The antimicrobial susceptibility of Borrelia burgdorferi isolated from human spinal fluid was determined in vitro and in vivo. A broth dilution technique was used to determine the MBCs of four antimicrobial agents. The Lyme disease spirochete was most susceptible to ceftriaxone (MBC, 0.04 ,ug/ml) and erythromycin (MBC, 0.05 ,ug/ml), then tetracycline (MBC, 0.8 ,ug/ml), and finally penicillin G (MBC, 6.4 ,g/ml). Syrian hamsters were used to determine the 50% curative doses (CD_0s) of the four antimicrobial agents. Ceftriaxone and tetracycline had the highest activities, with CD50s of 240 and 287 mg/kg, respectively. Both erythromycin and penicillin G possessed low activities. The CD50 of erythromycin was 2,353 mg/kg, and the CD50 of penicillin G was >1,975 mg/kg.

Syrian hamsters were shown to be susceptible to infection by the Lyme disease spirochete Borrelia burgdorferi. Although these spirochetes did not cause any outward signs of illness in these animals, they did cause a generalized infection. Spirochetemia was present during the first 6 days of infection. At 14 days postinfection, spirochetes could be isolated from one or more of the following organs: spleen, eyes, kidneys, liver, testes, and brain. Spirochetes were isolated from the eyes and kidneys of one animal 52 days postinfection, suggesting that these organisms may cause a persistent infection. Virulence of B. burgdorferi is maintained by animal passage but is lost upon prolonged in vitro cultivation.

Hamsters passively immunized with as little as 0.0125 ml of immune rabbit serum (indirect fluorescentantibody titer, 1:8,192) were protected from challenge with 1,000 50% infective doses of Borrelia burgdorferi. Cross-protection studies with Minnesota and Connecticut isolates of B. burgdorferi indicated no major differences in their capacity to elicit mutually protective antibodies in rabbits.

The immunogenicity of a single dose of a whole-cell vaccine of inactivated Borrelia burgdorferi1HSF (human spinal fluid isolate) was evaluated. Syrian hamsters were vaccinated subcutaneously and challenged by the intraperitoneal injection of 1,000 50% infectious doses of B. burgdorferi HSF 30 or 90 days postvaccination.Animals were sacrificed 14 days after challenge, and the kidneys and spleens were examined for spirochetes by cultural procedures. At 30 days postvaccination, 86 to 100% protection against infection was achieved in hamsters receiving 50 and 100 ,ug (dry weight) of vaccine. Protection was decreased to 60% with 25 ,ug of vaccine and was absent with 10 ,ug of vaccine. Resistance to infection decreased to 25, 40, and 5% for the 100-, 50-, and 25-,Lg vaccine doses, respectively, at 90 days postvaccination.

The correlation of plasmid profiles with infectivity was investigated by using five clones of Borrelia burgdorferi sensu stricto strain B31 (ATCC 35210). Plasmid profiles were determined by pulsed-field and two-dimensional gel electrophoresis. The 50% infectious dose (ID 50) in hamsters was determined. The ID 50 of the clone that possessed a full complement of eight linear and three circular plasmids was 10 3 cells. The loss of the 27.5-and 40-kb linear plasmids did not decrease the infectivity of these cells. Rather, the loss of the 27.5-kb linear plasmid was associated with a more disseminated infection. A moderate decrease of the ID 50 from 10 3 to 10 5 cells correlated with the loss of the 9.0-kb circular plasmid and the 27.5-kb linear plasmid. A major loss of infectivity (ID 50 > 10 8 cells) occurred with cells that lost the 24.7-and 27.5-kb linear plasmids and the 9.0-kb circular plasmid. A 3.0-kb HindIII fragment of the 24.7-kb linear plasmid was used as a probe to determine the presence of the homologous sequences in the three genospecies of Lyme disease spirochetes. An analysis of 21 infectious strains of B. burgdorferi sensu stricto, B. garinii, and B. afzelii revealed a consistent association of infectivity with strains possessing a linear plasmid (size range, 24 to 36 kb) that hybridized with the HindIII fragment. Western immunoblotting with hamster antisera against infectious B31 clone C-3 revealed two proteins with molecular masses of 28 and 43 kDa that were absent in the noninfectious B31 clone C-1. Additionally, a 14-kDa protein was absent in C-1 but present in infectious clone C-9 as shown by twodimensional polyacrylamide gel electrophoresis. Lyme disease is a multisystem disorder caused by three closely related Borrelia genospecies: Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii (1, 10, 12, 26, 37). B. burgdorferi sensu stricto will be referred to as B. burgdorferi hereafter in this work. The Borrelia genome is composed of one linear chromosome of about 950 kb with multiple linear and circular plasmids (3-5, 18, 24, 43, 45). Considerable diversity in plasmid profile among Lyme disease spirochetes obtained from different biological sources and geographical locations has been observed (3, 23, 45, 46). The function of plasmids in the virulence of B. burgdorferi is unknown. Genes specifying the outer surface lipoproteins (OspA,-B,-C,-D,-E, and-F) are located on plasmids. The genes ospAB, ospEF, and ospD are present on 49-, 45-, and 38-kb linear plasmids, respectively (7, 28, 32), while ospC is located on a 26-to 27-kb circular plasmid (19, 29, 38). Recently, the gene coding for exported plasmid protein A (EppA) was cloned from a 9-kb circular plasmid (15). The functions of these proteins in the virulence of B. burgdorferi remain to be elucidated. Serial in vitro cultivation of B. burgdorferi results in plasmid profile changes which are accompanied by the loss of infectivity in experimental animals (25, 31, 42). Previous reports (25, 31, 42) suggested a potential role for plasmids in ...