An enzootic transmission cycle of Lyme borreliosis spirochetes in the southeastern United States J. H. Oliver, Jr.*†, T. Lin*, L. Gao*, K. L. Clark‡, C. W. Banks*, L. A. Durden*, A. M. James§, and F. W. Chandler, Jr.¶ *Institute of Arthropodology and Parasitology and Department of Biology, Georgia Southern University, Statesboro, GA 30460-8056; ‡Department of Health Sciences, University of North Florida, Jacksonville, FL 32224; §United States Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services, Centers for Epidemiology and Animal Health, 555 South Howes Street, Fort Collins, CO 80525; and ¶Department of Pathology, Medical College of Georgia, Augusta, GA 30912-3605 Communicated by William C. Campbell, Drew University, Madison, NJ, July 18, 2003 (received for review April 17, 2003) Lyme borreliosis, or Lyme disease (LD), is a tick-borne zoonotic birds. In fact, in the northeast and north central states, I. infection of biomedical significance, caused by Borrelia burgdorferi scapularis is also the main enzootic vector. In contrast, in sensu lato (s.l.) spirochetes and transmitted by Ixodes species ticks. California, Ixodes spinipalpis appears to be more important than It usually circulates among wildlife vertebrate reservoirs and vector I. pacificus in maintaining the enzootic cycle of the spirochete, ticks but may infect humans, causing multisystem problems. In far although it rarely bites humans (11). There is greater spirochete western and northern North America, the host reservoirs, tick and tick vector diversity in California and the southeastern U.S. vectors, and genospecies of Borrelia are well known but not so in than in the northern states (8, 9, 11), which prompted us to the southern U.S., where there is controversy as to the presence of hypothesize that an analogous situation regarding enzootic tick ‘‘true’’ LD. Here we report the presence of the LD spirochete B. vectors in California might exist in the South. We also wanted to burgdorferi sensu stricto (s.s.) and Borrelia bissettii, three main determine the main vertebrate reservoirs of B. burgdorferi in this reservoir hosts, and two enzootic tick vectors in the southeastern region. U.S. The two enzootic tick vectors, Ixodes affinis and Ixodes minor, rarely bite humans but are more important than the human biting Materials and Methods ‘‘bridge’’ vector, Ixodes scapularis, in maintaining the enzootic Rodent Collections, Spirochete Isolation, and Characterizations. A spirochete cycle in nature. We also report extraordinary longevities variety of rodents and ticks were collected from nine sites in and infections in the reservoir rodents Peromyscus gossypinus, Georgia, seven in South Carolina, and five in Florida from 1991 Sigmodon hispidus, and Neotoma floridana. through 1999 and inspected for the presence and prevalence of B. burgdorferi s.l. (8, 9, 12–16). Rodents were live-trapped by yme borreliosis (LB) is an infection of public health impor- using an assortment of traps. Ticks were removed from the Ltance with endemic foci in North America and Eurasia. It is rodents, counted, and identified. They were also collected from the most common vector-borne infectious disease in the U.S. (1) vegetation via dragging or flagging a 1-m-square white flannel and Europe (2). The disease may affect the skin, joints, and cloth over low vegetation. We used the most conservative cardiovascular and nervous systems. It may range from severe to method available to determine infection, i.e., isolation of spiro- mild or even asymptomatic and may become chronic if not chetes in culture medium from tissues of collected vertebrates treated promptly. The causative agent of Lyme disease (LD) is and ticks. Urinary bladders and͞or ear biopsies from the rodents a spirochete, Borrelia burgdorferi sensu lato (s.l.), which is trans- and tick tissues were inoculated into Barbour–Stoenner–Kelly H mitted primarily by Ixodes spp. ticks (3). LB is a zoonosis in which medium and incubated at 32–34°C. Cultures were subsequently humans and domestic animals are incidental to its usual wildlife examined for spirochetes by dark-field microscopy for 6 weeks at reservoir hosts (4). The primary reservoir hosts in hyperendemic ϫ400. Before inoculation of tissues, they were processed as foci of the spirochete in the northeastern and north central U.S. reported (12). Spirochetal isolates were analyzed by using several are species of Peromyscus mice (5), although several other methods, including indirect immunofluorescence with several mammalian and avian species are reservoir-competent to vari- mAbs, polyclonal Abs, and Western blotting (17). Isolates were ous degrees and can be important locally throughout the U.S. (6) also screened by PCR for five known DNA target sequences and globally (7). It is important to identify the main reservoir specifically found in B. burgdorferi reference strain B-31, and host species in each particular geographic area, because the SDS͞PAGE protein profiles. Additionally, restriction