycles involving Neotoma fuscipes wood rats and Ixodes pacificus ticks in California, and Neotoma mexicana and Ixodes spinipalpis in Colorado. The preferred host of B. californiensis sp. nov. appears to be the California kangaroo rat, Dipodomys californicus; Ixodes jellisoni, I. spinipalipis and I. pacificus ticks are naturally infected with it. Thus, the ecological associations of the two genomospecies and their genetic distance from all other known Borrelia genomospecies species justify their description as separate genomospecies: B. bissettiae sp. nov. (type strain DN127T = DSM 17990T = CIP 109136T) and B. californiensis (type strain CA446T = DSM 17989T = ATCC BAA-2689T).

Nuclear Markers Reveal Predominantly North to South Gene Flow in Ixodes scapularis, the Tick Vector of the Lyme Disease Spirochete.

Ixodes scapularis, the tick vector of the Lyme disease spirochete, is distributed over most of the eastern United States, but >80% of all Lyme disease cases occur in the northeast. The role that genetic differences between northern and southern tick populations play in explaining this disparate distribution of Lyme disease cases is unclear. The present study was conducted with 1,155 SNP markers in eight nuclear genes; the 16S mitochondrial gene was examined for comparison with earlier studies. We examined 350 I. scapularis from 7 states covering a representative area of the species. A demographic analysis using Bayesian Extended Skyline Analysis suggested that I. scapularis populations in Mississippi and Georgia began expanding 500,000 years ago, those in Florida and North Carolina 200,000 years ago and those from Maryland and New Jersey only during the past 50,000 years with an accompanying bottleneck. Wisconsin populations only began expanding in the last 20,000 years. Analysis of current migration patterns suggests large amounts of gene flow in northern collections and equally high rates of gene flow among southern collections. In contrast there is restricted and unidirectional gene flow between northern and southern collections, mostly occurring from northern into southern populations. Northern populations are characterized by nymphs that quest above the leaf litter, are easy to collect by flagging, frequently feed on mammals such as rodents and shrews, commonly attach to people, and about 25% of which are infected with B. burgdorferi. If there is a genetic basis for these behaviors, then the patterns detected in this study are of concern because they suggest that northern I. scapularis populations with a greater ability to vector B. burgdorferi to humans are expanding south.

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.

Comparison of Tick Feeding Success and Vector Competence for Borrelia burgdorferi Among Immature Ixodes scapularis (Ixodida: Ixodidae) of Both Southern and Northern Clades.

Northern and southern Ixodes scapularis Say populations differ greatly in density, host utilization, and especially questing behavior of the immatures. Haplotypes of I. scapularis in North America can be divided into two major clades-the All American Clade (haplotypes A through J) and the Southern Clade (M through O). This genetic variation may affect feeding success and vector competence. This study compared feeding success of larval I. scapularis measured by time-to-drop-off and subsequent transmissibility success of Borrelia burgdorferi to mice using ticks from Mississippi, Connecticut (both F haplotype), and Louisiana (haplotype O). Northern ticks (CT) fed to repletion much faster than MS and LA ticks: overall, 73.6% of CT ticks had dropped off mice at Day 3 compared to only 1.7% and 6.6% of ticks dropped off for MS and LA ticks at that same time point. As for vector competence, 4 of the 4 mice in each case (MS or CT) that had been fed on by infected nymphs tested positive for B. burgdorferi. In a second experiment, 5 of the 6 mice tested positive for B. burgdorferi after exposure to infected LA ticks as compared with 3 of the 4 mice exposed to infected CT ticks. These data demonstrate that there is no difference in northern and southern populations of I. scapularis in their ability to transmit B. burgdorferi, but the ability of the northern populations to feed rapidly on rodents exceeds that of southern populations.

Transgene expression in tick cells using Agrobacterium tumefaciens.

