INFECTION AND IMMUNITY, Apr. 1998, p. 1803–1805 0019-9567/98/$04.0010 Copyright © 1998, American Society for Microbiology
Vol. 66, No. 4
Granulocytic Ehrlichiosis in Tick-Immune Guinea Pigs SUBRATA DAS,1 KATHLEEN DEPONTE,1 NANCY L. MARCANTONIO,1 JACOB W. IJDO,1 EMIR HODZIC,2 PAULA KATAVOLOS,3 STEPHEN W. BARTHOLD,2 SAM R. TELFORD III,3 FRED S. KANTOR,1 AND EROL FIKRIG1* Department of Internal Medicine, Yale University, New Haven, Connecticut1; The Center for Comparative Medicine, Schools of Medicine and Veterinary Medicine, University of California, Davis, Davis, California2; and Department of Tropical Public Health, Harvard University School of Public Health, Boston, Massachusetts3 Received 18 September 1997/Returned for modification 10 November 1997/Accepted 12 January 1998
We investigated whether Ixodes scapularis-mediated host immunity interrupts transmission of the agent of human granulocytic ehrlichiosis (aoHGE) to guinea pigs. Ticks infected with aoHGE readily transmitted aoHGE to tick-immune guinea pigs, despite incomplete tick engorgement and host attachment. Although tick immunity can prevent Lyme borreliosis, protection is not afforded against granulocytic ehrlichiosis. 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 ml 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 retroorbital 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 ml 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. To determine the infectivity and viability of aoHGE derived from the guinea pigs, we used a recently developed mouse model of granulocytic ehrlichiosis (14, 19). Blood from sensitized and naive guinea pigs that were challenged with infected
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 vectorborne 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
* Corresponding author. Mailing address: Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8031. Phone: (203) 785-2454. Fax: (203) 785-7053. E-mail:
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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 6 and standard deviation (error bars) of six observations.
ticks was injected intraperitoneally (50 ml) into C3H mice. At 12 days, blood samples were examined for aoHGE by PCR. A blood smear from each mouse was also prepared. PCR. Total DNA from 50 ml 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 ml of distilled water. Aliquots (5 ml) of each blood DNA sample or aliquots containing DNA from 10 salivary glands (1.2 mg of DNA) or one gut (3.1 mg of DNA) were added to 50-ml PCR mixtures containing Ehrlichia sp. 16S rRNA gene (rDNA)-specific primers Ehr 521 (59-TGTAGGCGGTTCGGTAAGTTAAAG-39) and Ehr 747 (59-GCACTCATCGTTTACAGCGTG-39) (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). As expected, aoHGE-infected ticks fed to repletion on naive guinea pigs (Fig. 1). In contrast, tick engorgement on sensitized animals was impaired. The average time the ticks fed on naive guinea pigs was 4 to 5 days, whereas ticks started to detach from the sensitized animals on day 2 and most of the ticks had fallen off the sensitized animals by 3 days after infestation (a statistically significant difference by Kaplan-Meier analysis [P , 0.05]). The average weight of ticks that fed on naive guinea pigs was 3.24 6 0.24 mg (average 6 standard deviation [n 5 60]) compared to 0.86 6 0.69 mg for ticks that detached from sensitized guinea pigs (P , 0.01 [Student’s t test]). Most of the nymphs (85%) that fed on naive animals molted and became adults, but all of the ticks that fed on sensitized guinea pigs died before molting. Several criteria were used to document ehrlichiosis in guinea pigs: PCR amplification of an aoHGE 16S rDNA target from blood, seroconversion to the aoHGE-specific 44-kDa antigen, and infectivity of the guinea pig blood in mice. Both naive and
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tick-immune guinea pigs became infected with the aoHGE, as determined by PCR and seroconversion (Fig. 2). All six naive and five of six tick-immune guinea pigs remained infected for at least 23 days (the final time point examined), based on PCR. Immunoglobulin G directed towards the 44-kDa aoHGE antigen was detectable in five of six naive and four of six immune guinea pigs from day 12 and beyond. Leukocyte counts and splenic weights of all experimental and control animals were similar. Cytoplasmic aoHGE clusters (morulae) were not identified in the neutrophils of any of the guinea pigs, but five of six mice inoculated with blood from individual naive animals and five of six mice with blood from tick-immune guinea pigs developed