iolog ter y & c P a a B r f a o s Scott et al., J Bacteriol Parasitol 2017, 8:1 i l Journal of Bacteriology and t o a l n o r DOI: 10.4172/2155-9597.1000304 g u y o J Parasitology ISSN: 2155-9597 Research Article Open Access Lyme Disease Bacterium, Borrelia burgdorferi Sensu Lato, Detected in Multiple Tick Species at Kenora, Ontario, Canada John D Scott1*, Kerry L Clark2, John F Anderson3, Janet E Foley4, Monica R Young5 and Lance A Durden6 1Research Division, Lyme Ontario, 365 St. David Street South, Fergus, Ontario, Canada N1M 2L7 2Environmental Epidemiology Research Laboratory, Department of Public Health, University of North Florida, 1 UNF Drive, Jacksonville, Florida 32224 3Department of Entomology and Center for Vector Ecology and Zoonotic Diseases. The Connecticut Agricultural Experiment Station, 123 Huntington St., New Haven, Connecticut 06511 4Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, 1320 Tupper Hall, Davis, California 95616 5Centre for Biodiversity Genomics, University of Guelph, Guelph, Ontario, Canada N1G 2W1 6Department of Biology, Georgia Southern University, 4324 Old Register Road, Statesboro, Georgia 30458, USA Abstract We detected the Lyme disease bacterium, Borrelia burgdorferi sensu lato (s.l.), in 8 species of ixodid ticks (Acari: Ixodidae) collected from mammalian hosts, including humans, at Kenora, Ontario, Canada. These 8 tick species include Ixodes angustus, Ixodes banksi, Ixodes cookei (groundhog tick), Ixodes gregsoni, Ixodes muris (mouse tick), Ixodes scapularis (blacklegged tick), Haemaphysalis leporispalustris (rabbit tick), and Dermacentor albipictus (winter tick). Based on PCR amplification, 39 (41%) of 94 ticks tested were positive for B. burgdorferi s.l. DNA sequencing of the flagellin B (flaB) gene of B. burgdorferi s.l. complex revealed the presence of B. burgdorferi sensu stricto (s.s.), which is pathogenic to humans, and causes diverse neurological manifestations in patients. Notably, we provide the first record ofB. burgdorferi s.l. in I. gregsoni, and reveal a new distribution record for this tick in eastern and central Canada by extending the known range westward by 200 km. Our findings indicate that there may be a wide-ranging enzootic transmission cycle of B. burgdorferi s.l. within the ecosystem throughout the Kenora area. The health-care profession must be fully cognisant that Lyme disease is present in the Kenora area, and is a public health risk. Keywords: Ticks; Mammalian hosts; Lyme disease; Lyme disease area has the highest infection prevalence of Borrelia burgdorferi sensu lato; Kenora; Ontario; B. burgdorferi s.l. ever reported in Canada. It is evident that Epidemiology; Public health the climate in the Kenora area provides an environment that is conducive for I. scapularis reproduction. Abbreviations: PCR: Polymerase Chain Reaction; CNS: Central Nervous System Across North America, several Ixodes species are vector competent for B. burgdorferi s.l., including I. affinis, Introduction I. angustus, I. dentatus, I. jellisoni, I. minor, I. muris, I. The Lyme disease bacterium,Borrelia burgdorferi scapularis, and I. spinipalpis [7]. Of these ticks, immature sensu lato (s.l.), is typically transmitted to mammalian stages of I. affinis, I. dentatus, I minor, I. muris, I. hosts, including humans, by ixodid (hard-bodied) ticks scapularis, and I. spinipalpis, and all host-seeking stages of (Acari: Ixodidae) [1]. Each tick species has its own set of I. muris can parasitize birds, and can be transported long suitable hosts, and Lyme disease, a zoonosis, is incidental distances into Canada during northward spring migration to humans and domestic animals [2,3]. Left untreated or [8-17]. Each of these ixodid ticks has the ability to acquire inadequately treated, Lyme disease can inflict a myriad of spirochetes while feeding on an infected host and, during multisystemic clinical manifestations. the moult, pass them transstadially to the next life stage. The first isolation ofB. burgdorferi s.l. in mainland Ontario [4] was from a blacklegged tick, Ixodes scapularis *Corresponding author: John D Scott, Research Division, Lyme Ontario, 365 (northern populations formerly considered as Ixodes St. David Street South, Fergus, Ontario N1M 2L7, Tel: 519-843-3646; E-mail: dammini), collected from a dog with no history of travel [email protected] Received February 20, 2017; Accepted March 13, 2017; Published March 20, that was living in Kenora, Ontario. A 10-year tick-host 2017 study (1993-2002), which was conducted across Ontario, Citation: Scott JD, Clark KL, Anderson JF, Foley JE, Young MR, et al. (2017) Lyme revealed several B. burgdorferi s.l.