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Ticks transmit the agents of important human diseases, such as Lyme disease, babesiosis, Rocky Mountain spotted fever, ehrlichiosis, and tick-borne encephalitis, among others (4, 5, 16). Therefore, the study of the host immunity to ticks and host immunity's possible role in disease prevention has been a focus of extensive investigation. Trager demonstrated host immunity to ticks in 1939 (20), and several authors subsequently reproduced and extended these findings (21). Tick immunity, induced by repeated tick exposures, has been shown to interfere with tick feeding and molting in rabbits, cattle, dogs, and guinea pigs (1, 3, 10, 12, 13). Even a natural host like Clethrionomys glareolus, the bank vole, develops resistance to Ixodes ricinus with repeated infestations (6).

Tick immunity can interfere with pathogen transmission. Rabbits preexposed to uninfected Dermacentor andersoni were shown to be partially protected when exposed to Francisella tularensis-infected nymphs (2). Transmission of tick-borne Babesia argentina was shown to be impaired in tick-immune cattle (11). Moreover, recent reports indicate that tick immunity prevents transmission of the Lyme disease spirochete, Borrelia burgdorferi. Immunity to I. ricinus in C. glareolus reduced the efficiency of I. ricinus-mediated B. burgdorferi transmission (7). Although laboratory mice, compared to larger animals, do not readily develop immunity to ticks after repeated exposures, a tick infestation-induced partial host resistance to tick-borne B. burgdorferi transmission has been reported in BALB/c mice (22).

Recently, we showed that transmission of B. burgdorferi was prevented in tick-immune guinea pigs (17). To determine whether tick immunity interrupts the transmission of other vector-borne pathogens, we now examine the effect of tick immunity on the transmission of the agent of human granulocytic ehrlichiosis (aoHGE). aoHGE, like B. burgdorferi, is present in Ixodes scapularis: the reported vector coinfection rate may be as high as 26% at a focus of Lyme disease hyperendemicity (18). We chose aoHGE because this organism resides in the salivary glands of ticks (19), whereas B. burgdorferi is present in the guts of unfed ticks and only migrates to the salivary glands following prolonged feeding.

Ticks and guinea pigs. C3H/HeN (C3H) female mice (3 to 4 weeks old) were infected by intraperitoneal inoculation of 50 µl of aoHGE (NCH-1 strain)-infected SCID mouse blood (15). Mated adult female Ixodes scapularis ticks were collected from the field. The egg masses were laid in the laboratory. Hatched larvae were fed on either uninfected or aoHGE-infected C3H mice to produce pathogen-free or aoHGE-infected nymphs. The molted nymphs were checked for the presence of aoHGE by Fuelgen staining (19). Batches of exposed ticks with infection rates of >50% were used in these experiments. Female Hartley guinea pigs weighing 300 to 500 g were housed in individual stainless steel wire cages fitted in a rack with bottoms hanging on water pans. Guinea pigs were sensitized to ticks by repeated infestation (three times) with at least 10 nymphs, with a resting period of about 21 days before rechallenge with a similar number of ticks (17).

Infection and disease. Two sets of guinea pigs (three tick sensitized and three naive in each set) were used in the tick challenge studies. We also included in each set one control guinea pig, which was housed in the same facility but not infested with ticks. The control guinea pig was used to determine the normal neutrophil counts and splenic weights in uninfected animals. In addition, blood and sera from uninfected guinea pigs served as internal negative controls for our PCR and immunoblot studies. The day of challenge with the aoHGE-infected ticks was designated day 0, and after challenge, the guinea pigs were coded in a double-blind manner for the remainder of the study. About 2 days after infestation, ticks started to detach from sensitized animals. Three hundred microliters of blood was collected from each guinea pig by retro-orbital puncture on days 4, 7, 12, 17, and 23. The guinea pigs were sacrificed on day 23. Splenic weights were recorded, bone marrow and spleen impression smears and blood smears were made, sera were collected, and 200 µl of blood from each guinea pig was sent to Antech Diagnostics (Farmingdale, N.Y.) for the quantification of leukocytes. The smears were stained with Diff-Quick (Baxter Healthcare Corp., Miami, Fla.) and checked for morulae within the neutrophils.

PCR. Total DNA from 50 µl of guinea pig or mouse blood or from 20 pairs of salivary glands or 20 guts of unfed nymphs was extracted (8) and dissolved in 50 µl of distilled water. Aliquots (5 µl) of each blood DNA sample or aliquots containing DNA from 10 salivary glands (1.2 µg of DNA) or one gut (3.1 µg of DNA) were added to 50-µl PCR mixtures containing Ehrlichia sp. 16S rRNA gene (rDNA)-specific primers Ehr 521 (5'-TGTAGGCGGTTCGGTAAGTTAAAG-3') and Ehr 747 (5'-GCACTCATCGTTTACAGCGTG-3') (4, 19). Denaturing, annealing, and extension temperatures and intervals used for the PCR were 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min, respectively, for 40 cycles.

Immunoblotting. The antigen used for immunoblots was from aoHGE-infected promyelocytic cell line HL-60 (15). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated aoHGE-infected HL-60 cell lysates were transferred to nitrocellulose strips, blocked with 2% bovine serum albumin in Tris-buffered saline, and then incubated for 1 h with guinea pig serum (1:100 dilution). Alkaline phosphatase-conjugated goat anti-guinea pig secondary antibody (1:2,000 dilution) was used to visualize the aoHGE-specific 44-kDa antigen, which has been shown to be a specific indicator of exposure (15).

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Effect of host tick immunity on duration of tick attachment. Ten aoHGE-infected nymphs were placed on the neck of each of the six tick-immune or six naive guinea pigs (from two sets of experiments), and tick attachment was monitored. Each point represents the average ± and standard deviation (error bars) of six observations.

View larger version (61K): [in this window] [in a new window]   FIG. 2.   aoHGE infection in naive and tick-immune guinea pigs. (A) PCR amplification of 16S aoHGE rDNA fragment from blood from one experiment (comprising three naive and three tick-immune guinea pigs). Lane 1, blood from an aoHGE-infected mouse (positive control); lanes 2 to 4, blood from tick-immune guinea pigs; lanes 5 to 7, blood from naive guinea pigs; lane 8, blood from an uninfected guinea pig (negative control). (B) Sera from the animals described above were used to probe aoHGE lysates in the immunoblot. Lane 1, serum from an aoHGE-infected mouse (positive control); lanes 2 to 4, sera from tick-immune guinea pigs; lanes 5 to 7, sera from naive guinea pigs; lane 8, serum from an uninfected guinea pig (negative control).

View larger version (73K): [in this window] [in a new window]   FIG. 3.   aoHGE is present in the salivary glands of unfed Ixodes nymphs. PCR amplification of aoHGE-specific 16S rDNA from gut and salivary gland total DNA was performed. Lane 1, molecular weight markers; lane 2, DNA (1.2 µg) from uninfected salivary glands; lane 3, DNA (1.2 µg) from aoHGE-infected salivary glands; lane 4, DNA (3.1 µg) from uninfected tick guts; lane 5, DNA (3.1 µg) from aoHGE-infected tick guts; lane 6, aoHGE DNA (positive control).