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Summer-weight clothing articles impregnated with permethrin are available as a personal protective measure against human-biting ticks in the United States. However, very few studies have addressed the impact of contact with summer-weight permethrin-treated textiles on tick vigor and behavior. Our aim was to generate new knowledge of how permethrin-treated textiles impact nymphal Ixodes scapularis ticks, the primary vectors in the eastern United States of the causative agents of Lyme disease, human anaplasmosis, and human babesiosis. We developed a series of bioassays designed to: (i) clarify whether permethrin-treated textiles impact ticks through non-contact spatial repellency or contact irritancy; (ii) evaluate the ability of ticks to remain in contact with vertically oriented permethrin-treated textiles, mimicking contact with treated clothing on arms or legs; and (iii) determine the impact of timed exposure to permethrin-treated textiles on the ability of ticks to move and orient toward a human finger stimulus, thus demonstrating normal behavior. Our results indicate that permethrin-treated textiles provide minimal non-contact spatial repellency but strong contact irritancy against ticks, manifesting as a "hot-foot" effect and resulting in ticks actively dislodging from contact with vertically oriented treated textile. Preliminary data suggest that the contact irritancy hot-foot response may be weaker for field-collected nymphs as compared with laboratory-reared nymphs placed upon permethrin-treated textile. We also demonstrate that contact with permethrin-treated textiles negatively impacts the vigor and behavior of nymphal ticks for >24h, with outcomes ranging from complete lack of movement to impaired movement and unwillingness of ticks displaying normal movement to ascend onto a human finger. The protective effect of summer-weight permethrin-treated clothing against tick bites merits further study., (Published by Elsevier GmbH.)

The recently recognized Lyme disease spirochete, Borrelia mayonii, has been detected in host-seeking Ixodes scapularis Say ticks and is associated with human disease in the Upper Midwest. Although experimentally shown to be vector competent, studies have been lacking to determine the duration of time from attachment of a single B. mayonii-infected I. scapularis nymph to transmission of spirochetes to a host. If B. mayonii spirochetes were found to be transmitted within the first 24 h after tick attachment, in contrast to Borrelia burgdorferi spirochetes (>24 h), then current recommendations for tick checks and prompt tick removal as a way to prevent transmission of Lyme disease spirochetes would need to be amended. We therefore conducted a study to determine the probability of transmission of B. mayonii spirochetes from single infected nymphal I. scapularis ticks to susceptible experimental mouse hosts at three time points postattachment (24, 48, and 72 h) and for a complete feed (>72-96 h). No evidence of infection with or exposure to B. mayonii occurred in mice that were fed upon by a single infected nymph for 24 or 48 h. The probability of transmission by a single infected nymphal tick was 31% after 72 h of attachment and 57% for a complete feed. In addition, due to unintended simultaneous feeding upon some mice by two B. mayonii-infected nymphs, we recorded a single occasion in which feeding for 48 h by two infected nymphs resulted in transmission and viable infection in the mouse. We conclude that the duration of attachment of a single infected nymphal I. scapularis tick required for transmission of B. mayonii appears to be similar to that for B. burgdorferi: transmission is minimal for the first 24 h of attachment, rare up to 48 h, but then increases distinctly by 72 h postattachment., (Published by Oxford University Press on behalf of Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US.)

Borrelia miyamotoi sensu lato relapsing fever group spirochetes are emerging as causative agents of human illness (Borrelia miyamotoi disease) in the United States. Host-seeking Ixodes scapularis ticks are naturally infected with these spirochetes in the eastern United States and experimentally capable of transmitting B. miyamotoi. However, the duration of time required from tick attachment to spirochete transmission has yet to be determined. We therefore conducted a study to assess spirochete transmission by single transovarially infected I. scapularis nymphs to outbred white mice at three time points post-attachment (24, 48, and 72h) and for a complete feed (>72-96h). Based on detection of B. miyamotoi DNA from the blood of mice fed on by an infected nymph, the probability of spirochete transmission increased from 10% by 24h of attachment (evidence of infection in 3/30 mice) to 31% by 48h (11/35 mice), 63% by 72h (22/35 mice), and 73% for a complete feed (22/30 mice). We conclude that (i) single I. scapularis nymphs effectively transmit B. miyamotoi relapsing fever group spirochetes while feeding, (ii) transmission can occur within the first 24h of nymphal attachment, and (iii) the probability of transmission increases with the duration of nymphal attachment., (Published by Elsevier GmbH.)

