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Internal and external mycoflora of the American dog tick, Dermacentor variabilis (Acari: Ixodidae), and its ecological implications.

Scopulariopsis brevicaulis, the anamorph of Microascus brevicaulis (Microascaceae, Ascomycota), has been identified in the body contents of the tick Dermacentor variabilis. After topical application of the fungal inoculum, tick mortality was marked. This is the first account describing the internal mycoflora of D. variabilis with a novel technique used to recover potential biological control agents.

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Sequence and expression analysis of the ompA gene of Rickettsia peacockii, an endosymbiont of the Rocky Mountain wood tick, Dermacentor andersoni.

The transmission dynamics of Rocky Mountain spotted fever in Montana appears to be regulated by Rickettsia peacockii, a tick symbiotic rickettsia that interferes with transmission of virulent Rickettsia rickettsii. To elucidate the molecular relationships between the two rickettsiae and glean information on how to possibly exploit this interference phenomenon, we studied a major rickettsial outer membrane protein gene, ompA, presumed to be involved in infection and pathogenesis of spotted fever group rickettsiae (SFGR) but which is not expressed in the symbiont. Based on PCR amplification and DNA sequence analysis of the SFGR ompA gene, we demonstrate that R. peacockii is the most closely related of all known SFGR to R. rickettsii. We show that R. peacockii, originally described as East Side agent in Dermacentor andersoni ticks from the east side of the Bitterroot Valley in Montana, is still present in that tick population as well as in D. andersoni ticks collected at two widely separated locations in Colorado. The ompA genes of R. peacockii from these locations share three identical premature stop codons and a weakened ribosome binding site consensus sequence relative to ompA of R. rickettsii. The R. peacockii ompA promoter closely resembles that of R. rickettsii and is functional based on reverse transcription-PCR results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting showed that OmpA translation products were not detected in cultured tick cells infected with R. peacockii. Double immunolabeling studies revealed actin tail structures in tick cells infected with R. rickettsii strain Hlp#2 but not in cells infected with R. peacockii.

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Reactivation of Rickettsia rickettsii in Dermacentor andersoni ticks: an ultrastructural analysis.

Virulent Rickettsia in Dermacentor andersoni lose their pathogenicity and virulence for guinea pigs when subjected to physiological stresses, such as starvation (overwintering), of its tick vector. However, incubation of infected ticks at an elevated temperature (37 degrees C) for 24 to 48 h or feeding for a time (usually greater than 10 h) induces R. rickettsii to revert to a virulent state, a phenomenon defined as "reactivation." Electron microscopy reveals that the microcapsular and slime layers of R. rickettsii undergo changes dependent upon the physiological conditions within the tick vector. In engorged ticks, the microcapsular layer is readily identified as a discrete layer, approximately 16 nm thick, composed of globular subunits that have a periodicity of approximately 10 nm. The slime layer external to the microcapsular layer forms a discrete electron-lucent zone around the rickettsia. In starved ticks, neither the microcapsular layer nor slime layer remains a discrete entity. Instead, they are shed and form stringy, shredded, and somewhat flocculent strands of low electron density without periodicity. Incubation at 37 degrees C or feeding of starved infected ticks results in the restoration of a discrete microcapsular and slime layer. These reversible structural modifications are linked to physiological changes in the tick host and correlate with reactivation, i.e., restoration of pathogenicity and virulence of R. rickettsii.

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Identification of endemic foci of Lyme disease: isolation of Borrelia burgdorferi from feral rodents and ticks (Dermacentor variabilis).

Borrelia burgdorferi, the etiological agent of Lyme disease, was isolated from the blood, kidneys, spleens, eyes, or livers of white-footed mice (Peromyscus leucopus) (n = 19 of 22) and from the blood, kidneys, or spleens of eastern chipmunks (Tamias striatus) (n = 2 of 2) captured at three foci for Lyme disease in eastern Connecticut. These bacteria were cultured most frequently from spleens (n = 19) and kidneys (n = 15). B. burgdorferi persisted in one mouse for at least 60 days. One spirochetemic mouse had infected Ixodes dammini and Dermacentor variabilis larvae attached, suggesting that these ticks may have acquired spirochetes from the host. Spirochetes isolated from P. leucopus, T. striatus, and D. variabilis larvae were serologically and genetically indistinguishable from reference B. burgdorferi isolates. We conclude that isolation of spirochetes from feral rodents is a method for identifying endemic areas of Lyme disease.

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Expression, inheritance, and linkage relationships among eight enzyme genes in Dermacentor albipictus (Packard) (Acarina: Ixodidae).

