Journal of Medical Microbiology (2003), 52, 909–911 DOI 10.1099/jmm.0.05101-0 05101 & 2003 SGM Printed in Great Britain 909 Short Communication Correspondence S. C. Sehgal pblicmr@sancharnet.in Received 16 October 2002 Accepted 14 May 2003 Antigenic and genetic relatedness of Leptospira strains isolated from the Andaman Islands in 1929 and 2001 Subarna Roy, D. Biswas, P. Vijayachari, A. P. Sugunan and S. C. Sehgal National Leptospirosis Reference Centre, Regional Medical Research Centre (Indian Council of Medical Research), Post Bag no. 13, Port Blair 744101, Andaman & Nicobar Islands, India Leptospirosis is a major public health problem in Andaman Islands. Several strains of Leptospira have been isolated from the Andamans over the years. Leptospires isolated recently from human cases were compared with one of the earliest available isolates from these islands, dating back to 1929, to study their serological and genetic relatedness. Randomly amplified polymorphic DNA (RAPD) fingerprints of the isolates, generated with a primer used previously to differentiate between Leptospira species and serovars, revealed that some of the recent isolates were genetically identical to the 1929 isolate. The antigenic properties of these strains, as revealed by microscopic agglutination tests with group-specific rabbit antisera and mAbs, were also similar. These findings suggest that a Leptospira strain originally isolated in 1929 has possibly persisted in these islands for over 70 years and continues to cause acute leptospirosis in humans. Introduction Leptospirosis is emerging as one of the world’s most wide- spread zoonotic diseases (Plank & Dean, 2000; Yang et al., 2001; Levett, 2001). The earliest authentic report of lepto- spirosis from India dates back to 1929, when Taylor & Goyle (1931) isolated 28 strains of leptospires from 78 suspected cases showing typical signs and symptoms of Weil’s syn- drome among convict labourers in the Andaman Islands. No further information was available regarding the status of leptospirosis in these islands until 1988, when post-monsoon outbreaks of a febrile illness with haemorrhagic tendencies started to appear. The mortality rate among patients was very high. Despite several efforts, the aetiology could not be established and, hence, the name ‘Andaman haemorrhagic fever’ (AHF) was given to this clinical entity. The clinical presentation during these outbreaks was characterized by sudden onset of fever, body ache, myalgia, cough, with or without haemoptysis and other haemorrhagic tendencies. Based on serological evidence, Sehgal et al. (1995) confirmed the cause of these outbreaks as leptospirosis. Bacteriological confirmation was subsequently established (Sehgal et al., 2000). Since then, several isolates of leptospires have been obtained and attempts have been made to characterize these strains serologically and by using molecular techniques. The present study was undertaken with the specific objective of examining any serological and genetic relatedness between the recent isolates and the isolates obtained in 1929. Methods Strains and isolates. Strain CH31, belonging to serogroup Grippo- typhosa, was originally isolated from a patient in the Andaman Islands in 1929 (Taylor & Goyle, 1931). This strain was obtained from the WHO/FAO Collaborating Centre for Reference and Research on Leptospirosis, Amsterdam, The Netherlands. Four isolates, designated D22, Mg47, Mg51 and Mg100, were recovered from suspected cases attending a primary health centre on South Andaman in recent years. All cultures were maintained in EMJH culture medium (Difco) at 30 8C. Serotyping Microscopic agglutination test (MAT) with group sera. All five isolates were screened against 36 group-specific rabbit antisera representing 23 serogroups following standard procedures (Wolff, 1954). An isolate was considered to belong to the serogroup of the group serum that gave the highest titre (Dikken & Kmety, 1978). MAT with mAbs. A panel of four mouse mAbs (F71C3, F71C9, 165C3 and 165C8) developed at the Dutch Royal Tropical Institute (Amster- dam, The Netherlands) that distinguish serovars of serogroup Grippo- typhosa was used. Microscopic agglutination was performed using each strain against all the mAbs in the panel. The antigenic profile was constructed by plotting the reciprocal titre against the mAb on a semi- logarithmic scale. This pattern for each strain was then compared with that of CH31. Preparation of genomic DNA. Genomic DNA of the bacterial strains was extracted following the method described by Boom et al. (1990). DNA was dissolved in Milli-Q water and used for randomly amplified polymorphic DNA (RAPD) analysis. RAPD analysis. The reference primer PB1 (59-GCGCTGGCTCAG- 39), used previously to differentiate between Leptospira species and serovars (Brown & Levett, 1997; Ramdass et al., 2002), was used to Abbreviations: MAT, microscopic agglutination test; RAPD, randomly amplified polymorphic DNA.