We describe a 2-yr trial to evaluate the ability of SELECT Tick Control System (TCS) host-targeted bait boxes to reduce numbers of host-seeking Ixodes scapularis nymphs in a residential neighborhood. After four successive 9-wk deployments, nymphal and larval I. scapularis infestation prevalence and intensity were significantly reduced on target small mammals. In addition, these deployments resulted in 87.9% and 97.3% control of host-seeking nymphs in treatment sites at 1 yr and 2 yr postintervention, respectively. Installation of a protective metal cover around the SELECT TCS bait boxes eliminated nontarget wildlife damage to bait boxes that resulted in failure of previous bait box types. The results are discussed in the context of the residential environment and future research needs., (© The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The causal agents of Lyme disease in North America, Borrelia burgdorferi and Borrelia mayonii, are transmitted primarily by Ixodes scapularis ticks. Due to their limited metabolic capacity, spirochetes rely on the tick blood meal for nutrients and metabolic intermediates while residing in the tick vector, competing with the tick for nutrients in the blood meal. Metabolomics is an effective methodology to explore dynamics of spirochete survival and multiplication in tick vectors before transmission to a vertebrate host via tick saliva. Using gas chromatography coupled to mass spectrometry, we identified statistically significant differences in the metabolic profile among uninfected I. scapularis nymphal ticks, B. burgdorferi-infected nymphal ticks and B. mayonii-infected nymphal ticks by measuring metabolism every 24 hours over the course of their up to 96 hour blood meals. Specifically, differences in the abundance of purines, amino acids, carbohydrates, and fatty acids during the blood meal among the three groups of nymphal ticks suggest that B. mayonii and B. burgdorferi may have different metabolic capabilities, especially during later stages of nymphal feeding. Understanding mechanisms underlying variable metabolic requirements of different Lyme disease spirochetes within tick vectors could potentially aid development of novel methods to control spirochete transmission.

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., (Published by Oxford University Press on behalf of Entomological Society of America 2016. This work is written by US Government employees and is in the public domain in the US.)

A novel species within the Borrelia burgdorferi sensu lato complex, Borrelia mayonii, was recently described and found to be associated with Lyme borreliosis in the Upper Midwest of the United States. The blacklegged tick, Ixodes scapularis, is naturally infected with B. mayonii in the Upper Midwest and has been experimentally demonstrated to serve as a vector for this spirochete. Natural vertebrate reservoirs for B. mayonii remain unknown. In this study, we demonstrate that an experimental spirochete host, the CD-1 strain outbred white mouse, can maintain active infection with B. mayonii for up to 1year: infected mice consistently yielded ear biopsies containing motile spirochetes from 29 to 375days after they were first infected via tick bite. Infection rates in resultant nymphal ticks varied greatly both over time for larvae fed on the same individual mouse at different time points after infection (2-42%) and for larvae fed on different mice at a given time point up to 8 months after infection (0-48%). Infection rates were lower in nymphs fed as larvae on mice 10-12 months after infection (2-3% for 5 mice and 9.8% for 1 mouse). In addition to ear biopsies, B. mayonii was detected from bladder, heart, and spinal cord of infected mice when they were sacrificed 163-375days after initial infection via tick bite. Examination of blood from mice determined to be infected with B. mayonii by ear biopsy did not produce evidence of B. mayonii DNA in blood taken 8-375days after the mice were first infected via tick bite., (Published by Elsevier GmbH.)

Borrelia mayonii, a recently recognized species within the Borrelia burgdorferi sensu lato complex, has been detected in host-seeking Ixodes scapularis Say ticks and found to be associated with Lyme disease in the Upper Midwest. This spirochete has, to date, not been documented from the Northeast, but we previously demonstrated that I. scapularis ticks originating from Connecticut are capable of serving as a vector of B. mayonii In this follow-up study, we compared the vector efficiency for B. mayonii (strain MN14-1420) of I. scapularis ticks originating from Minnesota in the Upper Midwest and Connecticut in the Northeast. CD-1 outbred white mice previously infected with B. mayonii via tick bite were exposed to simultaneous feeding by Minnesota and Connecticut larvae contained within separate feeding capsules. We found no difference in the ability of Minnesota and Connecticut larvae to acquire B. mayonii from infected mice and pass spirochetes to the nymphal stage (overall nymphal infection rates of 11.6 and 13.3%, respectively). Moreover, the efficiency of transmission of B. mayonii by single infected nymphs was similar for the Minnesota and Connecticut ticks (33 and 44%, respectively). We conclude that the examined I. scapularis ticks from the Upper Midwest and Northeast did not differ in their efficiency as vectors for B. mayonii., (Published by Oxford University Press on behalf of Entomological Society of America 2016 This work is written by US Government employees and is in the public domain in the US.)

Reducing exposure to ticks can help prevent Lyme disease and other tickborne diseases. Although it is currently recommended to dry clothes on high heat for one hour to kill ticks on clothing after spending time outdoors, this recommendation is based on a single published study of tick survival under various washing conditions and a predetermined one-hour drying time. We conducted a series of tests to investigate the effects of temperature, humidity, and drying time on killing nymphal and adult blacklegged ticks (Ixodes scapularis). Muslin bags containing 5 ticks each were washed then dried or dried only with six cotton towels during each drying cycle. All nymphal and adult ticks were killed when exposed to wash cycles when the water temperature reached ≥54°C (≥130°F); however, 50% of ticks survived hot water washes when the water temperature was <54°C. The majority (94%) of ticks survived warm washes [temperature range, 27-46°C (80-115°F)] and all ticks survived cold washes [15-27°C (59-80°F)]. When subsequently dried on high heat setting [54-85°C (129-185°F)], it took 50min to kill all ticks (95% confidence limit, 55min). Most significantly, we found that all adult and nymphal ticks died when placed directly in the dryer with dry towels and dried for 4min on high heat (95% confidence limit, 6min). We have identified effective, easily implemented methods to rid clothing of ticks after spending time outdoors. Placing clothing directly in a dryer and drying for a minimum of 6min on high heat will effectively kill ticks on clothing. If clothing is soiled and requires washing first, our results indicate clothing should be washed with water temperature ≥54°C (≥130°F) to kill ticks. When practiced with other tick-bite prevention methods, these techniques could further reduce the risk of acquiring tickborne diseases., (Published by Elsevier GmbH.)

