Gabrielle Dietrich

Repellent Activity of Fractioned Compounds from Chamaecyparis nootkatensis Essential Oil Against Nymphal Ixodes scapularis (Acari: Ixodidae)

Abstract 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.

The bba64 gene of Borrelia burgdorferi, the Lyme disease agent, is critical for mammalian infection via tick bite transmission

The spirochetal agent of Lyme disease, Borrelia burgdorferi, is transmitted by bites of Ixodes ticks to mammalian reservoir hosts and humans. The mechanism(s) by which the organism is trafficked from vector to host is poorly understood. In this study, we demonstrate that a B. burgdorferi mutant strain deficient in the synthesis of the bba64 gene product was incapable of infecting mice via tick bite even though the mutant was (i) infectious in mice when introduced by needle inoculation, (ii) acquired by larval ticks feeding on infected mice, and (iii) able to persist through tick molting stages. This finding of a B. burgdorferi gene required for pathogen transfer and/or survival from the tick to the susceptible host represents an important breakthrough toward understanding transmission mechanisms involved for the Lyme disease agent.

Coxiella Symbionts in the Cayenne Tick Amblyomma cajennense

Members of the Coxiella genus are intracellular bacteria that can infect a variety of animals including humans. A symbiotic Coxiella was recently described in Amblyomma americanum ticks in the Northern Hemisphere with no further investigations of other Amblyomma species in other geographic regions. These ixodid ticks represent a group of important vectors for human infectious agents. In the present work, we have demonstrated that symbiotic Coxiella (SCox) are widespread, occurring in South America and infecting 100% of all life stages and eggs of the Cayenne ticks Amblyomma cajennense from Brazil and the USA. Using light microscopy, in situ hybridization, and PCR, we demonstrated SCox in salivary glands, ovaries, and the intestines of A. cajennense. These symbionts are vertically and transtadially transmitted in laboratory reared A. cajennense, and quantitative PCR analyses indicate that SCox are more abundant in adult female ticks, reaching values corresponding to an 11×, 38×, and 200× increase in SCox 16S rRNA gene copy number in unfed females, compared to unfed nymphs, larvae, and eggs, respectively. Phylogenetic analyses showed distinct SCox subpopulations in the USA and Brazil and demonstrated that SCox bacteria do not group with pathogenic Coxiella burnetii.

Biocidal Activity of Three Wood Essential Oils Against Ixodes scapularis (Acari: Ixodidae), Xenopsylla cheopis (Siphonaptera: Pulicidae), and Aedes aegypti (Diptera: Culicidae)

Abstract 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.

Kinetics of Borrelia burgdorferi Infection in Larvae of Refractory and Competent Tick Vectors

Abstract The acquisition of Borrelia burgdorferi by the larvae of competent and refractory ixodid ticks was assessed by quantitative polymerase chain reaction (PCR). Larvae were fed on infected mice, and the spirochete loads were determined during feeding and up to 93 d postfeeding. Amblyomma americanum (L.) was refractory to B. burgdorferi infection, with almost no detection of spirochete DNA during or postfeeding. In contrast, Ixodes scapularis Say supported high loads of spirochetes (103–104 per larva). In Dermacentor variabilis (Say), B. burgdorferi uptake was reduced, with an average of 16 spirochetes per larvae acquired after 4 d of feeding, representing 1/195 of the counts in I. scapularis. However, during the first day postfeeding, the spirochete growth rate in D. variabilis reached 0.076 generations per hour, 7.7 times greater than the highest growth rate detected in I. scapularis. D. variabilis supported intense spirochete growth up to the fourth day postinfection, when the counts increased to an average of 282 spirochetes per larvae or 1/8.5 of the I. scapularis counts 4 d postfeeding. The kinetics of spirochete growth was unstable in D. variabilis compared with I. scapularis, and transmission of B. burgdorferi by D. variabilis could not be demonstrated. A cofeeding experiment indicated that I. scapularis feeding increased A. americanum spirochete uptake. These collective results indicate suboptimal conditions for B. burgdorferi uptake and colonization within A. americanum or the presence of anti-Borrelia factor(s) in this nonpermissive tick species.

