INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Oct. 1997, p. 958-968 Vol. 47, No. 4 0020-7713/97/$04.00+ 0 Copyright 0 1997, International Union of Microbiological Societies Borrelia recurrentis Characterization and Comparison with Relapsing-Fever, Lyrne-Associated, and Other Borrelia spp. S. J. CUTLER,'" J. MOSS,2 M. FUKUNAGA,3 D. J. M. WRIGHT,' D. FEKADE,4 AND D. WARRELLs Department of Medical Microbiology' and Department of Histopathology, Charing Cross Hospital, London, W6 8W, and Centre for Tropical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom; Department of Molecular Microbiology, Fukuyama University, Hiroshima, Japan3; and Department of Internal Medicine, Black Lion Hospital, Addis Ababa, Ethiopia4 Borrelia recurrentis, the cause of louse-borne relapsing fever, has until recently been considered nonculti- vable, which has prevented characterization of this spirochete. We successfully cultivated 18 strains from patients with louse-borne relapsing fever and present the initial characterization of these isolates. Electron microscopy revealed spirochetal cells with pointed ends, an average wavelength of 1.8 pm, an amplitude of 0.8 pm, and 8 to 10 periplasmic flagella. The G+C ratio was 28.4 mol%.Whole DNA-DNA hybridizations showed similarity between the isolates of B. recurrentis but not with Borrelia hermsii, Borrelia parkeri, Borrelia turicatae, or the Lyme-associated borreliae. Sequencing studies of both the flagellin and 16s RNA genes revealed that the greatest similarity was between B. recurrentis and Borrelia duttonii. Analysis of the sodium dodecyl sulfate- polyacrylamide gel electrophoresis profiles of strains revealed four groups based on the position of a major protein band (one of the groups showed some heterogeneity and was subdivided into four subgroups). Pulsed-field gel electrophoresis revealed five distinct patterns. In the last century, successive epidemics of louse-borne re- with hemolymph from a crushed infected louse. Clinically, lapsing fever spread from Asiatic Russia into Europe, causing louse-borne relapsing fever is characterized a 5- to 7-day incu- death in from 2 to 40% of the cases. Now, this disease has all bation period, one to five relapses of fever, and spirochetemia but vanished in all areas except the Andean foothills, the Ethi- (notably fewer spirochetemias than in tick-borne relapsing fe- opian highlands, and nearby Rwanda. Even in the turmoil in ver). Febrile episodes typically last 2 to 9 days, with an afebrile the former Yugoslavia, the appearance of louse-borne typhus interval of about 9 days. The initial fever is usually the longest was not accompanied by relapsing fever. Why the disease has and most severe and is followed by milder relapses. Very high persisted in only a few isolated foci and has become invisible spirochete counts are usually associated with a poorer progno- elsewhere remains an enigma. sis. Mortality is higher in louse-borne relapsing fever (up to The vector of this disease is the infected human body louse 40% in untreated cases and 10% in treated cases) than in the (Pediculus humanus humanus). Lice become infected by feed- tick-borne variety; this may be related to higher counts of ing on a spirochetemic patient. The ingested spirochetes enter circulating spirochetes. It is possible that as seen in outbreaks, the gut and cross the epithelium into the hemolymph. Trans- there is a spectrum of severity. mission to humans occurs by contamination of abraded skin We were able to culture Borrelia recurrentis for the first time since it was described in 1867 by Obermeier (12). Preliminary characterization of B. recurrentis and a comparison of this or- TABLE 1. Strains used in this study ganism with relapsing-fever, Lyme-associated, and other bor- relial species are described in this paper. Organism Strain(s) Location B. recurrentis A1 to A18" Addis Ababa, Ethiopia B. burgdo$eri sensu stricto B3 1 United States B. gurinii 20047 France TABLE 2. B. recurrentis strains grouped according to B. ufzelii VS46 1 France plasmid patterns and protein profiles B. juponicu H014 Japan Group VS116 UK United Kingdom Protein profile(s) Group PotiB2 PotiB2 Portugal Protein Protein Protein Protein Protein Protein Protein B. miyumotoi HT3 1 Japan group group group group subgroup subgroup subgroup subgroup B. hermsii HS 1 United States 1 2 3 4a 4b 4c 4d B. purkeri United States B. turicutue United States Type 1 Al, A2, A5 A3 Strain A1 has been deposited in the American Type Culture Collection as Type 2 A4 A6, A7, A10 strain ATCC 700241. A8, A9 Type 3 All, A12 * Corresponding author. Mailing address: Department of Medical Type 4 A13, A14, Microbiology, Charing Cross Hospital, Fulham Palace Road, London, A15, A16 W6 8RF, United Kingdom. Phone: 0181 846 7570. Fax: 0181 846 7261. Type A17, A18 E-mail: [email protected]. 