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Previous studies from the late 1980s defined the risk of human Lyme disease by determining the prevalence of Borrelia burgdorferi infection in Ixodes scapularis ticks and Peromyscus sp. mice captured from areas around La Crosse, Wis. High percentages of B. burgdorferi-infected I. scapularis ticks and P. leucopus mice were common in areas located north of Interstate 90 but were not detected in areas south of this major east-west thoroughfare. In this study, we reevaluated the extent of B. burgdorferi infection. High percentages of mice captured from sites north of the interstate were still infected with B. burgdorferi. In addition, B. burgdorferi was recovered from 12 (67%) of 18 mice captured from a site well south of the highway. However, none of 104 mice or 713 I. scapularis ticks captured from the study sites were infected with Ehrlichia spp. The results confirmed the continued high risk for humans to contract infection with B. burgdorferi and the significant southward expansion of the area in which Lyme disease is endemic. In contrast, the risk of acquiring human granulocytic ehrlichiosis remains minimal despite the abundance of appropriate vector ticks and reservoir rodents.
Ixodes scapularis ticks infected with Borrelia burgdorferi, the causative agent of Lyme disease, are endemic throughout the upper Midwest. During the late 1980s, we (8) and others (2) confirmed that high percentages of Peromyscus leucopus mice and I. scapularis ticks captured from areas north and east of La Crosse, Wis., were infected with B. burgdorferi, and most Lyme disease cases were contracted by residents of these regions (1). At that time, Interstate 90 appeared to be an effective barrier limiting the southward spread of Lyme disease, since B. burgdorferi-infected ticks and rodents were only rarely found south of this major east-to-west thoroughfare (8) and Lyme disease cases were relatively rare (1).
During the past several years, however, an increasing number of Lyme disease cases have been diagnosed in patients residing in regions south of La Crosse (10). In addition, researchers have documented the ability of I. scapularis to transmit other human pathogens—most notably Ehrlichia phagocytophila-like organisms (4) that cause human granulocytic ehrlichiosis (HGE). The HGE agent is nearly identical genetically to E. phagocytophila and Ehrlichia equi and typically causes a nonspecific febrile illness, often with thrombocytopenia, leukopenia, and elevated hepatic enzymes (6).
B. burgdorferi and the HGE agent share the same or a similar enzootic cycle. I. scapularis ticks become infected while feeding on a reservoir host such as P. leucopus mice (3, 8, 21). Thus, it is not surprising that HGE cases have been primarily detected in patients residing in Lyme disease foci (11, 20). Pancholi et al. (17) showed that E. phagocytophila-like organisms were detectable in 10% of I. scapularis ticks collected from northwestern Wisconsin and coinfection with B. burgdorferi was not uncommon. Belongia et al. (6) also showed that more than 10% of Lyme disease patients from northern Wisconsin had evidence of concurrent HGE. These findings highlight the necessity of examining previously described Lyme disease foci to also determine the geographic risk of acquiring HGE. In this study, we reevaluated areas surrounding La Crosse, Wis., for B. burgdorferi infection by culturing spirochetes from wild-caught P. leucopus mice. We also examined the mice and captured adult I. scapularis ticks for infection with E. phagocytophila-like organisms.
The study sites were located near La Crosse, Wis., and encompassed an area of approximately 1,400 square miles (Fig. (Fig.1).1). Site HX was approximately 4 miles west of Hixton, Wis., in Jackson County. Site FV bordered Ferryville, Wis., in Crawford County. Site CT was 6 miles east of Cataract, Wis., in Monroe County. Site FC, in Buffalo County, was 8 miles east of Fountain City, Wis. Site SP, located 2 miles west of Sparta, Wis., in Monroe County, bordered Interstate 90 and was near the residence of an individual previously diagnosed with HGE.
Mice were captured during June, July, or August 1997 using homemade live traps baited with unsalted peanuts. Eighty to 100 traps were set at dusk and collected early the next morning. The traps were set in pairs 20 to 30 feet apart along approximately straight-line transects chosen to sample a variety of woodland habitats. Captured mice were transported immediately to the laboratory.
Adult I. scapularis ticks were collected by flagging the underbrush from April to June and September to November 1997. The ticks were separated by sex (13) into 50-ml polypropylene tubes (Fisher Scientific, Pittsburgh, Pa.) and transported to the laboratory, where they were stored in groups of 5 to 15 at 8°C and 100% humidity in mesh-covered vials containing about one-half inch of plaster of paris. The vials were moistened weekly with several drops of distilled water.