morulae in peripheral blood neutrophils and were PCR positive. Using all these criteria to determine infection, we found that all six of the sensitized and six of the naive guinea pigs became infected with aoHGE. To determine whether aoHGE was specifically present in the salivary glands and/or in the gut of unfed nymphs, total DNA from these organs was used to identify aoHGE by PCR. Salivary glands, but not guts, of unfed nymphs harbored aoHGE, as evidenced by the amplification of the aoHGEspecific 16S rDNA band only from salivary gland DNA (Fig. 3). Encouraged by our and other researchers’ demonstration of the interruption of B. burgdorferi transmission in tick-sensitized animals (17, 22), we explored the efficacy of tick immunity in preventing transmission of other I. scapularis-borne pathogens. We focused on aoHGE because it resides in tick salivary glands, whereas B. burgdorferi is present within the gut of unfed ticks, and because pathogen location is likely to influence transmission (5, 17). We established a guinea pig model of aoHGE infection in which HGE infection in animals can be confirmed by several parameters, including PCR, immunoblot,
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 tickimmune 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).
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growth and retard its transport to salivary glands. Overall, these experiments demonstrate that guinea pigs can be experimentally infected with aoHGE and that I. scapularis-mediated host immunity is not sufficient to prevent aoHGE transmission. This work was supported by grants from the National Institute of Health (AI30548, AI37993, AI41440, and AI39002). S. Das is an Arthritis Foundation Postdoctoral Fellow. J. IJdo is a Daland Fellow of the American Philosophical Society and a Postdoctoral Fellow of the Markey Foundation. REFERENCES
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 mg) from uninfected salivary glands; lane 3, DNA (1.2 mg) from aoHGE-infected salivary glands; lane 4, DNA (3.1 mg) from uninfected tick guts; lane 5, DNA (3.1 mg) from aoHGE-infected tick guts; lane 6, aoHGE DNA (positive control).
and transfer of ehrlichiae to mice. In contrast to our observations with mice and humans, we did not observe aoHGE morulae in peripheral neutrophils of infected guinea pigs. Nevertheless, we used guinea pigs because tick immunity develops more readily in these animals than in mice (17, 20, 22). As we have shown with the aoHGE, tick immunity does not always prevent pathogen transmission to the host. For example, transmission of Theileria parva bovis, which is also localized in the salivary glands, was not blocked in cattle resistant to the tick Rhipicephalus appendiculatus (9). The reasons why tick immunity may protect against transmission of some pathogens but not others, particularly those carried by the same vector, are likely to be multifactorial. The proximity of the pathogens to the host tissues, the time required for transmission, and the duration of tick attachment may influence blockage of transmission. A pathogen residing in the salivary gland may be readily transmitted to the host even if the vector detaches quickly from the host without complete feeding. The aoHGE has previously been detected by Feulgen-staining in the salivary glands of unfed infected Ixodes nymphs (19). We have now shown by PCR that aoHGE is present in the salivary glands but not in the gut, suggesting that the salivary glands are the major, if not the only, location of residence. In contrast, B. burgdorferi resides in the guts of unfed ticks, and migrates to the salivary glands during tick engorgement (5). While B. burgdorferi usually requires 48 to 72 h for transmission to the host (5), the duration of tick attachment needed for aoHGE transmission is approximately 30 h (unpublished observation). Organisms that reside in salivary glands may move quickly to the host. Thus, it is possible that infected nymphs transmit the aoHGE within 2 days of initial host attachment before they reject the tick-sensitized host. In contrast, a pathogen such as B. burgdorferi (5), which resides in the gut and requires activation by a blood meal to multiply and migrate to the tick salivary glands, may not get either enough stimulation or time to migrate for transmission to tick-immune animals. Alternatively, contact of host blood from sensitized animals may lead to immune reactions between tick gut antigens and host serum, which could interfere with the dynamics of B. burgdorferi Editor: R. N. Moore
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