Clinical and Diagnostic Laboratory Immunology, January 2000, p. 68-71, Vol. 7, No. 1
1071-412X/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Human Granulocytic Ehrlichiosis Agent Infection in a Pony Vaccinated with a Borrelia burgdorferi Recombinant OspA Vaccine and Challenged by Exposure to Naturally Infected Ticks

Department of Population Medicine and Diagnostic Science,¹ Department of Biomedical Sciences,² and Department of Clinical Sciences,³ College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

Received 22 July 1999/Returned for modification 30 September 1999/Accepted 13 October 1999

	ABSTRACT

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A pony was vaccinated with recombinant OspA vaccine (rOspA) and then exposed 3 months later to Borrelia burgdorferi-infected ticks (Ixodes scapularis) collected in Westchester County, N.Y. At 2 weeks after tick exposure, the pony developed a high fever (105°F). Buffy coat smears showed that 20% of neutrophils contained ehrlichial inclusion bodies (morulae). Flunixin Meglumine (1 g daily) was given for 2 days, and the body temperature returned to normal. PCR for ehrlichial DNA was performed on blood samples for 10 consecutive days beginning when the pony was first febrile. This pony was monitored for another 3.5 months but developed no further clinical signs. The 44-kDa immunodominant human granulocytic ehrlichiosis antigen gene was amplified by PCR and cloned into a pCR2.1 vector. DNA sequence analysis of this gene showed it was only 8 bp different (99% identity) from the results reported by others (J.W. Ijdo et al., Infect. Immun. 66:3264-3269, 1998). Western blot analysis, growth inhibition assays, and repeated attempts to isolate B. burgdorferi all demonstrated the pony was protected against B. burgdorferi infection. These results highlight the potential for ticks to harbor and transmit several pathogens simultaneously, which further complicates the diagnosis and vaccination of these emerging tick-borne diseases.

	INTRODUCTION

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Lyme borreliosis and ehrlichiosis caused by Borrelia burgdorferi and members of the Ehrlichia phagocytophila group (including E. phagocytophila, E. equi, and the human granulocytic ehrlichiosis [HGE] agent) are tick-borne infectious agents for both humans and animals (8-10, 12, 13, 26, 27, 31). It is now generally agreed that E. equi, which causes equine granulocytic ehrlichiosis (EGE), and HGE agent are the same organism (1, 6a, 35, 38). These pathogens are mainly transmitted by ticks, either Ixodes scapularis (deer ticks) or I. pacificus (black-legged tick) in the United States and I. ricinus in Europe (27, 28, 29, 31, 34). Deer ticks often harbor both B. burgdorferi and E. equi (HGE agent), and coinfection with both pathogens has been reported in humans, dogs, and horses (21-23, 26, 35). In a previous study, we reported that 9% of ticks from Westchester county, N.Y. are infected with HGE agent (6). HGE agent (or E. equi) is an intracellular bacterium that resides within phagosomes of neutrophils or eosinophils, whereas E. chaffeenesis and E. canis infect only monocytes (35). Since clinical disease caused by either HGE agent or B. burgdorferi may initially present as a nonspecific febrile illness, it is sometimes difficult to distinguish between infection by either agent solely on the basis of clinical signs.

Recently, a human patient died of fatal pancarditis associated with HGE agent infection (19). Fatal pancarditis was reported in another human patient coinfected with B. burgdorferi and Babesia microti (25). B. burgdorferi by itself can also cause carditis in people and mice (3). The concurrent presence of B. microti, HGE agent, B. burgdorferi, and viral pathogens, such as tick-borne encephalitis virus, in ticks makes the diagnosis or vaccination of these tick-borne diseases a complicated issue in both humans and animals (25, 32). EGE was first reported in 1969 in California (15). EGE has been recently reported in British Columbia in Canada and in Wisconsin, Minnesota, New York, Connecticut, and Florida (20, 24, 30, 33).

Efforts have been made in the past several years to prevent Lyme disease by vaccination with either an outer surface protein A (OspA) recombinant vaccine (humans, dogs, and horses) or whole-cell bacterins (dogs) (4, 5, 11). Recently, we demonstrated that a recombinant OspA vaccine could protect ponies from infection when the animals were exposed to naturally infected ticks (5). Here we report on one pony in that study that was infected with HGE agent. This case shows that humans or animals vaccinated against Lyme disease must still take precautions in areas where the disease is endemic to prevent the transmission of other tick-borne pathogens. Additionally, we believe a vaccine to prevent HGE agent infection is also needed for use in areas where people and animals are at high risk of infection.

	MATERIALS AND METHODS

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Animal and vaccination protocol. The pony described in this report was part of a larger group vaccinated with recombinant OspA vaccine three times at days 0, 20, and 82 as previously described (5).