Captured mice were etherized and exsanguinated by intracardiac puncture using 1-ml Luer tip tuberculin syringes (Becton Dickinson & Co., Franklin Lakes, N.J.) and 23-gauge needles (Sherwood Medical, St. Louis, Mo.). Approximately 200 to 300 μl of blood was collected into Vacutainer purple-top tubes (Becton Dickinson & Co.) containing EDTA. The mice were then soaked with disinfectant (UKG; Dalco Inc., Minneapolis, Minn.), and the spleens and left kidneys were removed and forced through 1-ml tuberculin syringes into tubes containing 6 ml of Barbour-Stoenner-Kelly (BSK) medium (9). Urinary bladders were removed and placed directly into BSK medium. After mixing, a 600-μl aliquot was transferred to an additional tube containing 5.4 ml of fresh BSK medium and rifampin (40 μg/ml; Sigma Chemical Co., St. Louis, Mo.). The cultures were then incubated in the dark at 33°C for 3 weeks and examined weekly by dark-field microscopy for the presence of spirochetes. When the cultures were contaminated with other microorganisms, the spirochetes were recovered by passing a 1-ml volume of the culture through a sterile 0.45-μm-pore-size low protein binding filter (Acrodisc; Gelman Sciences, Ann Arbor, Mich.) into a test tube containing 4 ml of fresh BSK medium (15) and reincubating the culture for 1 week at 33°C.
Recovered spirochetes were pelleted by centrifugation at 7,000 × g (Marathon 21K/R; Fisher Scientific) for 10 min. The supernatant was discarded and the spirochetes were washed two times by resuspending the organisms in a 1 ml volume of phosphate-buffered saline (PBS) (pH 7.2) and repelleting the organisms by centrifuging for 10 min at 15,000 × g (Beckman Instruments Inc., Palo Alto, Calif.). After washing, the spirochetes were resuspended in 50 μl of PBS, and 10-μl amounts were fixed onto individual wells of Teflon-coated microscope slides (Fisher Scientific).
Mouse monoclonal antibody H5332, which reacts with the 31-kDa outer surface protein A (OspA) of B. burgdorferi (5), was diluted 1:40 in PBS and 50 μl was overlaid onto each well of the slides. After incubation in a moisture chamber at 37°C for 20 min, slides were rinsed with PBS and overlaid with rabbit anti-mouse fluorescein isothiocyanate-labeled immunoglobulin G antibody (Organon Teknika Corp., Durham, N.C.) diluted 1:500 in PBS. Slides were then reincubated in a moisture chamber at 37°C for 20 min. After incubation, slides were rinsed with PBS, air dried, and examined by fluorescence microscopy.
After sufficient numbers of adult ticks were collected from each study site, the ticks were removed from the incubator and processed at the same time. Each tick was sliced in half with a sterile scalpel, and the halves were placed into a microcentrifuge tube, homogenized with an aerosol-resistant pipette tip into 40 μl of buffer containing proteinase K (10 mM Tris [pH 8.3], 50 mM KCl, 1.75 mM MgCl2, 0.01% bovine serum albumin, 0.45% Tween 20, 0.45% Nonidet P-40, 100 μg of proteinase K), and centrifuged at 2,000 × g for 1 min. The supernatants (25 μl) were then transferred to clean microcentrifuge tubes and heated at 50°C for 5 min followed by an additional incubation at 95°C for 10 min. Subsequently, 5 μl of StrataClean resin (Stratagene, La Jolla, Calif.) was added to remove inhibitory compounds (22), and the suspensions were vortexed for 15 s and then left at room temperature for 1 min. The samples were centrifuged at 12,000 × g for 1 min, and the supernatants were removed and treated again with 5 μl of StrataClean resin as described. The treated supernatants were stored at −20°C until used.
