Computer simulation of management strategies for American dog ticks (Acari: Ixodidae) and Rocky Mountain spotted fever

Computer models were developed to simulate the effects of management technologies on populations of the American dog tick, Dermacentor variabilis (Say), principal vector of Rocky Mountain spotted fever (RMSF) in eastern North America. The technologies modeled were area-wide acaricide application, acaricide-food-baited tubes for self-treatment by small mammals, dipping of dogs in acaricides, acaricide-impregnated plastic dog collars, reduction of small mammal host populations (host management), and removal of vegetation that protects free-living tick stages (vegetative management). Submodels for each of these technologies were incorporated into a model (ADTSIM) for the population dynamics of the tick and RMSF transmission. Comparisons of simulated and observed data were used to verify reasonable accuracy of the submodels. Repetitive simulations were made to identify levels and timing of each control method (alone or combined) required to reduce tick populations below a RMSF transmission threshold of 252 unfed adults/ha. Eight to 30 acaricide applications, depending on acaricide and percentage of population treated, were needed during a 10-yr period to reduce densities of ticks below the threshold. The baited-tube method, host management, and vegetative management (depending on level and frequency of treatment) also were capable of reducing tick density below the threshold. However, acaricide-impregnated plastic dog collars did not reduce tick density below the threshold unless at least 50% of the hosts of adult ticks were domestic dogs. Integrated strategies were developed for management of ticks and RMSF in six selected states. These strategies reduced numbers of human cases of RMSF 90% or more by year 20 by maintaining tick densities between 100 and 252 unfed adults/ha.     

Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) in Amblyomma americanum (Acari: Ixodidae) from Kansas

The role of lone star ticks as vectors for Rocky Mountain spotted fever (RMSF) remains poorly described. We compared the entomological inoculation rates (EIRs) for Rickettsia spp. for representative sites in Missouri and Kansas, states that frequently report RMSF each year. Host-seeking ticks were collected during 2006 and pooled tick homogenates analyzed by polymerase chain reaction to detect probable R. rickettsii, with confirmation for multiple gene targets performed on individual ticks from pools that screened positive. Of 870 adult and nymphal lone star ticks, Amblyomma americanum (L.), 0.46% contained DNA of Rickettsia rickettsii. Interestingly, two of these positive ticks were concurrently infected by R. amblyommii. More than 90% of lone star tick pools contained R. amblyommii DNA. Of 169 dog ticks that were analyzed, none were infected by R. rickettsii. The entomological inoculation rate for spotted fever group (SFG) rickettsiae within lone star ticks was an order of magnitude greater than that for dog ticks. We conclude that lone star ticks may be epidemiologically significant vectors of Rocky Mountain spotted fever and of spotted fever group rickettsiae.     

Computer simulation of population dynamics of the American dog tick (Acari: Ixodidae)

A comprehensive computer model was developed for simulation of the population dynamics of the American dog tick (ADT), Dermacentor variabilis Say, in North America. The model simulates the effects of major environmental variables, such as ambient temperature, saturation deficit, kind of habitat, and host density, on ADT population dynamics in ecosystems with small mammals as hosts for immature ticks and medium-sized mammals or domestic dogs as hosts for adult ticks. General validity of the model was established by comparisons between simulated and actual population densities for a series of years at locations in Virginia, Maryland, and Massachusetts using actual weekly weather data for each year as a model input. Using historical-average weather data for 11 locations within the known geographic range of ADT and 3 locations outside this range, the model produced acceptable values for initial population growth rate and generation time, as well as realistic equilibrium population densities and seasonal activity patterns. This model can be used as a framework for additional modeling efforts to simulate the transmission of Rocky Mountain spotted fever and to study various strategies for management of ADT populations.     

Failure of ticks to transmit Scopulariopsis brevicaulis (Deuteromycota), a common filamentous fungal commensal of ticks

The capacity of ticks to transmit a fungus was examined by analyzing tick saliva, host tissue from feeding sites, and host blood for presence of Scopulariopsis brevicaulis (Sacc.) Bainier, an internal mycosymbiont of the American dog tick, Dermacentor variabilis (Say), and lone star tick, Amnblyomma americanum (L.). Although >85% of ticks were infected with S. brevicaulis, conidia presence was low (0-5% of samples) in microscopic observations and mycological culturing of saliva expressed from larvae, nymphs, and adults. Additionally, the recovery of S. brevicaulis from blood and tissue feeding sites from a rabbit where S. brevicaulis-positive adult ticks had attached and fed was not increased compared with control tissue where no feeding occurred, indicating that transmission does not occur by the blood-feeding route. Tick mouthparts were found to contain S. brevicaulis in addition to Penicillium glabrum (Wehmer) Westling, but these agents were sparse in isolations from the feeding sites, which makes it unlikely that ticks act frequently as a mechanical fungal vector.     