A novel species within the Borrelia burgdorferi sensu lato complex, provisionally named Borrelia mayonii, was recently found to be associated with Lyme borreliosis in the Upper Midwest of the United States. Moreover, B. mayonii was detected from host-seeking Ixodes scapularis, the primary vector of B. burgdorferi sensu stricto in the eastern United States. We therefore conducted a study to confirm the experimental vector competence of I. scapularis for B. mayonii (strain MN14-1420), using colony ticks originating from adults collected in Connecticut and CD-1 white mice. Larvae fed on mice 10 weeks after needle-inoculation with B. mayonii acquired spirochetes and maintained infection through the nymphal stage at an average rate of 12.9%. In a transmission experiment, 40% of naïve mice exposed to a single infected nymph developed viable infections, as compared with 87% of mice fed upon by 2-3 infected nymphs. Transmission of B. mayonii by one or more feeding infected nymphs was uncommon up to 48h after attachment (one of six mice developed viable infection) but occurred frequently when nymphs were allowed to remain attached for 72-96h or feed to completion (11 of 16 mice developed viable infection). Mice infected via tick bite maintained viable infection with B. mayonii, as determined by ear biopsy culture, for at least 28 weeks. Our results demonstrate that I. scapularis is capable of serving as a vector of B. mayonii. This finding, together with data showing that field-collected I. scapularis are infected with B. mayonii, indicate that I. scapularis likely is a primary vector to humans of this recently recognized Lyme borreliosis spirochete., (Published by Elsevier GmbH.)

Prior exposure of vertebrate hosts to tick salivary proteins can induce specific immunity to tick infestation, as well as affording protection against tick-transmitted Borrelia burgdorferi infection in the mammalian host. Vaccination using an adenovirus expression system to deliver 4 tick salivary proteins (Ad-Salps) derived from Ixodes scapularis, Salp15, Salp25A, Salp25D, and Isac, was explored. Results indicate that vaccination with tick salivary proteins in an adenoviral vector can be used to modulate a Th1 response in the host and partially control spirochete load in immunized mice after infected tick challenge., (Published by Elsevier GmbH.)

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.

A field trial was conducted in a Lyme disease-endemic area of New Jersey to determine the efficacy of a doxycyline hyclate rodent bait to prophylactically protect and cure small-mammal reservoirs and reduce infection rates in questing Ixodes scapularis ticks for Borrelia burgdorferi and Anaplasma phagocytophilum. The doxycycline-laden bait was formulated at a concentration of 500 mg/kg and delivered during the immature tick feeding season in rodent-targeted bait boxes. The percentage of infected small mammals recovered from treated areas after 2 years of treatment was reduced by 86.9% for B. burgdorferi and 74% for A. phagocytophilum. Infection rates in questing nymphal ticks for both B. burgdorferi and A. phagocytophilum were reduced by 94.3% and 92%, respectively. Results from this study indicate that doxycycline-impregnated bait is an effective means of reducing infection rates for B. burgdorferi and A. phagocytophilum in both rodent reservoirs and questing I. scapularis ticks.

We evaluated the ability of dual applications of natural, plant-derived acaricides to suppress nymphal Ixodes scapularis Say and Amblyomma americanum (L.) (Acari: Ixodidae) in a Lyme disease endemic area of New Jersey. An aqueous formulation of 2% nootkatone provided >90% control of I. scapularis through 7 d. Control declined to 80.9% at 14 d, and a second application was made that provided >95% control through the remaining 4 wk of the nymphal season. Nootkatone provided >90% control of A. americanum through 35 d postapplication. Applications of 2% carvacrol and EcoTrol T&O resulted in rapid knockdown of both tick species, but control declined significantly to 76.7 and 73.7%, respectively, after 14 d when a second application was made that extended control of both tick species to between 86.2 and 94.8% at 21 d. Subsequently, control declined steadily in all plots by 42 d postapplication except for I. scapularis in carvacrol-treated plots, where levels of control >90% were observed through 35 d. Of the three compounds tested, 2% nootkatone provided the most consistent results, with 96.5 and 91.9% control of I. scapularis and A. americanum through 42 and 35 d, respectively. The ability of plant-derived natural products to quickly suppress and maintain significant control of populations of these medically important ticks may represent a future alternative to the use of conventional synthetic acaricides. In addition, the demonstrated efficacy of properly-timed backpack sprayer application may enable homeowner access to these minimal-risk acaricides.

