Leptospire Cultivation and Identification

Culture of leptospires from human blood was performed using EMJH supplemented with 3% rabbit serum and 0.1% agarose, as described previously [9] . Positive cultures were sent to the WHO/FAO/OIE Collaborating Center for Reference & Research on Leptospirosis, Australia for serovar identification using the cross agglutinin absorption test (CAAT) [10] . Definitive identification of species was undertaken by amplification and sequencing of the near full-length 16S rRNA gene. Primers were designed to anneal to conserved regions of genes from pathogenic species L. interrogans, L. kirschneri, L. borgpetersenii, L. santarosai, L. alexanderi and L. fainei. The primers (f - 5′ GTTTGATCCTGGCTCAG 3′ and r -5′CCGCACCTTCCGATAC 3′) amplified a 1,483 bp PCR product which was sequenced in its entirety using internal primer pairs (primers available on request).