Isolation of a Spotted Fever Group Rickettsia, Rickettsia peacockii, in a Rocky Mountain Wood Tick, Dermacentor andersoni, Cell Line

An embryonic cell line (DAE100) of the Rocky Mountain wood tick, Dermacentor andersoni, was observed by microscopy to be chronically infected with a rickettsialike organism. The organism was identified as a spotted fever group (SFG) rickettsia by PCR amplification and sequencing of portions of the 16S rRNA, citrate synthase, Rickettsia genus-specific 17-kDa antigen, and SFG-specific 190-kDa outer membrane protein A (rOmpA) genes. Sequence analysis of a partial rompA gene PCR fragment and indirect fluorescent antibody data for rOmpA and rOmpB indicated that this rickettsia was a strain (DaE100R) of Rickettsia peacockii, an SFG species presumed to be avirulent for both ticks and mammals. R. peacockii was successfully maintained in a continuous culture of DAE100 cells without apparent adverse effects on the host cells. Establishing cell lines from embryonic tissues of ticks offers an alternative technique for isolation of rickettsiae that are transovarially transmitted.     

Isolation and characterization of symbiotes from the Rocky Mountain wood tick, Dermacentor andersoni

Wolbachia-like symbiotes in the Rocky Mountain wood tick, Dermacentor andersoni, were isolated repeatedly by injection of ovarial tissues into 5-day-old chick embryos. In Giemsa-stained smears of infected embryo tissues, the organisms appeared as blueish or pinkstained coccal bodies indistinguishable from those seen in the ovaries of ticks, where they are located in the luminal epithelium and funicle cells, as well as in oocytes.Electron microscopy revealed that these symbiotes are highly pleomorphic and vary in size from 0.6 to 3.4 μm in diameter. Their fine structure in tissue cells is differentiated into a granular, cortical region, which contains densely stained ribosomes, and a medullary region consisting of a diffuse reticulum partially or completely devoid of granular material or ribosomes. Multiplication is by binary fission. Each organism is delimited by a distinct plasmalemma; a cell wall as in bacterial and rickettsial agents was not observed in organisms from ovarial tissues.Symbiotes cultivated in chick embryos and then injected intracoelomically into adult D. andersoni, developed rapidly and produced massive infestations in hemocytes, hypodermal tissues, salivary glands, and in connective tissues surrounding midgut, Malpighian tubules, and ovary. In hypodermal tissue, organisms with a distinct bilayered cell envelope were occasionally detected. The massive invasion of tissues by injected symbiotes invariably proved fatal for ticks.Results of complement-fixation tests and of fluorescent antibody staining indicated that symbiotes in D. andersoni are closely related to Wolbachia persica, previously isolated from Argas arboreus.     

Factors influencing in vitro infectivity and growth of Rickettsia peacockii (Rickettsiales: Rickettsiaceae), an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni (Acari, Ixodidae)

Rickettsia peacockii, a spotted fever group rickettsia, is a transovarially transmitted endosymbiont of Rocky Mountain wood ticks, Dermacentor andersoni. This rickettsia, formerly known as the East Side Agent and restricted to female ticks, was detected in a chronically infected embryonic cell line, DAE100, from D. andersoni. We examined infectivity, ability to induce cytopathic effect (CPE) and host cell specificity of R. peacockii using cultured arthropod and mammalian cells. Aposymbiotic DAE100 cells were obtained using oxytetracycline or incubation at 37 degrees C. Uninfected DAE100 sublines grew faster than the parent line, indicating R. peacockii regulation of host cell growth. Nevertheless, DAE100 cellular defenses exerted partial control over R. peacockii growth. Rickettsiae existed free in the cytosol of DAE100 cells or within autophagolysosomes. Exocytosed rickettsiae accumulated in the medium and were occasionally contained within host membranes. R. peacockii multiplied in other cell lines from the hard ticks D. andersoni, Dermacentor albipictus, Ixodes scapularis, and Ixodes ricinus; the soft tick Carios capensis; and the lepidopteran Trichoplusia ni. Lines from the tick Amblyomma americanum, the mosquito Aedes albopictus, and two mammalian cell lines were non-permissive to R. peacockii. High cell densities facilitated rickettsial spread within permissive cell cultures, and an inoculum of one infected to nine uninfected cells resulted in the greatest yield of infected tick cells. Cell-free R. peacockii also were infectious for tick cells and centrifugation onto cell layers enhanced infectivity approximately 100-fold. The ability of R. peacockii to cause mild CPE suggests that its pathogenicity is not completely muted. An analysis of R. peacockii-cell interactions in comparison to pathogenic rickettsiae will provide insights into host cell colonization mechanisms.     

Abnormal morphology of an adult Rocky Mountain wood tick, Dermacentor andersoni (Acari: Ixodidae)

During a collection of ticks from vegetation in March 2006, a single adult male Rocky Mountain wood tick, Dermacentor andersoni (Stiles, 1908), was collected that exhibited unique morphological anomalies, including the absence of a leg on the right side of the body. Coxa IV on the right side also was missing in this specimen. Such teratological changes have not been reported previously for D. andersoni.     

Experimental infection of the tick Amblyomma cajennense, Cayenne tick, with Rickettsia rickettsii, the agent of Rocky Mountain spotted fever

In the laboratory, Amblyomma cajennense (Acari: Ixodidae) (Fabricius) larvae, nymphs and adults were exposed to Rickettsia rickettsii by feeding on needle-inoculated animals, and thereafter reared on uninfected guinea pigs or rabbits. Regardless of the tick stage that acquired the infection, subsequent tick stages were shown to be infected (confirming transstadial and transovarial transmissions) and were able to transmit R. rickettsii to uninfected animals, as demonstrated by serological and molecular analyses. However, the larval, nymphal and adult stages of A. cajennense were shown to be partially refractory to R. rickettsii infection, as in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. In addition, less than 50% of the infected engorged females transmitted rickettsiae transovarially, and when they did so, only part of the offspring became infected, indicating that vertical transmission alone is not enough to maintain R. rickettsii in A. cajennense for multiple generations. Finally, the R. rickettsii-infected tick groups had lower reproductive performance than the uninfected control group. Our results indicate that A. cajennense have a low efficiency to maintain R. rickettsii for successive generations, as R. rickettsii-infection rates should decline drastically throughout the successive tick generations.     