Ticks transmit infectious agents to humans and other animals. Genetic manipulation of vectors like ticks could enhance the development of alternative disease control strategies. Transgene expression using the phytopathogen Agrobacterium tumefaciens has been shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for A. tumefaciens to mediate transgene expression in HeLa cells as well as Rhipicephalus microplus tick cells. Translational fusions eGfp:eGfp or Salp15:eGfp, including the enhanced-green fluorescent protein and the Ixodes scapularis salivary factor SALP15 genes, were constructed using the CaMV 35S (cauliflower mosaic virus) promoter, "PBm" tick promoter (R. microplus pyrethroid metabolizing esterase gene) or the Simian Virus SV40 promoter. Confocal microscopy, RT-PCR and Western-blot assays demonstrated transgene(s) expression in both cell lines. Transgene expression was also achieved in vivo, in both R. microplus and I. scapularis larvae utilizing a soaking method including the A. tumefaciens donor cells and confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNA(s). This strategy opens up a new avenue to express exogenous genes in ticks and represents a potential breakthrough for the study of tick-host pathophysiology.

Detection of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti, with two different multiplex PCR assays.

We have developed 2 real-time multiplex PCR assays for detection of Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. The efficiency and sensitivity of each multiplex PCR assay was evaluated using field-collected Ixodes scapularis ticks that were positive for each of the pathogens, cloned plasmids harboring each of the PCR targets, and laboratory I. scapularis infected with B. burgdorferi B31. There was no difference in efficiency or sensitivity when comparing the multiplex PCR with the individual PCR reactions. If the 2 multiplex PCR assays are used in the same analysis, field-collected ticks that only harbor B. miyamotoi can also be identified. The multiplex assays are fast and cost-effective methods for screening and detecting pathogens in ticks, when compared to single-target PCR.

Efficacy of an experimental azithromycin cream for prophylaxis of tick-transmitted lyme disease spirochete infection in a murine model.

As an alternative to oral prophylaxis for the prevention of tick transmission of Borrelia burgdorferi, we tested antibiotic cream prophylactic formulations in a murine model of spirochete infection. A 4% preparation of doxycycline cream afforded no protection, but a single application of 4% azithromycin cream was 100% protective when applied directly to the tick bite site at the time of tick removal. Indeed, the azithromycin cream was 100% effective when applied at up to 3 days after tick removal and protected 74% of mice exposed to tick bite when applied at up to 2 weeks after tick removal. Azithromycin cream was also protective when applied at a site distal to the tick bite site, suggesting that it was having a systemic effect in addition to a local transdermal effect. Mice that were protected from tick-transmitted infection did not seroconvert and did not infect larval ticks on xenodiagnosis. Azithromycin cream formulations appear to hold promise for Lyme disease prophylaxis.

Borrelia kurtenbachii sp. nov., a widely distributed member of the Borrelia burgdorferi sensu lato species complex in North America.

Lyme borreliosis group spirochaetes are parasitic bacteria transmitted by vector ticks of the genus Ixodes and distributed mainly between 40° and 60° northern latitudes. Since Borrelia burgdorferi sensu stricto (hereinafter, B. burgdorferi) was described in the north-eastern USA during the early 1980s, an increasing diversity has been noted within the species complex. Here, we describe a novel genomic species, Borrelia kurtenbachii sp. nov. (type strain 25015(T) = ATCC BAA-2495(T) =  DSM 26572(T)), that is prevalent in transmission cycles among vector ticks and reservoir hosts in North America. Confirmation of the presence of this species in Europe awaits further investigation.

High SNP density in the blacklegged tick, Ixodes scapularis, the principal vector of Lyme disease spirochetes.

Single-nucleotide polymorphisms (SNPs) are the most widespread type of sequence variation in genomes. SNP density and distribution varies among different organisms and genes. Here, we report the first estimates of SNP distribution and density in the genome of the blacklegged tick (Ixodes scapularis), an important vector of the pathogens causing Lyme disease, human granulocytic anaplasmosis and human babesiosis in North America. We sampled 10 individuals from each of 4 collections from New Jersey, Virginia, Georgia, and Mississippi and analyzed the sequences of 9 nuclear genes and the mitochondrial 16S gene. SNPs are extremely abundant (one SNP per every 14 bases). This is the second highest density so far reported in any eukaryotic organism. Population genetic analyses based either on haplotype frequencies or the 372 SNPs in these 9 genes showed that the 40 ticks formed 3 genetic groups. In agreement with earlier population genetic studies, northern ticks from New Jersey and Virginia formed a homogeneous group with low genetic diversity, whereas southern ticks from Georgia and Mississippi consisted of 2 separate groups, each with high genetic diversity.

