Four distinct genetic groups of leptospiras were demonstrated among selected pathogenic and "biflexa" serological types. Pathogenic leptospiras could be divided into two groups on the basis of per cent guanine + cytosine (GC) in their deoxyribonucleic acid (DNA). One group had 36 1%, the other 39 4 1%. The biflexa strains had DNA of 39 + 1 % GC, but were further separated into two groups on the basis of DNA-annealing tests. Strains within groups had a high degree of specific duplex formation (75 % binding or more with reference to the homologous DNA). There was little or no genetic relatedness between strains of the four groups (less than 10% DNA homology). The thermal elution midpoint of heterologous DNA duplexes was always lower than the homologous reaction. The serological relationships among strains were not meaningful in terms of relatedness determined by specific duplex formation.
During the course of a periodic epidemiological survey for leptospirosis of a herd of bulls at the University of Illinois Agricultural Research Center, Dixon Springs, a new leptospiral strain was isolated from the urine of a clinically healthy Hereford bull, no. 3055. The strain was found to be antigenically unrelated t o known pathogenic (Leptospira interrogans) and "saprophytic" (Leptospira bilflexa) serotypes. Phenotypically, it most closely resembled L. biflexa strains; however, in view of the reported distinct genetic characteristics of strain 3055, its species classification is not recommended at this time.A periodic evaluation of leptospiral antibodies in a herd of 60 2-year-old Hereford bulls at the Dixon Springs Agricultural Center indicated recent Lep tospira interrogans serotype pomonu infection in the spring of 1965. (Information available since submission of the manuscript indicates "serovar" is the new official term for "serotype.") As no clinical signs of disease were evident, an attempt was made to isolate leptospiras from the urine of selected seropositive and seronegative bulls to determine whether serotype pomona was present in the herd. The isolation and characterization of a new leptospiral serotype from a clinically healthy bull (no. 3055) is described. MATERIALS AND METHODSUrine was collected from each animal in a sterile container, and approximately 0.1 ml was transferred into 5 ml of liquid and semisolid bovine albumin polysorbate 80 (4) medium. The inoculated medium was incubated at 30 C and examined weekly over a 6-week period with dark-field microscopy for the presence of leptospires.The leptospiral culture and the serum from the bull from which it was isolated were sent to the WHOFA0 Leptospirosis Reference Laboratory at Walter Reed Army Institue of Research (WRAIR) for typing and additional serological tests, respectively. The submitted culture was cloned by the use of plating medium. The cloned culture used as antigen was tested for cross-agglutination reactions with 85 different serotype-specific antisera selected from all known pathogenic serogroups, and with all except one (serotype tororo) of the antisera to the 55 classified Leptospira bij7exa serotypes (2). Additional tests were made with antisera prepared against strain 3055 and against eight other unidentified "bijlexa-like" isolates, four of which were from the same geographic region as strain 3055. One of the latter strains, A-177, isolated from a turtle (R. D. Andrews, Ph.D. thesis, Univ. of Illinois, 1966), had the same growth, physiological, and phage characteristics as strain 3055 (H. E. Ellinghausen and A. Ritchie, personal communication), which it also resembled in genetic characteristics (unpublished data, WRAIR). The microscopic agglutination technique was used for serological tests (1). Homologous titers of test antisera ranged from 1 :10,000 to 1 :500,000.Several differential tests, e.g., growth in 2,6diaminopurine (8), growth at 13 C (7), hemolysis of mouse erythrocytes (9), and egg yolk reaction test ( 5 ) , we...
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