HIGH-INFECTIVITY AND LOW-INFECTIVITY PHENOTYPES OF CLONAL POPULATIONSOF IN VITRO-CULTURED BORRELIA-BURGDORFERI

Borrelias that cause Lyme disease lose the ability to infect and cause disease in laboratory animals following 10 to 16 passages of in vitro culture. In this study, clonal populations of the Sh-2-82 (Sh2) and B 31 strains of Borrelia burgdorferi were isolated by subsurface plating on BSK-II agar plates and examined for infectivity in the C3H/HeN mou se model. Mice were injected intradermally with 10(5) B. burglorferi o rganisms, and the tibiotarsal joint, heart, and bladder were cultured 2 to 4 weeks postinfection to determine whether viable organisms were present. clones exhibited either a high-infectivity phenotype, in whic h cultures were consistently positive at all organ sites, or a low-inf ectivity phenotype, in which a low proportion of cultures were positiv e (5 of 40 in a representative experiment). In an Sh2 population that had undergone five in vitro passages, 7 of 10 clones were of the high- infectivity phenotype, and the remaining clones were of the low-infect ivity phenotype. The proportion of high-infectivity clones decreased w ith continued in vitro passage, with only 1 of 10 clones exhibiting th e high-infectivity phenotype after 10 passages and 0 bf 10 clones yiel ding positive cultures after 20 passages. Representative high- and low -infectivity clones from passage 5 Sh2 cultures had 50% infectious dos es of 1.8 x 10(2) and 1 x 10(5), respectively. Subclones consistently reflected the same infectivity phenotypes as those of the parent clone s. The protein profiles and plasmid contents of the high- and low-infe ctivity clones were compared and exhibited few discernible differences . On the basis of these results, the loss of infectivity during in vit ro culture results from the outgrowth of low-infectivity clones and be gins to occur within the first five in vitro passages. Further examina tion of clonal populations may lead to the identification of genetic a nd protein factors important in the virulence and pathogenicity of Lym e disease borrelias.