Successful in vitro cultivation of Borrelia duttonii and its comparison with Borrelia recurrentis.

Abstract

Borrelia duttonii, the cause of East African tick-borne relapsing fever, has until now been refractory to growth in laboratory media. This spirochaete has only be propagated in mice or by tissue culture, restricting both yield and purity of cells available for research. The successful isolation of five clinical isolates of B. duttonii from patients in Central Tanzania and their comparison with Borrelia recurrentis is reported. Electron microscopy revealed spirochaetal cells with pointed ends, a mean wavelength of 1.8 microns with an amplitude of 0.8 micron, similar to the findings for B. recurrentis. Cells contained 10 periplasmic flagella inserted at each end of the spirochaete, again comparable with the counts of 8-10 flagella found in B. recurrentis. PFGE revealed a chromosome of approximately 1 Mb, a large plasmid of approximately 200 kb, and a small plasmid of 11 kb in all strains of B. duttonii and in B. recurrentis. B. duttonii possessed a further 7-9 plasmids with sizes ranging from 20 to 90 kb. In two isolates of B. duttonii, the profiles were identical. In contrast, all 18 isolates of B. recurrentis fell into one of five plasmid patterns with 3-4 plasmids ranging from 25 to 61.5 kb in addition to those of 11 and 200 kb described above. Analysis of the SDS-PAGE profiles of B. duttonii strains revealed a high-molecular-mass band of 33.4-34.2 kDa in four strains (variable large protein, VLP) and a low-molecular-mass band of 22.3 kDa in the remaining strain (variable small protein, VSP). This resembles the protein profiles found in B. recurrentis. The G + C ratio of B. duttonii was 27.6 mol%. Nucleotide sequence of the rrs gene (16S rRNA) from four B. duttonii isolates revealed 100% identity among these strains and 99.7% homology with three strains deposited by others in GenBank. The rrs gene of eight representative clinical isolates of B. recurrentis confirmed their close similarity with B. duttonii.