Computer simulation of Babesia bovis (Babes) and B. bigemina (Smith & Kilborne) transmission by Boophilus cattle ticks (Acari: Ixodidae).

A computer model was developed to simulate the processes involved in transmission of the cattle fever parasites Babesia bovis (Babes) and Babesia bigemina (Smith & Kilborne) between cattle and Boophilus ticks. The model of Babesia transmission was combined with a dynamic life history model for population dynamics of the tick vectors, Boophilus microplus (Canestrini) and B. annulatus (Say). Epidemiological parameters and relationships in the model include the reduction in fecundity of infected ticks, rate of transovarial transmission, effect of cattle type and inoculation rate on infectivity of cattle, variation of infected cattle recovery rate with age of infection, inoculation rate, and species of parasite. Some parameters in the model were fitted by iterative simulations to produce realistic rates of Babesia infection in larval ticks. Comparisons of simulated and reported epidemiological data from one location in Australia indicated a reasonable level of validity for the model. Theoretical tick density thresholds for maintenance of Babesia in cattle and for inoculation of greater than or equal to 99.5% calves were determined by iterative simulations at 10 locations with B. microplus and six locations with B. annulatus. The model and transmission thresholds can serve as the basis for further simulation studies on strategies for control or eradication of babesiosis.     

Infestation of Peromyscus leucopus and Tamias striatus by Ixodes scapularis (Acari: Ixodidae) in relation to the abundance of hosts and parasites

The risk of humans acquiring Lyme disease is a function of the local density of nymphal and adult ticks that are infected with Lyme disease spirochetes. This in turn, will be related to host-use patterns of ticks and to the densities of both juvenile ticks and their hosts. At a forested site in Dutchess County, NY, we quantified host-use patterns of larval and nymphal Ixodes scapularis Say infesting the 2 dominant vertebrate hosts, white-footed mice and eastern chipmunks, during a 3-yr period. Larval tick burdens were 2-3 times higher on mice than they were on chipmunks, whereas nymphal tick burdens were > 3 times higher on chipmunks than they were on mice. We used multiple regression analysis to examine juvenile tick and host densities as independent variables influencing tick burdens. The density of questing larval ticks was positively correlated with larval tick burdens on mice, whereas the density of questing nymphs was weakly related to nymphal burdens on either host. Effects of the densities of mice and chipmunks on tick burdens were strong in some years, but weak in others. Moreover, the sign of the regression coefficients changed from one year to the next. We argue that these results are inconsistent with a passive encounter model of host selection, and suggest instead that either tick behavior or host responses cause strong biases in the distribution of juvenile ticks on their hosts.     

Molecular typing of isolates of Rickettsia rickettsii by use of DNA sequencing of variable intergenic regions

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is found throughout the Americas, where it is associated with different animal reservoirs and tick vectors. No molecular typing system currently exists to allow for the robust differentiation of isolates of R. rickettsii. Analysis of eight completed genome sequences of rickettsial species revealed a high degree of sequence conservation within the coding regions of chromosomes in the genus. Intergenic regions between coding sequences should be under less selective pressure to maintain this conservation and thus should exhibit greater nucleotide polymorphisms. Utilizing these polymorphisms, we developed a molecular typing system that allows for the genetic differentiation of isolates of R. rickettsii. This typing system was applied to a collection of 38 different isolates collected from humans, animals, and tick vectors from different geographic locations. Serotypes 364D, from Dermacentor occidentalis ticks, and Hlp, from Haemaphysalis leporispalustris ticks, appear to be distinct genotypes that may not belong to the species R. rickettsii. We were also able to differentiate 36 historical isolates of R. rickettsii into three different phylogenetic clades containing seven different genotypes. This differentiation correlated well, but not perfectly, with the geographic origin and likely tick vectors associated with the isolates. The few apparent typing discrepancies found suggest that the molecular ecology of R. rickettsii needs more investigation.     