We evaluated the ability of the natural, plant-derived acaricides nootkatone and carvacrol to suppress Ixodes scapularis Say and Amblyomma americanum (L.) (Acari: Ixodidae). Aqueous formulations of 1 and 5% nootkatone applied by backpack sprayer to the forest litter layer completely suppressed I. scapularis nymphs through 2 d. Thereafter, the level of reduction gradually declined to < or =50% at 28 d postapplication. Against A. americanum nymphs, 1% nootkatone was less effective, but at a 5% concentration, the level of control was similar or greater to that observed with I. scapularis through 21 d postapplication. Initial applications of 0.05% carvacrol were ineffective, but a 5% carvacrol formulation completely suppressed nymphs of both species through 2 d and resulted in significant reduction in I. scapularis and A. americanum nymphs through 28 and 14 d postapplication, respectively. Backpack sprayer applications of 5% nootkatone to the shrub and litter layers resulted in 100% control of I. scapularis adults through 6 d, but the level of reduction declined to 71.5% at 28 d postapplication. By contrast, high-pressure applications of 2% nootkatone to the litter layer resulted in 96.2-100% suppression of both I. scapularis and A. americanum nymphs through 42 d, whereas much lower control was obtained from the same formulation applied by backpack sprayer. Backpack sprayer application of a 3.1% nootkatone nanoemulsion resulted in 97.5-98.9 and 99.3-100% reduction in I. scapularis and A. americanum nymphs, respectively, at 1 d postapplication. Between 7 d and 35 d postapplication, the level of control varied between 57.1% and 92.5% for I. scapularis and between 78.5 and 97.1% for A. americanum nymphs. The ability of natural products to quickly suppress and maintain significant control of populations of these medically important ticks at relatively low concentrations may represent a future alternative to the use of conventional synthetic acaricides.

In a recent study, the combined use of 4-Posters and Maxforce TMS bait boxes along with a barrier application of deltamethrin resulted in accelerated control of Ixodes scapularis Say by sequentially attacking each postembryonic life stage. We report the results of a follow-up study to test the ability of 4-Posters used alone to sustain the high levels of control achieved through the integrated tick management (ITM) approach after withdrawal of the bait boxes. In the first year after withdrawal, we observed declines in the level of control of larvae on small mammals, as well as of numbers of host-seeking larvae in the treatment area. There was no difference in the level of control of host-seeking adults in the treatment area after 2 yr. Within 2 yr, we observed a decline in control of subadult ticks infesting small mammals, but continued to see significant control of both host-seeking nymphs (85.9%) and larvae (89.0%) in the treatment area. The inconsistency that we observed between the apparent ability of 4-Posters to sustain high levels of control of host-seeking ticks, although having less effect on tick burdens on small mammal hosts, may be explained by the host-seeking ecology of immature I. scapularis.

The biocidal activity of three steam distilled wood essential oils-incense cedar, Calocedrus decurrens (Torr.) Florin; Port-Orford-cedar, Chamaecyparis lawsoniana (A. Murr.) Parl.; and western juniper, Juniperus occidentalis (Hook)--were evaluated against adult Aedes aegypti (L.) (Diptera: Culicidae) and Xenopsylla cheopis (Rothchild) (Siphonaptera: Pulicidae) and nymphal Ixodes scapularis Say (Acari: Ixodidae). In vitro laboratory bioassays were conducted to establish baseline dose-mortality data through 24 h. Incense cedar heartwood was the most toxic to all three vector species followed in order of activity by western juniper and Port-Orford-cedar based on LC50 and LC90 values. Ae. aegypti were substantially more susceptible to the oils than either I. scapularis or X. cheopis.

Preliminary repellent activity of 14 natural products isolated from essential oil components extracted from the heartwood of Alaska yellow cedar, Chamaecyparis nootkatensis (D. Don) Spach., were evaluated against nymphal Ixodes scapularis Say in a laboratory bioassay and compared with technical grade N,N-diethyl-3-methylbenzamide (deet). Four hours after treatment, nootkatone and valencene-13-ol had repellent concentration (RC)50 values of 0.0458 and 0.0712% (wt:vol), respectively; two additional Alaska yellow cedar compounds, nootkatone 1 --> 10 epoxide and carvacrol had reported RC50 values of 0.0858 and 0.112%, respectively. The observed RC50 value for deet was 0.0728% (wt:vol). Although not statistically significantly more active than deet, the ability of these natural products to repel ticks at relatively low concentrations may represent a potential alternative to synthetic commercial repellents.

Ixodes scapularis transmits several pathogens including Borrelia burgdorferi. Bioactive compounds in tick saliva support tick feeding and influence pathogen transmission to the mammalian host. These studies utilized oral delivery of dsRNA to silence an anticomplement gene (isac) in I. scapularis nymphs. Silencing of isac significantly reduced fed-tick weight compared to delivery of control lacZ dsRNA, and immunoblots specific for FlaB protein indicated a reduction in spirochete load in isac-silenced infected nymphs. SDS-PAGE demonstrated that isac gene silencing affected expression of a number of salivary and non-salivary gland proteins in ticks. Finally, multiple isac cDNA homologues were cloned, and these may represent a new gene family coexpressed during tick feeding. This work presents a novel oral delivery approach for specific gene silencing in I. scapularis nymphs and characterizes the effect of isac on blood-feeding in an attempt to block transmission of B. burgdorferi.

Blood fed nymphal Ixodes scapularis Say infected with Borrelia burgdorferi were dissected to obtain salivary gland and midgut extracts. Extracts were inoculated into C3H/HeJ mice, and ear, heart, and bladder were cultured to determine comparative infectivity. Aliquots of extracts were then analyzed by quantitative polymerase chain reaction to determine the number of spirochetes inoculated into mice. A comparative median infectious dose (ID50) was determined for both salivary gland and midgut extract inoculations. Our data demonstrated a statistically significant difference (P < 0.002) in the ID50 derived from salivary gland (average = 18) versus midgut (average = 251) extracts needed to infect susceptible mice. A rationale for the differential infectivity of salivary and midgut derived spirochetes is discussed.