Starch gel electrophoresis was used to examine the inheritance, expression, and linkage relationships among eight enzyme genes in the winter tick, Dermacentor albipictus. A fructose-specific hexokinase (FHK), adenylate kinase (ADK), and two forms of aconitase (ACON-A, ACON-C) appeared to have monomeric quaternary structures. A glycylleucine peptidase (PEP), isocitrate dehydrogenase (IDH), and anodally migrating malate dehydrogenase (MDH-A) were apparently dimers. The quaternary structure of glucose phosphate isomerase (GPI) could not be determined because of the similarity in relative mobility of the two available electromorphs. The genes for GPI, FHK, and ADK are located on the X chromosome in the following order: Adk - 37.4 - Gpi - 24.6 - Fhk, with Adk - Fhk being 46.5 map units apart. The remaining five genes were autosomally inherited. Of the 10 possible paired combinations of these genes, only the data for two pairs, Idh-Mdh (44.5% recombinants) and Acon-A--Acon-C (46.4% recombinants), suggested statistically significant linkage.

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Prevalence data of Rickettsia slovaca and other SFG Rickettsiae species in Dermacentor marginatus in the southeastern Iberian peninsula.

In southern Spain, Dermacentor marginatus ticks can be infected with several genospecies of spotted fever Group (SFG) Rickettsia. We developed a nested polymerase chain reaction assay by using a species-specific probe targeting the ompA gene to detect and differentiate between the two groups of rickettsiae previously described in D. marginatus. SFG rickettsia has been detected in 85.15% of ticks studied (26.7% of positives have been to R. slovaca, the causative agent of TIBOLA-DEBONEL, and 73.3% to SFG rickettsia closely related to strains RpA4-JL-02-DnS14-DnS28).

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Detection of equine Babesia spp. gene fragments in Dermacentor nuttalli Olenev 1929 infesting mongolian horses, and their amplification in egg and larval progenies.

Babesia equi (EMA-1) and Babesia caballi (BC48) gene fragments were amplified by polymerase chain reaction (PCR), in blood samples, and partially fed-females and egg and larval progenies of Dermacentor nuttalli, collected from horses in Altanbulag, Tuv Province, Mongolia. While Babesia parasite DNA was detected in some horse blood samples during the first PCR, all positive cases in partially fed-female ticks, eggs and larvae were confirmed by nested PCR. Present study reinforces earlier similar findings in unfed D. nuttalli ticks collected from an open space vegetation in Bayanonjuul, Tuv Province in Central Mongolia, pointing to the most likely important role of D. nuttalli in the transmission of equine babesiosis in Mongolia. The detection of parasite DNA in eggs and larval progenies is likewise suggestive of transovarial parasite transmission in this tick species.

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Relative efficiency of biological transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni (Acari: Ixodidae) compared with mechanical transmission by Stomoxys calcitrans (Diptera: Muscidae).

Anaplasma marginale Theiler is a tick-borne intraerythrocytic rickettsial pathogen of cattle that also can be mechanically transmitted by biting flies. Rickettsemia during the acute phase of infection may reach as high as 10(9) infected erythrocytes (IEs) per milliliter of blood. Animals that survive acute infection develop a life-long persistent infection that cycles between 10(2.5) and 10(7) IE/ ml of blood. We compared stable fly Stomoxys calcitrans (L.) -borne mechanical transmission during acute infection with Rocky Mountain wood tick, Dermacentor andersoni Stiles-borne biological transmission in the persistent phase of infection to demonstrate quantitatively that biological transmission by ticks is considerably more efficient than mechanical transmission by biting flies. Stable flies that partially fed on an acutely infected calf and were immediately transferred to susceptible calves to complete their bloodmeals failed to transmit A. marginale. Ticks that fed on the original acquisition host after it reached the persistent phase of infection (>300-fold lower rickettsemia) successfully transmitted A. marginale after transfer to the same calves that failed to acquire infection after fly feeding. Failure of fly-borne mechanical transmission at a rickettsemia >300-fold higher than that from which ticks transmit with 100% efficiency demonstrates that tick-borne biological transmission is at least 2 orders of magnitude more efficient than direct stable fly-borne mechanical transmission.

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County-level surveillance of white-tailed deer infestation by Ixodes scapularis and Dermacentor albipictus (Acari: Ixodidae) along the Illinois River.

From 1998 to 2003, 4,935 hunter-killed deer in northern and central Illinois were examined for ticks; 4,066 blacklegged ticks, Ixodes scapularis Say, and 6,530 winter ticks, Dermacentor albipictus (Packard) (Acari: Ixodidae), were collected. I. scapularis was the predominant tick species in the northern portion of the study area, with a decreasing north-to-south prevalence gradient. In contrast, D. albipictus was more common in the south with a decreasing south-to-north prevalence gradient. Compared with previous studies, the geographic range for both species expanded into the central portion of the Illinois River. Prevalence and intensity of both tick species were greater on bucks, and infested bucks were geographically more widespread than infested does and fawns. These findings indicate that blacklegged tick and winter tick distributions remain dynamic in the north central United States

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A Canadian bison isolate of Anaplasma marginale (Rickettsiales: Anaplasmataceae) is not transmissible by Dermacentor andersoni (Acari: Ixodidae), whereas ticks from two Canadian D. andersoni populations are competent vectors of a U.S. strain.