Exposure to ticks. Field-collected adult ticks (20 female and 10 male) were applied to the left trunk 112 days after the first vaccination as previously described (5, 7). Of these ticks, 12 were fully engorged.

Serology. Kinetics-ELISA (KELA) and Western blot analysis were performed as previously described (4, 5, 7). KELA for measuring the relative quantity of serum antibody to B. burgdorferi was performed as described previously. Briefly, diluted serum was added to duplicate wells in microtiter plates containing antigens of French-pressed B. burgdorferi lysate. Bound antibody was detected by using horseradish peroxidase-conjugated goat anti-horse immunoglobulin G (Cappel Research Products, Durham, N.C.). Color development with the chromogen tetramethylbenzidine with H₂O₂ as a substrate was measured kinetically and expressed as the slope of the reaction rate between enzyme and substrate solution. Each unit of slope was designated as a KELA unit.

The procedure for the Western blot analysis was done as previously described (4, 5, 7). French-pressed B. burgdorferi lysate was used as an antigen and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4, 5, 7). Western blot analysis was performed in a miniblotter (4, 5, 7). Test sera from experimental animals were used as a first antibody, followed by goat anti-horse immunoglobulin G conjugated to horseradish peroxidase as a second antibody.

PCR and sequence of 44-kDa protein gene for HGE agent. Venous blood was collected into ACD tubes. DNA template derived from the blood samples was prepared by using DNAzol according to the manufacturer's instructions (Life Technologies, Rockville, Md.). PCR with the HGE agent-specific Ger3 and Ger4 primer set for 16 S RNA gene was performed on blood obtained when the pony was febrile, as previously described (14). Based on published data, two additional primers (sense, 5'-ATGTCTATGGCTATAGTCATGGCT-3'; antisense, 5'-CTTAAAAAGCAAACCTAACACC-3') were designed to amplify the 44-kDa immunodominant protein gene (17). The amplified PCR product was ligated into a pCR2.1 vector (Invitrogen), and both strands of the cloned DNA were completely sequenced by using the Applied Biosystems model 373A automated DNA sequencing system. The thermal cycling of the sequencing reactions utilized the Tag Dyedeoxy terminator cycle sequencing kit.

Pathology and histopathology. As part of the overall rOspA vaccine study, this pony was euthanized 3.5 months after tick exposure and then necropsied. The following tissues were fixed in 10% neutral buffered formalin: joint capsules (right and left elbow, shoulder, stifle, carpus, tarsus, and fetlock), cerebellum, cerebrum, meninges, spinal cord, myocardium, urinary bladder, thyroid, liver, spleen, kidney, lung, stomach, intestine, skeletal muscles, aorta, eyes, nerves (left and right brachial plexus, trigeminal ganglion, cervical and thoracic nerve roots, median, ulnar, radial, tibial, fibular, sciatic, and facial), and lymph nodes (axillary, prescapular, and popliteal). Tissues were embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin by conventional methods for histopathologic evaluation.

	RESULTS

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Clinical signs and examination of buffy coats. Two weeks after tick exposure, this pony had a high fever (105°F). Examination of Giemsa-stained buffy coats on the first day of fever revealed that 20% of the neutrophils had visible morulae, a finding compatible with HGE agent infection (Fig. 1). For the next 3 days, morulae remained visible in about 10% of the neutrophils, but Giemsa-stained buffy coats were negative when examined 10 days after the initial fever.

View larger version (93K): [in this window] [in a new window]   FIG. 1.   Blood smear. Ehrlichia inclusion bodies (arrows) in equine neutrophils during a febrile episode 14 days after tick exposure.

Serology. Western blots confirmed that the pony developed an appropriate antibody response to rOspA vaccination, while unvaccinated controls developed typical Western blot patterns seen only with infection. Western blot analysis with B. burgdorferi B31 whole-cell lysate showed that OspA antibody appeared 3 weeks after the first vaccination (Fig. 2, lane 2). Bands became denser after the second vaccination (Fig. 2, lane 5). Bands also appeared in the 20- and 60-kDa regions (Fig. 2). Western blot analysis with HGE agent whole-cell lysate (NCH-1 strain) showed antibody reactivity at approximately 29, 37, 38, and 44 kDa and faintly at 110 kDa (Fig. 3, lanes 12 and 13), but no bands formed when DH82 cell lysate was used as a control (data not shown).

View larger version (69K): [in this window] [in a new window]   FIG. 2.   Western blot analysis of antibody responses to B. burgdorferi (B31) whole-cell antigens. The pony was vaccinated three times with 100 µg of rOspA in adjuvant. Lane 1, preimmune serum; lanes 2 to 12, 2-week intervals (except week 6, no sample) after the first vaccination. Lane 6 (*) indicates the first day of tick exposure. The numbers at the right indicate molecular sizes (biotinylated SDS-PAGE standard broad-range molecular markers; Bio-Rad Laboratories, Richmond, Calif.) in kilodaltons.

