Genotyping of Razi vaccinal Leptospira Serovars based on VNTR(MLVA)method

Abstract: Leptospirosis is an emergency zoonotic diseases which caused by pathogenic Leptospires. The incidence of the disease is higher in tropical and subtropical climates. In tropical regions of the world, leptospirosis is a widespread public health problem. Detection and identification of Leptospires has conventionally been performed by cultural and serological methods. These methods are tedious, time consuming, and potentially biohazardous. Recently several PCR based techniques have been demonstrated to provide useful tools in identification of leptospiral serovars but most these techniques have drawbacks. The VNTRs can provide good information relating to both the evolutionary and epidemiology of bacterial diversity. The present study was carried out to genotyping of vaccinal Leptospira serovars by VNTR method. Five vaccinal Leptospira serovars including Leptospira interrogans serovars Grippotyphosa, Canicola, Hardjo, Icterohemoragaea and Pomona and one saprophytic L.biflexa serovar Semaranga were used in this study obtained from the Leptospira Reference Laboratory, Razi Vaccine and Serum Research Institute, Karaj, Iran. The bacteria were inoculated into selective EMJH medium. Genomic DNA of leptospiral serovars were extracted using the phenol-chloroform method. PCR was performed with the 10 selected VNTR loci (4, 10, 7, lb5, 8, 19, 29, 30, 50 and 36). The amplified products were analyzed by 1.5% agarose gel electrophoresis. All loci successfully amplified in all leptospiral serovars with the primers. Although the sizes of the amplified products displayed a wide range of polymorphisms among the serovars. No PCR products for non- pathogenic serovars at VNTR-4, lb5, 19, 30 were observed. VNTR-4 was unable to discriminate between L. grippotyphosa and L. hardjo, L. icterohemoragaea and L. pomona, this locus was thoroughly differentiated between pathogenic and non-pathogenic. VNTR-10 had an appropriate discrimination to differentiate among these serovars but was unable to differentiate between L. pomana and L. semaranga. The VNTR-36 separated L. Pomona from other serovars. VNTR-lb5 succeed in distinguish between L.canicola and L.hardjo. VNTR 7 and 30 were only amplified in non- pathogenic serovars. The combination of the VNTR-4, VNTR-10, and VNTR-36 loci were useful for typing of L. interrogans serovars. The results showed high range of polymorphisms for these VNTR loci among the leptospiral serovars. No locus could differentiate all serovars and combination of them was useful for typing L.interrogans. The VNTR method provides rapid molecular typing as well as a highly discriminate to identify L.interrogans serovars. The method based on VNTR polymorphism provides rapid typing as well as a highly discriminant assay to identify L. interrogans serovars. In addition, VNTR typing could be widely accessible for research and public health laboratories, particularly in developing countries. Key words: Leptospirosis, Leptospira.spp, VNTR technique, PCR