Nucleic acids were recovered from mouse blood samples using an IsoQuick kit (Orca Inc., Bothell, Wash.). Briefly, 100 μl of blood was mixed with an equal volume of guanidine thiocyanate lysis solution. After mixing, 500 μl of extraction matrix and 400 μl of extraction buffer were added to the blood solution. Samples were then incubated at 65°C for 5 min, vortexed at high speed for 10 s, and heated for an additional 5 min before centrifugation at 12,000 × g for 5 min (Eppendorf centrifuge 5415C; Brinkmann Instruments Inc., Westbury, N.Y.). The supernatant was transferred to a clean tube, and an additional 500 μl of extraction matrix was added. The sample was then centrifuged for 5 min at 12,000 × g. After centrifugation, the supernatant was transferred to a clean tube and sodium acetate was added to a final concentration of 0.3 M. An equal volume of isopropanol was added, and the sample was centrifuged at 12,000 × g for 10 min to pellet the nucleic acids. The supernatant was discarded, and the nucleic acids were washed by gentle inversion in a 1-ml volume of 70% ethanol. The recovered nucleic acids were air dried for 20 min, resuspended in 40 μl of nuclease-free water, and stored at −20°C until tested.
Ehrlichia DNA was detected by using a modification of a previously described PCR procedure to amplify a 293-bp region of the 16S rRNA gene of E. phagocytophila-genogroup organisms (16, 17). Three microliters of DNA from each blood sample was combined with 500 pmol of the positive-strand primer Ehr521 (5′-TGTAGGCGGTTCGGTAAGTTAAAG-3′), 500 pmol of the negative-strand primer Ehr790 (5′-CTTAACGCGTTAGCTACAACACAG-3′), 1.5 U of Taq DNA polymerase, and distilled water to a final volume of 25 μl. In addition, 25 μl of PreMix buffer D or G (Epicentre Technologies, Madison, Wis.) containing deoxynucleoside triphosphates, Tris-HCl (pH 8.3), KCl, MgCl2, and betaine (19) was added. The DNA was amplified in a thermal cycler (GeneAmp 2400, Perkin-Elmer, Norwalk, Conn.) as follows: an initial 5 min at 95°C and then 40 cycles of 45 s at 95°C, 45 s at 60°C, and 45 s at 72°C. A final extension was done for 7 min at 72°C to fully extend truncated DNA strands.
After amplification, 5 μl of 10× loading buffer was added to each PCR mixture and 20 μl of the reaction volume was removed and loaded onto 1× TAE-2.0% agarose gels and electrophoresed at 80 V for approximately 45 min. Included in each electrophoresis run was one lane containing 0.5 μg of DNA fragments from a ΦX174/HaeIII digest (Sigma) as a size standard. Electrophoresed gels were stained with 5 μl of Sybr-Green I DNA stain (Molecular Probes, Eugene, Oreg.) diluted 1:10,000 in 1× TAE overnight at 4°C. After staining, bands were illuminated with UV light (254 nm) and photographed.
In preliminary studies using tick extracts, inhibiting factors appeared to hinder the PCR. To address this and ensure the validity of negative PCR results, 12 nmol of a plasmid containing a 207-bp internal fragment of the 293-bp DNA target was added prior to amplification. The PCR was repeated if the fragment was not amplified. In addition, aerosol-resistant pipette tips were used (Molecular Bio-Products Inc., San Diego, Calif.), and amplifications were performed in a separate room. Counter surfaces and equipment were cleaned thoroughly with a DNA contaminant removal solution (DNA-Erase; ICN Biomedics, Inc., Aurora, Ohio) and 70% ethanol.
The plasmid containing the 293-bp 16S rRNA gene of E. phagocytophila-genogroup organism gene was amplified and purified as described previously. An 86-bp region was removed by digestion with MspI (Promega, Madison, Wis.), and the two remaining DNA fragments were religated using T4 DNA ligase (Promega). The resultant 207-bp E. phagocytophila-genogroup gene was then amplified as described above, ligated into a T vector (Promega), and transformed into competent E. coli JM109. The plasmid DNA was recovered (Wizard Plus MiniPreps DNA purification system; Promega) after incubating the transformed E. coli JM109 in 5 ml of 2× TY broth containing ampicillin (100 μg/ml) for 24 h at 37°C. Approximately 12 nmol of the plasmid could be added to the PCR without affecting the sensitivity.
A total of 104 P. leucopus mice were cultured for Lyme disease spirochetes. B. burgdorferi was recovered from 37 (36%) mice, which included rodents captured from all five study sites (Table (Table1).1). Infection rates ranged from 3 (13%) of 23 mice collected from site FC to 12 (67%) of 18 mice collected from site FV. Of the 37 B. burgdorferi-positive mice, 23 (62%), 22 (59%), and 11 (30%) isolates were obtained from the urinary bladder, spleen, and kidney, respectively.