View larger version (66K): [in this window] [in a new window]   FIG. 3.   Western blot analysis of antibody response to HGE agent whole-cell antigens. Lane 1, preimmune serum; lanes 2 to 12, 2-week intervals after first vaccination. Lane 7 (*) indicates the first day of tick exposure. The numbers at the right indicate molecular sizes (biotinylated SDS-PAGE standard broad-range molecular markers; Bio-Rad Laboratories) in kilodaltons.

PCR and DNA sequence. PCR was positive for ehrlichial DNA when the pony was first febrile at 14 days after tick exposure (Fig. 4). Ten days later, we could no longer detect the PCR product (Fig. 4, lane 24). The DNA sequence indicated only 8 bp differences from previously reported DNA sequence.

View larger version (56K): [in this window] [in a new window]   FIG. 4.   PCR amplification of a 151-bp 16S rRNA gene fragment from pony blood. Numbers indicate the days after tick exposure. The positive control (+) was derived from DNA obtained from blood of a person infected with the HGE agent. The negative control () included all reaction components plus deionized H2O but no template DNA.

Histopathology. No significant gross or histopathologic lesions were seen in this pony 3.5 months after infection.

	DISCUSSION

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Here we report HGE agent infection in a horse vaccinated with recombinant OspA vaccine and then exposed to field-collected adult I. scapularis ticks harboring both B. burgdorferi and the HGE agent. This case highlights the continued importance of vaccinating individuals against Lyme disease to avoid tick exposure, since ticks harbor multiple potential pathogens. Failure to do so may result in infection with pathogens other than the Lyme agent. Reportedly, horses that recovered from HGE agent infection are protected against reinfection (2). However, Horowitz et al. recently reported that a woman was reinfected with HGE agent 2 years after the first infection (16). Revaccination may be necessary to fully protect animals or humans. HGE agent infection may cause immunosuppression (36). However, Holmeister et al. reported that mice coinfected with B. burgdorferi and HGE agent only had greater spirochete distribution without increasing the severity of Lyme disease (E. K. Holmeister, M. H. Moro, D. Mathiesen, S. W. Barthold, and D. H. Persing, Abstr. 7th Int. Cong. Lyme Borreliosis, abstr. F1008, 1996). Whether this is the case in humans, dogs, and horses is currently unknown.

Western blot analysis with whole B. burgdorferi cell lysates also showed bands in the 20- and 60-kDa regions, which were probably degraded products and dimers of OspA as previously described (4). Western blot analysis with HGE agent whole-cell lysate (NCH-1) indicated that there were strong bands at 10 weeks after tick challenge (17, 18, 23), but no bands were present when uninfected DH82 cell lysates were used as antigens (data not shown). Thus, the bands seen in the Western blot with HGE agent-infected cell lysates as antigens were specific for the HGE agent. The 44-kDa immunodominant protein is heterogeneous among different HGE agent isolates (1, 38). It has been reported that a minimum copy number of this 44-kDa protein gene is 18 and that five different mRNAs are transcribed from 44-kDa protein genes in HL-60 cell cultures (37). Two different 44-kDa homologous proteins are expressed in the HL-60 cell cultures, which are regulated at the transcriptional level (37). It is currently unknown if antibodies against the 44-kDa immunodominant protein are protective. Further study is required to ascertain if this antigen could be used in combination with OspA as a recombinant vaccine to protect people and animals against both diseases.

No histopathologic lesions were found at the time of necropsy in this pony. Lesions associated with HGE agent infection would most likely be found when the patient is febrile (15). However, we monitored the pony for 3.5 months after B. burgdorferi-infected tick exposure in order to determine if this pony was protected from Lyme disease after vaccination with an OspA vaccine.

In conclusion, EGE occurred in a pony vaccinated with a recombinant OspA vaccine and challenged by exposure to field-collected adult ticks. Caution still needs to be taken to prevent tick bites in order to avoid transmission of other tick-borne diseases.

	ACKNOWLEDGMENTS

We are grateful to Helen Bell for administrative assistance. We are grateful to Jacob Ijdo, Yale University, for his generous donation of the HGE agent NCH-1 strain and to Allyn Vondercheck, Dale Strickland, David Dietterich, and John Daley for animal care.

This work was supported by grants from the Zweig fund from Cornell University and the Cornell Biotechnology Program.

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Population Medicine and Diagnostic Science, College of Veterinary medicine, Cornell University, Ithaca, NY 14853. Phone: (607) 253-3675. Fax: (607) 253-2943. E-mail: yc42{at}cornell.edu.

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