Laboratory bioassays were conducted to determine the activity of 15 natural products isolated from essential oil components extracted from the heartwood of Alaska yellow cedar, Chamaecyparis nootkatensis (D. Don) Spach., against Ixodes scapularis Say nymphs, Xenopsylla cheopis (Rothchild), and Aedes aegypti (L.) adults. Four of the compounds from the essential oil have been identified as monoterpenes, five as eremophilane sesquiterpenes, five as eremophilane sesquiterpene derivatives from valencene and nootkatone, and one as a sesquiterpene outside the eremophilane parent group. Carvacrol was the only monoterpene that demonstrated biocidal activity against ticks, fleas, and mosquitoes with LC50 values after 24 h of 0.0068, 0.0059, and 0.0051% (wt:vol), respectively. Nootkatone from Alaska yellow cedar was the most effective of the eremophilane sesquiterpenes against ticks (LC50 = 0.0029%), whereas the nootkatone grapefruit extract exhibited the greatest biocidal activity against fleas (LC50 = 0.0029%). Mosquitoes were most susceptible to one of the derivatives of valencene, valencene-13-aldehyde (LC50 = 0.0024%), after 24 h. Bioassays to determine residual activity of the most effective products were conducted at 1, 2, 4, and 6 wk after initial treatment. Residual LC50 values for nootkatone did not differ significantly at 4 wk posttreatment from the observations made at the initial 24-h treatment. The ability of these natural products to kill arthropods at relatively low concentrations represents an alternative to the use of synthetic pesticides for control of disease vectors.

A 3-yr community-based study was conducted on residential properties on Mason's Island, Mystic, CT, to determine the efficacy of a rodent-targeted acaricide (fipronil) to control immature Ixodes scapularis (Say) on Peromyscus leucopus. Results indicated that modified commercial bait boxes were effective as an acaricide delivery method for reducing nymphal and larval tick infestations on white-footed mice by 68 and 84%, respectively. Passive application of fipronil significantly reduced the infection rate of Borrelia burgdorferi among white-footed mice by 53%. Moreover, the abundance of questing I. scapularis adults on treated properties was reduced by 77% and fewer were infected with spirochetes (31%) compared with untreated sites (47%) after 3 yr of treatment. Likewise, the abundance of host-seeking nymphs was significantly reduced on treated properties by >50%. Finally, infection rates in flagged nymphal ticks for both B. burgdorferi and Anaplasma phagocytophilum were reduced by 67 and 64%, respectively, after only 2 yr of treatment. Results from this 3-yr trial indicate that the use of fipronil passively applied to reservoir animals by bait boxes is an environmentally acceptable means to control ticks, interrupt the natural disease transmission cycle, and reduce the risk of Lyme disease for residents of treated properties.

Clinical isolates of Borrelia burgdorferi sensu stricto have been categorized into disseminated and nondisseminated groups based on distinct ribosomal spacer restriction fragment length polymorphism genotypes (RSTs). In order to determine whether transmission by tick bite would alter the dissemination dynamics and disease produced by distinct genotypes, disseminated isolates (RST1), nondisseminated isolates (RST3), and a standard laboratory strain (B-31) were established in a murine cycle utilizing infections transmitted by ticks. B-31 spirochetes circulated in the blood of inbred C3H/HeJ mice longer than in the blood of outbred mice. The majority of C3H mice exposed to RST1-infected ticks contained cultivable spirochetes in their blood for up to 17 days; in contrast, mice exposed to RST3 isolates demonstrated a precipitous decline in infection after day 7 postexposure. A quantitative PCR (q-PCR) assay demonstrated that the densities of spirochetes in blood were similar for the RST1 and RST3 isolates, except during the 2nd week postexposure, when the RST1 isolates displayed a markedly higher density in blood. Spirochete load in the heart and bladder of infected mice was measured by q-PCR at 8 weeks postexposure; the numbers of spirochetes in these tissues were similar for mice infected with either disseminated or nondisseminated strains. Similarly, histopathology samples of heart, bladder, and joint tissue obtained at 8 weeks postexposure did not reveal greater pathology in mice infected with the disseminated isolates. We conclude that although the spirochetemia induced by tick-transmitted disseminated isolates was more intense and of longer duration than that induced by nondisseminated isolates, the resultant pathologies produced by these strains were ultimately similar.

The first technique for repeatedly sampling individual Ixodes scapularis adult ticks was developed and validated. Gut samples from the same individual ticks were removed and analyzed at weekly intervals. Multiple analyses were conducted on each gut sample (e.g., total protein concentration, presence of viable B. burgdorferi spirochetes, and concentration of outer surface protein A [OspA]). Female I. scapularis survived for up to 25 d after gut sampling. Seventy-five percent of females oviposited after the sampling procedure, with 14% of ticks laying >1,500 eggs. No significant differences in either fecundity or length of survival were found between B. burgdorferi-infected and uninfected I. scapularis. This technique will enable longitudinal studies on both tick-pathogen interactions and physiological studies that have hitherto not been attempted.