Prior to testing the internal tissues from the captured I. scapularis ticks and blood samples from the P. leucopus mice, the ability of the Ehrlichia PCR assay to detect the HGE agent was confirmed by evaluating a blood sample from a SCID mouse infected with the HGE agent (from Chris Kolbert, Mayo Medical Foundation, Rochester, Minn. ) and a wild-caught HGE agent-infected I. scapularis tick (from J. Stephen Dumler, Johns Hopkins University, Baltimore, Md.). E. phagocytophila genogroup DNA was amplified to easily detectable levels in both the tick extract and mouse blood (Fig. (Fig.2).2). The 104 blood samples from the captured mice and 713 I. scapularis ticks were then evaluated. At least 18 mice and 94 ticks were evaluated from each study site (Table (Table2).2). Ehrlichia sp. DNA was not detected in any tick or mouse samples.
The geographic area surrounding and including La Crosse, Wis., is a well-established region of endemicity for Lyme disease, and monitoring the percentage of B. burgdorferi-infected Peromyscus sp. mice has provided an accurate assessment of the boundaries of this focus. In 1986, Anderson et al. (2) isolated B. burgdorferi organisms from 15 (88%) of 17 P. leucopus and 54 (66%) of 82 immature I. scapularis ticks collected from Fort McCoy, Wis., an army installation located approximately 20 miles east of La Crosse. During the same time frame, we (8) determined the prevalence of B. burgdorferi infection in mice captured from several locations surrounding La Crosse to define the local boundaries of this midwestern Lyme disease focus. B. burgdorferi organisms were isolated from 28 (33%) of 86 mice collected north of Interstate 90 but only 1 (1%) of 75 mice captured south of this major east-west thoroughfare. Thus, the southward expansion of B. burgdorferi-infected ticks and mice appeared to be hampered by the interstate highway.
In this study, we again isolated B. burgdorferi organisms from 25 (29%) of the 86 mice captured from sites north of the interstate. Captured ticks were not cultured for B. burgdorferi in order to maximize the tissues tested for Ehrlichia spp. However, we have consistently detected similar infection rates for both I. scapularis and P. leucopus mice (8; unpublished data). Thus, these data reaffirm the continued high risk of acquiring Lyme disease in these areas. In addition, we also detected B. burgdorferi infection in 12 (67%) of the 18 mice captured from site FV, which was located approximately 40 miles south of the interstate in a region where B. burgdorferi spirochetes had not previously been detected (8). Thus, the Lyme disease focus expanded southward considerably during the past decade. These results are not surprising since white-tailed deer populations are at record high levels, the range of I. scapularis ticks has been increasing southward in Wisconsin (18), and the incidence of Lyme disease cases has increased during the last decade (10).
Prior to this study, Belongia et al. (7) had detected Ehrlichia sp. DNA in 12 (46%) of 26 blood samples collected from white-tailed deer at Fort McCoy. We had also confirmed HGE in an individual who resided on a farm within study site SP. Thus, it seemed likely that mice and ticks in these regions would also harbor Ehrlichia spp. In contrast to our findings with B. burgdorferi, however, we did not detect Ehrlichia sp. DNA in any captured mice or ticks.
These results should be accurate. Prior to testing, we demonstrated that the PCR could detect E. phagocytophila-like organisms in both mouse blood and tick tissue. In addition, the DNA added as a PCR control was consistently detected. A closer examination of the HGE patient's medical history also revealed that the individual had traveled to a region of Florida previously confirmed as a region of endemicity for Ehrlichia sp.-infected ticks (12) immediately prior to their illness. As further support, other investigators (14) also recently failed to detect Ehrlichia spp. in mice captured from Fort McCoy.
In conclusion, the risk of acquiring Lyme disease in the focus of endemicity surrounding La Crosse, Wis., remains high, and the range of B. burgdorferi-infected mice continues to expand southward. In contrast, the risk of acquiring HGE remains minimal despite the abundance of I. scapularis ticks and P. leucopus mice.
This work was sponsored by the Gundersen Lutheran Medical Foundation, Ltd.
We gratefully acknowledge Dean Jobe and Jennifer Marks for assistance with the collection of ticks and mice. We also thank Marc Rott for technical expertise.