American canine hepatozoonosis

American canine hepatozoonosis (ACH) is a tick-borne disease that is spreading in the southeastern and south-central United States. Characterized by marked leukocytosis and periosteal bone proliferation, ACH is very debilitating and often fatal. Dogs acquire infection by ingesting nymphal or adult Gulf Coast ticks (Amblyomma maculatum) that, in a previous life stage, ingested the parasite in a blood meal taken from some vertebrate intermediate host. ACH is caused by the apicomplexan Hepatozoon americanum and has been differentiated from Old World canine hepatozoonosis caused by H. canis. Unlike H. canis, which is transmitted by the ubiquitous brown dog tick (Rhipicephalus sanguineus), H. americanum is essentially an accidental parasite of dogs, for which Gulf Coast ticks are not favored hosts. The geographic portrait of the disease parallels the known distribution of the Gulf Coast tick, which has expanded in recent years. Thus, the endemic cycle of H. americanum involves A. maculatum as definitive host and some vertebrate intermediate host(s) yet to be identified. Although coyotes (Canis latrans) are known to be infected, it is not known how important this host is in maintaining the endemic cycle. This review covers the biology of the parasite and of the tick that transmits it and contrasts ACH with classical canine hepatozoonosis. Clinical aspects of the disease are discussed, including diagnosis and treatment, and puzzling epidemiologic issues are examined. Brief consideration is given to the potential for ACH to be used as a model for study of angiogenesis and of hypertrophic osteoarthropathy.     

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.     

Ticks parasitizing dogs in northwestern Georgia

From January 1998 through September 1999, 324 dogs in three northwestern Georgia counties were examined for ticks. Six species of ticks were recovered. The three most commonly collected ticks were the American dog tick, Dermacentor variabilis (Say) (310 male male, 352 female female; prevalence, 97%; mean intensity 2.1); the brown dog tick, Rhipicephalus sanguineus (Latreille) (118 male male, 119 female female, 38 nymphs; prevalence, 22%; mean intensity, 3.8); and the lone star tick, Amblyomma americanum (L.) (8 male male 26 female female, 2 nymphs; prevalence, 5%; mean intensity, 2.4). Other ticks recovered were Ixodes cookei Packard (3 female female); the Gulf Coast tick, Amblyomma maculatum Koch (2 female female); and the blacklegged tick, Ixodes scapularis Say (1 female). Another adult female specimen of I scapularis was recovered from a cat, further reinforcing that this medically important tick is present in northwestern Georgia.     

Hemocytic rickettsia-like organisms in ticks: serologic reactivity with antisera to Ehrlichiae and detection of DNA of agent of human granulocytic ehrlichiosis by PCR.

Ixodid ticks were collected from Connecticut, Massachusetts, Missouri, Pennsylvania, Rhode Island, and British Columbia (Canada) during 1991 to 1994 to determine the prevalence of infection with hemocytic (blood cell), rickettsia-like organisms. Hemolymph obtained from these ticks was analyzed by direct and indirect fluorescent antibody (FA) staining methods with dog, horse, or human sera containing antibodies to Ehrlichia canis, Ehrlichia equi, or Rickettsia rickettsii. Of the 693 nymphal and adult Amblyomma americanum, Dermacentor variabilis, Ixodes scapularis, and Ixodes pacificus ticks tested with dog anti-E. canis antiserum, 209 (32.5%) contained hemocytic bacteria. The prevalence of infected ticks varied greatly with species and locale. In parallel tests of duplicate hemolymph preparations from adult I. scapularis ticks, the hemocytic organisms reacted positively with E. canis and/or E. equi antisera, including sera from persons who had granulocytic ehrlichiosis. In separate PCR analyses, DNA of the agent of human granulocytic ehrlichiosis was detected in 59 (50.0%) of 118 adult and in 1 of 2 nymphal I. scapularis ticks tested from Connecticut. There was no evidence of Ehrlichia chaffeensis DNA in these ticks. In indirect FA tests of hemolymph for spotted fever group rickettsiae, the overall prevalence of infection was less than 4%. Specificity tests of antigens and antisera used in these studies revealed no cross-reactivity between E. canis and E. equi or between any of the ehrlichial reagents and those of R. rickettsii. The geographic distribution of hemocytic microorganisms with shared antigens to Ehrlichia species or spotted fever group rickettsiae is widespread.     