Transmission dynamics of Francisella tularensis subspecies and clades by nymphal Dermacentor variabilis (Acari: Ixodidae).

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.

An Acarologic Survey and Amblyomma americanum Distribution Map with Implications for Tularemia Risk in Missouri

In the United States, tickborne diseases occur focally. Missouri represents a major focus of several tickborne diseases that includes spotted fever rickettsiosis, tularemia, and ehrlichiosis. Our study sought to determine the potential risk of human exposure to human-biting vector ticks in this area. We collected ticks in 79 sites in southern Missouri during June 7-10, 2009, which yielded 1,047 adult and 3,585 nymphal Amblyomma americanum, 5 adult Amblyomma maculatum, 19 adult Dermacentor variabilis, and 5 nymphal Ixodes brunneus. Logistic regression analysis showed that areas posing an elevated risk of exposure to A. americanum nymphs or adults were more likely to be classified as forested than grassland, and the probability of being classified as elevated risk increased with increasing relative humidity during the month of June (30-year average). Overall accuracy of each of the two models was greater than 70% and showed that 20% and 30% of the state were classified as elevated risk for human exposure to nymphs and adults, respectively. We also found a significant positive association between heightened acarologic risk and counties reporting tularemia cases. Our study provides an updated distribution map for A. americanum in Missouri and suggests a wide-spread risk of human exposure to A. americanum and their associated pathogens in this region.

Functional Analysis of the Borrelia burgdorferi bba64 Gene Product in Murine Infection via Tick Infestation

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 mutants 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 hosts 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.

A sustained-release formulation of doxycycline hyclate (Atridox) prevents simultaneous infection of Anaplasma phagocytophilum and Borrelia burgdorferi transmitted by tick bite

Current prophylaxis for infected tick bites consists of personal protective measures directed towards ticks. This study compared the efficacy of a single oral dose of doxycycline with that of a single injection of sustained-release doxycycline in a model of Lyme borreliosis and Anaplasma phagocytophilum infection. Dosages of doxycycline were equilibrated based on previously determined peak plasma levels in mice [oral, 2.4 microg (ml plasma)(-1); sustained release, 1.9 microg (ml plasma)(-1)] determined 8 h after inoculation. In challenge experiments where five Borrelia burgdorferi-infected and five A. phagocytophilum-infected nymphs were used per mouse, only 20 and 30 % of mice were protected from B. burgdorferi and A. phagocytophilum infection, respectively, using oral doxycycline. In contrast, 100 % of mice receiving sustained-release doxycycline were protected from A. phagocytophilum infection, as indicated by real-time PCR of blood samples, quantitative PCR and culture isolation of spleen samples, and protected against B. burgdorferi infection as demonstrated by culture of ear, heart and bladder. Although 15-40 copies of A. phagocytophilum could be amplified from the spleens of mice treated with sustained-release doxycycline, no viable A. phagocytophilum from these spleens could be cultured in HL-60 cells. In contrast, 7/10 mice receiving oral doxycycline were PCR- and culture-positive for A. phagocytophilum, with copy numbers ranging from 800 to 10 000 within the spleen, as determined by quantitative PCR. Other correlates with A. phagocytophilum infection included a significant difference in spleen mass (mean of 110 mg for sustained-release treatment versus a mean of 230 mg for oral treatment) and the number of splenic lymphoid nodules (mean of 8 for sustained-release treatment versus mean of 12.5 for oral doxycycline) as determined by histopathology. These studies indicate that a single injection of a sustained-release formulation antibiotic may offer a viable prophylactic treatment option for multiple infectious agents in patients presenting with tick bites.

Multiple Francisella tularensis Subspecies and Clades, Tularemia Outbreak, Utah

In July 2007, a deer fly–associated outbreak of tularemia occurred in Utah. Human infections were caused by 2 clades (A1 and A2) of Francisella tularensis subsp. tularensis. Lagomorph carcasses from the area yielded evidence of infection with A1 and A2, as well as F. tularensis subsp. holarctica. These findings indicate that multiple subspecies and clades can cause disease in a localized outbreak of tularemia.