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

Abstract

Borreiia 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 pm with an amplitude of 0 8 pm, similar to the findings for B. recurrentis. Cells contained 10 periplasmic flagella inserted a t 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 highmolecular-mass band of 334-3402 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 mol0/o. Nucleotide sequence of the rrs gene (16s rRNA) from four B. duffonii isolates revealed 100% identity among these strains and 997 O/ O 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.

Typing African Relapsing Fever Spirochetes

“…Cultivating these fastidious microbes is challenging, with some, until recently, considered noncultivable ( 16 – 18 ). Furthermore, the complexity of coexistent mechanisms for antigenic variation with associated potential for major genomic reorganization presents a problem for many microbiologic typing approaches.…”

“…Furthermore, the complexity of coexistent mechanisms for antigenic variation with associated potential for major genomic reorganization presents a problem for many microbiologic typing approaches. Isolates that are homogeneous by gene sequence–based approaches may present vastly different pulsed-field electrophoretic profiles ( 16 , 17 , 26 ), possibly through extensive genetic duplication associated with antigenic variation of major outer membrane proteins, whose genes are carried on large linear plasmids ( 7 , 11 ). …”

“…Borrelial isolates were obtained from blood samples drawn from patients with clinical cases of relapsing fever as previously described ( 16 – 18 ). Borrelia spp.…”

A novel, fast-growing Borrelia sp. isolated from the hard tick Hyalomma aegyptium in Turkey

“…is 12-18 h (Kelly, 1984). The spirochaete reached high numbers in 2 days, which can be considered astonishing when compared to well-growing Borrelia spp., such as B. burgdorferi sensu stricto, and fastidious spirochaetes, such as Borrelia duttonii, which has recently been cultured in vitro (Cutler et al, 1999). …”

Relapsing Fever Borreliae in Africa

“…Because relapsing fever borreliae remained uncultured, they were classified on the basis of their vectors and virulence in animal models. 8 Indeed, Borrelia hispanica remained uncultured in axenic medium until 1976, 9 Borrelia recurrentis until 1994, 10 and Borrelia duttonii until 1999 11 ; Borrelia crocidurae, first described in musk shrew blood in Senegal in 1917, 12 was only cultured in axenic medium in 1999. 13 Other relapsing fever borreliae have been identified along the eastern border of Africa in Eurasia, chiefly Borrelia persica in Israel and the Palestinian territories, 14 Iran 15 and Uzbekistan and Tajikistan, 16 and Borrelia microti in Iran.…”