fragment vertebrate species composition may affect local B. burgdorferi length polymorphism and sequence analyses of rrf-rrl intergenic abundance in nature. It is also important to identify the main spacer amplicon, ospC, flaB, and rrs genes were performed on local vector tick species responsible for transmission of the spirochete isolates (8, 9, 12, 17). spirochete to humans and the tick enzootic vectors, as well as the local genospecies of B. burgdorferi s.l. Phylogenetic Tree. The neighbor-joining tree was constructed At least 11 genospecies comprise the B. burgdorferi s.l. com- with PAUP (Ver. 4.0) software (18) and is based on a comparison plex worldwide, and three of these occur in the southeastern of 258-bp nucleotides of the rrf-rrl intergenic spacer sequence. U.S.: Borrelia andersonii, Borrelia bissettii, and B. burgdorferi The tree was compared with trees produced by maximum sensu stricto (s.s.) (8, 9). Of the 11 genospecies described globally, likelihood, parsimony, and unweighted pair group method with only three have been confirmed to have been cultured from arithmetic mean methods with PAUP software, and the four humans: B. burgdorferi s.s. (North America and western Europe), methods produced similar results. Borrelia garinii, and Borrelia afzelii (Europe and Asia). Addi- tionally, B. bissettii was reportedly cultured from several patients Results in Slovenia (10), but this has yet to be confirmed. Reservoir and Tick Collections. Peromyscus gossypinus was the most In North America, two tick species are known to transmit B. frequently trapped small mammal during a period of Ͼ10 years burgdorferi s.s. to humans, Ixodes pacificus in the far west and Ixodes scapularis in the east. In the U.S., Ͼ80% of LD cases occur in the northeastern and mid-Atlantic states (1). Although these Abbreviations: LD, Lyme disease; s.s, sensu stricto; s.l., sensu lato. two tick species transmit B. burgdorferi to humans, they can also †To whom correspondence should be addressed. E-mail: [email protected]. serve as enzootic vectors among several species of mammals and © 2003 by The National Academy of Sciences of the USA 11642–11645 ͉ PNAS ͉ September 30, 2003 ͉ vol. 100 ͉ no. 20 www.pnas.org͞cgi͞doi͞10.1073͞pnas.1434553100 Downloaded by guest on September 28, 2021 Table 1. Prevalence of B. burgdorferi s.l. in three species of rodents, P. gossypinus, S. hispidus, and N. floridana P. gossypinus, no. S. hispidus, no. N. floridana, no. Location pos͞no. tested (%) pos͞no. tested (%) pos͞no. tested (%) Georgia Sapelo Island 2͞9 (22) 1͞9 (11) — St. Catherines Island 5͞113 (4) —— Bulloch County 12͞50 (24) 2͞20 (10) 5͞14 (35) St. Marys 1͞17 (6) —— Mistletoe State Park 2͞10 (20) 0͞17 (0) 1͞8 (13) Screven County — 2͞7 (28) — Florida Merritt Island 3͞63 (5) 6͞43 (13) — Faver-Dykes State Park 0͞12 (0) 1͞1 (100) — Amelia Island 0͞7 (0) 1͞11 (9) — Tall Timbers Res. Station 2͞38 (5) 0͞20 (0) 0͞5 (0) South Carolina Wedge Plantation 17͞26 (65) 2͞17 (12) 8͞14 (57) Mt. Pleasant 11͞16 (69) 8͞10 (80) 7͞9 (78) Hobcaw Barony 10͞15 (67) — 8͞13 (62) Sumter County 2͞25 (8) 0͞1 (0) — Jasper County 2͞6 (33) — 4͞22 (18) Chester County 3͞22 (14) —— Total 69͞407 (17) 26͞178 (15) 33͞85 (39) in our study areas in Georgia, Florida, and South Carolina. Of Florida, and 0% to 80% in South Carolina. Among N. floridana, the 485 mammals collected in Georgia, 236 (48%) were P. prevalences ranged from 13% to 35% in Georgia, zero in the one gossypinus; in Florida, 74 (42%) of 176; and in South Carolina, area from which we collected woodrats in Florida, and 18% to 97 (41%) of 237 (13). Sigmodon hispidus was the second most 78% in South Carolina. Interestingly, no spirochetes were iso- abundant species collected in Georgia (55 of 236, 23%); in lated from a series of other small and medium-size mammals Florida (64 of 176, 36%); and in South Carolina (60 of 237, 25%). from Georgia (144 animals of 13 species), South Carolina (18 The third most abundant species of the 485 mammals in Georgia animals of 5 species), or Florida (77 animals of 7 species). The was a virtual tie between the 54 (11%) house mice Mus musculus species can be found in Appendix B, which is published as and the 52 (11%) Neotoma floridana eastern woodrats. In supporting information on the PNAS web site. MEDICAL SCIENCES Florida, the third most abundant mammal was the Norway rat, A total of 128 spirochetal strains were isolated from P. Rattus norvegicus. In South Carolina the third most abundant was gossypinus (69 isolates), N. floridana (33 isolates), and S. hispidus N. floridana (21%) (13). (26 isolates) (Table 1). All were identified as B. burgdorferi s.l. Five species of ticks were commonly recovered from cotton There is considerable genetic diversity among these isolates, mice, hispid cotton rats, or eastern woodrats during this study: which includes B. burgdorferi s.s., B. bissettii, B. andersonii, and the Gulf Coast tick Amblyomma maculatum, the American dog possibly an undescribed genospecies.