Deoxyribonucleic acid hybridization (hydroxyapatite method, 55 and 70°C) was used to characterize 38 serovars from 22 named serogroups of Leptospira interrogans and Leptospira bijlexa, 6 serovars from 4 new unnamed serogroups of Leptospira interrogans, and single serovars of the proposed species Leptospira parva and Leptonema illini. Deoxyribonucleic acid relatedness confirmed the validity of Leptospira parva and Leptonema illini. The well-accepted species Leptospira interrogans and Leptospira bijlexa, as currently defined, were extremely heterogeneous. Relatedness results revealed at least five new species among the parasitic serovars formerly included in Leptospira interrogans and two new species among the saprophytic serovars formerly included in Leptospira bijlexa. Serogrouping did not equate with species identification, as serovars from several different subserogroups belonged to different species. The new species named in this paper are Leptospira, noguchii, Leptospira weilii, Leptospira santarosai, Leptospira borgpetersenii, Leptospira meyeri, Leptospira wolbachii, and Leptospira inadai.Leptospiraceae is a family in the Spirochaetales whose type genus is Leptospira (7, 12, 14). The three Leptospira species are Leptospira interrogans, which contains a large number of serogroups whose strains are parasitic or pathogenic for humans and animals (14); Leptospira bijlexa, which contains a large number of serogroups whose strains are primarily found in fresh surface waters and moist soil and are rarely isolated from humans or animals (14); and Leptospira parva, which was isolated from tap water, is not pathogenic for hamsters, and is biochemically intermediate between L . interrogans and L . bijlexa (13). Leptonema, with the single species Leptonema illini, was proposed as a second genus in the Leptospiraceae (12). Leptonema illini deoxyribonucleic acid (DNA) has a guanine plus cytosine (G+C) content of 51 to 53 mol% (12), compared with 47.4 mol% for Leptospira parva (13), 36 to 39 mol% for Leptospira bijlexa (14), and 35 to 41 mol% for Leptospira interrogans (14). Leptonema possesses cytoplasmic tubules which are absent in Leptospira, and the structure of the basal complex on its flagella resembles that of gram-positive bacteria, whereas this structure in Leptospira resembles that of gram-negative bacteria (12). Leptonema strains were isolated from the urine of a healthy bull, a turtle, and water. They are not pathogenic for animals and are biochemically similar to Leptospira bijexa except for their ability to grow in Trypticase soy broth (1, 12). In addition to pathogenicity, Leptospira interrogans differs from Leptospira bijlexa by its inability to grow in the presence of 8-azaguanine and its inability to grow at 13°C (14-17). Two biogroups within Leptospira interrogans differ in their ability to grow in the presence of 2,6-diaminopurine (14). Leptospira parva does not grow in the presence of either of the above chemicals and does grow at 13°C (13). In general, the phenotypic characteristics that reportedly d...

Meningitis and Special of Bacterial and MycoticDiseases, Bldg 1-2226 Mailstop D11, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA DNA relatedness was determined among 303 strains of Lepfospira and Leptonema. Included in the analysis were reference strains from 228 wellcharacterized and recognized serovars. The study included 268 serovars from 29 named and one or more unnamed serogroups. The strains clustered into 17 DNA hybridization groups, representing 12 previously described species (292 strains) and five new genomospecies (1 1 strains). The largest groups included Lepfuspira inferrogans (91 strains from 82 serovars), Lepfospira sanfarosai (65 strains from 59 serovars), Lepfospira borgpefersenii (49 strains from 43 serovars), Lepfospira kirschneri (29 strains from 26 serovars) and Lepfospira noguchii (20 strains from 20 serovars). The new genomospecies include Lepfospira genomospecies 1 (two strains, serovars pinagchang and sichuan), Lepfospira genomospecies 2 (six strains, serovars lushui, manhao 3, manzhuang, nanding, mengla and yunnan), Lepfospira genomospecies 3 (one strain, serovar holland), Lepfospira genomospecies 4 (one strain, serovar hualin) and Lepfospira genomospecies 5 (one strain, serovar saopaulo). With the exception of BalCum, all serogroups with greater than one serovar studied were genetically heterogeneous. Phenotypic tests, including optimal growth temperature, lipase activity and growth inhibition by copper sulfate or 2,6-diaminopurine, were of little use in differentiating DNA relatedness groups. The name Lepfospira alexanderi sp. nov. is proposed for Lepfospira genomospecies 2 (type strain L 60T = ATCC 7O052OT, serovar manhao 3). Division

