Molecular Characterisation Of Leptospiral Isolates Identified In Agricultural Labours Of Salem District Of Tamilnadu

摘要

In the present study, RAPD analysis of the two Leptospire isolates identified (K1 and K2) were subjected to random amplified polymorphic DNA fingerprinting analysis and compared with 5 genomospecies. Genotype characters of isolated K1 and K2 have poor resemblance with reference strain Panama CZ 14M, LSU 1945 and Sarmin CZ 390. The genomic profile of K1 and K2 were very similar and resemble the genomic profile of Rachmat belongs to the genomospecies Leptospira interrogans. The present study reports the prevalence of the leptospire belongs to Rachmat strain of the genomospecies Leptospira interrogans. Introduction Traditionally leptospires are classified and identified in serological techniques such as the cross-absorption tests (Gerritsen et al., 1995). However, these techniques are laborious and show poor inter-laboratory reproducibility (Kobayashi et al., 1985), but still cannot distinguish all serovars. The DNA based methods for the identification of Leptospira holds good results and these method include restriction fragment length polymorphism (RFLP) analysis (Terpstra et al., 1978; Hookey et al., 1987; Ellis et al., 1991); DNA-DNA hybridisation (Perolat et al., 1993); Pulsed field gel electrophoresis (PFGE) (Herrmann et al., 1992); Polymerase chain reaction followed by RFLP analysis (Ralph et al., 1993) and random amplified polymorphic DNA (RAPD) fingerprinting (Corney et al., 1993; Natarajaseenivasan et al., 2004). RFLP analysis is generally more sensitive and discriminatory than serotyping, but some serovars are not readily distinguished. (Thiermann et al., 1986), and the complex banding patterns are difficult to interpret. Hybridization of labelled DNA probes to RFLP blots greatly simplifies the visualised banding patterns facilitating interpretation. (Van Eys et al., 1991) and allows serovars with similar RFLP banding pattern to be differentiated (Zuerner et al., 1993). RFLP analysis, DNA-DNA hybridization and PFGE are slow and require large amounts of purified DNA. In contrast, PCR - based methods are rapid and require only small amounts of DNA. (Van Eys et al., 1989; Gerritsen et al., 1991; Merien et al., 1992; Gravekamp et al., 1993). PCR-based fingerprinting systems have been well developed for a range of bacteria. (Corney et al., 1993; Giesendorf et al., 1993; Ralph et al., 1993; Van Belkum et al., 1993; Woodward et al., 1993). Because of some disadvantage in the application of cross-absorption agglutination (CAA) technique and in restriction endonuclease analysis (REA) alternative methods were searched. Corney et al. (1993) stressed the need to develop a rapid and simple typing method which can distinguish different genotype without the disadvantages of CAA and REA. Rapid identification of the isolates would allow farmers to start appropriate vaccination regimens with minimal delays. Welsh and McClelland (1990) and Williams et al. (1990) developed a DNA fingerprinting technique based on the random amplification of genomic sequences by using a single primer at low stringency in a polymerase chain reaction (PCR). Gerritsen et al. (1995) investigated the use of leptospiral and non leptospiral primers in RAPD fingerprinting of leptospirosis. Perolat et al. (1998), Brenner et al. (1999) and Levett (2001) reported more than 16 genomospecies of leptospires based on genotypic characters. The molecular analysis of new isolates from different regions may increase genomospecies number of leptospires. With this background the leptospires isolates collected in the present study were subjected to random amplification of polymorphic DNA finger printing analysis to find out whether they are new genomospecies or related to the already existing genomospecies. Material and Methods Isolation of genomic DNA from Leptospiral isolates Blood samples from Leptospiral infected patients were inoculated into sterile EMJH medium and the isolated leptospiral strains (K1 and K2) were grown at 300C in EMJH medium for 7 days to a density of 108