Survival of host blood proteins in Ixodes scapularis (Acari: Ixodidae) ticks: a time course study.

Ixodes scapularis Say, 1821 larvae were fed on mice and allowed to molt under laboratory conditions. A liquid chromatography-tandem mass spectrometry-based proteomic study was conducted to identify the type of mammalian proteins present in the derived nymphal ticks at different time intervals after molting. Albumin was present for 85 d; transferrin was present for 29 d; and, more importantly, hemoglobin remained detectable for up to 309 d postmolting. Peptides of actin, keratin, and tubulin are highly similar between mouse and tick, and therefore, unambiguous assignment of these proteins to different species was not possible. Establishing a time line for the persistence of hemoglobin, one of the most abundant blood proteins, at detectable levels in ticks after the bloodmeal and molting advances our efforts to use this protein to identify the host species.

A prevalent alpha-proteobacterium Paracoccus sp. in a population of the Cayenne ticks (Amblyomma cajennense) from Rio de Janeiro, Brazil.

As Rocky Mountain Spotted Fever is the most common tick-borne disease in South America, the presence of Rickettsia sp. in Amblyomma ticks is a possible indication of its endemicity in certain geographic regions. In the present work, bacterial DNA sequences related to Rickettsia amblyommii genes in A. dubitatum ticks, collected in the Brazilian state of Mato Grosso, were discovered. Simultaneously, Paracoccus sp. was detected in aproximately 77% of A. cajennense specimens collected in Rio de Janeiro, Brazil. This is the first report of Paracoccus sp. infection in a specific tick population, and raises the possibility of these bacteria being maintained and/or transmitted by ticks. Whether Paracoccus sp. represents another group of pathogenic Rhodobacteraceae or simply plays a role in A. cajennense physiology, is unknown. The data also demonstrate that the rickettsial 16S rRNA specific primers used forRickettsia spp. screening can also detect Paracoccus alpha-proteobacteria infection in biological samples. Hence, a PCR-RFLP strategy is presented to distinguish between these two groups of bacteria.

Geographic variation in the relationship between human Lyme disease incidence and density of infected host-seeking Ixodes scapularis nymphs in the Eastern United States.

Prevention and control of Lyme disease is difficult because of the complex biology of the pathogen's (Borrelia burgdorferi) vector (Ixodes scapularis) and multiple reservoir hosts with varying degrees of competence. Cost-effective implementation of tick- and host-targeted control methods requires an understanding of the relationship between pathogen prevalence in nymphs, nymph abundance, and incidence of human cases of Lyme disease. We quantified the relationship between estimated acarological risk and human incidence using county-level human case data and nymphal prevalence data from field-derived estimates in 36 eastern states. The estimated density of infected nymphs (mDIN) was significantly correlated with human incidence (r = 0.69). The relationship was strongest in high-prevalence areas, but it varied by region and state, partly because of the distribution of B. burgdorferi genotypes. More information is needed in several high-prevalence states before DIN can be used for cost-effectiveness analyses.

Protective value of prophylactic antibiotic treatment of tick bite for Lyme disease prevention: an animal model.