We investigated the experimental vector competence of Ixodes pacificus Cooley and Kohls and Ixodes spinipalpis Hadwen and Nuttall, and the reservoir competence of the dusky-footed woodrat (Neotoma fuscipes Baird) and the deer mouse (Peromyscus maniculatus [Wagner]), for Borrelia bissettii Postic, Marti Ras, Lane, Hendson, and Baranton. Both rodent species are capable reservoirs for B. bissettii; infection rates for I. pacificus or I. spinipalpis nymphs fed as larvae on infected animals ranged from 50 to 57%. Moreover, both I. pacificus and I. spinipalpis are efficient vectors of B. bissettii. Viable infections were recorded from all rodents known to be exposed to one or more infected nymphs of I. spinipalpis (seven deer mice, two white mice) or I. pacificus (seven deer mice). In contrast, none of four New Zealand white rabbits fed upon by 90 I. pacificus nymphs with a probable B. bissettii infection rate of >50% became infected or seroconverted. The attachment and feeding success of laboratory-reared nymphs similarly confined with deer mice in muslin-covered wire-mesh cages for 24 h ranged from 0% for I. pacificus to 17-73% for I. spinipalpis. Notably, the I. pacificus nymphs were physiologically capable of feeding; nymphs failing to attach to rodents fed readily when placed in feeding capsules upon rabbits. We conclude that the dusky-footed woodrat and the deer mouse are capable experimental reservoir hosts of B. bissettii, and that both I. spinipalpis and I. pacificus are efficient experimental vectors of B. bissettii. However, the reluctance of I. pacificus nymphs to feed on certain rodents may limit its importance as an enzootic vector of B. burgdorferi sensu lato spirochetes.

A linkage map of the Ixodes scapularis genome was constructed, based upon segregation amongst 127 loci. These included 84 random amplified polymorphic DNA (RAPD) markers, 32 Sequence-Tagged RAPD (STAR) markers, 5 cDNAs, and 5 microsatellites in 232 F1 intercross progeny from a single, field-collected P1 female. A preliminary linkage map of 616 cM was generated across 14 linkage groups with one marker every 10.8 cM. Assuming a genome size of approximately 109 bp, the relationship of physical to genetic distance was found to be approximately 300 kb/cM in the I. scapularis genome.

Public health recommendations for Lyme disease prevention generally include daily tick checks and prompt removal of attached ticks as a means of decreasing the risk of acquiring Lyme disease in highly endemic regions. In the current study, we determined whether crushing nymphal ticks during removal with forceps increased the risk of B. burgdorferi transmission, what degree of protection from transmission of B. burgdorferi was provided by removal of nymphal Ixodes scapularis Say at specific intervals, and whether commercial devices marketed for tick removal worked when tested against nymphal I. scapularis. Both removal via gentle pressure (26% transmission) or crushing the tick (30% transmission) caused a significant decrease in transmission as compared with the sham control (70% transmission). The degree of protection provided via tick removal decreased steadily up to 60 h of attachment; between 60 and 66 h, a dramatic falloff in protection occurred to the point where no protection was observed at 66 h. Finally, commercial tick removal devices varied widely in their efficacy for the removal of attached nymphal I. scapudaris.

A linkage map of the Ixodes scapularis genome was constructed based upon segregation amongst 127 loci. These included 84 random amplified polymorphic DNA (RAPD) markers, 32 Sequence-Tagged RAPD (STAR) markers, 5 cDNAs, and 5 microsatellites in 232 F1 intercross progeny from a single, field-collected P1 female. A preliminary linkage map of 616 cM was generated across 14 linkage groups with one marker every 10.8 cM. Assuming a genome size of approximately 10(9) bp, the relationship of physical to genetic distance is approximately 300 kb/cM in the I. scapularis genome.

A potent inhibitor of mitogen-stimulated T cell proliferation exists in the saliva of several species of hard ticks, including the Lyme disease vector tick, Ixodes scapularis. Our characterization of this phenomenon has led to the identification of a possible mechanism for the T cell inhibitory activity of I. scapularis saliva. The T cell inhibitor can overcome stimulation of mouse spleen cells with anti-CD3 mAb; however, a direct and avid interaction with T cells does not appear to be necessary. Tick saliva inhibits a mouse IL-2 capture ELISA, suggesting that a soluble IL-2 binding factor is present in the saliva. This hypothesis was verified by using a direct binding assay in which plate-immobilized tick saliva was shown to bind both mouse and human IL-2. Elimination of the IL-2 binding capacity of saliva in the in vitro assays by trypsin digestion demonstrated that the IL-2 binding factor is a protein. These experiments comprise the first demonstration of the existence of such a secreted IL-2 binding protein from any parasite or pathogen. This arthropod salivary IL-2 binding capacity provides a simple mechanism for the suppression of T cell proliferation as well as for the activity of other immune effector cells that are responsive to IL-2 stimulation. Relevance of the tick T cell inhibitory activity to the human immune system is demonstrated by the ability of tick saliva to inhibit proliferation of human T cells and CTLL-2 cells grown in the presence of human IL-2.