Indicators for elevated risk of human exposure to host-seeking adults of the Rocky Mountain wood tick (Dermacentor andersoni) in Colorado

The human-biting adult stage of the Rocky Mountain wood tick (Dermacentor andersoni) can cause tick paralysis in humans and domestic animals and is the primary tick vector in the intermountain west of the pathogens causing Colorado tick fever, Rocky Mountain spotted fever, and tularemia. We conducted drag sampling studies in Poudre Canyon and Rocky Mountain National Park of Larimer County, CO, to determine microhabitat use patterns by host-seeking D. andersoni adults and find environmental factors signaling elevated risk of tick exposure. Big sagebrush (Artemisia tridentata) was found to serve as a general indicator of areas with elevated risk of exposure to host-seeking D. andersoni adults; this likely results from a shared climate tolerance of big sagebrush and D. andersoni. Grass was the favored substrate for host-seeking ticks. Drag sampling of open grass or grass bordering rock or shrub produced abundances of D. andersoni adults significantly higher than sampling of brush. Sampling sites in Rocky Mountain National Park, relative to Poudre Canyon, were characterized by more intense usage by elk (Cervus elaphus) but decreased brush coverage, smaller brush size, and lower abundances of host-seeking D. andersoni adults. There has been a tremendous increase in the population of elk in Rocky Mountain National Park over the last decades and we speculate that this has resulted in an ecological cascade where overgrazing of vegetation by elk is followed by suppression of rodent populations, decreased tick abundance, and, ultimately, reduced risk of human exposure to D. andersoni and its associated pathogens.     

Antibodies to Rickettsia rickettsii in Peromyscus leucopus from a focus of Rocky Mountain spotted fever in Connecticut

During 1980-1982, white-footed mice (Peromyscus leucopus) were captured in Newtown, Connecticut, an area where Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, is thought to be enzootic. An indirect microimmunofluorescence test identified specific antibodies to this organism in 16 of 237 (7%) sera: titration end points for 14 samples were relatively high (1:128-1:2048). Antibodies were detected in mice during 1980 and 1981 with monthly prevalences varying from 8 to 22%. These results suggest that P. leucopus may be involved in the ecology of R. rickettsii and that these rodents can be included along with other mammals to monitor spotted fever rickettsial infections in nature.     

Developmental profiles in tick water balance with a focus on the new Rocky Mountain spotted fever vector, Rhipicephalus sanguineus

Recent reports indicate that the common brown dog tick, or kennel tick, Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) is a competent vector of Rocky Mountain spotted fever in the U.S.A. This tick is of concern to public health because of its high frequency of contact, as it has a unique ability to thrive within human homes. To assess the moisture requirements necessary for survival, water balance characteristics were determined for each developmental stage, from egg to adult. This is the first time that water relations in ticks have been assessed throughout the complete lifecycle. Notably, R. sanguineus is differentially adapted for life in a dry environment, as characterized by a suppressed water loss rate distinctive for each stage that distinguishes it from other ticks. Analysis of its dehydration tolerance limit and percentage body water content provides no evidence to suggest that the various stages of this tick can function more effectively containing less water, indicating that this species is modified for water conservation, not desiccation hardiness. All stages, eggs excepted, absorb water vapour from the air and can drink free water to replenish water stores. Developmentally, a shift in water balance strategies occurs in the transition from the larva, where the emphasis is on water gain (water vapour absorption from drier air), to the adult, where the emphasis is on water retention (low water loss rate). These results on the xerophilic-nature of R. sanguineus identify overhydration as the primary water stress, indicating that this tick is less dependent upon a moisture-rich habitat for survival, which matches its preference for a dry environment. We suggest that the controlled, host-confined conditions of homes and kennels have played a key role in promoting the ubiquitous distribution of R. sanguineus by creating isolated arid environments that enable this tick to establish within regions that are unfavourable for maintaining water balance.     

Fine Structure of Rickettsia canada in Tissues of Dermacentor andersoni Stiles

Preliminary observations on growth and developmental fine structure of Rickettsia canada in various organs and tissues of the hard tick, Dermacentor andersoni Stiles, are reported. R. canada is typically rod-shaped, being delimited with a three-layered wall having a velvety coating adsorbed to its exterior surface. A finely reticulated cytoplasmic matrix containing prominent ribosomes is delimited with a three-layered unit membrane. Average length and width of these organisms are 1.6 by 0.4 mum. Although R. canada produces a generalized infection in D. andersoni, hypodermal and muscle tissues experience heaviest growth. Three morphologically distinct rickettsial forms were observed in individual hypodermal cells: (i) typical growth forms with a finely reticulated cytoplasmic matrix and distinct ribosomes; (ii) atypical forms with lightly to densely staining cytoplasm and a coagulated appearance in which ribosomes cannot be distinguished from the matrix; and (iii) forms with crystalline bodies that have a striated to beaded lattice structure and, at times, a fibrillar body in the cytoplasm as well. Occasional finger-like to irregular invaginations of the plasma membrane are noted. Intranuclear growth was demonstrated by electron microscopy in gut epithelial cells only. Growth and development of R. canada were manifest in all tissues examined.