White-tailed deer (Odocoileus virginianus) develop spirochetemia following experimental infection with Borrelia lonestari

Veterinary Microbiology 115 (2006) 229–236 www.elsevier.com/locate/vetmic

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White-tailed deer (Odocoileus virginianus) develop spirochetemia following experimental infection with Borrelia lonestari P.L. Moyer a, A.S. Varela a, M.P. Luttrell b, V.A. Moore IV a, D.E. Stallknecht b,c, S.E. Little a,d,* a

Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, United States b Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, United States c Department of Population Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, United States d Department of Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74078, United States Received 18 October 2005; received in revised form 15 December 2005; accepted 20 December 2005

Abstract Borrelia lonestari is considered a putative agent of southern tick-associated rash illness (STARI) and is known to occur naturally only in lone star ticks (Amblyomma americanum) and white-tailed deer (Odocoileus virginianus). We used a low passage isolate of B. lonestari (LS-1) to inoculate white-tailed deer, C3H mice, Holstein cattle, and beagles. Animals were monitored via examination of Giemsa and acridine orange stained blood smears, polymerase chain reaction (PCR), indirect fluorescent antibody (IFA) test, and/or culture isolation. Spirochetes were visualized in blood smears of both deer on days postinoculation (DPI) 6, 8, 12 and one deer on DPI 15. Whole blood collected from deer tested PCR positive starting on DPI 4 and remained positive as long as DPI 28. Both deer developed antibody titers of >64, with a maximum IFA titer of 1024. The organism was reisolated from the blood of both deer on DPI 6 and one deer on DPI 12. All isolation attempts from mice, calves, or dogs were negative, although one of seven mice was transiently PCR positive. Mice and dogs developed an IFA titer 64, while calves lacked a detectable antibody response. These preliminary experimental infection trials show that white-tailed deer are susceptible to infection with B. lonestari and develop a spirochetemia following needle-inoculation, while C3H mice, calves, and dogs do not. Results suggest that deer may serve as a vertebrate reservoir host. Tick transmission studies are needed to confirm that this organism can be maintained in a natural cycle involving deer and A. americanum. # 2006 Elsevier B.V. All rights reserved. Keywords: Borrelia lonestari; Reservoir host; STARI

* Corresponding author. Tel.: +1 405 744 8523; fax: +1 405 744 5275. E-mail address: [email protected] (S.E. Little). 0378-1135/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2005.12.020

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1. Introduction Lyme disease, the most commonly diagnosed vector-borne illness in people in the United States, is rare in the southern U.S. (Orloski et al., 2000; CDCP, 2003). However, a Lyme disease-like illness has been described in patients from this region (Masters and Donnell, 1995; Campbell et al., 1995; Kirkland et al., 1997; Masters et al., 1998; Felz et al., 1999; Orloski et al., 2000; James et al., 2001; Wormser et al., 2005). Due to confusion about the etiology, this Lyme-like disease is alternatively referred to as southern tick-associated rash illness (STARI), southern Lyme disease, or Masters’ disease (James et al., 2001; Masters et al., 1998). The etiological agent of STARI is unknown. However, Barbour et al. (1996) described a novel Borrelia sp. in Amblyomma americanum, the suspected vector tick, from Texas and New Jersey by sequence of the flagellin and 16S rRNA genes. Since first described, DNA of Borrelia lonestari has been detected in A. americanum across the south-central and southeastern United States (Bacon et al., 2003; Burkot et al., 2001; James et al., 2001; Stegall-Faulk et al., 2003; Stromdahl et al., 2003; Varela et al., 2004a,b). Direct evidence suggesting B. lonestari may be a causative agent of STARI was obtained when the organism was identified by polymerase chain reaction (PCR) from a skin biopsy from a STARI patient and from a lone star tick that was attached to that patient (James et al., 2001). Although a recent study failed to detect evidence of B. lonestari in a series of patients from Missouri (Wormser et al., 2005), B. lonestari currently remains the only potential etiologic agent described from a STARI patient. We recently obtained the first culture isolate of B. lonestari from naturally infected wild A. americanum ticks (Varela et al., 2004a). To evaluate different vertebrates as potential reservoir hosts for B. lonestari, we experimentally inoculated white-tailed deer, C3H mice, Holstein cattle, and beagles with this isolate (LS-1), and then monitored them for evidence of infection. Although comprehensive surveys of other species are lacking, we chose white-tailed deer because they are known to be naturally infected with B. lonestari and may serve as a reservoir host capable of infecting A. americanum ticks, which feed on deer as larvae, nymphs, and adults (Kollars et al., 2000;

Lockhart et al., 1997b; Luckhart et al., 1992; Moore et al., 2003). Although an infrequent host of A. americanum in nature, and thus unlikely to serve as a reservoir host, C3H mice were inoculated because they are known to be highly susceptible to infection with B. burgdorferi, the causative agent of Lyme borreliosis in North America (Barthold et al., 1990). Cattle were inoculated with B. lonestari because of this agent’s close phylogentic relationship with B. theileri, an agent of bovine borreliosis (Rich et al., 2001). Finally, infection was also attempted in dogs, a species known to be susceptible to infection with B. burgdorferi (Chang et al., 2001).

