High- and low-infectivity phenotypes of clonal populations of in vitro-cultured Borrelia burgdorferi

Abstract

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 B31 strains of Borrelia burgdorferi were isolated by subsurface plating on BSK-II agar plates and examined for infectivity in the C3H/HeN mouse model. Mice were injected intradermally with 10 5 B. burgdorferi organisms, 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 which cultures were consistently positive at all organ sites, or a low-infectivity phenotype, in which a low proportion of cultures were positive (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 lowinfectivity phenotype. The proportion of high-infectivity clones decreased with continued in vitro passage, with only 1 of 10 clones exhibiting the high-infectivity phenotype after 10 passages and 0 of 10 clones yielding positive cultures after 20 passages. Representative high-and low-infectivity clones from passage 5 Sh2 cultures had 50% infectious doses of 1.8 ؋ 10 2 and 1 ؋ 10 5 , respectively. Subclones consistently reflected the same infectivity phenotypes as those of the parent clones. The protein profiles and plasmid contents of the high-and low-infectivity clones were compared and exhibited few discernible differences. On the basis of these results, the loss of infectivity during in vitro culture results from the outgrowth of low-infectivity clones and begins to occur within the first five in vitro passages. Further examination of clonal populations may lead to the identification of genetic and protein factors important in the virulence and pathogenicity of Lyme disease borrelias.

Interaction ofArcobacterspp. with human and porcine intestinal epithelial cells

“…However, upon repeated testing, the invasiveness of the porcine A. cryaerophilus strain 1038 was lost. Apparently, in vitro subculture of the strain had resulted in a change in virulence; this has also been reported for other pathogenic bacteria, including Helicobacter and Campylobacter (Norris et al, 1995;On, 1998;Kim et al, 2002;Gaynor et al, 2004).…”

Molecular Adaptation of Borrelia burgdorferi in the Murine Host

“…B. burgdorferi has coevolved with the mammalian (immunocompetent) host and tick vector for a long period of time. The in vitro artificial conditions probably constitute an aberrant environment where B. burgdorferi changes protein expression profiles and can more easily lose plasmids, infectivity, and pathogenicity (26).…”

A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi

“…After the isolation of clone 4a from a solid agar colony, passage through a BALB/c mouse for 4 weeks and re-isolation from the mouse (see Casjens et al, 1997a), it carried all of the plasmids whose sequences are known except for cp9, lp5, lp28-3 and lp28-4 (data not shown). The plasmids missing in clone 4a may well have been lost during the cloning/mouse passage procedure because Borrelia strains have often been found to lose one or more plasmids during laboratory propagation and cloning procedures (for example Barbour, 1988;Schwan et al, 1988;Persing et al, 1994;Norris et al, 1995;Xu et al, 1996).…”