Clinical studies have demonstrated that prophylactic antibiotic treatment of tick bites by Ixodes scapularis in Lyme disease hyperendemic regions in the northeastern United States can be effective in preventing infection with Borrelia burgdorferi sensu stricto, the Lyme disease spirochete. A large clinical trial in Westchester County, NY (USA), demonstrated that treatment of tick bite with 200mg of oral doxycycline was 87% effective in preventing Lyme disease in tick-bite victims (Nadelman, R.B., Nowakowski, J., Fish, D., Falco, R.C., Freeman, K., McKenna, D., Welch, P., Marcus, R., Agúero-Rosenfeld, M.E., Dennis, D.T., Wormser, G.P., 2001. Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. N. Engl. J. Med. 345, 79-84.). Although this excellent clinical trial provided much needed information, the authors enrolled subjects if the tick bite occurred within 3 days of their clinical visit, but did not analyze the data based on the exact time between tick removal and delivery of prophylaxis. An animal model allows for controlled experiments designed to determine the point in time after tick bite when delivery of oral antibiotics would be too late to prevent infection with B. burgdorferi. Accordingly, we developed a tick-bite prophylaxis model in mice that gave a level of prophylactic protection similar to what had been observed in clinical trials and then varied the time post tick bite of antibiotic delivery. We found that two treatments of doxycycline delivered by oral gavage to mice on the day of removal of a single potentially infectious nymphal I. scapularis protected 74% of test mice compared to controls. When treatment was delayed until 24 h after tick removal, only 47% of mice were protected; prophylactic treatment was totally ineffective when delivered ≥2 days after tick removal. Although the dynamics of antibiotic treatment in mice may differ from humans, and translation of animal studies to patient management must be approached with caution, we believe our results emphasize the point that antibiotic prophylactic treatment of tick bite to prevent Lyme disease is more likely to be efficacious if delivered promptly after potentially infectious ticks are removed from patients. There is only a very narrow window for prophylactic treatment to be effective post tick removal.

Human risk of infection with Borrelia burgdorferi, the Lyme disease agent, in eastern United States.

The geographic pattern of human risk for infection with Borrelia burgdorferi sensu stricto, the tick-borne pathogen that causes Lyme disease, was mapped for the eastern United States. The map is based on standardized field sampling in 304 sites of the density of Ixodes scapularis host-seeking nymphs infected with B. burgdorferi, which is closely associated with human infection risk. Risk factors for the presence and density of infected nymphs were used to model a continuous 8 km×8 km resolution predictive surface of human risk, including confidence intervals for each pixel. Discontinuous Lyme disease risk foci were identified in the Northeast and upper Midwest, with a transitional zone including sites with uninfected I. scapularis populations. Given frequent under- and over-diagnoses of Lyme disease, this map could act as a tool to guide surveillance, control, and prevention efforts and act as a baseline for studies tracking the spread of infection.

Saliva, salivary gland, and hemolymph collection from Ixodes scapularis ticks.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) (1-8). To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick (3,9-13). For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle (14,15). Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva (9,16-19). As a tick feeds the host typically responds with a strong hemostatic and innate immune response (11,13,20-22). Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding (3,11,20,21,23). The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens (3,20,24-27). To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

What do we need to know about disease ecology to prevent Lyme disease in the northeastern United States?

Lyme disease is the most commonly reported vector-borne disease in the United States, with the majority of cases occurring in the Northeast. It has now been three decades since the etiological agent of the disease in North America, the spirochete Borrelia burgdorferi, and its primary North American vectors, the ticks Ixodes scapularis Say and I. pacificus Cooley & Kohls, were identified. Great strides have been made in our understanding of the ecology of the vectors and disease agent, and this knowledge has been used to design a wide range of prevention and control strategies. However, despite these advances, the number of Lyme disease cases have steadily increased. In this article, we assess potential reasons for the continued lack of success in prevention and control of Lyme disease in the northeastern United States, and identify conceptual areas where additional knowledge could be used to improve Lyme disease prevention and control strategies. Some of these areas include: 1) identifying critical host infestation rates required to maintain enzootic transmission of B. burgdorferi, 2) understanding how habitat diversity and forest fragmentation impacts acarological risk of exposure to B. burgdorferi and the ability of interventions to reduce risk, 3) quantifying the epidemiological outcomes of interventions focusing on ticks or vertebrate reservoirs, and 4) refining knowledge of how human behavior influences Lyme disease risk and identifying barriers to the adoption of personal protective measures and environmental tick management.