Identification of new repetitive element in Leptospira interrogans serovar copenhageni and its application to PCR-based differentiation of Leptospira serogroups

Abstract

A new repetitive DNA element was identified in an isolate of Leptospira interrogans serovar copenhageni from a patient in Salvador, Brazil. A Sau3A genomic library from this strain was constructed and screened for repetitive DNA elements. An insert of 438 bp (Rep1) from one library clone hybridized to multiple chromosomal DNA fragments resolved electrophoretically after digestion with BamHI, HindIII, and MfeI. A single oligonucleotide primer, designated iRepl, was designed to generate multiple PCR amplicons of various electrophoretic mobilities in a PCR typing method. The method distinguished strains belonging to the eight pathogenic and three saprophytic species of the genus Leptospira. Clinical isolates obtained during urban epidemics between 1996 and 1998 in Salvador, Brazil, were analyzed by this PCR method. Although the iRep1 primer was unable to discriminate strains among L. interrogans serovar copenhageni isolates, it was able to differentiate strains belonging to different species and serogroups of Leptospira identified in Salvador. This PCR-based method may provide a faster and less expensive alternative to serologic tests used in reference laboratories.

FIG. 1

Distribution of the repetitive element among reference strains representing 11 species of the genus Leptospira. (A) Genomic DNA from a clinical isolate of L. interrogans serovar copenhageni was digested with BamHI (lane 2), MfeI (lane 3), and HindIII (lane 4). (B) Genomic DNA from leptospiral species was digested with BamHI, separated on a 0.7% agarose gel electrophoresis, transferred to a positively charged nylon membrane, and hybridized with Dig-labeled pRep1. Genomic DNA was analyzed from the following species: L. interrogans serovar copenhageni strain Winjberg (lane 2), L. interrogans serovar copenhageni strain L1-130 (lane 3), L. borgpetersenii serovar castelloni strain Castellon 3 (lane 4), L. weilii serovar celledoni strain Celledoni (lane 5), L. noguchi serovar panama strain CZ 214 K (lane 6), L. santarosai serovar shermani strain 1342 K (lane 7), L. kirshneri serovar grippotyphosa strain Moskva V (lane 8), L. inadai serovar lyme strain 10 (lane 9), L. fainei serovar hurstbridge strain BUT6 (lane 10), L. meyeri serovar ranarum strain Ranae (lane 11), and L. biflexa serovar patoc strain Patoc1 (lane 12). The Dig-labeled molecular weight marker set VII is represented in lane 1.

FIG. 2

Sequence analysis of the repetitive element identified from a genomic library of L. interrogans serovar copenhageni strain L1-130. Structural features of the repetitive element are shown including the location of primer iRepl used for PCR genotyping. iRep1 primer is shown as the reverse complement of the target sequence.

FIG. 3

iRep1 PCR-based molecular typing of reference strains from the genus Leptospira. DNA from boiled culture pellets was used in PCRs with the iRep1 primer for L. interrogans serovar autumnalis strain Akiyami A (lane 2), serovar icterohaemorrhagiae strain RGA (lane 3), serovar copenhageni strain Winjberg (lane 4), serovar wolfii strain 3705 (lane 5), and serovar hardjo strain Hardjoprajitno (lane 6); L. borgpetersenii serovar hardjo strain Lely 607 (lane 7), serovar sejroe strain M84 (lane 8), and serovar castellonis strain Castellon 3 (lane 9); L. weilii serovar celledoni strain Celledoni (lane 10); L. noguchi serovar panama strain CZ 214 K (lane 11); L. santarosai serovar shermani strain 1342 (lane 12); L. kirshneri serovar grippotyphosa strain Moskva V (lane 13); L. wolbachi serovar codice strain CDC (lane 14); L. inadai serovar lyme strain 10 (lane 15); L. fainei serovar hurstbridge strain BUT6 (lane 16); L. meyeri serovar ranarum strain ranae (strain 17); and L. biflexa serovar patoc strain Patoc1 (lane 18). A negative control sample without DNA is shown in lane 19. The positions of the 100-bp size markers are represented in lanes 1 and 20.

FIG. 4

iRep1 PCR-based molecular typing of human and rat Leptospira strains isolated from an urban epidemic in Salvador, Brazil. DNA from boiled Leptospira culture pellets was amplified with the iRep1 primer. The following samples were identified as L. interrogans serovar copenhageni except for L1-133, shown in lane 3 (L. interrogans serovar canicola). The following clinical isolates of L. interrogans serovar copenhageni were evaluated: L1-130 (lane 5), L1-212 (lane 6), L8-38 (lane 7), L8-118 (lane 8), and L8-163 (lane 9). Rat isolates included R1-15 (lane 10), R1-98 (lane 11), and R1-147 (lane 12), and R1-152 (lane 13). Lanes 2 and 4 represent reference strains L. interrogans serovar canicola strain Hond Utrecht IV and L. interrogans serovar copenhageni strain Winjberg, respectively. The positions of the 100-bp size markers are represented in lanes 1 and 14.