2. Materials and methods 2.1. Animals Two, 6-month-old male white-tailed deer (Odocoileus virginianus) from a captive herd at the University of Georgia (Athens, GA); seven, 6week-old C3H/HeNHsd mice (two male and five female) purchased from a commercial vendor (Harlan, Indianapolis, IN); four 2–3-month-old male Holstein calves purchased from Reidsville State Penitentiary (Reidsville, GA); four adult male beagles donated by TRS Laboratories Inc. (Athens, GA) were used in these experimental infection trials. All animals were kept in climate-controlled, tick-free, animal housing facilities at the College of Veterinary Medicine, University of Georgia (Athens, GA) for the duration of these studies. Animal care was provided by the Animal Resources staff at the college, and all experimental procedures were approved by the Institutional Animal Care and Use Committee. Commercially available food and water were provided ad libitum. Prior to inoculation with B. lonestari, all deer, calves, and dogs were tested by PCR assay for Anaplasma phagocytophilum, an Anaplasma sp. of deer, Borrelia spp., Ehrlichia chaffeensis, E. canis, and E. ewingii as previously described (Barbour et al., 1996; Dawson et al., 1994; Little et al., 1997; Massung and Slater, 2003; Moore et al., 2003; Zeidner et al., 2000), and by indirect fluorescent antibody (IFA) testing for antibodies against A. phagocytophilum, B. lonestari, and E. chaffeensis (Dawson et al., 1991;

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Luckhart et al., 1991). In addition, dogs were tested for Dirofilaria immitis antigen and for antibodies to B. burgdorferi and E. canis using a commercially available test (Canine Snap 3Dx Test, IDEXX Laboratories, Westbrook, ME) according to manufacturer’s directions. All dogs were dewormed with a combination pyrantel, febantel, and praziquantel product (Drontal Plus, Bayer Animal Health, Shawnee Mission, KS) according to manufacturer’s instructions 1 week prior to inoculation. Mice were obtained from a commercial vendor and therefore were not tested for evidence of infection or exposure prior to inoculation.

of supernatant, containing predominately spirochetes, was transferred to a sterile 15 ml tube and centrifuged at 1118  g for 10 min. The pellet was resuspended in 1.0 ml of fresh L-15B300/BSK media, transferred to a syringe, and held at 34 8C until use. Uninfected ISE6 tick cells were prepared in a manner identical to the inoculum containing tick cells for the negative control animal dog (n = 1) and calf (n = 1); negative control deer and mice were not included in these studies.

2.2. Spirochetes

2.4.1. Deer Deer were immobilized with 7.5 mg/kg ketamine (100 mg/ml; Ketaset, Fort Dodge; Overland Park, Kansas) and 2.2 mg/kg xylazine (100 mg/ml; Fort Dodge; Overland Park, Kansas) prior to inoculation and each blood sample collection. A minimum of 4.4  105 spirochetes of B. lonestari (LS-1), passage 3, were inoculated intravenously, intradermally, and subcutaneously into each of two deer fawns; negative control animals were not included. Sedation of each deer was then reversed with 0.1 mg/kg yohimbine (2 mg/ml Ben Venue Laboratories; Bedford, OH). Blood samples were collected from each deer at 0, 4, 6, 8, 12, 15, 19, 22, 28, 35, 42, 49, and 56 days postinoculation (DPI).