Four laboratory-grown, low-passage isolates of Borrelia burgdorferi sensu stricto, B31, JD-1, 910255, and N40, were incorporated into Ixodes scapularis ticks to examine the pathogenesis of these isolates in mice after tick transmission. All isolates induced multifocal, lymphoid nodular cystitis, subacute, multifocal, necrotizing myocarditis, and a localized periostitis and arthritis of the femorotibial joint 6-18 weeks after tick infestation. In terms of the number of mice that demonstrated pathology in bladder, heart, and joint, the highest incidence of lesions occurred 12 weeks after tick bite. Utilizing the Taqman quantitative polymerase chain reaction (q-PCR) fluorogenic detection technology to amplify a conserved region of the flagellin gene, a trend was demonstrated between the number of spirochetes in tissue with duration of pathology. The q-PCR assay developed for this study was sensitive and could reliably measure as few as 1 to 10 spirochetes in the target tissues tested. A higher percentage of B31- and N40-infected mice (92 and 100%, respectively) developed myocarditis than JD-1- or 910255-infected mice (67 and 46%, respectively) 12 weeks after tick bite. The amount of spirochetal DNA that could be amplified for heart at this time point was not statistically different between isolates, indicating a difference in virulence between B31 and N40 relative to JD-1 and 910225. N40-infected mice demonstrated a significantly higher spirochete load (an average of 1.23 spirochetes/mg of tissue, p = 0.045) in femorotibial joints 18 weeks after infection, with 60% of these mice maintaining lesions compared with those infected with B31 (13%), JD-1 (25%), or 910255 (50%), which averaged <0.5 spirochetes/mg of tissue. This mouse model of Lyme borreliosis, including the ability to monitor lesion development and spirochete load, can facilitate the testing of therapeutic regimens for the later stages of tick-transmitted Lyme disease and help investigate aspects of the immunopathogenesis of lesion development.

To study interactions between Ixodes scapularis (Say) and Borrelia burgdorferi, an artificial feeding system was refined to allow controlled manipulation of single variables. The feeding system uses a mouse skin mounted on a water-jacketed glass membrane feeder. I. scapularis were infected using either BSK-H-cultured B. burgdorferi spirochetes or a B. burgdorferi-infected mouse skin as the source of spirochetes. Sixty-six percent of nymphs successfully fed to repletion using the artificial feeding systems with at least 75% of nymphs becoming infected with B. burgdorferi. Strain B31 B. burgdorferi spirochetes from passages 2-17 were equally infectious to nymphal ticks. At concentrations of one spirochete per microliter, 12% of nymphs acquired infection and 14 and 100 spirochetes per microliter resulted in 50 and 100% infection rates, respectively. Eighty-nine percent of nymphs fed by artificial feeding molted to the adult stage. When subsequently fed as adults, these I. scapularis successfully transmitted infectious B. burgdorferi spirochetes to mice.

The vector competence of Ixodes muris (Bishopp & Smith) was determined for Borrelia burgdorferi, the etiologic agent of Lyme disease. Larval I. muris were fed on ICR outbred mice infected with the B-31 laboratory strain of B. burgdorferi. Replete larvae, at 5 d after feeding, were assayed for infection by culture in Barbour-Stoner-Kelly (BSK-H) media. Infection frequency in I. muris replete larvae was 66%. Resultant nymphs were fed on naive ICR outbred mice to determine the ability of I. muris to transmit infection. Infection frequency in fed nymphs declined to 38% and only 1/5 mice was positive for B. burgdorferi on ear biopsy culture. We demonstrated that I. muris is capable of acquiring and transmitting B. burgdorferi but is a relatively poor vector compared with I. scapularis (Say).

Previous work described an enzootic cycle of Borrelia burgdorferi sensu lato (hereafter referred to as B. burgdorferi) maintained by the rodent Neotoma mexicana and the tick Ixodes spinipalpis in northern Colorado. We investigated the incidence of coinfection among rodents with the agent of human granulocytic ehrlichiosis (aoHGE). aoHGE was detected in 23.5% of 119 rodent spleens examined. Biopsy results indicated that 78 (65.5%) of the 119 rodents were positive for B. burgdorferi, whereas 22 (78.5%) of the 28 animals that harbored aoHGE were also infected with B. burgdorferi. In 14 of 25 I. spinipalpis tick pools, aoHGE was detected by amplifying both the 16s rRNA and p44 gene of aoHGE. The ability of I. spinipalpis to transmit aoHGE was examined in C3H/HeJ mice. aoHGE was detected in their blood 5 days after I. spinipalpis infestation. This study confirms that both B. burgdorferi and aoHGE can be transmitted by I. spinipalpis ticks and that there is a high incidence of coinfection in rodents, predominantly Peromyscus maniculatus and N. mexicana, that inhabit the foothills of northern Colorado.

Populations of adult Ixodes scapularis and Ixodes pacificus, the two principal vectors of Lyme disease spirochetes in the United States, were collected from 17 sites in 12 states. Female ticks were fed on experimental rabbits; ticks and rabbits were subsequently examined for infection with Borrelia burgdorferi. Fourteen rabbits were exposed to I. scapularis ticks from the northeastern states of Connecticut, New York, New Jersey, and Maryland; all 14 rabbits became infected with B. burgdorferi. A total of 165/226 (73%) of these northeastern ticks was infected. Similarly, ticks from the midwestern states of Michigan, Wisconsin, and Minnesota transmitted infection to all three exposed rabbits; 29/51 (57%) of these midwestern I. scapularis were infected. In marked contrast, none of the 12 rabbits exposed to I. scapularis ticks from the southeastern states of South Carolina, Georgia, Florida, and Mississippi acquired infection with B. burgdorferi, and 0/284 (0%) of these ticks contained spirochetes. Four rabbits were exposed to I. pacificus collected from one location in California; 2/4 of these rabbits acquired infection and 2/57 (4%) of the I. pacificus were infected with B. burgdorferi. The antigenic profiles of all 58 strains tested were consistent with an identity of B. burgdorferi sensu lato. The availability of a human Lyme disease vaccine adds urgency to our efforts to calculate the ecological transmission risk throughout the United States, as an aid to the judicious use of such a vaccine.