Detection,characterization, and prediction of tick-borne disease foci

Tick-borne disease (TBD) transmission foci need to be characterized in space and time, and are often discontinuous on both scales. An active TBD focus is dependent on the fulfillment of three conditions: tick survival, pathogen survival and opportunities for human exposure. The essentials for tick survival include food sources, reproduction, and protection from environmental extremes. The pathogen survival kit includes sufficient densities of ticks and suitable reservoir hosts, and opportunities for transmission between them in order to maintain infection. Opportunities for human exposure depend on sufficient number of encounters between ticks and humans. Because tick foci need to be described on a range of spatial and temporal resolutions, data for such characterization include a variety of surveillance data, field and laboratory experimental data, as well as results of statistical and mathematical analysis and modeling. The application of new tools from molecular biology, geographic information systems (GIS), and satellite imagery, in conjunction with appropriate analytical methods allow for detection of unknown foci and prediction of new ones. A long-term multi-scale study of Ixodes scapularis and Lyme disease in the north-central U. S. is reviewed. Diverse surveillance methods of ticks, rodents, deer, canids and humans were coupled with environmental characterization in situ to create a habitat profile for Lyme disease ticks. Incorporating various digitized databases, a statistical model was used to develop a risk map for tick distribution in the region. The process of introduction and establishment of new tick foci along the Illinois River is described in relation to the known tick distribution and predictions of invasion based on the risk model.     

Molting success of Ixodes scapularis varies among individual blood meal hosts and species

The blacklegged tick (Ixodes scapularis) is an important vector of emerging human pathogens. It has three blood-feeding stages, as follows: larva, nymph, and adult. Owing to inefficient transovarial transmission, at least for the Lyme disease agent (Borrelia burgdorferi), larval ticks rarely hatch infected, but they can acquire infection during their larval blood meal. Nymphal ticks are primarily responsible for transmitting pathogens to hosts, including humans. The transition from uninfected host-seeking larva to infectious host-seeking nymph is therefore a key aspect of human risk of infection. It can be divided into a series of steps, as follows: finding a host, taking a blood meal, becoming infected, molting, and overwintering. The chance of succeeding in each of these steps may depend on the species identity of the blood meal host. We used a Bayesian method to estimate the molting success of larval I. scapularis collected from four commonly parasitized species of birds and eight commonly parasitized small and mid-sized mammals found in the forests of Dutchess County, New York. We show that molting success varies substantially among host species; white-footed mice, veeries, and gray catbirds support particularly high molting success, whereas ticks feeding on short-tailed shrews, robins, and wood thrushes were less successful. We also show that larval molting success varies substantially between individual blood meal hosts, and that this intraspecific variability is much higher in some species than in others. The causes of both inter- and intraspecific variation in molting success remain to be determined.     

Ticks and tick-borne diseases in Africa: a disease transmission model

A general disease transmission model is developed for a tick-borne disease for cattle. Both the tick and cow are possible hosts for the disease parasite. The two species are therefore classified as infected or uninfected. The tick individual is further classified as sedentary (off-host) or feeding (on-host). First conditions for the persistence of the tick population are investigated. A threshold quantity for the disease is derived which is dependent on both the tick and host population parameters and the two transmission rates: from ticks to cattle and vice versa. From the analysis persistence and non-persistence conditions for both the tick population and the disease are investigated. The effect of the presence of a second host species is introduced as an additional feature in the analysis.     

A mathematical model of the ecology of Lyme disease

A mathematical model of enzootic Lyme-disease transmission ina natural focus is presented. This model is based on the lifehistory of the vector tick Ixodes scapularis Say and the primaryreservoir host Peromyscus leucopus. Using this model, the thresholdcondition for the disease to be able to invade a nonenzooticregion is determined as a function of the various possible transmissionchains operating throughout the year. These expressions showthat the transmission chain in which ticks acquire the diseasefrom mice in the fall and transmit it back to mice as nymphsin the spring is the most important chain (contributing approximately87 % of the elasticity of the threshold for the parameter choicesexamined). Equilibrium disease levels were examined under theassumption of a constant tick population; these levels weredetermined as a function of tick and mouse density, the verticaltransmission rate, the infectivity of mice, and the survivorshipparameters of the ticks and of the tick-host contact rates.Vertical transmission has a disproportionately large effect,since unfed infected larval ticks have two opportunities tofeed on mice, rather than only one opportunity (as for a newlyinfected unfed nymph). Finally, a global sensitivity analysisbased on Latin hypercube sampling is performed, in which isshown the importance of quantifying the natural history of infectionin mice, and of elucidating the contribution of other hostsfor I.scapularis than mice.     

Ticks and biting insects infected with the etiologic agent of Lyme disease, Borrelia burgdorferi.