5 958 VOL. 47, 1997 4 BORRELLARECURRENTIS CHARACTERIZATION 959 I 2 3 4 5 6 7 8 9101112131415 12345 6 7 89 FIG. 2. PFGE patterns comparing each plasmid type of B. recurrentis with tick-borne and Lyme-associated borreliae. Lane 1, B. recurrentis type 1; lane 2, B. recurrentis type 2; lane 3, B. recurrentis type 3; lane 4, B. recurrentis type 4; lane 5, B. recurrentis type 5; lane 6, B. hemzsii; lane 7, B. parkeri; lane 8, B. turicatae; lane 9, B. miyamotoi; lane 10, B. busdo$en sensu stricto; lane 11, B. garinii; lane 12, B. aftelii; lane 13, B. japonica; lane 14, B. burgdo$en UK (group VS116); lane 15, B. buigdo$eri PotiB2. Immunoblotting. SDS-PAGE gels as described above were blotted onto ni- trocellulose membranes as described elsewhere (1l). After these membranes had been blocked overnight in 5% dried milk (Marvel) in Tris-buffered saline (50 mM Tris, 150 mM sodium chloride; pH 7.5), they were incubated at room 10 ll 12 13 14 15 16 17 18 temperature overnight with 1:1,000 dilutions of monoclonal antibody H9724 (4). After washing, each membrane was incubated with a 1:3,000 dilution of goat FIG. 1. Coomassie blue-stained SDS-PAGE protein profiles of B. recurrentis anti-mouse alkaline phosphatase conjugate (Bio-Rad), washed, and developed A1 to AlX. Lanes 1 through 4, protein group 1 strains; lane 5, protein group 2 by using a solution containing p-nitroblue tetrazolium chloride (0.3 mgiml) and strain; lanes 6 through 9, protein group 3 strains; lane 10, protein subgroup 4a 5-bromo-4-chloro-3-indolylphosphatep-toluidine salt (0.15 mg/ml), and the re- strain; lanes 11 and 12, protein subgroup 4b strains; lanes 13 through 16, protein action was stopped by washing in distilled water (11). The molecular weight of subgroup 4c strains; lanes 17 and 18, protein subgroup 4d strains. the flagellin band could then be determined by comparison to molecular weight standards (Rainbow markers; Amersham International). Coomassie blue staining. Minigels were stained by using a Coomassie blue stain (2.5 g of Coomassie blue R250 in 1 liter of 95% [vol/vol] ethanol, filtered MATERIALS AND METHODS before use)-l0% (vol/vol) glacial acetic acid solution (1:l) overnight. The gels were destained twice for 1.5 h with 100 ml of 95% (vol/vol) ethanol and 150 ml Louse-borne relapsing-fever patients. All patients from which cultures were of 5% (vol/vol) glacial acetic acid, then with 33 ml of 95% (vol/vol) ethanol and obtained were demonstrated to be spirochetemic by blood film microscopy by 133 ml of 5% (vol/vol) glacial acetic acid for 2 h, and finally with 117 ml of 5% using Wright’s stain. One patient was female, and the remaining 17 patients were (vol/vol) glacial acetic acid and 50 ml of distilled water overnight. male. The ages of the patients ranged from <12 to 36 years. Most were laborers Pulsed-field gel electrophoresis (PFGE). Cells were harvested from BSK I1 or jobless. Body lice were found on the clothing in all cases. The duration of medium by centrifugation at 3,500 Xg for 20 min, washed twice with a wash clinical symptoms varied between 3 and 7 days. solution (75 mM NaCl, 25 mM EDTA; pH 74, and resuspended in 1 ml of the Bacterial strains and culture conditions. The strains used in this study are wash solution. The optical density at 600 nm was adjusted to 2. Blocks were listed in Table 1. All isolates were cultured in BSK I1 medium (3). Relapsing- prepared by using a 1:l dilution of cells and 1% (vol/vol) low-melting-point fever strains were maintained by thrice weekly subculturing, and the remaining agarose (Promega) poured into 100-p.1 molds. DNA was ext,-zted by incubation strains were subcultured on a weekly basis. The strains of Lyme-associated and at 50°C overnight with 1 ml of 0.5 M EDTA (pH 8.5)-1% (wt/vol) sarcosyl- tick-borne relapsing-fever borreliae were of unknown passage number. The proteinase K (10 pg/ml) per block. Following extraction, the blocks were washed strains of R. recurrentis had passage numbers between 8 and 15; this was the four times with TE (10 mM Tris-C1 [pH 7.6],1 mM EDTA) and stored at 4°C in TE. minimum number of passages necessary to obtain sufficient material to under- Two-hundred-milliliter 1.2% agarose gels were loaded with blocks, sealed with take the study. agarose, and run in 0.5% TBE (0.045 M Tris-borate, 0.001 M EDTA) by using Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). a Pharmacia Gene Navigator. Lambda ladder, delta 39 (Promega), 8- to 48-kb, Borrelial cells were harvested from BSK I1 medium by centrifugation at 3,500 X g, washed twice, and suspended in phosphate-buffered saline (Oxoid) to give a protein concentration of approximately 1.8 mg/ml (as determined with a Bio-Rad Bradford protein assay kit).