B. lonestari (LS-1) was originally isolated from A. americanum ticks collected in Clarke County, Georgia (Varela et al., 2004a). Four passage two isolates stored in liquid nitrogen from the original culture were thawed and transferred to flasks containing ISE6 cell monolayers (Kurtti et al., 1993; Munderloh et al., 1994). Cultures were fed every 3–4 days with L15B300 medium supplemented with either 10% Barbour-Stoenner-Kelly (BSK)-H (Sigma, St. 117 Louis, MO) or BSK-II as previously described (Varela et al., 2004a). 2.3. Preparation of inocula Inocula were prepared the day of inoculation and used within 4 h. To prepare the inocula used in deer (n = 2), mice (n = 5), calves (n = 3), and dogs (n = 3), 5–10 ml of B. lonestari culture grown in ISE6 cells were harvested, gently vortexed, and then centrifuged at 1118  g for 10 min. The supernatant was removed and the pellet resuspended in fresh media. Since LS-1 spirochetes are difficult to separate for counting, a direct count of viable organisms unattached to cells was made using 10 ml of inoculum under darkfield and thus estimating the minimum number of spirochetes inoculated into each animal. The inoculum was transferred to syringes and held at 34 8C until use; 0.1 ml of each inoculum was passed to a new flask of ISE6 cells to assess viability. A ‘‘cell minimized’’ inoculum was prepared for injection into two additional mice by harvesting 5 ml of B. lonestari culture grown in ISE6 cells, gently vortexing, and then centrifuging at 80  g for 10 min. The top 4.5 ml

2.4. Inoculation of animals

2.4.2. Mice A minimum of 4.24  105 spirochetes of B. lonestari (LS-1), passage 4 were inoculated into one male and two females, subcutaneously and intraperitoneally, and two females, intradermally and intraperitoneally. An additional two mice, one male and one female, were needle inoculated with a minimum of 5.3  105 spirochetes of B. lonestari (LS-1), passage 4, ‘‘cellminimized’’ culture subcutaneously and intraperitoneally. Blood samples were collected from each mouse at 0, 3, 7, 10, 14, 17, 21, 24, and 28 DPI. 2.4.3. Calves Three calves were manually restrained and inoculated with a minimum of 1.7  106 spirochetes of B. lonestari (LS-1), passage 3, intravenously and subcutaneously. One control calf was inoculated with uninfected ISE6 cells. Blood samples were collected from calves at 0, 4, 6, 8, 11, 13, 15, 18, and 21 DPI.

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2.4.4. Dogs Three dogs were inoculated intravenously and subcutaneously with a minimum of 3.4  106 spirochetes of B. lonestari (LS-1), passage 3. One control dog was inoculated with uninfected ISE6 cells. Blood samples were collected at 0, 2, 4, 7, 9, 11, 14, 16, 18, 21, 28, 36, 42, 49, and 56 DPI. 2.5. Detection of antibodies To detect antibodies to B. lonestari, two-fold dilutions of serum were used with a cut-off value of 1:64. ISE6 cells infected with LS-1 were fixed on microscope slides with acetone, incubated for 25 min at 37 8C with diluted serum collected from experimental animals. Slides were washed twice in 1 phosphate buffered saline (PBS; pH 7.4) for 5 min each, rinsed in distilled water for 5 min, and allowed to air dry. A 1:30 dilution of fluorescein isothiocyanatelabeled anti-deer, -mouse, -dog, or -cow, depending on the species considered, was applied to slides followed by incubation for 25 min at 37 8C. Afterwards, the slides were washed in PBS and counterstained with eriochrome black T. After drying in the dark, slides were examined with a compound microscope under UV illumination. Antibodies to the ISE6 cells were absorbed from the dog serum before beginning the IFA procedure. Unlike the dogs, deer, mice, and calves did not develop reactions to tick cells and thus absorption was not performed for the serum samples from these hosts. To absorb antibodies, 5 ml of uninfected ISE6 cells were harvested and transferred to a 15 ml tube, diluted 1:1 with sterile dPBS, and then sonicated on ice at 5 W for two, 1 min cycles. Cells were pelleted by centrifugation at 20,000  g for 30 min and the sonicate aliquoted and frozen until use. Dog serum was centrifuged at 12,000  g for 20 min, diluted 1:10 with the ISE6 cell sonicate, and then incubated at 37 8C for an hour. The serum and sonicate mixture was then used in the IFA procedure as described above. 2.6. Detection of organisms Thin blood smears were prepared using blood collected in ethylenediaminetetraacetic acid (EDTA)– tetrasodium salt tubes, air dried, and fixed in methanol. Fixed slides were stained using Giemsa or by flooding

