TICKS AND TICK-BORNE PATHOGENS AND PUTATIVE SYMBIONTS OF BLACK BEARS (URSUS AMERICANUS FLORIDANUS) FROM GEORGIA AND FLORIDA Michael J. Yabsley*§, Todd N. NimsÀ§, Mason Y. Savage*, and Lance A. Durden`§ D. B. Warnell School of Forestry and Natural Resources, University of Georgia, Athens, Georgia 30602. e-mail: myabsley@uga.edu ABSTRACT: Ticks were collected from 38 black bears (Ursus americanus floridanus) from northwestern Florida (n 5 18) from 2003 to 2005 and southern Georgia (n 5 20) in 2006. Five species (Amblyomma americanum, A. maculatum, Dermacentor variabilis, Ixodes scapularis, and I. affinis) were collected from Florida bears, and 4 species (A. americanum, A. maculatum, D. variabilis, I. scapularis) were collected from bears in Georgia. Ixodes scapularis was the most frequently collected tick, followed by D. variabilis, A. americanum, A. maculatum, and I. affinis. The collection of I. affinis from a Florida bear represents a new host record. A subset of ticks was screened for pathogens and putative symbionts by polymerase chain reaction (PCR). The zoonotic tick-borne pathogens Ehrlichia chaffeensis and Rickettsia parkeri were detected in 1 of 23 (4.3%) A. americanum and 1 of 12 (8.3%) A. maculatum, respectively. The putative zoonotic pathogen ‘‘Rickettsia amblyommii’’ was detected in 4 (17.4%) A. americanum and 1 (8.3%) A. maculatum. Other putative symbiotic rickettsiae detected included R. bellii and R. montanensis in D. variabilis,a Rickettsia cooleyi–like sp. and Rickettsia sp. Is-1 in I. scapularis, and Rickettsia TR39–like sp. in I. scapularis and A. americanum. All ticks were PCR-negative for Anaplasma phagocytophilum, Panola Mountain Ehrlichia sp., E. ewingii, Francisella tularensis, and Borrelia spp. The black bear, Ursus americanus Pallas, is widely distributed in North America and is an important mega-omnivore in several habitats (Larivie `re, 2001). Although ticks have previously been reported from black bears in California, Colorado, Florida, Michigan, Minnesota, Montana, New York, Wisconsin, and Nova Scotia (Taylor, 1951; Rogers, 1953; King et al., 1960; Dodds et al., 1969; Rogers, 1975; Rogers and Rogers, 1976; Manville, 1978; Furman and Loomis, 1984; Forrester, 1992), there are no published data on the presence of putative symbionts and zoonotic pathogens in ticks collected from bears. The black bear subspecies in northern Florida, southern Georgia, and southern Alabama is Ursus americanus floridanus Merriam, and it is currently listed as threatened in Florida. In an effort to determine the tick species parasitizing this black bear subspecies and the presence of pathogens and other putative symbionts in these ticks, we collected and analyzed ticks from this host in northern Florida and southern Georgia from 2003 to 2006. MATERIALS AND METHODS Ticks were collected from 18 black bears in northwestern Florida (Bay, Franklin, Gulf, Jefferson, Liberty, Madison, Okaloosa, and Wakulla counties) from December 2003 to September 2005 and from 20 black bears in southern Georgia (Charlton, Clinch, Echols, and Ware counties) from September to November 2006. Florida bears included in the study were mostly road-kills and some immobilized nuisance bears from the months of January (5 bears), March (1), April (1), May (1), June (2), July (1), August (2), September (2), October (1), and December (2). Georgia bears were lawfully harvested by various hunters and then examined at hunter check stations during the months of September (10 bears), October (9), and November (1). Ticks were collected from all bears but, because of time constraints, probably did not represent the entire tick population on each bear. Ticks were stored in 95% ethanol and later identified to species and stage using standard guides (Keirans and Litwak, 1989; Keirans and Durden, 1998). Voucher tick specimens from this study are deposited in the U.S. National Tick Collection at Georgia Southern University under accession numbers RML 123942 and RML 123943. DNA was extracted from the remaining ticks (n 5 128) for pathogen and putative symbiont screening. Each individual tick was washed with phosphate buffered saline (PBS) and placed into an individual micro- centrifuge tube (Sarstedt, Newton, North Carolina). The tubes were immersed in liquid nitrogen for ,15 sec. Each tick was then rapidly removed from the tube and placed into a second tube that contained 2 copper BBs. The tick was homogentized at 1/2 speed for 40 sec with a commercial beadbeater (Biospec Products, Bartlesville, Oklahoma). After homogenization, 225 ml of PBS was added to each tube, tubes were centrifuged at 2,500 g for 10 min, and then samples were frozen at 280 C until testing. DNA was extracted from 100 ml of homogenate using the DNeasy blood and tissue kit (Qiagen, Valencia, California) following the manufacturer’s supplemental protocol for extraction from insects. The following PCR techniques and gene targets were utilized for screening: 17 kDa for Rickettsia spp., msp2 for Anaplasma phagocytophilum, gltA for Panola Mountain Ehrlichia sp., 16S rRNA and VLPT for Ehrlichia chaffeensis, 16S rRNA for Ehrlichia ewingii, FopA for Francisella tularensis and fla for Borrelia spp. as previously described (Barbour, et al., 1996; Fulop et al., 1996; Zeidner et al., 2000; Labruna et al., 2004; Varela et al., 2004; Yabsley et al., 2008). Positive samples were sequenced and compared to published sequences in the GenBank database. RESULTS Five species of ixodid (hard) ticks were recorded from 38 black bears (Table I); all 5 tick species were recorded from bears (n 5 18) from Florida and 4 species from bears (n 5 20) in Georgia. Because ticks collected from individual bears probably did not represent total populations, the numbers shown in Table I should be considered as minimum prevalences and mean intensities. A total of 128 ticks from GA (n 5 34) and FL (n 5 94) was available for PCR testing. One of 23 (4.3%) A. americanum specimens was PCR-positive and sequence confirmed as Ehrlichia chaffeensis by VLPT (4 repeats) PCR assay. The E. chaffeensis– positive tick was from Charlton County, Georgia. A total of 42 (32.8%) ticks was PCR-positive for Rickettsia spp. (Table II). Both ‘‘R. amblyommii’’ and a Rickettsia TR39–like sp. were detected in A. americanum (Table II). Two of 12 (16.7%) A. maculatum were PCR-positive for Rickettsia spp., 1 for R. parkeri, and 1 for ‘‘R. amblyommii’’ (Table II). The R. parkeri–positive tick was from Charlton County, Georgia. R. montanensis was detected in D. variabilis from both Georgia and Florida and R. bellii was detected in a single D. variabilis from Florida (Table II). Twenty-six of 65 (40%) I. scapularis were PCR-positive for Rickettsia spp., and a Rickettsia TR39–like sp., a Rickettsia cooleyi–like sp., and Rickettsia sp. Is-1 sp. were identified } Contributed equally to this work. { Wildlife Resources Division, Georgia Department of Natural Resources, 2065 U.S. Highway 278 SE, Social Circle, Georgia 30025. { Department of Biology, 69 Georgia Avenue, Georgia Southern University, Statesboro, Georgia 30460-8042. DOI: 10.1645/GE-2111.1 *Southeastern Cooperative Wildlife Disease Study, 589 DW Brooks Drive, Department of Population Health, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602. J. Parasitol., 95(5), 2009, pp. 1125–1128 F American Society of Parasitologists 2009 1125