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Pontiac fever, a unique epidemiologic form of legionellosis, is characterized by a short (one- to two-day) incubation period and a self-limited grippe-like illness without pneumonia. In 1968, the first documented outbreak of this syndrome affected persons who had entered a health department building in Pontiac, Michigan. Epidemiologic analyses clearly implicated as airborne agent and suggested that evaporative condenser water aerosols being disseminated by a defective air conditioning system played a key role in the outbreak. Guinea pigs that were exposed in the building and to laboratory aerosols of evaporative condenser water developed bronchopneumonia. Legionella pneumophilia (serogroup 1) was isolated from the exposed guinea pigs' lungs. Paired acute and convalescent serum specimens from 37 patients were tested by the indirect fluorescent antibody technique using L. pneumophila serogroup 1 antigen, and 31 (84%) had rises in titer from less than 32 to greater than or equal to 64.

In 1993, the Aum Shinrikyo cult aerosolized Bacillus anthracis spores over Kameido, Japan. Spore samples were obtained from the release site, cultured, and characterized by molecular genetic typing. The isolates were consistent with strain Sterne 34F2, which is used in Japan for animal prophylaxis against anthrax.Strain identification in Bacillus anthracis has been problematic due to a lack of distinguishing features, both phenotypic and molecular (1). With the identification of variable-number tandem repeats (VNTRs), identification of strains (unique genotypes) by multiple-locus VNTR analysis (MLVA) is now possible, and worldwide clone-based diversity patterns have been demonstrated (2). The VNTR loci are hypervariable and have multiple allelic states that provide high discrimination capacity for differentiating among strains and for identifying evolutionary relationships. There are about six major worldwide clonal lineages and nearly 100 unique types now known.We recently applied this typing system to a forensic study of a B. anthracis strain associated with the Aum Shinrikyo cult's activities. Our objective was to determine whether the strain was similar to any other previously known types from our studies of B. anthracis worldwide diversity (2). This information could potentially identify the origin of the strain used in the bioterrorism attack and provide insights into how or why the attack was carried out.In June 1993, the Aum Shinrikyo cult sprayed a liquid suspension of B. anthracis from their headquarters building in Kameido, near Tokyo, Japan (3; H. Takahashi, A. Kaufmann, K. L. Smith, P. Keim, and K. Taniguchi, Program 4th Int. Anthrax Conf., p. 27, 2001). While this aerosolization went largely unnoticed, the cult's later (1995) sarin gas attack of a Tokyo subway attracted worldwide attention. It was only with testimony of cult members and a retrospective investigation (3; Takahashi et al., 4th Int. Anthrax Conf.) that the 1993 incident was recognized as an anthrax release. The cult had developed and constructed a delivery system that involved the pumping of a liquid bacterial suspension up eight floors of their headquarters building to an aerosol dispersal device on the roof. During the aerosol dispersal, health authorities received numerous public complaints concerning odors emanating from the building. Upon investigation, they observed and collected a fluid from the outside of the building. An archived (stored at 4°C) portion of this fluid was analyzed in this study.We examined the fluid collected from the Aum Shinrikyo headquarters for bacterial content. Microscopically, malachite green and safranin staining revealed stained spores, a large amount of debris, and other bacterial cells. Aliquots of the fluid were cultured by spreading on sheep blood agar plates and incubated at 37°C under ambient CO 2 concentrations. Approximately 4 ϫ 10 4 CFU per ml were observed, though most of the colonies grew only weakly. This weak growth was inconsistent with normal B. anthracis characteristics under these con...