for 2–3 min with acridine orange (AO) (Lauer et al., 1981), air dried, and examined under oil immersion using a light microscope or a fluorescent microscope. DNA was extracted from 100 ml of whole blood using the GFX Genomic Blood Purification kit (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. A 330-bp region of the flagellin gene ( flaB) was targeted using the external primers FLALL and FLARL and the internal primers FLALS and FLARS in a nested PCR assay previously described (Moore et al., 2003). Samples were also tested for the presence of B. lonestari DNA using a nested PCR designed to amplify a 706-bp region of the glycerophosphodiester phosphodiesterase (glpQ) gene using external primers GlpQx1 and GlpQx2 and internal primers GlpQi5 and GlpQi3a as previously described (Bacon et al., 2004). Sequencing was performed on positive control cultures and both blood and culture positive samples from deer. Amplicons were purified and concentrated with a Micron 100 microconcentrator (Amicon Inc., Beverly, MS) and submitted to MWG-Biotech Inc. (High Point, NC) for sequencing of both forward and reverse strands. The sequences were then aligned using the ClustalX multiple-alignment program and directly compared to the published flaB sequence of the B. lonestari culture (AY442142) in GenBank. 2.7. Recovery of organisms For isolation of B. lonestari from inoculated animals, sterilely collected heparinized whole blood samples (3–7 ml) were centrifuged at 711  g for 20 min and the buffy coat placed in 10 ml of L-15B cell culture media (catalog number 41300; Invitrogen, Carlsbad, CA), mixed gently, and then centrifuged at 711  g for another 20 min. The supernatant was removed and the pellet mixed with ISE6 cells freshly harvested from a single 5 ml flask suspended in L-15B media. An additional 10 ml of L-15B was added, the mixture incubated at room temperature for 15 min, and then centrifuged at 711  g for 30 min. The supernatant was removed, and the pellet was resuspended in 5 ml of L-15B300/BSKII, transferred to new 5 ml flask of ISE6 cells, and incubated at 34 8C. Cultures were fed twice weekly as previously described. Each flask was monitored for development of

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spirochetes at each feeding and maintained for at least 45 days (Varela et al., 2004a).

3. Results 3.1. Inoculum All control cultures of B. lonestari inocula grew after passage into fresh ISE6 cells and thus were viable.

Fig. 1. Borrelia lonestari in an acridine orange stained thin blood smear from an experimentally infected white-tailed deer fawn.

test PCR positive until DPI 28. B. lonestari was isolated in culture from both deer on DPI 6 and from one deer on DPI 12. On days when deer were positive by both blood Giemsa and AO blood smears they were also positive on both the primary and secondary PCR reactions. Sequence of amplicons from whole blood and culture from DPI 6 was 100% identical to the published sequence of B. lonestari LS-1 (AY442142). One deer had a maximum antibody titer of 1024 on DPI 15; maximum titer antibody in the other deer was 128.

3.2. Infection of deer Results from infection of white-tailed deer are summarized in Table 1. Prescreen results from deer showed no evidence of infection or previous exposure by PCR and IFA, except one deer with a weak positive IFA titer of 64 on DPI 0. AO was more sensitive than Giemsa for detecting spirochetes in blood smears. Spirochetes were visualized in Giemsa stained blood smears of both deer only on DPI 8 and in one deer on DPI 12 and 15. Spirochetes were directly observed in AO stained blood smears of both infected white-tailed deer on DPI 6, 8, and 12 and in one deer on DPI 15 (Fig. 1). PCR targeting the flagellin gene of Borrelia spp. identified B. lonestari in the blood of both deer from DPI 4 through DPI 22, and one deer continued to

3.3. Infection of mice No spirochetes were observed in any of the thin blood smears from mice. Only one mouse, a male injected with the ‘‘cell minimized’’ inoculum, was

Table 1 Giemsa (G) and acridine orange (AO) blood smears, flagellin (FlaB) and glpQ (GlpQ) polymerase chain reaction (PCR), culture, and indirect fluorescent antibody (IFA) results for white-tailed deer experimentally-inoculated with ISE6 tick cell culture derived Borrelia lonestari DPIa

0 4 6 8 12 15 19 22 28 35 42 49 56 a b

WTD 151

WTD 152

Blood smears G/AO

FlaB PCR 18/28

GlpQ PCR 18/28

/ / /+ +/+ +/+ +/+ / / / / /ND b /ND /ND

/ /+ /+ +/+ +/+ +/+ /+ / / / / / /

ND /+ /+ +/+ +/+ +/+ /+ /+ / / / / ND

Days post-inoculation. No data.

Culture

+

IFA

Blood smears G/AO

FlaB PCR 18/28

GlpQ PCR 18/28

64 128 256 128 256 1024 512 512 256 128 128 64 64

/ / /+ +/+ /+ / / / / / /ND /ND /ND

/ /+ /+ +/+ +/+ /+ /+ /+ /+ / / / /

ND /+ /+ +/+ +/+ /+ /+ /+ /+ / / / ND

Culture

IFA

128 + +

128 128 128 128 64 64

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PCR positive for a flaB fragment on DPI 17 and 21. All other mice were PCR negative throughout the study. However, all mice had detectable antibodies following inoculation, with a maximum antibody titer of 512. B. lonestari was not recovered by culture from any of the mice. 3.4. Infection of calves and dogs All prescreen tests for cattle and dogs showed no evidence of prior infection, except one experimentally infected dog that had a weak IFA titer (64) to B. lonestari in tick cells. All cattle and dogs were consistently negative at all time points when evaluated by both culture and PCR of whole blood. All calves also remained negative for antibodies by IFA throughout the study. All inoculated dogs except the negative control dog showed an increase in antibody titer with a maximum titer of 512 between DPI 11 and 28 that decreased to 64 by DPI 56.