-infected I. scapularis Disease Bacterium, Borrelia burgdorferi Sensu Lato, Detected in Multiple Tick Species at Kenora, Ontario, Canada. J Bacteriol Parasitol 8: 304. doi:10.4172/2155- ticks on domestic animals and humans in the Kenora 9597.1000304 District [5]. Recently, Scott et al. [6] documented an Copyright: © 2017 Scott JD, et al. This is an open-access article distributed under established population of I. scapularis on Corkscrew Island, the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and which is 20 km southwest of Kenora. This hyperendemic source are credited. J Bacteriol Parasitol, an open access journal ISSN: 2155-9597 Volume 8 • Issue 1 • 1000304 Citation: Scott JD, Clark KL, Anderson JF, Foley JE, Young MR, et al. (2017) Lyme Disease Bacterium, Borrelia burgdorferi Sensu Lato, Detected in Multiple Tick Species at Kenora, Ontario, Canada. J Bacteriol Parasitol 8: 304. doi:10.4172/2155-9597.1000304 Page 2 of 10 Subsequently, Lyme disease spirochetes can be transmitted Dead and damaged ticks were put in 2 mL micro tubes to vertebrate hosts, including humans. containing 94% ethyl alcohol. Both live and dead ticks were sent to the tick identification lab (JDS). The aim of this study was to identify species of ticks in the Kenora District that may be involved in the enzootic Fully engorged immature ticks were held to molt to cycle of B. burgdorferi s.l. the next developmental life stage. These engorged ticks were placed in 8.5 mL polypropylene tubes with a slightly Materials and Methods moistened piece (5 cm × 5 cm) of chlorine-free paper Study area towel. The open end of the tube was covered with tulle Ticks were collected from humans, domestic and netting before the open-centered push cap was inserted. wildlife animals within 50 km of Kenora, Ontario, Canada The tubes were placed in a double-zipper plastic bag with (49° 46′ N, 94° 29′ W) on the northern edge of Lake of a slightly moistened paper towel to provide adequate the Woods. This geographic area lies within the Canadian humidity (~95%). A full spectrum, 12W LED, LifeLite shield, which is comprised of Precambian igneous rock. light bulb (LifeEnergy Systems), on a timer, was set for The coniferous-deciduous forest lies within the boreal a summertime photoperiod (16:8/L:D). Each plastic bag, forest region, and consists primarily of: red pine, Pinus which contained replete ticks, was checked every 3-5 days resinosa; eastern white pine, Pinus strobus; black spruce, to assess the progress of the moult and to ensure that Picea mariana; white spruce, Picea glauca; white birch, sufficient humidity was maintained. A log sheet was kept Betula papyrifera; trembling aspen, Populus tremuloides; for each tick to record the dates checked, advancement of bur oak, Quercus macrocarpa; and Manitoba maple, Acer the moult, and the number of days to complete the moult. negundo. Spirochete detection Tick collection Field-collected ticks were sent to 3 different Ticks were collected from injured wild mammals, laboratories for B. burgdorferi s.l. testing because the first domestic animals, and from local residents, and submitted two laboratories had policy changes during the study, and to local wildlife rehabilitators, wildlife biologists, and stopped testing ticks. The 3 laboratory periods are: May veterinarians. These ticks were removed using fine- 2013–June 2013 (JFA), July 2014–June 2015 (KLC), and pointed, stainless steel tweezers. Live ticks were placed in July 2015–November 2016 (JEF). At lab A (JFA), live round-bottom, 8.5 mL polypropylene tubes (15.7 mm × ticks from mammalian hosts were cultured in Barbour- 75 mm) (Sarstedt) with attached labels specifying host, Stoenner-Kelly (BSK) medium, and dead ticks were tested date collected, geographic location, and collector’s name. directly using DNA extraction and PCR testing. The A 7-mm hole in the polyethylene cap (15.7 mm) provided DNA detection protocols have been described previously ventilation for ticks and, to prevent the ticks from escaping, [18-20]. Although Persing et al. [18] employed both the fine tulle netting was stretched over the open end of the flagellin (fla) gene and the major outer surface protein tube before the push-cap was inserted. The tube, which (OspA) gene, lab A only used the OspA gene. Appropriate contained the ticks from a single host, was placed in a negative and positive controls were used. double-zippered plastic bag to maintain high humidity. In the second part of the study, ticks were placed in 94% No. of No. of Life stages of ticks No. ticks Bbsl-pos./ No. Tick species Host species (no.) hosts ticks L N M F of ticks tested (%) I. angustus dm (1) 1 1 0 0 0 1 1/1 (100) I. banksi bv (8) 8 11 0 8 0 3 6/11 (55) I. cookei gh (2), hm (1), mk (1) 4 17 0 12 0 5 6/17 (35) I. gregsoni er (2), mk (1) 3 17 0 14 0 3 4/12 (33) I. muris ct (2), dm (2), dg (5), mv (2), nss (2), srv (3) 16 16 2 8 0 6 7/16 (44) I. scapularis dg (9), hm (3) 12 15 0 0 2 13 5/15 (33) D.