Anaplasma marginale Theiler is a tick-borne rickettsial pathogen of cattle with a global distribution in both temperate and tropical regions. The pathogen is endemic in regions within the United States, whereas the Canadian cattle population is considered to be free ofA. marginale. Farmed bison, Bison bison L., in central Saskatchewan have been found to be infected with A. marginale; however, there is no evidence of transmission from bison to cattle. We tested a Saskatchewan bison isolate of A. marginale (SB1) to determine whether it is transmissible by the Rocky Mountain wood tick, Dermacentor andersoni Stiles. Colonized D. andersoni from the United States and Canada failed to transmit SB1. A separate transmission trial using D. andersoni adults reared from ticks collected in Alberta and British Columbia showed that ticks from these populations could successfully transmit the St. Maries, Idaho, strain of A. marginale. Although the Saskatchewan bison isolate of A. marginale seems not to be transmissible by D. andersoni, in the event of the introduction of a tick-transmissible strain, Canadian D. andersoni are likely to be competent vectors.

Anaplasma marginale↗

Climbing simulated vegetation to heights of ungulate hosts by larvae of Dermacentor albipictus (Acari: Ixodidae).

Larvae of winter ticks, Dermacentor albipictus (Packard), ascend vegetation in autumn and form clumps that attach to passing ungulate hosts. We tested the hypothesis that vegetation height determines the height of clumps. During the vegetation-to-ungulate transmission period (early September to mid-November), larvae were released at the base of simulated vegetation (nylon rods 245 cm tall) in outdoor and laboratory trials and in the absence of host cues. Rod height exceeded the height of the tallest ungulate host, which is the moose, Alces alces (L.). Most larvae stopped climbing and formed clumps 50-190 cm above ground, which coincided with torso heights of moose; elk, Cervus elaphus L.; and deer, Odocoileus spp. Rafinesque. More clumps formed in outdoor trials than in laboratory trials and clump heights tended to increase over the course of the experiment, but clump number, size, and height did not correlate with weather conditions. Winter tick larvae appear to determine their height above ground in the absence of external cues, but this mechanism may be modified by external conditions.

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Relationship between weights of the engorged nymphal stage and resultant sexes in Ixodes scapularis and Dermacentor variabilis (Acari: Ixodidae) ticks.

The relationship between weights of the nymphal stage and their resultant sexes in Ixodes scapularis Say and Dermacentor variabilis (Say) was examined under laboratory conditions. Nymphs were fed to repletion on laboratory white mice, weighed individually, and held until they molted. Fully engorged I. scapularis nymphs that subsequently molted to females weighed between 3.8 and 6.4 mg (n = 64). Those nymphs that molted to males weighed between 2.0 and 3.2 mg (n = 57). Body weights of engorged nymphs that became females were significantly greater than those of nymphs that became males (P < 0.05). Similarly, the range of body weights of engorged D. variabilis nymphs that subsequently became females was 9.1-15.1 mg (n = 32), whereas those nymphs that molted to males weighed between 3.9 and 13.0 mg (n = 39). Body weights of engorged nymphs that became females were significantly greater than those of nymphs that became males (P < 0.05). This suggests that nymphs of both species that become females presumably imbibe more blood than those that became males.

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Response of the tick Dermacentor variabilis (Acari: Ixodidae) to hemocoelic inoculation of Borrelia burgdorferi (Spirochetales).

When Borrelia burgdorferi B31 low passage strain spirochetes were directly injected into the hemocoel of Dermacentor variabilis (Say) females, the bacteria were cleared from the hemocoel within < 24 h. Viable spirochetes were not found in hemolymph, salivary gland, or ovary tissues by subculturing or by IFA. The hemocyte population increased approximately 6 times within the first 6 h after inoculation compared with the uninoculated controls. In contrast, the soluble total hemolymph protein content decreased inversely with the increase in hemocytes. Borreliacidal activity was demonstrated with cell-free hemolymph from D. variabilis. In vitro antimicrobial assays using hemolymph from borrelia-challenged and nonchallenged ticks resulted in 72% spirochete reductions compared with only 11.5%, respectively, within 1 h. Additional evidence of induced antimicrobial hemolymph protein activity was demonstrated by SDS-PAGE, which revealed upregulation of a lysozyme-like peptide (approximately 15 kDa) (22% increase) and the induction of a approximately 5.8 kDa peptide in the B. burgdorferi-challenged ticks. In contrast with the nonvector borne Bacillus subtilis, D. variabilis presented a rapid and robust response to challenge with cultured B. burgdorferi spirochetes and lead to their early elimination. The role of the tick immune system, including possible differences between vector and nonvector ticks, in determining the success of invasive bacteria is discussed.