4. Discussion Deer in this study that were experimentally infected with B. lonestari developed a spirochetemia detectable by direct examination of blood smears and/or by PCR of B. lonestari DNA in whole blood samples for as long as 16–25 days, indicating that the organism survived in infected deer and infected deer were able to maintain the infection. B. lonestari also was reisolated in tick cell culture from both deer, and both deer developed detectable antibody responses following inoculation. It seems somewhat unusual that deer were negative by culture on several days that they were obviously spirochetemic as seen by blood smears and PCR; however, because the majority of plasma and red blood cells were discarded during the culture process, it is possible our protocol was not ideally suited for recovery of organism in culture at every time point. Nonetheless, B. lonestari was isolated from whole blood and these results clearly demonstrate that white-tailed deer are susceptible to experimental infection with B. lonestari. Deer did not develop overt clinical signs of disease at any time during these studies. Naturally occurring B. lonestari infections have been described in wild white-tailed deer from several southeastern states (Moore et al., 2003). When

considered together with the natural history of the vector tick, A. americanum, which feeds on deer as a larva, nymph, and adult (Kollars et al., 2000; Lockhart et al., 1997a,b; Luckhart et al., 1992), the finding of B. lonestari infection in wild deer led to speculation that deer may serve as a reservoir host for this organism as they do for other A. americanum-vectored disease agents, including E. chaffeensis and E. ewingii (Lockhart et al., 1997a; Yabsley et al., 2002). The finding that deer developed spirochetemia offers further support for the hypothesis that the natural life cycle of this organism may involve white-tailed deer as reservoir host and A. americanum as tick vector. Although all of the mice in this study did not become infected with B. lonestari, they each developed a detectable antibody response. One male mouse inoculated with tick cell-minimized culture was transiently PCR positive on 2 study days, but was negative at all other time points tested; the other six mice were negative throughout the entire 1-month study period following inoculation. In contrast, others have shown that young C3H mice are highly susceptible to infection with B. burgdorferi (Barthold et al., 1990), and wild mice and other rodents are known to play vital roles in the maintenance of B. burgdorferi in nature by serving as a reservoir host capable of infecting larval or nymphal Ixodes scapularis ticks when the ticks feed (Levine et al., 1985). However, unlike I. scapularis, A. americanum is found infrequently on mice or other small rodents in nature, and studies focusing on other pathogens vectored by A. americanum have shown that mice are rarely exposed to these pathogens in nature (Kollars et al., 2000; Lockhart et al., 1997b; Magnarelli et al., 1997). Although this study supports the hypothesis that mice are not likely to be important in the natural history of B. lonestari, their role, if any, can only be completely understood through vector feeding studies. The experimentally inoculated calves and dogs did not become PCR positive nor did they mount a detectable antibody response as evaluated by IFA. The calves were monitored only for 21 days which may not have been long enough to detect an antibody response. A previous study using Ayrshire calves and Finnish B. burgdorferi sensu lato strains showed a low susceptibility of cattle to B. burgdorferi infection (Tuomi et al., 1998). Dogs are susceptible to infection with the

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Lyme disease agent and develop clinical signs (Chang et al., 2001). The results from this study provide evidence that white-tailed deer may play an important role in the maintenance of B. lonestari in nature, while mice, cattle, and dogs likely do not, although tick feeding studies are required before any definitive conclusions about true reservoir status of these potential reservoir host species can be drawn. Nonetheless, the data described here as well as from field surveys support the assertion that B. lonestari is maintained in a cycle involving white-tailed deer as a reservoir host and lone star ticks as a vector. Additional tick transmission trials using infected ticks to transmit B. lonestari from infected to naı¨ve deer are needed to confirm this natural history model.

Acknowledgements The authors thank Dr. David Osborn (Forest Resources, UGA) and TRS Labs Inc. (Athens, GA) for donating the white-tailed deer fawns and beagles, respectively. We also thank the numerous personnel of Animal Resources and the Southeastern Cooperative Wildlife Disease Study for their assistance with animal care/handling and laboratory studies. This work was supported by the University of Georgia Research Foundation and the College of Veterinary Medicine.

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