Members of 18 species of ticks, mosquitoes, horse flies, and deer flies were collected in southeastern Connecticut and tested by indirect fluorescent-antibody staining methods for Borrelia burgdorferi, the etiologic agent of Lyme disease. An infection rate of 36.2% (116 tested), recorded for immature Ixodes dammini, exceeded positivity values for all other arthropod species. Prevalence of infection for hematophagous insects ranged from 2.9% of 105 Hybomitra lasiophthalma to 14.3% of seven Hybomitra epistates. Infected I. dammini larvae and nymphs coexisted with infected Dermacentor variabilis (American dog tick) immatures on white-footed mice (Peromyscus leucopus), but unlike I. dammini, none of the 55 adult American dog ticks collected from vegetation harbored B. burgdorferi. Groups of 113 field-collected mosquitoes of Aedes canadensis and 43 Aedes stimulans were placed in cages with uninfected Syrian hamsters. Of these, 11 females of both species contained B. burgdorferi and had fed fully or partially from the hamsters. No spirochetes were isolated from the hamsters, but antibodies were produced in one test animal.     

Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.     

Persistently infected horses are reservoirs for intrastadial tick-borne transmission of the apicomplexan parasite Babesia equi

Tick-borne pathogens may be transmitted intrastadially and transstadially within a single vector generation as well as vertically between generations. Understanding the mode and relative efficiency of this transmission is required for infection control. In this study, we established that adult male Rhipicephalus microplus ticks efficiently acquire the protozoal pathogen Babesia equi during acute and persistent infections and transmit it intrastadially to naïve horses. Although the level of parasitemia during acquisition feeding affected the efficiency of the initial tick infection, infected ticks developed levels of ≥10⁴ organisms/pair of salivary glands independent of the level of parasitemia during acquisition feeding and successfully transmitted them, indicating that replication within the tick compensated for any initial differences in infectious dose and exceeded the threshold for transmission. During the development of B. equi parasites in the salivary gland granular acini, the parasites expressed levels of paralogous surface proteins significantly different from those expressed by intraerythrocytic parasites from the mammalian host. In contrast to the successful intrastadial transmission, adult female R. microplus ticks that fed on horses with high parasitemia passed the parasite vertically into the eggs with low efficiency, and the subsequent generation (larvae, nymphs, and adults) failed to transmit B. equi parasites to naïve horses. The data demonstrated that intrastadial but not transovarial transmission is an efficient mode for B. equi transmission and that persistently infected horses are an important reservoir for transmission. Consequently, R. microplus male ticks and persistently infected horses should be targeted for disease control.     

Transmission efficiency of Francisella tularensis by adult american dog ticks (Acari: Ixodidae)

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.     

A call for renewed research on tick-borne Francisella tularensis in the Arkansas-Missouri primary national focus of tularemia in humans

Arkansas-Missouri has emerged as the primary U.S. focus of tularemia, which is caused by the National Institute of Allergy and Infectious Diseases Category A Priority Pathogen Francisella tularensis, over the past 30 yr. There are several pieces of indirect evidence suggesting that a key role of ticks in the transmission of F. tularensis to humans in Arkansas-Missouri is the primary reason why tularemia has remained a prominent disease of humans in this two-state area while fading away from other central or eastern states after a general decline in rabbit-associated tularemia cases. The primary tick vector(s) in Arkansas-Missouri can, based on a comparison of seasonal patterns of human tularemia cases and peak host-seeking activity of commonly human-biting tick species and life stages, be narrowed down to Amblyomma americanum (L.) nymphs, A. americanum adults, or Dermacentor variabilis (Say) adults. Unfortunately, currently available data cannot be used to further elucidate the relative roles of these ticks as vectors of F. tularensis to humans in Arkansas-Missouri. To address the fact that we do not know which tick species is the primary vector of F. tularensis to humans in the most prominent U.S. focus of tularemia, we need to determine (1) relative contributions of different tick species and life stages as human biters in Arkansas-Missouri; (2) natural rates of infection with F. tularensis tularensis (type A) and F. tularensis holarctica (type B) of the most prominent human-biting ticks in areas of Arkansas-Missouri hyperendemic for tularemia; (3) experimental vector efficiency of these ticks for both F. tularensis tularensis and F. tularensis holarctica; and (4) presence of infection with F. tularensis tularensis or F tularensis holarctica in ticks collected from humans in Arkansas-Missouri.