Genetic variability and expression of sphA and sphB genes and immunogenicity of SphA and SphB of Leptospira borgpetersenii serovar hardjo

Abstract

Leptospira borgpetersenii serovar hardjo isolates, (n=200), were examined by hybridization for restriction fragment length polymorphisms (RFLPs) in sphA and sphB. These genes, sphA and sphB, were previously cloned from L. borgpetersenii serovar hardjo (strain Sponselee) and encode a sphingomyelinase gene and a sphingomyelinase-related gene, respectively. A single RFLP was noted in sphA, while nine were detected in sphB. The RFLP detected in sphA was found in a single isolate and may be due to introduction of an insertion element. RFLPs detected in sphB were found in a region of tandem repeats within the open reading frame. Differing numbers of repeats of a 45 bp sequence were found in some isolates of serovar hardjo, while other isolates had a single copy of the 45 bp sequence followed by differing numbers of repeats of a 30 bp sequence. The sequence of the 30 bp element suggested deletion of 15 bp from the 45 bp sequence. Tests using reverse transcriptase-PCR showed that sphA and sphB were transcribed during growth of serovar hardjo in culture. Polyclonal antisera prepared against recombinant SphA and SphB reacted with a Triton X-114 outer membrane extract of serovar hardjo (strain Sponselee), which indicated that both sphA and sphB are transcribed, translated, and transported to the periplasmic space or outer membrane. Hamsters were immunized with recombinant SphA and SphB and challenged with a highly virulent isolate of serovar hardjo (strain 197). Hamsters were not protected from infection or disease when immunized with either SphA.