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.

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.

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.

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 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.

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.

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.

In the 1980s, the blacklegged tick, Ixodes scapularis Say, and rodents were recognized as the principal vector and reservoir hosts of the Lyme disease spirochete Borrelia burgdorferi in the eastern United States, and deer were incriminated as principal hosts for I. scapularis adults. These realizations led to pioneering studies aiming to reduce the risk for transmission of B. burgdorferi to humans by attacking host-seeking ticks with acaricides, interrupting the enzootic transmission cycle by killing immatures infesting rodent reservoirs by means of acaricide-treated nesting material, or reducing deer abundance to suppress tick numbers. We review the progress over the past three decades in the fields of: 1) prevention of human-tick contact with repellents and permethrin-treated clothing, and 2) suppression of I. scapularis and disruption of enzootic B. burgdorferi transmission with environmentally based control methods. Personal protective measures include synthetic and natural product-based repellents that can be applied to skin and clothing, permethrin sprays for clothing and gear, and permethrin-treated clothing. A wide variety of approaches and products to suppress I. scapularis or disrupt enzootic B. burgdorferi transmission have emerged and been evaluated in field trials. Application of synthetic chemical acaricides is a robust method to suppress host-seeking I. scapularis ticks within a treated area for at least 6-8 wk. Natural product-based acaricides or entomopathogenic fungi have emerged as alternatives to kill host-seeking ticks for homeowners who are unwilling to use synthetic chemical acaricides. However, as compared with synthetic chemical acaricides, these approaches appear less robust in terms of both their killing efficacy and persistence. Use of rodent-targeted topical acaricides represents an alternative for homeowners opposed to open distribution of acaricides to the ground and vegetation on their properties. This host-targeted approach also provides the benefit of the intervention impacting the entire rodent home range. Rodent-targeted oral vaccines against B. burgdorferi and a rodent-targeted antibiotic bait have been evaluated in laboratory and field trials but are not yet commercially available. Targeting of deer-via deer reduction or treatment of deer with topical acaricides-can provide area-wide suppression of host-seeking I. scapularis These two deer-targeted approaches combine great potential for protection that impacts the entire landscape with severe problems relating to public acceptance or implementation logistics. Integrated use of two or more methods has unfortunately been evaluated in very few published studies, but additional field evaluations of integrated tick and pathogen strategies are underway.

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 degrees C (≥130 degrees F); however, 50% of ticks survived hot water washes when the water temperature was <54 degrees C. The majority (94%) of ticks survived warm washes [temperature range, 27-46 degrees C (80-115 degrees F)] and all ticks survived cold washes [15-27 degrees C (59-80 degrees F)]. When subsequently dried on high heat setting [54-85 degrees C (129-185 degrees 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 degrees C (≥130 degrees F) to kill ticks. When practiced with other tick-bite prevention methods, these techniques could further reduce the risk of acquiring tickborne diseases.

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 naive 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.

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrAS226A). Although inoculation with either BbHtrA or BbHtrAS226A produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.

Zoonotic pathogens that cause devastating morbidity and mortality in humans may be relatively harmless in their natural reservoir hosts. The tick-borne bacterium Borrelia burgdorferi causes Lyme disease in humans but few studies have investigated whether this pathogen reduces the fitness of its reservoir hosts under natural conditions. We analyzed four years of capture-mark-recapture (CMR) data on a population of white-footed mice, Peromyscus leucopus, to test whether B. burgdorferi and its tick vector affect the survival of this important reservoir host. We used a multi-state CMR approach to model mouse survival and mouse infection rates as a function of a variety of ecologically relevant explanatory factors. We found no effect of B. burgdorferi infection or tick burden on the survival of P. leucopus. Our estimates of the probability of infection varied by an order of magnitude (0.051 to 0.535) and were consistent with our understanding of Lyme disease in the Northeastern United States. B. burgdorferi establishes a chronic avirulent infection in their rodent reservoir hosts because this pathogen depends on rodent mobility to achieve transmission to its sedentary tick vector. The estimates of B. burgdorferi infection risk will facilitate future theoretical studies on the epidemiology of Lyme disease.

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 up to 3 days after tick removal and protected 74% of mice exposed to tick bite when applied up to 2 weeks after tick removal. Azithromycin cream was also protective when applied at a site distal to the tick-bite-site, suggesting 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.

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.

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.

The American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae), has been implicated as a potential bridging vector to humans of Francisella tularensis, the etiological agent of tularemia. Since the initial studies evaluating vector competency of D. variabilis were conducted, F. tularensis has been subdivided into subspecies and clades that differ in their geographical distribution in the United States and in the severity of infections caused in humans. Here, we demonstrate that D. variabilis nymphs efficiently acquire, transtadially maintain, and transmit each of the strains tested (clades A1b and A2, and type B). Transmission efficiency by adult females was similarly high among infection groups and ranged from 58% for type B to 89% for A2 infections. In addition, we demonstrated that transmission can occur shortly after tick attachment. These findings support the concept that D. variabilis adults may play a significant role in epizootic transmission of F. tularensis, and as a bridging vector to humans.

Borrelia burgdorferi, the causative agent of Lyme borreliosis, is transmitted to humans from the bite of Ixodes spp. ticks. During the borrelial tick-to-mammal life cycle, B. burgdorferi must adapt to many environmental changes by regulating several genes, including bba64. Our laboratory recently demonstrated that the bba64 gene product is necessary for mouse infectivity when B. burgdorferi is transmitted by an infected tick bite, but not via needle inoculation. In this study we investigated the phenotypic properties of a bba64 mutant strain, including 1) replication during tick engorgement, 2) migration into the nymphal salivary glands, 3) host transmission, and 4) susceptibility to the MyD88-dependent innate immune response. Results revealed that the bba64 mutant's attenuated infectivity by tick bite was not due to a growth defect inside an actively feeding nymphal tick, or failure to invade the salivary glands. These findings suggested there was either a lack of spirochete transmission to the host dermis or increased susceptibility to the host's innate immune response. Further experiments showed the bba64 mutant was not culturable from mouse skin taken at the nymphal bite site and was unable to establish infection in MyD88-deficient mice via tick infestation. Collectively, the results of this study indicate that BBA64 functions at the salivary gland-to-host delivery interface of vector transmission and is not involved in resistance to MyD88-mediated innate immunity.

In the United States, the American dog tick, Dermacentor variabilis (Say) is considered an important biological vector of Francisella tularensis, the etiologic agent of tularemia. In this study, we evaluated the vector efficiency of nymphal D. variabilis infected as larvae with differing clades and subspecies (A1b, A2, and type B) of F. tularensis. In all cases, D. variabilis larvae were able to acquire, maintain, and transstadially transmit F. tularensis. Significant replication of the bacteria also occurred in infected nymphs. Transmission of F. tularensis to Swiss Webster mice was not observed with A1b, and low rates were observed with A2 (8.0%) and type B (13.5%). Negative effects on tick survivorship were also observed for A1b, A2, and type B infections. Our results provide evidence of a high fitness cost and low transmission rates during the immature stages, suggesting that D. variabilis may play a limited role in enzootic maintenance of F. tularensis.

Two new p-cymene based diphenols (1-2) were isolated from the heartwood of Calocedrus decurrans. Structures were elucidated by ID and 2D NMR techniques and HRMS. Libocedroquinone (3) was also isolated as a natural product for the first time. A new system of nomenclature is proposed for description of such oligomeric p-cymene derivatives.

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.