Molecular and pathogenic characterization of Borrelia burgdorferi sensu lato isolates from Spain

Abstract

Fifteen Borrelia burgdorferi sensu lato isolates from questing ticks and skin biopsy specimens from erythema migrans patients in three different areas of Spain were characterized. Four different genospecies were found (nine Borrelia garinii, including the two human isolates, three B. burgdorferi sensu stricto, two B. valaisiana, and one B. lusitaniae), showing a diverse spectrum of B. burgdorferi sensu lato species. B. garinii isolates were highly variable in terms of pulsed-field gel electrophoresis pattern and OspA serotype, with four of the seven serotypes described. One of the human isolates was OspA serotype 5, the same found in four of seven tick isolates. The second human isolate was OspA serotype 3, which was not present in ticks from the same area. Seven B. garinii isolates were able to disseminate through the skin of C3H/HeN mice and to cause severe inflammation of joints. One of the two B. valaisiana isolates also caused disease in mice. Only one B. burgdorferi sensu stricto isolate was recovered from the urinary bladder. One isolate each of B. valaisiana and B. lusitaniae were not able to disseminate through the skin of mice or to infect internal organs. In summary, there is substantial diversity in the species and in the pathogenicity of B. burgdorferi sensu lato in areas in northern Spain where Lyme disease is endemic.

FIG. 1

Phylogenetic tree of B. burgdorferi strains based on the sequence of the 16S rRNA gene as described in the text. The scale under the tree measures the distance between sequences.

FIG. 2

PFGE separation of MluI-digested (A) and SmaI-digested (B) genomic DNA. Lanes: L, DNA lambda concatemers (48.5 to 485 kb); 1, strain TI-1; 2, Esp1; 3, PV2; 4, PV1; 5, PBi; 6, Rio4; 7, PV6; 8, PV4; 9, Rio1; 10, Rio2; 11, Rio3; 12, Rio5; 13, PV7; 14, Rio6; 15, PV3; 16, PV5; 17, CL1. The arrowhead indicates the 135-kbp band characteristic of B. burgdorferi sensu stricto, and the arrows indicate the 220- and 80-kbp bands characteristic of B. garinii.

FIG. 3

PFGE separation of the total undigested genome of the B. burgdorferi sensu lato isolates and reference strains. Lanes: M, DNA molecular size markers of 8.3, 8.6, 10.1, 12.2, 15.0, 17.1, 19.4, 22.6, 24.8, 29.9, 33.5, 38.4, and 48.5 kb; 1, Rio4; 2, Rio3; 3, Rio5; 4, Rio2; 5, CL1; 6, Rio6; 7, PV7; 8, PV6; 9, PV4; 10, PV5; 11, Esp1; 12, PV2; 13, PV1; 14, PV3.

FIG. 4

SDS-PAGE and Coomassie blue staining of B. burgdorferi sensu lato isolates. In each panel, the protein profiles of B31^T (B. burgdorferi sensu stricto), PBi (B. garinii), and VS461^T (B. afzelii) are also given. Lanes M contain molecular mass markers of the sizes shown. (A) Isolates from Basque Country. Lanes: 1, B31; 2, Esp1; 3, PV2; 4, PV1; 5, PV3; 6, PBi; 7, PV6; 8, PV4; 9, PV5; 10, VS461; 11, PV7. (B) Isolates from La Rioja. Lanes: 1, B31; 2, VS461; 3, Rio6; 4, PBi; 5, Rio4; 6, Rio2; 7, Rio3; 8, Rio5.

FIG. 5

Phylogenetic tree of B. burgdorferi strains based on the sequence of the ospA gene as described in the text. The scale under the tree measures the distance between sequences. OspA serotypes of each strain are given in parentheses.

FIG. 6

Reactivity of sera from the B. burgdorferi PV1, PV3, PV7, and Rio6 isolates by Western blotting to the respective homologous strain. M, molecular mass markers of the sizes shown.