Genospecies identification and characterization of Lyme disease spirochetes of genospecies Borrelia burgdorferi sensu lato isolated from rodents in Taiwan

Abstract

Lyme disease spirochetes of the genospecies Borrelia burgdorferi sensu lato were identified and characterized for the first time in Taiwan. Seven isolates, designated TWKM1 to TWKM7, were purified from the ear tissues of three species of rodents captured from seven localities of Taiwan. The immunological characteristics of these Taiwan isolates were compared with those of other genospecies of Lyme disease spirochetes by analyzing the protein profiles and reactivities with B. burgdorferi-specific monoclonal antibodies (MAbs). The genospecies of these Taiwan isolates were also identified by the similarities in their plasmid profiles and differential reactivities with genospecies-specific PCR primers. Although two distinct protein profiles were observed among the seven Taiwan isolates, the MAb reactivities against the outer surface proteins of B. burgdorferi of all of these isolates were consistent with those of B. burgdorferi sensu lato. The similarities of the plasmid profiles also confirmed the identities of these Taiwan isolates. PCR analysis indicated that all of these Taiwan isolates were genetically related to the genospecies B. burgdorferi sensu stricto. These results demonstrate the first identification of Lyme disease spirochetes in Taiwan and also highlight the increasing demand for defining the reservoirs and vector ticks of B. burgdorferi. A serosurvey for Lyme disease infection in the human population of Taiwan may also be required.

FIG. 1

SDS-PAGE of whole-cell lysates of Borrelia isolates. The 12.5% gel was revealed by Coomassie brilliant blue staining. Lane B, American type strain B31 (control); lanes 1 to 7, Taiwan isolates TWKM1 to TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii. Molecular mass standards (M) are provided on the left (in kilodaltons). Arrows identify the OspA, OspB, and flagellin proteins of B. burgdorferi sensu lato.

FIG. 2

Western immunoblot analysis of Borrelia isolates with MAbs against OspA (H5332 and H3TS), OspB (H6831 and H614), flagellin (H9724), and p39 (anti-p39) proteins of B. burgdorferi sensu lato. Lane B, American type strain B31; lanes 1 to 7, Taiwan isolates TWKM1 to TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii.

FIG. 3

Plasmid profiles of Borrelia isolates from Taiwan and three genospecies of Lyme disease spirochetes. Lane B, American type strain B31; lanes 1 to 7, Taiwan isolates TWKM1 to TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii; lanes M, the high-molecular-mass DNA markers (Gibco BRL).

FIG. 4

Amplification specificities of the universal-type (a) and B31 (b) primer sets with Borrelia isolates from Taiwan and three genospecies of Lyme disease spirochetes. Lane B, B31 isolate; lanes 1 to 7, Taiwan isolates TWKM1 to TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii; lanes M, 100-bp DNA ladder (GIBCO BRL). The amplification products for the universal-type (a) and B31 (b) primers were DNA fragments with size of 127 and 374 bp, respectively.

FIG. 5

Amplification specificities of the primer sets of BB (a), BG (b), and VS461 (c) with Borrelia isolates from Taiwan and three genospecies of Lyme disease spirochetes. Lane B, B31 isolate; lanes 1 to 7, Taiwan isolates TWKM1 TWKM7, respectively; lane J, JD1 isolate of B. burgdorferi sensu stricto; lane K, K48 isolate of B. garinii; lane V, VS461 isolate of B. afzelii; lanes M, 100-bp DNA ladder (Gibco BRL). The expected amplification products for primer sets BB, BG, and VS461 were DNA fragments with sizes of 575, 575, and 590 bp, respectively.