Borrelia burgdorferi, the spirochetal agent of Lyme disease, is transmitted by Ixodes ticks. When an infected nymphal tick feeds on a host, the bacteria increase in number within the tick, after which they invade the tick's salivary glands and infect the host. Antibodies directed against outer surface protein A (OspA) of B. burgdorferi kill spirochetes within feeding ticks and block transmission to the host. In the studies presented here, passive antibody transfer experiments were carried out to determine the OspA antibody titer required to block transmission to the rodent host. OspA antibody levels were determined by using a competitive enzyme-linked immunosorbent assay that measured antibody binding to a protective epitope defined by monoclonal antibody C3.78. The C3.78 OspA antibody titer (>213 microgram/ml) required to eradicate spirochetes from feeding ticks was considerably higher than the titer (>6 microgram/ml) required to block transmission to the host. Although spirochetes were not eradicated from ticks at lower antibody levels, the antibodies reduced the number of spirochetes within the feeding ticks and interfered with the ability of spirochetes to induce ospC and invade the salivary glands of the vector. OspA antibodies may directly interfere with the ability of B. burgdorferi to invade the salivary glands of the vector; alternately, OspA antibodies may lower the density of spirochetes within feeding ticks below a critical threshold required for initiating events linked to transmission.

The vector competence of 2 tick species, Ixodes ricinus (L.) and Ixodes scapularis Say, was determined and compared for 3 genospecies of Borrelia burgdorferi. The 3 genospecies of B. burgdorferi used in the following experiments were Borrelia burgdorferi sensu stricto (B-31 and B-31.D1 clone), Borrelia afzelii (strain Pgau. C3), and Borrelia garinii (strain VS286 and VSBP). Spirochetes from all 5 strains were inoculated intradermally into outbred mice; larval ticks of both species were subsequently fed on those mice and replete larvae were assayed for infection by culture in BSK-H media every 7 d for 4 wk. Infection frequencies in I. scapularis exposed to the 5 strains were as follows: B-31 (90%), B-31.D1 (83%), Pgau.C3 (87%), VS286 (10%), and VSBP (5%). The comparable infection frequencies for I. ricinus were B-31 (3%), B-31.D1 (3%), Pgau.C3 (90%), VS286 (5%), and VSBP (3%). Resultant nymphal I. scapularis successfully transmitted B-31, B-31,D1, Pgau.C3, and VS286 to outbred mice. I. ricinus nymphs transmitted Pgau.C3 and VS286. Both species failed to transmit strain VSBP.

This report describes the vector competence of 3 ixodid tick species, Ixodes scapularis (Say), I. spinipalpis (Nuttall), and Dermacentor andersoni (Stiles), for Borrelia burgdorferi in Colorado. The study was based on preliminary field work performed in 6 Colorado counties, where rodents and ticks were collected and assayed for the presence of B. burgdorferi. Four of the 6 counties produced 52 rodent and 39 I. spinipalpis isolates of B. burgdorferi. Two B. burgdorferi isolates were tested under laboratory conditions and found to be infective to Imperial Cancer Research Fund (ICRF) outbred mice. The 1st, a low-passage strain originating from New York (B-31, passage 6) was used as a control, and the 2nd was isolated from ear tissue of a Neotoma mexicana (Baird) (Mexican wood rat) that was trapped in Colorado. Larvae of I. scapularis, I. spinipalpis. and D. andersoni were fed on infected mice and cultured in Barbour-Stoner-Kelly media to assay for infection at 1, 2, 3, and 4 wk after repletion. The infection rates in replete larvae. were 75, 69, and 8.5%, respectively, whereas transstadial nymphal infection rates were 80, 75, and 0%, respectively. Both I. scapularis and I. spinipalpis were shown to be competent vectors that acquired the infection from the host reservoir mice and subsequently transmitted it to naive mice. Given that I. scapularis are not found in Colorado, I. spinipalpis are restricted to the nests and burrows of rodents, and because of the semiarid environment in Colorado, the risk of human contact with B. burgdorferi appears to be low.

Chickens were used as a laboratory model to determine the conditions affecting the ability of birds to infect ticks with Lyme disease spirochetes. Chicks (Gallus gallus) were exposed to 12 nymphal Ixodes scapularis at one week or three weeks of age. Xenodiagnostic larval ticks fed these birds at weekly intervals thereafter. Chicks exposed to infected nymphs at one week of age infected 87% of larvae at three weeks of age, but only infected 3% of larvae at four weeks and 0% of larvae at five weeks. Chicks exposed to nymphs at three weeks of age infected only 12% of larvae at four weeks, and 0% thereafter. Thus, experimentally infected chicks can infect larval ticks, but only for a brief interval after exposure. Young chicks are more infectious than older chickens. The immune response of infected chicks was rapid and directed against diverse antigens.