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Contrasts in tick innate immune responses to Borrelia burgdorferi challenge: immunotolerance in Ixodes scapularis versus immunocompetence in Dermacentor variabilis (Acari: Ixodidae).

The blacklegged tick, Ixodes scapularis Say, transmits the Lyme disease spirochete Borrelia burgdorferi, whereas the American dog tick, Dermacentor variabilis (Say), is unable to transmit the bacterium. We compared the innate immune response of these ticks against spirochetes directly inoculated into the hemocoel cavity of ticks. In I. scapularis, some Borrelia were found associated with hemocytes, while numerous other spiral-shaped, intact bacteria remained free in the hemolymph. In contrast, in D. variabilis only remnants of the bacteria were evident in the hemolymph, indicating lysis; intact spirochetes were rare. Spirochetes were observed bound to or within the organs of both tick species, although many more spirochetes were found associated with the I. scapularis organs. The few spirochetes observed with the D. variabilis organs appeared to be dead because D. variabilis tissues rarely contained culturable bacteria, unlike I. scapularis tissues. When spirochetes were incubated with I. scapularis hemolymph plasma in vitro, bacterial survival and motility were not reduced. In contrast, incubation of spirochetes with D. variabilis hemolymph plasma resulted in > 50% of the spirochetes becoming nonmotile by 45 min. The differences in the responses of the two different tick species indicate that I. scapularis is immunotolerant when challenged with B. burgdorferi and dependent on a slow phagocytic response to clear Borrelia from the hemolymph. In contrast, D. variabilis is highly immunocompetent (i.e., innate immunity), using plasma borreliacidal factors and a rapid increase in phagocytic cells to clear the infection and limit tissue invasion.

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Spotted fever group Rickettsia in Dermacentor variabilis (Acari: Ixodidae) infesting raccoons (Carnivora: Procyonidae) and opossums (Marsupialia: Didelphimorphidae) in Tennessee.

Spotted fever group Rickettsia were present in 13 of 1,171 (1.1%) of adult Dermacentor variabilis Say removed from raccoons (Procyon lotor L.) and Virginia opossums (Didelphis virginiana Kerr) in western Tennessee. Spotted fever group Rickettsia were detected by amplification of a 617-bp segment of the citrate synthase gene by polymerase chain reaction.

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Rickettsial infection in Dermacentor variabilis (Acari: Ixodidae) inhibits transovarial transmission of a second Rickettsia.

This study examined the ability of ticks to maintain multiple species of spotted fever group rickettsiae via transovarial transmission. Using a capillary feeding method, previously established Ricketisia montana- and Rickettsia rhipicephali-infected cohorts of Dermacentor variabilis (Say) were exposed to R. rhipicephali and R. montana, respectively, in two reciprocal challenge experiments. Eggs collected from individual females, for two successive generations, of each cohort were assessed for rickettsial infection by polymerase chain reaction for each challenge experiment. Assessment of the eggs from challenged ticks identified that both B. montana- and R. rhipicephali-infected ticks were refractory to their respective challenge rickettsiae. The prechallenged infection rate for both F1 and F2 generations (100%) of the B. montana-infected cohort was resistant to transovarial transmission of the second rickettsia species, and only R. montana was detected in the eggs of FL = (50%) and F2 = (74%) challenged females. The R. rhipicephali-infected cohort maintained a lower level of infection (20%) in the population and did not transovarially transmit the challenge species, however, detectable levels of infection were lost after the first generation. Second-generation ticks, no longer infected with B. rhipicephali, became susceptible to infection with R. montana and female ticks (approximately 4%) were able to transmit R. montana to their progeny. The resistance of the ovaries to co-infection and apparent host-specific nature of infection suggests that rickettsial infection of tick ovaries may alter the molecular expression of the oocytes so as to preclude secondary infection with other rickettsiae.

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Identification of 2,4-dichlorophenol in females of the American dog tick, Dermacentor variabilis (Acari: Ixodidae), and its possible role as a component of the attractant sex pheromone.

Presence of 2,4-dichlorophenol (2,4-DCP) in extracts of fed and nonfed adult female Dermacentor variabilis (Say) by gas chromatography and mass spectroscopy (GC/MS) is reported. 2,4-DCP was detected as part of a mixture with 2,6-dichlorophenol, which is the attractant sex pheromone in this species in a 1:9 ratio, respectively. Extracts did not contain the monochlorinated phenols, 2-chlorophenol and 4-chlorophenol. None of these chlorophenols were detected in eggs. 2,4-DCP is comparable in efficacy to 2,6-DCP as an attractant sex pheromone, prompting attraction, arrestment and feeding posture behavior by fed males. It is also present in the female adult.

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